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V " X 9 G 0 w V j k J k D 6W k V 6W t ! FL $ ! ! ! D D K ^ ! ! ! w P# P# P# P# s Committee Certification of Approved Version
HIDDEN TEXT: NOTE: this page in hard copy with all original signatures must be submitted with the dissertation to the Graduate School; this is required whether the document is in electronic format or on paper. This page is bound into any bound copies as the first page in the preliminary pages and it is counted in the page numbering. This page also is included in an electronic copy, but there are no signatures --- the signed copy is in the students file in the graduate school office.
The committee for Melanie Cree Green certifies that this is the approved version of the following dissertation:
The interaction between glucose and fat metabolism and insulin resistance in severely burned children and the metabolic effects of Fenofibrate
Committee:
Robert R. Wolfe, PhD SupervisorAsle Aarsland, MD, PhDRandall Urban, MDKarl Anderson, MDDavid Chinkes, PhD
Lynis G. Dohm, PhD
______________________________
Dean, Graduate School
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The interaction between glucose and fat metabolism and insulin resistance in severely burned children and the metabolic effects of Fenofibrate
by
Melanie Cree Green, BA
HIDDEN TEXT: name should be identical to the name on your UTMB transcript. Abbreviate previous degrees earned.
Dissertation
Presented to the Faculty of The University of Texas Graduate School of
Biomedical Sciences at Galveston
in Partial Fulfillment of the Requirements
for the Degree of
Clinical Science
Approved by the Supervisory Committee
Robert R. Wolfe, PhD
Randall Urban, MD
Lynis G. Dohm, PhD
Asle Aarsland, MD, PhD
David Chinkes, PhD
Karl Anderson, MD
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August 2005
Galveston, Texas
Key words:
Insulin resitance, aging, burn trauma, glucose metabolism, fat metabolsim
2005, Melanie Cree Green
This work is dedicated to the children described herein, and their families.
Acknowledgements
First to my husband Justin Green, and my family, Holly, Jonathan and Jeremy Cree for all of their support.
The collaborators on this project: Ting Qian, MD, Beatrice Moriro, PhD, Bradley Newcomer, PhD, Jennifer Zwetsloot, BS, Lynis Dohm, PhD, and Jun Martini, PhD.
This project was only made possible through the combined effort of numerous people, to whom I am indebted for their time, dedication and friendship. The technical staff in the metabolism lab have been invaluable, including Mary Finn, Shelter Dziya, Ann Hightower, Natalie Ngyden, Stephanie Blas, Chris Danesi, Farai B, Melissa Bailey, Tara Cocke, Dayoung Sun, Ming Zheng, Guy Jones, and Jariwala Guarang.
The metabolism nurses, Dan Creson, Lyzanne Sargeant, Scott Schutzler, Deanne Jordan, and the 8th floor nurses including Charles Mitchell, Veronica Honc, Wesley Benjamin, Sylvia Ojeda, Lupe Jecker, Phyllis Calabrese and Rosa Chappa.
Michael Buffalo, Mario Celias, and Peter Yen for providing the expertise for conducting muscle biopsies and maintaining patient safety.
The multiple physicians involved in the care of the patients, including David Herndon, Art Sanford, Jong Lee, Walter Meyers, Steve Wolf, Dennis Gore, Carlos Angel, the burn fellows Orlando Beckham, Amalia Chochran, Warren Gold, Ricki Fram, and the numerous residents that have rotated through the Shriners hospital.
The 2 east clinical staff and the Shriners respiratory staff for conducting the metabolic carts and liver ultrasound measurements, and providing assistance with MRIs on intubated patients.
Thank you all!!
The funding for this project was provided by
Robert R. Wolfe: NIH R01DK041317 and Shriners 8490
David Herndon: NIH R01 GM 56687
Randall Urban: Stark Foundation Grant
James Goodwin: Pepper Center Grant P60 AG17231-01
GCRC: NIH USPHS M01-RR-00073 The interaction between glucose and fat metabolism and insulin resistance in severely burned children and the metabolic effects of Fenofibrate
Publication No._____________
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Melanie Cree Green, PhD
The University of Texas Graduate School of Biomedical Sciences at Galveston, 2005
Supervisor: Robert R. Wolfe
fillin \* mergeformat Insulin resistance occurs in conjunction with disturbances in fat and glucose metabolism and mitochondrial dysfunction. Four human studies were conducted to better understand this relationship in the settings of aging and burn trauma.
The first two studies focused on aging. Insulin resistance commonly occurs in aging, but it was unknown if this was accompanied by changes in fat metabolism. The first study examined tissue fat in healthy young and elderly in relation to insulin sensitivity. The second study attempted to decrease insulin sensitivity by increasing the mitochondrial oxidation rate of intracellular fat, and lower plasma lipids with the peroxisome proliferator activating receptor alpha agonist fenofibrate.
The final two studies focused on burn trauma in children. Children who experience severe flame burn rapidly develop insulin resistance and hyper-metabolic fat, protein and glucose regulation. This insulin resistance has been linked to increases in mortality and morbidities such as infection and delayed wound healing. It is hypothesized that the insulin resistance and metabolic dysregulation develop cumulatively for several weeks following a burn and that treatment between weeks 1 and 3 post-burn with fenofibrate will increase lipid oxidation and improve insulin sensitivity, as compared to changes seen in placebo treated children. Study three examined glucose kinetics including a) whole body glucose uptake during a hyperinsulinemic-euglycemic clamp, b) endogenous glucose production, c) glucose uptake across the leg and d) fasting glucose and insulin levels measured at 1 and 3 weeks post-burn, before and after treatment. Additionally, lipid kinetics, as reflected by a) free fatty acid oxidation b) fatty acid release c) intracellular triglyceride in the liver and muscle, and intracellular diacyglycerol and fatty acyl CoA were measured at these time points. Finally, the activity of the insulin signaling pathway in muscle was also measured, as was mitochondrial function. The last study served to quantitate the level of mitochondrial dysfunction following burn, by comparing the mitochondrial function in the burn trauma patients to that in healthy children.
Table of Contents
Page
TOC \h \z \t "Heading 1,2,Heading 2,3,Heading 3,4,Chapter Title,1" HYPERLINK \l "_Toc114382104" List of Tables PAGEREF _Toc114382104 \h xii
HYPERLINK \l "_Toc114382105" List of Figures PAGEREF _Toc114382105 \h xiv
HYPERLINK \l "_Toc114382106" List of Figures PAGEREF _Toc114382106 \h xiv
HYPERLINK \l "_Toc114382107" IntroducTion PAGEREF _Toc114382107 \h 1
HYPERLINK \l "_Toc114382108" Overview PAGEREF _Toc114382108 \h 1
HYPERLINK \l "_Toc114382109" The relationship between IntraCellular Lipids and Insulin Resistance PAGEREF _Toc114382109 \h 2
HYPERLINK \l "_Toc114382110" Insulin resistance in aging PAGEREF _Toc114382110 \h 7
HYPERLINK \l "_Toc114382111" Hypermetabolism and Insulin resistance in burns PAGEREF _Toc114382111 \h 11
HYPERLINK \l "_Toc114382112" Prevalence PAGEREF _Toc114382112 \h 11
HYPERLINK \l "_Toc114382113" Development of hyperglycemia PAGEREF _Toc114382113 \h 12
HYPERLINK \l "_Toc114382114" Effect of burns on specific fates of glucose PAGEREF _Toc114382114 \h 13
HYPERLINK \l "_Toc114382115" Effect of burns on fat metabolism PAGEREF _Toc114382115 \h 14
HYPERLINK \l "_Toc114382116" Mitochondrial function in burns PAGEREF _Toc114382116 \h 17
HYPERLINK \l "_Toc114382117" Intensive insulin therapy in burns PAGEREF _Toc114382117 \h 17
HYPERLINK \l "_Toc114382118" Other medications to prevent insulin resistance in burns PAGEREF _Toc114382118 \h 19
HYPERLINK \l "_Toc114382119" PPAR-( agonist manipulation of fat and glucose metabolism PAGEREF _Toc114382119 \h 20
HYPERLINK \l "_Toc114382120" PPAR-( agonists in animals PAGEREF _Toc114382120 \h 20
HYPERLINK \l "_Toc114382121" PPAR-( agonists in humans PAGEREF _Toc114382121 \h 22
HYPERLINK \l "_Toc114382122" Aims and Hypothesis of Studies Conducted PAGEREF _Toc114382122 \h 22
HYPERLINK \l "_Toc114382123" Study 1: Young vs. Elderly PAGEREF _Toc114382123 \h 22
HYPERLINK \l "_Toc114382124" Study 2: 60 days of fenofibrate in the elderly PAGEREF _Toc114382124 \h 23
HYPERLINK \l "_Toc114382125" Study 3: 14 days of fenofibrate in severely burned children PAGEREF _Toc114382125 \h 23
HYPERLINK \l "_Toc114382126" Study 4: Mitochondrial function in healthy children PAGEREF _Toc114382126 \h 24
HYPERLINK \l "_Toc114382127" Summary PAGEREF _Toc114382127 \h 24
HYPERLINK \l "_Toc114382128" Protocol DesignS PAGEREF _Toc114382128 \h 25
HYPERLINK \l "_Toc114382129" Study 1: Young vs. Elderly PAGEREF _Toc114382129 \h 25
HYPERLINK \l "_Toc114382130" Study 2: Fenofibrate in the Elderly PAGEREF _Toc114382130 \h 25
HYPERLINK \l "_Toc114382131" Study 3: Fenofibrate in Burns PAGEREF _Toc114382131 \h 27
HYPERLINK \l "_Toc114382132" Overall study design PAGEREF _Toc114382132 \h 27
HYPERLINK \l "_Toc114382133" Tracer study design PAGEREF _Toc114382133 \h 29
HYPERLINK \l "_Toc114382134" Glucose Study Design PAGEREF _Toc114382134 \h 30
HYPERLINK \l "_Toc114382135" Fat Study Design PAGEREF _Toc114382135 \h 31
HYPERLINK \l "_Toc114382136" Other measurements: PAGEREF _Toc114382136 \h 31
HYPERLINK \l "_Toc114382137" Muscle biopsy measurements: PAGEREF _Toc114382137 \h 32
HYPERLINK \l "_Toc114382138" Study 4: Mitochondrial Function in Healthy Children PAGEREF _Toc114382138 \h 33
HYPERLINK \l "_Toc114382139" Methods PAGEREF _Toc114382139 \h 35
HYPERLINK \l "_Toc114382140" Sample analysis for All studies PAGEREF _Toc114382140 \h 35
HYPERLINK \l "_Toc114382141" Blood samples PAGEREF _Toc114382141 \h 35
HYPERLINK \l "_Toc114382142" Plasma glucose concentration PAGEREF _Toc114382142 \h 35
HYPERLINK \l "_Toc114382143" Serum Insulin Concentrations PAGEREF _Toc114382143 \h 35
HYPERLINK \l "_Toc114382144" Plasma total FFA concentrations - Study 1 and 2 only PAGEREF _Toc114382144 \h 35
HYPERLINK \l "_Toc114382145" Imaging PAGEREF _Toc114382145 \h 36
HYPERLINK \l "_Toc114382146" MRS soleus: PAGEREF _Toc114382146 \h 36
HYPERLINK \l "_Toc114382147" MRS liver PAGEREF _Toc114382147 \h 37
HYPERLINK \l "_Toc114382148" Dual X-Ray absorptometry (DEXA) PAGEREF _Toc114382148 \h 38
HYPERLINK \l "_Toc114382149" Calculations PAGEREF _Toc114382149 \h 39
HYPERLINK \l "_Toc114382150" Matsuda Model-Study 1 and 2 only PAGEREF _Toc114382150 \h 39
HYPERLINK \l "_Toc114382151" sample analysis for study 3 only PAGEREF _Toc114382151 \h 39
HYPERLINK \l "_Toc114382152" Whole blood and plasma analysis PAGEREF _Toc114382152 \h 39
HYPERLINK \l "_Toc114382153" Glucose enrichment PAGEREF _Toc114382153 \h 39
HYPERLINK \l "_Toc114382154" Free Fatty Acid Concentration and Enrichment PAGEREF _Toc114382154 \h 40
HYPERLINK \l "_Toc114382155" Carbon Dioxide Concentration and Enrichment PAGEREF _Toc114382155 \h 41
HYPERLINK \l "_Toc114382156" Plasma Glycerol PAGEREF _Toc114382156 \h 41
HYPERLINK \l "_Toc114382157" Muscle Tissue PAGEREF _Toc114382157 \h 41
HYPERLINK \l "_Toc114382158" Diacyglycerol (DAG) PAGEREF _Toc114382158 \h 41
HYPERLINK \l "_Toc114382159" Fatty Acyl CoA PAGEREF _Toc114382159 \h 43
HYPERLINK \l "_Toc114382160" Fatty Acyl Carnitine PAGEREF _Toc114382160 \h 44
HYPERLINK \l "_Toc114382161" Mitochondrial enzyme activity -Cytochrome C oxidase, (-HAD, Citrate synthase PAGEREF _Toc114382161 \h 46
HYPERLINK \l "_Toc114382162" Mitochondrial oxidation rates PAGEREF _Toc114382162 \h 46
HYPERLINK \l "_Toc114382163" Intracellular signaling PAGEREF _Toc114382163 \h 46
HYPERLINK \l "_Toc114382164" Resting energy expenditure PAGEREF _Toc114382164 \h 49
HYPERLINK \l "_Toc114382165" CALCULATIONS PAGEREF _Toc114382165 \h 49
HYPERLINK \l "_Toc114382166" Plasma PAGEREF _Toc114382166 \h 49
HYPERLINK \l "_Toc114382167" Glucose Kinetics PAGEREF _Toc114382167 \h 49
HYPERLINK \l "_Toc114382168" FFA Kinetics PAGEREF _Toc114382168 \h 51
HYPERLINK \l "_Toc114382169" Muscle PAGEREF _Toc114382169 \h 54
HYPERLINK \l "_Toc114382170" DAG, TAG, Fatty acyl CoA, and Fatty acyl carnitine concentrations PAGEREF _Toc114382170 \h 54
HYPERLINK \l "_Toc114382171" Imaging PAGEREF _Toc114382171 \h 55
HYPERLINK \l "_Toc114382172" MRS of soleus and liver PAGEREF _Toc114382172 \h 55
HYPERLINK \l "_Toc114382173" STATISTICS PAGEREF _Toc114382173 \h 55
HYPERLINK \l "_Toc114382174" Study 1 PAGEREF _Toc114382174 \h 55
HYPERLINK \l "_Toc114382175" Study 2 PAGEREF _Toc114382175 \h 56
HYPERLINK \l "_Toc114382176" Study 3 PAGEREF _Toc114382176 \h 56
HYPERLINK \l "_Toc114382177" Study 4 PAGEREF _Toc114382177 \h 56
HYPERLINK \l "_Toc114382178" Patient Demographics and clinical labs PAGEREF _Toc114382178 \h 57
HYPERLINK \l "_Toc114382179" Study 1: Young vs. Elderly PAGEREF _Toc114382179 \h 57
HYPERLINK \l "_Toc114382180" Study 2: 60 Days of Fenofibrate in Elderly PAGEREF _Toc114382180 \h 59
HYPERLINK \l "_Toc114382181" Baseline Parameters PAGEREF _Toc114382181 \h 59
HYPERLINK \l "_Toc114382182" Study 3: Fenofibrate in burns PAGEREF _Toc114382182 \h 62
HYPERLINK \l "_Toc114382183" Standard of Care PAGEREF _Toc114382183 \h 64
HYPERLINK \l "_Toc114382184" Medications PAGEREF _Toc114382184 \h 64
HYPERLINK \l "_Toc114382185" Diet PAGEREF _Toc114382185 \h 65
HYPERLINK \l "_Toc114382186" Physiologic measurements PAGEREF _Toc114382186 \h 65
HYPERLINK \l "_Toc114382187" Blood Flow PAGEREF _Toc114382187 \h 67
HYPERLINK \l "_Toc114382188" Study 4 PAGEREF _Toc114382188 \h 69
HYPERLINK \l "_Toc114382189" Relationship between insulin resistance and fat metabolism in Young Vs. Elderly PAGEREF _Toc114382189 \h 70
HYPERLINK \l "_Toc114382190" Results PAGEREF _Toc114382190 \h 70
HYPERLINK \l "_Toc114382191" MRS Results PAGEREF _Toc114382191 \h 70
HYPERLINK \l "_Toc114382192" Insulin Sensitivity Measures PAGEREF _Toc114382192 \h 71
HYPERLINK \l "_Toc114382193" Discussion PAGEREF _Toc114382193 \h 76
HYPERLINK \l "_Toc114382194" Conclusions PAGEREF _Toc114382194 \h 83
HYPERLINK \l "_Toc114382195" 60 days of Fenofibrate in elderly PAGEREF _Toc114382195 \h 84
HYPERLINK \l "_Toc114382196" Results PAGEREF _Toc114382196 \h 84
HYPERLINK \l "_Toc114382197" Changes over treatment duration: PAGEREF _Toc114382197 \h 84
HYPERLINK \l "_Toc114382198" Discussion PAGEREF _Toc114382198 \h 86
HYPERLINK \l "_Toc114382199" Conclusions PAGEREF _Toc114382199 \h 90
HYPERLINK \l "_Toc114382200" Glucose Metabolism in pediatric burns Patients PAGEREF _Toc114382200 \h 92
HYPERLINK \l "_Toc114382201" Results PAGEREF _Toc114382201 \h 92
HYPERLINK \l "_Toc114382202" Fasted glucose and insulin PAGEREF _Toc114382202 \h 92
HYPERLINK \l "_Toc114382203" Hyperinsulinemic-euglycemic clamp measurements PAGEREF _Toc114382203 \h 93
HYPERLINK \l "_Toc114382204" Muscle glucose sensitivity PAGEREF _Toc114382204 \h 97
HYPERLINK \l "_Toc114382205" Liver glucose sensitivity PAGEREF _Toc114382205 \h 99
HYPERLINK \l "_Toc114382206" Discussion PAGEREF _Toc114382206 \h 102
HYPERLINK \l "_Toc114382207" Fatty Acid Metabolism in pediatric burns patients PAGEREF _Toc114382207 \h 109
HYPERLINK \l "_Toc114382208" Results: PAGEREF _Toc114382208 \h 109
HYPERLINK \l "_Toc114382209" Plasma Lipid Profiles PAGEREF _Toc114382209 \h 109
HYPERLINK \l "_Toc114382210" Percent Body Fat PAGEREF _Toc114382210 \h 110
HYPERLINK \l "_Toc114382211" Indirect Calorimetry Results PAGEREF _Toc114382211 \h 110
HYPERLINK \l "_Toc114382212" Labeled Palmitate Studies PAGEREF _Toc114382212 \h 112
HYPERLINK \l "_Toc114382213" Raw Data PAGEREF _Toc114382213 \h 112
HYPERLINK \l "_Toc114382214" Calculated whole body data PAGEREF _Toc114382214 \h 114
HYPERLINK \l "_Toc114382215" Calculated leg data PAGEREF _Toc114382215 \h 117
HYPERLINK \l "_Toc114382216" Glycerol kinetics PAGEREF _Toc114382216 \h 120
HYPERLINK \l "_Toc114382217" Intracellular Data PAGEREF _Toc114382217 \h 123
HYPERLINK \l "_Toc114382218" Spectroscopy Results PAGEREF _Toc114382218 \h 123
HYPERLINK \l "_Toc114382219" Discussion PAGEREF _Toc114382219 \h 131
HYPERLINK \l "_Toc114382220" Whole Body Changes PAGEREF _Toc114382220 \h 131
HYPERLINK \l "_Toc114382221" Liver and muscle results PAGEREF _Toc114382221 \h 137
HYPERLINK \l "_Toc114382222" Conclusions PAGEREF _Toc114382222 \h 142
HYPERLINK \l "_Toc114382223" Muscle enzyme activity and signaling in pediatric burns PAGEREF _Toc114382223 \h 143
HYPERLINK \l "_Toc114382224" Results PAGEREF _Toc114382224 \h 143
HYPERLINK \l "_Toc114382225" Mitochondrial oxidation rates PAGEREF _Toc114382225 \h 143
HYPERLINK \l "_Toc114382226" Mitochondrial enzymes PAGEREF _Toc114382226 \h 147
HYPERLINK \l "_Toc114382227" Insulin Signaling PAGEREF _Toc114382227 \h 150
HYPERLINK \l "_Toc114382228" Discussion PAGEREF _Toc114382228 \h 155
HYPERLINK \l "_Toc114382229" Mitochondrial Data PAGEREF _Toc114382229 \h 155
HYPERLINK \l "_Toc114382230" Insulin signaling PAGEREF _Toc114382230 \h 158
HYPERLINK \l "_Toc114382231" Conclusions PAGEREF _Toc114382231 \h 160
HYPERLINK \l "_Toc114382232" Discussion PAGEREF _Toc114382232 \h 162
HYPERLINK \l "_Toc114382233" Different mechanisms of insulin resistance in trauma and aging PAGEREF _Toc114382233 \h 162
HYPERLINK \l "_Toc114382234" Insulin resistance in Aging PAGEREF _Toc114382234 \h 163
HYPERLINK \l "_Toc114382235" Insulin Resistance in Burns PAGEREF _Toc114382235 \h 165
HYPERLINK \l "_Toc114382236" Conclusions PAGEREF _Toc114382236 \h 173
HYPERLINK \l "_Toc114382237" Refrences PAGEREF _Toc114382237 \h 175
HIDDEN TEXT: If you choose to place the chapter number (Chapter 1) and the chapter title (Introduction) on different lines, the automatically generated table of contents will reflect that format. After creating a new table of contents, set them on the same line by deleting the page number and paragraph marker at the end of each chapter number line.
List of Tables
TOC \h \z \t "Heading 7,1" HYPERLINK \l "_Toc114382047" Findings in insulin resistant patients in different populations. PAGEREF _Toc114382047 \h 1
HYPERLINK \l "_Toc114382048" Study 1 Volunteer Baseline Laboratory Measurements PAGEREF _Toc114382048 \h 58
HYPERLINK \l "_Toc114382049" Study 2: Volunteer Baseline Demographics PAGEREF _Toc114382049 \h 60
HYPERLINK \l "_Toc114382050" Study 2: Laboratory Values in Elderly Volunteers PAGEREF _Toc114382050 \h 60
HYPERLINK \l "_Toc114382051" Study 3: Patient demographics PAGEREF _Toc114382051 \h 64
HYPERLINK \l "_Toc114382052" R2 values from Correlations of LFAT and IMCL to Obesity and Insulin Resistance PAGEREF _Toc114382052 \h 76
HYPERLINK \l "_Toc114382053" Measures of Insulin Sensitivity PAGEREF _Toc114382053 \h 85
HYPERLINK \l "_Toc114382054" Measurement of intra-cellular lipids PAGEREF _Toc114382054 \h 86
HYPERLINK \l "_Toc114382055" Fasted Concentrations of Blood Glucose and Insulin PAGEREF _Toc114382055 \h 92
HYPERLINK \l "_Toc114382056" Insulin clearance during the clamp PAGEREF _Toc114382056 \h 95
HYPERLINK \l "_Toc114382057" Arterial and venous plasma glucose measurements PAGEREF _Toc114382057 \h 98
HYPERLINK \l "_Toc114382058" Leg glucose net balance PAGEREF _Toc114382058 \h 98
HYPERLINK \l "_Toc114382059" Endogenous Ra of glucose PAGEREF _Toc114382059 \h 100
HYPERLINK \l "_Toc114382060" Plasma Cholesterol PAGEREF _Toc114382060 \h 109
HYPERLINK \l "_Toc114382061" Metabolic Cart details PAGEREF _Toc114382061 \h 111
HYPERLINK \l "_Toc114382062" Palmitate and CO2 concentration and enrichment PAGEREF _Toc114382062 \h 113
HYPERLINK \l "_Toc114382063" Whole body palmitate release PAGEREF _Toc114382063 \h 114
HYPERLINK \l "_Toc114382064" Whole body fat oxidation and percent PAGEREF _Toc114382064 \h 115
HYPERLINK \l "_Toc114382065" Palmitate oxidation in the leg PAGEREF _Toc114382065 \h 118
HYPERLINK \l "_Toc114382066" Release, uptake and net balance of palmitate across the leg PAGEREF _Toc114382066 \h 119
HYPERLINK \l "_Toc114382067" Glycerol release PAGEREF _Toc114382067 \h 121
HYPERLINK \l "_Toc114382068" Glycerol Ra vs. FFA Ra PAGEREF _Toc114382068 \h 122
HYPERLINK \l "_Toc114382069" Fatty Acyl CoA and Carnitine Enrichments PAGEREF _Toc114382069 \h 131
HYPERLINK \l "_Toc114382070" Rabbit experiment results PAGEREF _Toc114382070 \h 141
HYPERLINK \l "_Toc114382071" Mechanism of Insulin resistance in different populations PAGEREF _Toc114382071 \h 173
List of Figures
TOC \h \z \t "Heading 8,1" HYPERLINK \l "_Toc114379933" Intracellular insulin signaling PAGEREF _Toc114379933 \h 4
HYPERLINK \l "_Toc114379934" Key enzymes for mitochondrial oxidation of fats PAGEREF _Toc114379934 \h 10
HYPERLINK \l "_Toc114379935" Study Design PAGEREF _Toc114379935 \h 28
HYPERLINK \l "_Toc114379936" Tracer Infusion Study Design PAGEREF _Toc114379936 \h 30
HYPERLINK \l "_Toc114379937" Plasma Glucose during baseline OGTT PAGEREF _Toc114379937 \h 61
HYPERLINK \l "_Toc114379938" Insulin During Baseline OGTT PAGEREF _Toc114379938 \h 62
HYPERLINK \l "_Toc114379939" IMCL measurements from MRS in young and elderly. PAGEREF _Toc114379939 \h 70
HYPERLINK \l "_Toc114379940" LFAT in Young and Elderly PAGEREF _Toc114379940 \h 71
HYPERLINK \l "_Toc114379941" Changes in plasma total FFA during a 2-hour OGTT PAGEREF _Toc114379941 \h 74
HYPERLINK \l "_Toc114379942" Plasma total cholesterol PAGEREF _Toc114379942 \h 84
HYPERLINK \l "_Toc114379943" Plasma total triglycerides PAGEREF _Toc114379943 \h 85
HYPERLINK \l "_Toc114379944" Whole blood glucose during hyperinsulinemic-euglycemic clamp PAGEREF _Toc114379944 \h 93
HYPERLINK \l "_Toc114379945" Plasma insulin during hyperinsulinemic-euglycemic clamp PAGEREF _Toc114379945 \h 94
HYPERLINK \l "_Toc114379946" Glucose infusion rate during hyperinsulinemic-euglycemic clamp (M-value) PAGEREF _Toc114379946 \h 96
HYPERLINK \l "_Toc114379947" Glucose clearance during the basal and clamp states PAGEREF _Toc114379947 \h 97
HYPERLINK \l "_Toc114379948" Insulin-stimulated leg glucose uptake PAGEREF _Toc114379948 \h 99
HYPERLINK \l "_Toc114379949" Insulin stimulated decrease in hepatic glucose output compared to basal PAGEREF _Toc114379949 \h 100
HYPERLINK \l "_Toc114379950" Total glucose uptake during the clamp PAGEREF _Toc114379950 \h 101
HYPERLINK \l "_Toc114379951" Percent body fat PAGEREF _Toc114379951 \h 110
HYPERLINK \l "_Toc114379952" Change in basal whole body oxidation PAGEREF _Toc114379952 \h 117
HYPERLINK \l "_Toc114379953" Change in fat oxidation in the leg PAGEREF _Toc114379953 \h 118
HYPERLINK \l "_Toc114379954" LFAT as assessed by MRS PAGEREF _Toc114379954 \h 123
HYPERLINK \l "_Toc114379955" IMCL as assessed by MRS PAGEREF _Toc114379955 \h 124
HYPERLINK \l "_Toc114379956" Muscle palmitate TAG as measured from muscle biopsies from the vastus lateralus PAGEREF _Toc114379956 \h 125
HYPERLINK \l "_Toc114379957" Muscle DAG palmitate as measured from muscle biopsies from the vastus lateralus PAGEREF _Toc114379957 \h 126
HYPERLINK \l "_Toc114379958" DAG and TAG enrichment PAGEREF _Toc114379958 \h 127
HYPERLINK \l "_Toc114379959" Palmatyl Fatty Acyl CoA Concentrations PAGEREF _Toc114379959 \h 128
HYPERLINK \l "_Toc114379960" Fatty Acyl Carnitine Concentration PAGEREF _Toc114379960 \h 129
HYPERLINK \l "_Toc114379961" Fatty Acyl CoA/Fatty Acyl Carnitine Ratio PAGEREF _Toc114379961 \h 130
HYPERLINK \l "_Toc114379962" Mitochondrial oxidation of pyruvate PAGEREF _Toc114379962 \h 144
HYPERLINK \l "_Toc114379963" Mitochondrial oxidation of palmatyl-CoA PAGEREF _Toc114379963 \h 145
HYPERLINK \l "_Toc114379964" ATP production after pyruvate PAGEREF _Toc114379964 \h 146
HYPERLINK \l "_Toc114379965" ATP production after palmatyl CoA PAGEREF _Toc114379965 \h 147
HYPERLINK \l "_Toc114379966" Change in citrate synthase activity PAGEREF _Toc114379966 \h 148
HYPERLINK \l "_Toc114379967" Change in cytochrome C-oxidase activity PAGEREF _Toc114379967 \h 149
HYPERLINK \l "_Toc114379968" Change in (-HAD activity PAGEREF _Toc114379968 \h 149
HYPERLINK \l "_Toc114379969" Insulin receptor tyrosine phosphorylation PAGEREF _Toc114379969 \h 150
HYPERLINK \l "_Toc114379970" IRS-1 tyrosine phosphorylation PAGEREF _Toc114379970 \h 151
HYPERLINK \l "_Toc114379971" PI3 K Co-precipitated with IRS-1 phosphorylated tyrosine PAGEREF _Toc114379971 \h 152
HYPERLINK \l "_Toc114379972" AKT phosphorylation PAGEREF _Toc114379972 \h 153
HYPERLINK \l "_Toc114379973" Protein Kinase C-( PAGEREF _Toc114379973 \h 154
HYPERLINK \l "_Toc114379974" Protein Kinase C-( PAGEREF _Toc114379974 \h 155
HYPERLINK \l "_Toc114379975" The proposed relationship between aging and insulin resistance PAGEREF _Toc114379975 \h 163
HYPERLINK \l "_Toc114379976" The proposed relationship between the first 24 hours of trauma and insulin resistance PAGEREF _Toc114379976 \h 167
HYPERLINK \l "_Toc114379977" The proposed relationship between 24 hours to 21 days post trauma and insulin resistance PAGEREF _Toc114379977 \h 168
HYPERLINK \l "_Toc114379978" The proposed relationship affect of fenofibrate on insulin resistance in trauma patients PAGEREF _Toc114379978 \h 170
LIST OF ABBREVIATIONS
ACC Acetyl CoA Carboxylase
AKT-
ASR actual synthetic rate
(-HAD- Betahydroxyactyhydrogenase
Ca concentration in the artery
CS- Citrate Synthase
CoA- CoAcyl
COX- Cytochrome C Oxidase
CPT-I Carnitine Palmotyl Transferase
Cv- concentration in the vein
DAG- Diacylglycerol
DEXA Dual X-ray Absormetry
FEN- the fenofibrate treatment group
FBR fractional breakdown rate
FFA- Free Fatty Acids
FSR fractional synthetic rate
HPLC high pressure liquid chromatography
GCMS Gas Chromatography Mass Spectrometry
GCRC- General Clinical Research Center
IMCL- Intramuscular Triglyceride
IRS-1 Insulin Receptor Substrate - 1
IRMS Infrared Mass Spectrometry
LCMS Liquid Chromatography Mass Spectrometry
LFAT- Liver triglycerides
LPL- Lipoprotein Lipase
MRS magnetic resonance spectroscopy
NB- Net Balance
PI3-K Protein Tyrosine Kinase 3
PLA- the placebo treatment group
Ra- rate of appearance
Rd rate of disappearance
REE- Resting Energy Expenditure
RIA radioactive immuno assay
RQ- Respiratory Quotient
TAG- Triacylglycerol, also triglycerides
Chapter 1
IntroducTion
Overview
Hyperglycemia, or increased blood glucose, from insulin resistance is a serious health problem that affects millions of individuals world-wide. Insulin resistance is defined as the inability of insulin to adequately stimulate glucose uptake in peripheral tissue or inhibit gluconeogenesis in the liver, leading to hyperglycemia. Insulin resistance is most commonly seen in type 2 diabetes, but is also seen in aging individuals and trauma patients. Recent research in type 2 diabetics has focused on the relationship between fat metabolism and insulin resistance. As shown in Table 1.1, impaired glucose uptake, or insulin resistance, has been associated with increased intracellular lipids in both muscle and liver and mitochondrial dysfunction in type 2 diabetics, but it is not known whether this occurred in elderly and trauma patients..
Table 1.1
Findings in insulin resistant patients in different populations.Type 2 diabetesAgingBurns/
TraumaImpaired peripheral glucose uptakeYesYesYesIncreased intracellular lipidsYesUnknownUnknownMitochondrial DysfunctionYesMaybeUnknown
Impaired peripheral glucose uptake has been documented in elderly individuals, and also in trauma patients. However, the studies performed in type 2 diabetics that have established a role of fat and mitochondrial dysfunction have not been performed in either of these populations. The overall goal of this work was two-fold:
To determine if tissue fat metabolism contributes to insulin resistance in ambulatory elderly and burn trauma patients
To determine if altering fat metabolism alters insulin resistance and improves clinical outcomes in ambulatory elderly and trauma patients.
The relationship between IntraCellular Lipids and Insulin Resistance
The muscle plays a crucial role in insulin sensitivity, since muscle tissue is responsible for up to 80% of insulin stimulated glucose uptake ADDIN EN.CITE DeFronzo1992564elderlypaper2-Converted.enlEndNote56417(DeFronzo 1992). Recent advances in magnetic resonance spectroscopy have enabled non-invasive measurements of the amount of intramyocellular lipids (IMCL) ADDIN EN.CITE Bachmann OP200123EndNote017(Bachmann OP). While these measurements have been conducted in muscle biopsy samples for a number of years, the difficulty in removing all of the adipocytes from the sample greatly increases variability ADDIN EN.CITE Machann2004563elderlypaper2-Converted.enlEndNote56317(Machann 2004). Studies of IMCL measured with MRS have given new perspectives on the relationship between lipid metabolism and insulin resistance, since IMCL has been found to be associated with insulin resistance and/or obesity ADDIN EN.CITE Perseghin G199913EndNote017Pan DA199715EndNote017Goodpaster BH200357EndNote017Ferrannini199631EndNote017(Ferrannini 1996; Pan DA 1997; Perseghin G 1999; Goodpaster BH 2003). In healthy men with no family history of diabetes, high levels of IMCL were found to correlate with lower levels of insulin-stimulated glucose uptake during a euglycemichyper insulinemic clamp. This method is considered to be the gold standard for measuring insulin sensitivity, where hyper-insulinemia is induced, and euglycemia is maintained by infusion of exogenous glucose ADDIN EN.CITE Virkamaki A20016EndNote017(Virkamaki A 2001). Women with a history of gestational diabetes had increased levels of IMCL that were associated with body fat mass ADDIN EN.CITE Kautzky-Willer A200347EndNote017(Kautzky-Willer A 2003). On the other hand, IMCL was not elevated in obese individuals with normal glucose tolerance ADDIN EN.CITE Perseghin G200212EndNote017(Perseghin G 2002). Thus, IMCL may be an indicator of dysfunctional muscle lipid metabolism and may be related to insulin resistance but not obesity.
Despite these associative studies, the role of IMCL in causing insulin resistance through alterations in the insulin signaling cascade and the mechanism involved are not clear. Whereas the IMCL per se may not cause insulin resistance, it may be associated with increases in triglyceride metabolites such as diacylglycerol (DAG) and long chain fatty acyl CoA ADDIN EN.CITE Shulman2004454elderlypaper2-Converted.enlEndNote45417Hulver2004517elderlypaper2-Converted.enlEndNote51717(Hulver 2004; Shulman 2004). The insulin signaling pathway has been well characterized and in diabetics several dysfunctional regulatory sites are known (Figure 1.1). Insulin causes the insulin receptor to auto-phosphorylate and this leads to a cascade of phosphorylation and activations of the insulin substrate receptor 1 (IRS-1) protein, protein kinase -3 (PIK-3), then AKT before eventually causing translocation of GLUT-4 to the cell surface, in addition to inducing gene expression and production of more GLUT-4 protein. A disruption in the pathway at any point can prevent translocation of GLUT-4, and lead to apparent insulin resistance ADDIN EN.CITE Goodyear1995515elderlypaper2-Converted.enlEndNote51517(Goodyear 1995). DAG activates protein kinase C proteins, which inhibit the insulin receptor and cause inactivation of IRS-1 through serine phosphorylation instead of tyrosine phosphorylation ADDIN EN.CITE Shulman2004454elderlypaper2-Converted.enlEndNote45417Hulver2004517elderlypaper2-Converted.enlEndNote51717(Hulver 2004; Shulman 2004). After lipid infusions in animals and rats, increased DAG, PKC activity and decreased IRS-1 associated PI3 K were found, in addition to insulin resistance. ADDIN EN.CITE Itani2000516elderlypaper2-Converted.enlEndNote51617Griffin1999476elderlypaper2-Converted.enlEndNote47617(Griffin 1999; Itani 2000).
Figure 1.1
Intracellular insulin signaling EMBED PowerPoint.Slide.8 The insulin signaling pathway is shown above in blue. The potential inhibition of the insulin signaling by fat is shown in black dashes and the normal metabolism of fat shown in double black lines.
Many of the above studies also found that increased plasma free fatty acids (FFA) are associated with increased IMCL. The increased IMCL can result from either increased delivery or synthesis of lipids, or decreased breakdown. To better understand the role of muscle delivery and synthesis of FFA on insulin sensitivity, the effects several acute perturbations of FFA and/or glucose concentrations have been studied. Infusions of intralipid and/or heparin, both of which increase plasma FFA concentrations, have been shown to rapidly induce insulin resistance in several populations, including healthy lean and obese, diabetics and healthy offspring of diabetics ADDIN EN.CITE Griffin1999476elderlypaper2-Converted.enlEndNote47617Jucker1997477elderlypaper2-Converted.enlEndNote47717Boden1994482elderlypaper2-Converted.enlEndNote48217Boden1995483elderlypaper2-Converted.enlEndNote48317Homko2003486elderlypaper2-Converted.enlEndNote48617(Boden 1994; Boden 1995; Jucker 1997; Griffin 1999; Homko 2003). The acute increase in insulin resistance following acute elevations in FFAs has been associated with increases in IMCL in some studies, but often this parameter was not measured ADDIN EN.CITE Boden2001487elderlypaper2-Converted.enlEndNote48717(Boden 2001). Furthermore, studies where plasma FFAs in volunteers and type 2 diabetics were decreased with drugs such as nicotinic acid, improvement in insulin sensitivity developed as quickly as within three days, and remained sustained over 7 days ADDIN EN.CITE Bajaj2004484elderlypaper2-Converted.enlEndNote48417Paolisso1998490elderlypaper2-Converted.enlEndNote49017Worm1994491elderlypaper2-Converted.enlEndNote49117(Worm 1994; Paolisso 1998; Bajaj 2004). These studies indicate that increased plasma FFA concentrations are a component of insulin resistance.
Despite a clear association between elevated plasma FFAs and insulin resistance, conflicting results have been reported regarding changes in intracellular signaling within muscle tissues following intra-lipid infusions ADDIN EN.CITE Storgaard2004492elderlypaper2-Converted.enlEndNote49217Griffin1999478elderlypaper2-Converted.enlEndNote47817(Griffin 1999; Storgaard 2004). High fatty acid infusion rates in healthy adults and diabetic offspring decreased glucose uptake and oxidation, but did not affect IRS-1 tyrosine phosphorylation, PI3-kinase activity, nor AKT activation in either patient population ADDIN EN.CITE Storgaard2004492elderlypaper2-Converted.enlEndNote49217(Storgaard 2004). Results from a similar infusion protocol in rats also reported decreased glucose oxidation and uptake, but in conjunction with decreased IRS-1 tyrosine phosphorylation, PI3-kinase activity and increased PKC activity ADDIN EN.CITE Griffin1999476elderlypaper2-Converted.enlEndNote47617(Griffin 1999). Another study in healthy humans found that 4 hours of fatty acid infusion at three different infusion rates induced dose dependent increases in insulin resistance and decreased insulin receptor, IRS-1 and AKT phosphorylation ADDIN EN.CITE Belfort2005519elderlypaper2-Converted.enlEndNote51917(Belfort 2005). Thus the relationship between plasma FFAs and insulin signaling in muscle may be mediated through PKC, but this is not conclusive.
Muscle intracellular lipid may also be increased in type 2 diabetics due to decreased breakdown of fats. Studies have shown that diabetics, and subjects with a family history of diabetes, have decreased rates of (-oxidation of fats ADDIN EN.CITE Kelley1994494elderlypaper2-Converted.enlEndNote49417(Kelley 1994). These patients also have fewer and smaller mitochondria, that are located further from lipid stores ADDIN EN.CITE Ritov2005521elderlypaper2-Converted.enlEndNote52117(Ritov 2005). These may represent genetic defects that are present in diabetics and contribute to the development of obesity and insulin resistance. Subsequent weight gain further exacerbates the insulin resistance, leading eventually to diabetes.
The liver also plays a primary role in plasma glucose and lipid regulation, and may also be affected by plasma FFA concentration. Correlation studies conducted in patients with non-alcoholic steatohepatitis (NASH) and polymetabolic syndrome X have found a correlation between the degree of liver fat (LFAT) and insulin resistance ADDIN EN.CITE Marchesini G199940EndNote017Marceau P199942EndNote017(Marceau P 1999; Marchesini G 1999). Further, patients with chronic hepatitis C infections often develop fatty liver and type 2 diabetes ADDIN EN.CITE Caronia S199965EndNote017Knobler H200063EndNote017Konrad T200062EndNote017(Caronia S 1999; Knobler H 2000; Konrad T 2000). In type 2 diabetics, the amount of insulin taken daily and the ability of intravenous insulin to suppress endogenous glucose production were significantly correlated with the amount of liver fat ADDIN EN.CITE Ryysy L200011EndNote017(Ryysy L 2000). In patients treated with nicotinic acid to lower plasma FFAs, improvement in insulin stimulated glucose uptake was associated with increased glycogen synthesis and insulin suppression of hepatic endogenous glucose production ADDIN EN.CITE Bajaj2004484elderlypaper2-Converted.enlEndNote48417(Bajaj 2004). Furthermore, as in muscle tissue, FFA infusion in rats increased hepatic PKC translocation and activation ADDIN EN.CITE Lam2002520elderlypaper2-Converted.enlEndNote52017(Lam 2002). It appears that both the liver and the muscle play a central role in interrelating glucose and fat metabolsim.
Because insulin resistance is becoming more prevalent, the relationships between fat and glucose metabolism are intriguing, and offer new approachs for treatment of insulin resistance. Insulin resistance also occurs in the settings of both aging and trauma, and it was not known if the relationships between increased plasma lipid and insulin resistance exist also in these populations.
Insulin resistance in aging
Insulin resistance increases after the age of 50. Approximately 7 million Americans over the age of 65 had type 2 diabetes in 1999, representing 20 % of this age group ADDIN EN.CITE Harris MI199849EndNote017(Harris MI 1998). Additionally, the NIH currently estimates that approximately 16 million Americans between the ages of 45 and 70 are glucose intolerant ADDIN EN.CITE Harris MI199849EndNote017(Harris MI 1998). Thus, nearly 50% of those over 65 in the United States are estimated to have some degree of insulin resistance related to peripheral glucose metabolism. Furthermore, resistance to the action of insulin on muscle protein may be even more widespread in the elderly because insulin resistance is associated with obesity and higher rates of obesity in people over 60 have been documented since 1960 ADDIN EN.CITE Volpi E200051EndNote017(Volpi E 2000); ADDIN EN.CITE Flegal KM200248EndNote017(Flegal KM 2002). The most recent data from the Centers for Disease Control indicate that 25% of people aged 60-69 years are severely obese, ( body mass index of > 30) compared to 14% of people aged 18-29 ADDIN EN.CITE Mokdad AH2003112EndNote017(Mokdad AH 2003).
Previous research indicates that the insulin resistance of aging may be related not only to increased FFA delivery due to adiposity, but also decreases in the (-oxidation of fats ADDIN EN.CITE Levadoux2001469elderlypaper2-Converted.enlEndNote46917(Levadoux 2001). (-oxidation of fats occurs within the mitochondria, and recent studies in elderly individuals have found that mitochondrial dysfunction develops in conjunction with increased IMCL and LFAT ADDIN EN.CITE Petersen KF2003201elderlypaper2-Converted.enlEndNote20117(Petersen KF 2003). Some attribute this mitochondrial dysfunction to cumulative age-related mitochondrial DNA damage ADDIN EN.CITE Wang2001457elderlypaper2-Converted.enlEndNote45717(Wang 2001). Others have proposed that the mitochondrial dysfunction is simply a manifestation of reduced physical activity levels in the elderly that can be reversed with regular physical activity ADDIN EN.CITE Rimbert2004470elderlypaper2-Converted.enlEndNote47017Pruchnic2004522elderlypaper2-Converted.enlEndNote52217(Pruchnic 2004; Rimbert 2004). A decrease in mitochondrial function, and thus (-oxidation regardless of the mechanims, may cause intracellular fat to accumulate, thereby contributing to the development of insulin resistance ADDIN EN.CITE Shulman2004454elderlypaper2-Converted.enlEndNote45417Petersen KF2003201elderlypaper2-Converted.enlEndNote20117(Petersen KF 2003; Shulman 2004). Therefore in elderly, the accumulation of tissue lipids may not only be due to increased delivery of plasma triglycerides and FFA, but also to a decrease in the oxidation rate.
In question is the timing of the development of the decreased (-oxidation in mitochondria in relation to insulin resistance. Acetyl CoA carboxylase (ACC) converts acetyl CoA into malonyl CoA in the process of de-novo fatty acid synthesis when it is simulated by insulin or citrate ( Figure 1.2). Malonyl CoA inhibits carnitine palmatyl transferase -1, the key enzyme responsible for the transport of long chain fatty acids into the mitochondria ADDIN EN.CITE Rasmussen BB2002207elderlypaper2-Converted.enlEndNote20717(Rasmussen BB 2002). Presumably in the fed state with ample glucose availability, glucose can be used for energy through the TCA cycle in mitochondria. This would increase the amount of citrate, and thus inhibit fat oxidation in favor of fat storage, since glucose is readily available. However, in the insulin resistant state insulin signaling cannot be transmitted to ACC, and there is not an abundance of glucose. Thus it may not be the CPT-1 activity that regulates (-oxidation in the insulin resistant state.
The mitochondrial activity itself may be more important in regulating the rate of (-oxidation. Once a fat molecule is within the mitochondria, it is rapidly broken down by (-hyrdoxy-acetydehydrogenase ((-HAD) into acetyl-CoA. There are multiple enzymes involved in the TCA cycle and the subsequent oxidative phosphorylation pathway, including citrate synthase, and cytochrome c oxidase. Both of these enzymes have been shown to be increased in elderly, although they can be further up-regulated with exercise ADDIN EN.CITE Morio2001468elderlypaper2-Converted.enlEndNote46817(Morio 2001). Further, the electron chain is involved in diffusing reactive oxygen species. These have been shown to increase in aging, and may damage the mitochondria, and account for the decreased (-oxidation, which in turn leads to increased fatty acyl CoA and PKC activity ADDIN EN.CITE Sarkar2005565elderlypaper2-Converted.enlEndNote56517(Sarkar 2005).
Figure 1.2
Key enzymes for mitochondrial oxidation of fats SHAPE \* MERGEFORMAT The Figure depicts some of the key enzymes involved in the B-oxidation of fats, namely CPT-1 and CPT2, on the membrane, B-HAD in the breakdown of long chain Acyl-CoA, citrate synthase in the TCA cycle, and Cytochrome C oxidase in the electron transport chain. Double lines indicate direct metabolism, dotted lines are negative feedback, and solid lines are positive feedback.
Research conducted thus far indicates that the insulin resistance in aging is very similar to that seen in type 2 diabetes. It appears that the increased adiposity associated with aging, in conjunction with a decrease in the (-oxidative capacity of the mitochondria may increase IMCL, LFAT and also insulin resistance. It is unknown if this mechanism is also present in trauma patients.
Hypermetabolism and Insulin resistance in burns
Prevalence
Multiple studies have documented hyperglycemia and insulin resistance post-trauma ADDIN EN.CITE Howard1955376Proposal-Converted.enlEndNote37617Frayn1975372Proposal-Converted.enlEndNote37217Wolfe198561Proposal-Converted.enlEndNote6117(Howard 1955; Frayn 1975; Wolfe 1985). Several studies have used hyperinsulinemic-euglycemic clamps to quantify the level of insulin resistance in trauma patients during the second phase of the trauma response. Adult trauma patients had half the glucose uptake during hyperinsulinemia when compared to that of normal controls ADDIN EN.CITE Little1987365Proposal-Converted.enlEndNote36517(Little 1987). Black et al. found that glucose uptake in response to 4 varied levels of insulin approximately one week post-injury was impaired by at least a third compared to controls ADDIN EN.CITE Black1982326Proposal-Converted.enlEndNote32617(Black 1982). The insulin resistance seen with injury is not due to the inactivity imposed by the injury, as patients undergoing surgery had decreased whole glucose uptake and increased hepatic glucose release during a hyper-insulinemic clamp when compared to healthy bed rest subjects ADDIN EN.CITE Nygren1997370Proposal-Converted.enlEndNote37017(Nygren 1997). Therefore, severe insulin resistance in these patients is a direct consequence of the trauma.
The insulin resistance in these patients is of serious clinical concern, as several studies indicate that insulin resistance and related liver dysfunction may be implicated in increasing the morbidity and mortality of surgical and burned patients ADDIN EN.CITE Thorell1993300Proposal-Converted.enlEndNote30017Thorell1994301Proposal-Converted.enlEndNote30117Thorell1996302Proposal-Converted.enlEndNote30217van den Berghe2001341Proposal-Converted.enlEndNote34117Garcia-Avello1998352Proposal-Converted.enlEndNote35217(Thorell 1993; Thorell 1994; Thorell 1996; Garcia-Avello 1998; van den Berghe 2001). Retrospective reviews have indicated that burned patients with higher plasma glucose levels over the duration of care have increased mortality ADDIN EN.CITE Holm2004390Proposal-Converted.enlEndNote39017Gore200193Proposal-Converted.enlEndNote9317(Gore 2001; Holm 2004) Further, the degree of hyperglycemia in the first 48 hours is also correlated with mortality ADDIN EN.CITE Holm200437pc540.enlEndNote3717(Holm 2004). In burn patients, higher plasma glucose levels are associated with increased graft loss and sepsis ADDIN EN.CITE Gore200193Proposal-Converted.enlEndNote9317Mowlavi200043pc540.enlEndNote4317(Mowlavi 2000; Gore 2001). These outcome studies underscore the importance of a better understanding insulin resistance in this patient population.
Development of hyperglycemia
The hypermetabolic response phase to injury appears to occur in two stages. The first phase occurs within the first 48 hours from injury and has classically been called the ebb phase ADDIN EN.CITE Cuthbertson1942380Proposal-Converted.enlEndNote38017Wolfe1981381Proposal-Converted.enlEndNote38117(Cuthbertson 1942; Wolfe 1981). This phase of the response appears to be related to the mahnitude of the trauma and the acute response hormones, including norepinephrine (NE), epinephrine (EPI), dopamine and cortisol ADDIN EN.CITE Thorell1993300Proposal-Converted.enlEndNote30017Frayn1985377Proposal-Converted.enlEndNote37717Smith1997342Proposal-Converted.enlEndNote34217Wolfe197785Proposal-Converted.enlEndNote8517Stoner1979410Proposal-Converted.enlEndNote41017(Wolfe 1977; Stoner 1979; Frayn 1985; Thorell 1993; Smith 1997). Although the response of these hormones in children may not be as robust as it is in adults, hyperglycemia is seen in pediatric trauma patients ADDIN EN.CITE Sedowofia1998298Proposal-Converted.enlEndNote29817(Sedowofia 1998). In 30 children, plasma glucose, cortisol, EPI, and NE levels were all elevated post-trauma ADDIN EN.CITE Childs1990297Proposal-Converted.enlEndNote29717(Childs 1990). The initial rise in plasma glucose after injury is seen within 30 minutes in guinea pigs, 2 hours in rats, and one hour in severely burned children ADDIN EN.CITE Childs1990297Proposal-Converted.enlEndNote29717Frayn1985377Proposal-Converted.enlEndNote37717Wolfe197785Proposal-Converted.enlEndNote8517(Wolfe 1977; Frayn 1985; Childs 1990). During this acute period, the hyperglycemia is not accompanied by an increase in insulin, perhaps due to the suppressive effect of epinephrine on insulin release ADDIN EN.CITE Childs1990297Proposal-Converted.enlEndNote29717(Childs 1990). This time-specific hyperglycemia after burn injury appears to be due to increased gluconeogenesis and glycogenolysis in the liver, mediated by the increased catecholamines ADDIN EN.CITE Wolfe197785Proposal-Converted.enlEndNote8517(Wolfe 1977). The increased gluconeogenesis may be induced by the stimulation of EPI and NE on cAMP in the liver, since cAMP dependent protein kinase inactivates pyruvate kinase, preventing glycolysis, and a shift in liver metabolism towards gluconeogenesis ADDIN EN.CITE Champe1994523elderlypaper2-Converted.enlEndNote5236(Champe 1994).
The second phase of the metabolic response, lasting several weeks after injury, has been termed the flow phase ADDIN EN.CITE Cuthbertson1942380Proposal-Converted.enlEndNote38017Wolfe1981381Proposal-Converted.enlEndNote38117Hart200020Proposal-Converted.enlEndNote2017Thorell1994301Proposal-Converted.enlEndNote30117(Cuthbertson 1942; Wolfe 1981; Thorell 1994; Hart 2000), although more recent studies indicate that this phase may continue in children for at least a year post-burn ADDIN EN.CITE Hart200089elderlypaper2-Converted.enlEndNote8917(Hart 2000). Initial studies in burned animals indicated that glucose uptake in response to insulin injections was decreased ADDIN EN.CITE Turinsky1977375Proposal-Converted.enlEndNote37517(Turinsky 1977). Additionally, insulin release in trauma patients during this time period was twice that of controls in response to a glucose load ADDIN EN.CITE Black1982326Proposal-Converted.enlEndNote32617Galster1984296Proposal-Converted.enlEndNote29617(Black 1982; Galster 1984). In burned children, although plasma glucose decreases after the initial spike, it remains above normal levels, and insulin begins to increase, indicating an insulin resistant state ADDIN EN.CITE Childs1990297Proposal-Converted.enlEndNote29717(Childs 1990). While catecholamines may play a role in this response, plasma cortisol levels begin to decline shortly after the initial injury ADDIN EN.CITE Matsui199149pc540.enlEndNote4917(Matsui 1991). Thus, it is not clear what mediates the insulin resistance in this phase.
Effect of burns on specific fates of glucose
The insulin resistance in burns affects in multiple tissues, but primarily liver and muscle. Glucose uptake in the liver is modulated by the GLUT-2 transporter and is concentration dependent, rather than being insulin sensitive ADDIN EN.CITE DeFronzo1988394Proposal-Converted.enlEndNote39417(DeFronzo 1988). However, the liver is still responsible for approximately 40% of glucose uptake during hyperglycemia ADDIN EN.CITE Sidossis1999401Proposal-Converted.enlEndNote40117(Sidossis 1999). Instead, insulin resistance is seen in other aspects of hepatic metabolism, primarily glucose release. The hyperglycemia seen after 48 hours post-burn is due to EPI induced increased glucose release by the liver, mainly through gluconeogenesis ADDIN EN.CITE Black1982326Proposal-Converted.enlEndNote32617Wolfe19799pc1D4.enlEndNote917(Wolfe 1979; Black 1982). Increased gluconeogenesis may be due to increased levels of glucagon, relative to insulin although this cannot be the only stimulus ADDIN EN.CITE Spitzer1979374Proposal-Converted.enlEndNote37417Jahoor198660Proposal-Converted.enlEndNote6017Wolfe1985413Proposal-Converted.enlEndNote41317(Spitzer 1979; Wolfe 1985; Jahoor 1986). High levels of exogenous insulin fail to suppress the release of glucose in trauma patients ADDIN EN.CITE Black1982326Proposal-Converted.enlEndNote32617Carter2004369Proposal-Converted.enlEndNote36917(Black 1982; Carter 2004). Hence, the treatment goal for improving insulin sensitivity in the liver is to reduce the endogenous production and release of glucose.
The insulin resistance at the level of muscle tissue is evident in the inability of insulin to stimulate glucose uptake through the insulin sensitive GLUT-4 transporter. Burned rats studied with a hyperinsulinemic-euglycemic clamp had inadequate insulin stimulated glucose uptake and muscle IRS-1 was not phosphorylated in response to the insulin ADDIN EN.CITE Carter2004369Proposal-Converted.enlEndNote36917(Carter 2004). Another study with burned rats found a 30% decrease in insulin stimulated glucose uptake on post-burn day 3, concomitant with non-existent PI3-K activation, decreased tyrosine phosphorylaton of IRS-1, and decreased autophosphorylation of the insulin receptor ADDIN EN.CITE Ikezu1997393Proposal-Converted.enlEndNote39317(Ikezu 1997). It is likely that these disruptions in insulin signaling were also present in the insulin resistant burn patients described previously.
Effect of burns on fat metabolism
Fat metabolism is less well understood than glucose metabolism in burn patients. Plasma triglycerides have been documented as being within the normal range in burn patients given low fat feedings ADDIN EN.CITE Aarsland199830Proposal-Converted.enlEndNote3017(Aarsland 1998). Patients who underwent major abdominal surgery experienced no change in the VLDL cholesterol nor TG concentrations, although total, LDL and HDL fractions decreased significantly ADDIN EN.CITE Thorne2005407Proposal-Converted.enlEndNote40717(Thorne 2005). Plasma FFA concentrations in adults with large burns were within the same range as controls, although glycerol concentrations were elevated above normal for the first 20 days post-burn ADDIN EN.CITE Harris1982409Proposal-Converted.enlEndNote40917(Harris 1982). FFAs are bound to albumin since they are hydrophobic, and due to the decreased concentrations of albumin the ratio of FFA to albumin was elevated 3 to 4 fold in these burned adults compared to healthy controls ADDIN EN.CITE Harris1982409Proposal-Converted.enlEndNote40917(Harris 1982). It may be that the decreased albumin concentration limits the release of FFAs, whereas glycerol can more freely enter the plasma.
Despite the variable concentrations of FFAs, free fatty acid flux is increased in burns at all sites of regulation ADDIN EN.CITE Frayn1985377Proposal-Converted.enlEndNote37717Wolfe198757Proposal-Converted.enlEndNote5717(Frayn 1985; Wolfe 1987). The FFA flux represents the futile cycle involving the breakdown of adipose and muscle TGs into FFAs, their subsequent re-esterification into VLDL or TG in the liver, and ultimate reincorporation into adipocytes or muscle TG. In burn patients, the rate of FFA release far exceeds the amount needed for energy use, so that much of the FFA is recycled in the liver and re-secreted as VLDL-TG ADDIN EN.CITE Aarsland199635Proposal-Converted.enlEndNote3517Wolfe1997395Proposal-Converted.enlEndNote39517(Aarsland 1996; Wolfe 1997). In healthy adults, the re-esterification of FFAs account for 65% of VLDL-TG release after 96 hours of hyperglycemia ADDIN EN.CITE Aarsland1996396Proposal-Converted.enlEndNote39617(Aarsland 1996). After bariatric surgery, the clearance of infused TGs by the periphery in obese patients was increased two-fold, and was accompanied by increased plasma glucose and insulin levels ADDIN EN.CITE Thorne2005407Proposal-Converted.enlEndNote40717(Thorne 2005). It appears that all sites of regulation are increased in trauma patients.
Total fat flux is represented both by the FFA and glycerol and may influenced by catecholamines effects on hormone sensitive lipase ADDIN EN.CITE Wolfe1997395Proposal-Converted.enlEndNote39517(Wolfe 1997). For each intracellular TG molecule, hormone sensitive lipase facilitates the release of one glycerol and three FFAs ADDIN EN.CITE Oscai1990397Proposal-Converted.enlEndNote39717(Oscai 1990). When the release of both is in the ratio of 3 FFA to 1 glycerol, all FFAs are released directly from muscle cells and adipocytes, and not re-esterified within the cell. (-adrenergic stimulus of muscle strips from burned animals increases glycerol release three fold, but does not affect FFA release, indicating not only an increased breakdown but also increased FFA recycling within muscle cells ADDIN EN.CITE Enoksson2005104pc540.enlEndNote10417(Enoksson 2005). Glycerol concentrations and turnover were increased significantly 6 days following total hip-replacement surgery ADDIN EN.CITE Carpentier1978412Proposal-Converted.enlEndNote41217(Carpentier 1978). Basal plasma palmitate turnover was significantly increased in trauma patients compared to controls, yet there was no relationship between EPI or NE levels and palmitate turnover ADDIN EN.CITE Galster1984296Proposal-Converted.enlEndNote29617(Galster 1984). In trauma patients, increased glycerol concentrations, but not FFA concentrations, correlated with the severity of the injury ADDIN EN.CITE Stoner1979410Proposal-Converted.enlEndNote41017(Stoner 1979). So in muscle and adipocytes of trauma pateints, the products of TG breakdown are not all leaving the cell, and this may account for the lack of increased plasma FFA concentrations despite the increase glycerol concentrations.
Some studies have found that TG accumulation occurs in the liver in burn patients. This is due in part to decreased (-oxidation of FFA and decreased VLDL-TG secretion ADDIN EN.CITE Morio20027Proposal-Converted.enlEndNote717(Morio 2002). Acute hyperglycemia in healthy volunteers decreases free fatty acid oxidation in the liver and increases VLDL-TG secretion. When these defects are coupled with the increased FFA release, hepatomegaly due to increased TGs ensues. In healthy volunteers, large increases in carbohydrate feeding lead to increased fat accumulation in adipocytes, but in the liver, VLDL-TG synthesis increases, and there is no significant de-novo fat synthesis in the liver ADDIN EN.CITE Aarsland1997404Proposal-Converted.enlEndNote40417(Aarsland 1997).
It is not known if TGs accumulate in muscle of burned children. There is increased delivery of fat to muscle cells in the form of de novo synthesized TGs. However, it has been shown that fatty acid oxidation is increased in burn patients relative to healthy controls ADDIN EN.CITE Herndon2004535elderlypaper2-Converted.enlEndNote53517(Herndon 2004). The degree of the increase in delivery relative to the oxidation is in question. If the delivery rate is higher than the oxidation rate, this would lead to increased intracellular TG accumulation.
Mitochondrial function in burns
A limited number of studies have examined mitochondrial function in skeletal muscle, liver and cardiac muscle in burned animals. Genes for glycolytic enzymes were down regulated in rat hind leg muscle 3 days post-burn, as were TCA cycle genes including citrate synthase, and electron transport chain genes, including cytochrome c oxidase (complex IV) ADDIN EN.CITE Padfield2005371Proposal-Converted.enlEndNote37117(Padfield 2005). NMR studies in these animals also indicated that ATP synthesis in muscle was dramatically reduced compared to sham burn control animals. The skeletal muscle of burned rats released cytochrome C from the mitochondria within fifteen minutes of burn and the mitochondria membrane was severely damaged at 1 hour post-burn ADDIN EN.CITE Yasuhara2000526elderlypaper2-Converted.enlEndNote52617(Yasuhara 2000). Thirty minutes after a 20% burn in rats liver cytochrome C was decreased significantly ADDIN EN.CITE Wang1986524elderlypaper2-Converted.enlEndNote52417(Wang 1986). ATP and cytochrome C production in heart cell was significantly depressed in heart myocytes for 3-24 hours post-burn in rats ADDIN EN.CITE Liang2002525elderlypaper2-Converted.enlEndNote52517(Liang 2002). These studies indicate that at least in animals, muscle insulin resistance in burns may be relate to mitochondrial defects. However, no studies of this nature have been completed in humans.
Intensive insulin therapy in burns
Intensive insulin therapy has been used to control hyperglycemia and improve morbidity in trauma and burn patients in an attempt to decrease mortality. In critically ill non-surgical patients, intensive insulin therapy was correlated with a 46% reduction in bloodstream infections, a 41% reduction in kidney failure requiring dialysis, a 50% reduction in blood transfusions and a decrease in time spent on mechanical ventilation ADDIN EN.CITE van den Berghe2001341Proposal-Converted.enlEndNote34117(van den Berghe 2001). Furthermore, the mortality rate was lowered to 4.6 % from 8.0 % ADDIN EN.CITE van den Berghe2001341Proposal-Converted.enlEndNote34117(van den Berghe 2001). In burn patients, time spent in intensive care is dependent on the severity of the burn and the covering of burned areas with donor skin through auto-grafting or from cadavers. Very high-level exogenous insulin administration in burn patients to maintain a plasma insulin level of 400-900(U/mL was shown to improve the donor site wound healing from 6.51.0 days to 4.21.2 days ADDIN EN.CITE Pierre1998325Proposal-Converted.enlEndNote32517(Pierre 1998). In burned children, moderate dose insulin therapy decreased acute phase proteins and cytokines TNF-(, IL-1 and IL-6 from week 1 to week 4 post-burn, whereas there was no decrease in these measures in placebo-treated burn children ADDIN EN.CITE Wu2004346Proposal-Converted.enlEndNote34617(Wu 2004). The two current leading causes of death in burned children at the Shriners of Galveston hospital are kidney failure and severe infection (Jonathan Espana Denorio, MD, Shiners Burn Fellows Meeting Presentation, Galveston, TX Nov 2003). and, a reduction in these co-morbidities could lead to a reduction in mortality.
While there are documented benefits, insulin therapy is not without risk. Its administration requires intensive monitoring to avoid hypoglycemia. Furthermore, in order to meet the glucose demands during exogenous insulin administration, caloric intake must be increased far beyond the metabolic need of the patient, contributing to increased adiposity and thus increased plasma FFA due to fat deposition ADDIN EN.CITE Hart2002311Proposal-Converted.enlEndNote31117Burke197978Proposal-Converted.enlEndNote7817(Burke 1979; Hart 2002). Additionally, while insulin lowers plasma glucose levels, studies have shown that the insulin resistance still exists in terms of fat metabolism. High levels of exogenous insulin, given for seven days to severely burned patients (638% TBSA) did not suppress the release of endogenous free fatty acids from adipocytes ADDIN EN.CITE Aarsland199830Proposal-Converted.enlEndNote3017(Aarsland 1998). The combination of increased calories and high plasma FFAs can lead to increases in liver size ADDIN EN.CITE Burke197978Proposal-Converted.enlEndNote7817(Burke 1979). Indeed, three days of insulin infusion in type 2 diabetics leads to decreased plasma glucose, in conjunction with increased muscle and liver fat ADDIN EN.CITE Anderwald2002527elderlypaper2-Converted.enlEndNote52717(Anderwald 2002). Insulin also prevents the transcription of CPT-1 in the liver, which would have the potential of decreasing fat oxidation, and increasing fat accumulation ADDIN EN.CITE Morio2003400Proposal-Converted.enlEndNote40017(Morio 2003). Furthermore, exogenous insulin exacerbates hyperinsulinemia and associated high plasma FFAs.
If insulin resistance itself could be reduced in this population, it is likely that not only would mortality decrease and time in the ICU be shortened, but also that major complications such as infection, muscle wasting and renal failure would be reduced.
Other medications to prevent insulin resistance in burns
In addition to insulin, several other therapies have been tried to alleviate insulin resistance. Adrenergic blockers such as propranolol or phentolamine have been studied as potential therapeutics. However, while these drugs reduce overall energy expenditure, they do not cause a normal pattern in lipid oxidation, glucose production or glucose clearance in burned humans or animals ADDIN EN.CITE Breitenstein1990306Proposal-Converted.enlEndNote30617Durkot198172Proposal-Converted.enlEndNote7217Wolfe198758Proposal-Converted.enlEndNote5817Wolfe198757Proposal-Converted.enlEndNote5717Herndon199438Proposal-Converted.enlEndNote3817Breitenstein1990306Proposal-Converted.enlEndNote30617(Durkot 1981; Wolfe 1987; Wolfe 1987; Breitenstein 1990; Herndon 1994). Moreover, plasma palmitate turnover in burns is associated with size of burn, but not with the increased levels of NE or Epi ADDIN EN.CITE Galster1984296Proposal-Converted.enlEndNote29617(Galster 1984). Metformin was used in patients to increase the uptake of glucose by muscle and adipose cells. However, the cells were not able to metabolize the glucose fully, and lactic acidosis ensued ADDIN EN.CITE Gore20034Proposal-Converted.enlEndNote417(Gore 2003). Thus, none of these therapies adequately alleviates insulin resistance in the burn population.
In summary, burned patients have abnormal glucose and fat metabolism but the mechanism of insulin resistance has not been identified. Many metabolic processes are affected, yet to date no drug therapy has successfully treated all abnormal processes.
PPAR-( agonist manipulation of fat and glucose metabolism
PPAR-( agonists in animals
Peroxisome proliferator activating receptors (PPAR) are nuclear receptors that, when stimulated by endogenous lipids, activate specific genes involved in fat metabolism. High levels of PPAR-( receptors are found in the liver, and lower levels in muscle and fat tissue ADDIN EN.CITE Su1998448elderlypaper2-Converted.enlEndNote44817(Su 1998). In animals, PPAR-( activators such as WY-14643, a potent PPAR-( agonist, have been shown to affect multiple aspects of fat and glucose metabolism ADDIN EN.CITE Boden G200120elderlypaper2-Converted.enlEndNote2017Guerre-Millo M200085elderlypaper2-Converted.enlEndNote8517(Guerre-Millo M 2000; Boden G 2001). Of interest is the apparent ability of PPAR-( agonists to lower plasma triglycerides, increase fat oxidation and improve insulin sensitivity. PPAR-( agonist administration in insulin resistant animals increased peripheral glucose metabolism and FFA (-oxidation ADDIN EN.CITE Sugden2003319Proposal-Converted.enlEndNote31917Guerre-Millo M2000224Proposal-Converted.enlEndNote22417Lee2002312Proposal-Converted.enlEndNote31217Chou CJ2002213Proposal-Converted.enlEndNote21317Ye JM2003295Proposal-Converted.enlEndNote29517Ye JM2001294Proposal-Converted.enlEndNote29417Furuhashi2002339Proposal-Converted.enlEndNote33917(Guerre-Millo M 2000; Ye JM 2001; Chou CJ 2002; Furuhashi 2002; Lee 2002; Sugden 2003; Ye JM 2003). Significant decreases in fasting plasma glucose, insulin and triglycerides, with concomitant decreases in IMCL and LFAT, have been reported in lipoatrophic mice, mice given high fat diets and diabetic Zucker rats after treatment with PPAR-( agonists ADDIN EN.CITE Guerre-Millo M200085elderlypaper2-Converted.enlEndNote8517Chou CJ200230elderlypaper2-Converted.enlEndNote3017(Guerre-Millo M 2000; Chou CJ 2002). PPAR-( agonists decreased intracellular fatty acyl coA and malonyl CoA and increased fatty acid oxidation in rodents ADDIN EN.CITE Young2001502elderlypaper2-Converted.enlEndNote50217Furuhashi200259elderlypaper2-Converted.enlEndNote5917(Young 2001; Furuhashi 2002). PPAR( agonists have also been shown to upregulate expression of mitochondrial proteins involved in fat breakdown including HMG CoA synthase, liver long chain acyl CoA thioesterase, peroxisomal bifunctional enzyme FAT/CD36 and CPT-1 ADDIN EN.CITE White2003314Proposal-Converted.enlEndNote31417Lee2002312Proposal-Converted.enlEndNote31217Guerre-Millo M2000224Proposal-Converted.enlEndNote22417Zhang2004328Proposal-Converted.enlEndNote32817Furuhashi2002339Proposal-Converted.enlEndNote33917(Guerre-Millo M 2000; Furuhashi 2002; Lee 2002; White 2003; Zhang 2004). In human muscle, endogenous levels of PPAR-( protein correlated with mRNA levels of CD36, lipoprotein lipase, CPT-1 and mitochondrial uncoupling proteins 2 and 3 ADDIN EN.CITE Zhang2004328Proposal-Converted.enlEndNote32817(Zhang 2004). PPAR-( agonist treatment in human myocytes increased (-oxidation of oleate and decreased oleate incorporation into TAG. Corresponding with these changes, mRNA levels of carnitine palmityltransferese 1 (CPT-1), malonyl-CoA decarboxylase (MCD) and pyruvate dehydrogenase kinase (PDHK) increased ADDIN EN.CITE Muoio2002452elderlypaper2-Converted.enlEndNote45217(Muoio 2002). Recent studies suggest that PPAR( agonists may also affect intracellular insulin signaling ADDIN EN.CITE Lee2002312Proposal-Converted.enlEndNote31217Frederiksen2003335Proposal-Converted.enlEndNote33517(Lee 2002; Frederiksen 2003).
These studies show a direct effect of PPAR( agonists on rodent fat and glucose metabolism. They improve whole body insulin sensitivity and fat oxidation. This appears to happen because of an increase in expression of mitochondrial oxidative genes, a decrease in inhibitory molecules such as DAG and improvement in intracellular insulin signaling. Thus, in certain models, the PPAR-( agonists affect fat metabolism and improve insulin sensitivity by increasing mitochondrial oxidative capacity.
PPAR-( agonists in humans
Despite the encouraging results in animal and in-vitro studies, results of PPAR-( agonist treatment in young and middle aged humans have not been as clear-cut. Some studies have found that PPAR-( drugs lower plasma triglycerides and fasting concentrations of insulin, and thus improve insulin sensitivity as assessed by HOMA scores, or improve insulin area under the curve following an oral glucose tolerance test (OGTT) ADDIN EN.CITE Idzior-Walus2000117elderlypaper2-Converted.enlEndNote11717Tan2001464elderlypaper2-Converted.enlEndNote46417Yong1999336elderlypaper2-Converted.enlEndNote33617(Yong 1999; Idzior-Walus 2000; Tan 2001). Other studies have also found a decrease in fasting plasma glucose concentrations, and HbA1C following treatment ADDIN EN.CITE Damci2003453elderlypaper2-Converted.enlEndNote45317Kobayashi1988465elderlypaper2-Converted.enlEndNote46517(Kobayashi 1988; Damci 2003). In contrast, some studies failed to identify an effect of PPAR-( agonists on insulin sensitivity in varied populations ADDIN EN.CITE Vega2003446elderlypaper2-Converted.enlEndNote44617Forcheron2002449elderlypaper2-Converted.enlEndNote44917(Forcheron 2002; Vega 2003). Thus the role of PPAR-( in improving insulin sensitivity in humans is not well established.
Aims and Hypothesis of Studies Conducted
In light of these recently established associations between intracellular lipids and glucose metabolism, four complimentary studies were conducted in an attempt to better understand the relationship between lipid metabolism and insulin resistance. Two unique models of insulin resistance were studied: aging and burn trauma.
Study 1: Young vs. Elderly
The aim of the first study was to examine the relationship between LFAT, IMCL and insulin sensitivity in healthy young and elderly. We hypothesized that elderly, who have increased insulin resistance, would also have an increase in intracellular fat. An important component of this study was the development of the magnetic resonance spectroscopy method at UTMB for the non-invasive measurement of the intracellular lipids.
Study 2: 60 days of fenofibrate in the elderly
The aim of the second study was to examine the effect of PPAR( agonist administration on the (-oxidation of fats in elderly with or without insulin resistance, but with normal lipids profiles. We hypothesized that the accumulation of intracellular lipid was due to decreased (-oxidation in the mitochondria. Treatment with a PPAR( agonist increases the rate of (-oxidation, and therefore should decrease intracellular lipids and improve insulin sensitivity.
Study 3: 14 days of fenofibrate in severely burned children
The aim of the third and largest study was to examine the effect of the PPAR( agonist fenofibrate on insulin resistance in burned pediatric patients. It was hypothesized that PPAR( agonist treatment in burned patients would improve insulin sensitivity by increasing the (-oxidation of fats. We hypothesized that burned children had mitochondrial damage, and that the PPAR( agonist would restore mitochondrial functioning. Furthermore, we hypothesized that the improved insulin signaling at least in part would be due to a decrease in fatty acid metabolites such as DAG. This decrease in FFA metabolites would further increase CPT-1 activity and (-oxidation leading to a decrease in PKC activity and ultimately increase signaling activity and insulin sensitivity.
Study 4: Mitochondrial function in healthy children
The last study was a small control study. The aim was to examine the mitochondrial function in normal children, as compared to burn victims. We hypothesized that the mitochondrial function in burned children was lower than that in healthy children.
Summary
Studies in type 2 diabetics have indicated that insulin resistance in this population is mediated by increased intracellular fat storage due to decreased fat oxidation and increased fat delivery. Further, metabolites of the intracellular fat may directly prevent insulin signaling. It is unknown if these same mechanism are present in other forms on insulin resistance, namely aging and burn trauma. Studying this mechanism in these two populations is novel, and contributes to this field of knowledge. PPAR-( agonists were used as treatment modalities in both populations to not only create a dynamic system of study, but also to study a new therapeutic approach for these patients.
Chapter 2
Protocol DesignS
Study 1: Young vs. Elderly
This was a cross-sectional design. All volunteers were screened by laboratory and physical exam prior to the study and found to be healthy for inclusion. Exclusion criteria included a total cholesterol >220 mg/dL, abnormal serum thyroid stimulating hormone level, palpable liver enlargement, positive Hepatitis B, C or HIV tests, anemia or elevation of more than one of the following: alkaline phosphatase >122 U/l, ALT > 51U/I, AST >40 U/I. Subjects were not taking lipid lowering medications, diabetes medications, anticoagulants, illicit drugs and were not to consume alcohol in excess (>1 drink/day or 6/wk).
Volunteers were admitted to the General Clinical Research Center at the University of Texas Medical Branch in the evening. Following an overnight fast, MRS was conducted, followed by a full body dual x-ray absorptiometry (DEXA) scan to measure body composition. At approximately 8 am, a 20 gauge IV catheter was inserted in the antecubital vein for blood sampling. After 2 baseline samples, blood was sampled every 30 minutes. A 2-hour oral glucose tolerance test (OGTT) with a 75 mg dextrose load was performed. The circumferences of the waist and hip were measured.
Study 2: Fenofibrate in the Elderly
To assess the effects of fenofibrate in an ambulatory healthy elderly population, a longitudinal cohort study was performed. All volunteers were healthy by history and physical exam, and none participated in regular aerobic or resistance training routines. Exclusion criteria included a total cholesterol >300 mg/dL (6.5 mmol/L), abnormal thyroid stimulating hormone level, palpable liver enlargement, positive Hepatitis B, C or HIV tests, anemia or elevation of more than one of the following: alkaline phosphatase >122 U/l, ALT > 51U/I, AST >40 U/I. Subjects were not taking lipid lowering medications, diabetes medications, anticoagulants, illicit drugs or consuming alcohol in excess (>1 drink/day or 6/wk).
All volunteers started fenofibrate treatment (160mg micronized Tablets once daily) on day one, following the completion of the baseline study. Subjects were given the full 60 days dose in a special pill pack, with each day and date clearly labeled. Subjects were required to bring the pill pack with them to each visit in order to assess compliance.
The subjects were studied on five occasions three as inpatients and two as outpatients. On inpatient days (days 1, 11 and 61 of the study) volunteers were admitted to the General Clinical Research Center (GCRC) at the University of Texas Medical Branch. Following a 12 hour overnight fast, IMCL and LFAT were measured by magnetic resonance spectroscopy (MRS) and body composition with a dual x-ray absorptiometry (DEXA) scan. A 20 gauge IV catheter was inserted in the antecubital vein for blood sampling. After 2 baseline samples, a 2-hour OGTT with a 75 mg dextrose load was performed. Waist and hip measurements were taken, and a 24 hour food record was collected. The two outpatient visits occurred on day 6 and day 35. The volunteers were fasting for both the outpatient visits, and OGTT was performed on day 6, and liver function tests were performed on day 35.
Study 3: Fenofibrate in Burns
Overall study design
Assessment of the acute affect of fenofibrate in pediatric children was assessed with a placebo controlled, prospective randomized clinical trial. Children between the ages of 4-16 and greater than 20 kg at admission were studied in the acute burn period. In order to provide homogeneity, enrollment was limited to children who had suffered flame burns over greater than 40% of their total body surface area and were admitted to the Shriners Burns Hospital within 96 hours of burn. A parent or legal guardian provided permission for participation of the child in the study and assent was obtained from children who were over the age of seven years of age when medically and ethically possible. Children with major electrical burns, renal failure, iodine allergies, severe sepsis or who had required cardiac resuscitation were excluded.
Two 8 hour tracer studies were conducted approximately 2 weeks apart, and
approximately 4 days after the childrens first and third operations, to avoid acute surgery-induced metabolic changes, but using existing arterial and venous access. Children with greater than 40% burns generally have at least 3 or more surgeries, each approximately 7 days apart. The first study was conducted after the first excision and grafting operation, ollowing admission, with a few exceptions. Since only children admitted to the hospital within 96 hours of burn were enrolled, the majority of the first infusion studies occurred 5 to 7 days post-burn. A few patients, the first surgery comprised of only excision and homo-grafting rather than auto-grafting, as the skin of the child was too infected to do successful auto-grafting. In these cases the children returned to surgery 48 hours later for auto-grafting grafting procedures. The study was then conducted 4 days after this second procedure. However, all initial
Figure 2.1
Study Design EMBED PowerPoint.Slide.8 The overall study design is shown above. Briefly, after injury children received an operation, and then a metabolic study four days later. Then after their second operation they were randomized to fenofibrate or placebo until four days after their third operation, when the second metabolic study was performed.metabolic studies were conducted within 10 days of the injury. The second studies were conducted after the third operation, Which was done approximately 18-21 days post-burn.
MRS analysis was conducted the day following the tracer studies. At this time, all staples had been removed, so the children could safely enter the MRI. Following MRS, a DEXA scan was performed to measure the body composition of the children. The children generally returned to the operating room for their second operation within 48 hours of the MRS, and during the operation a muscle sample was taken.
Following the post-study operation, the children were randomized to either fenofibrate (Tricor) or a placebo, as pre-determined by a randomization schedule. For the children receiving fenofibrate, a 5 mg/kg dose was ground up in 1-2 cc of 200 proof ethyl alcohol and suspended in standard pharmacy drug suspension agent with a flavoring agent. The dosage of 5 mg/kg is that commonly used for fenofibrate administration in children in Europe, as the drug is not approved for use in children in the United States. An FDA exemption for this protocol was granted to allow administration of the fenofibrate to children in this study. The placebo consisted of the suspension and flavoring agents. The medications were administered daily at 13:00 through a naso-gastric tube or orally, and liver enzymes were drawn every other day at 1:00. The medication was given following the second surgery until prior to the fourth surgery. This allowed for a mean treatment period of 11 days between studies.
Tracer study design
The tracer study was performed in association with a hyperinsulinemic- euglycemic clamp to measure glucose and fat metabolism. To achieve a basal fasting state within reasonable clinical limits, each study was preceded by a 4 hour fast with IV fluids of only 0.9% or 0.45% saline, and 8 hours of hemodynamic stability i.e. no blood or albumin transfusions. Prior to the start of the tracer infusions, blood was taken for background concentration and enrichments for glucose and FFA. Cholesterol and cortisol concentrations were also obtained at this time. The 4 hour basal period was followed by a 4 hour hyper-insulinemic-euglycemic clamp.
Figure 2.2
Tracer Infusion Study Design
The tracer study used 1 tracer for glucose and 1 for fat. The 6,6-2d glucose ran for all 8 hours, and the U-13C Palmitate for the last two hours of each period. Blood samples are described in the text.
Glucose Study Design
The hyper insulinemic- euglycemic clamp was used to assess glucose uptake in response to insulin, and to measure the change in fat metabolism in response to insulin. Insulin was infused at a rate of 1.5 mU(min-1(kg-1 into a vein and D20 was simultaneously infused in the same line to maintain a plasma glucose concentration between 85-95 mg/dL. Whole blood glucose was measured every 10 min from a different venous site and adjustments were made to the rate of D20 infusion to obtain the desired plasma concentration. Whole blood glucose measurements are 5% lower than plasma, so the whole blood glucose target during the clamp was 80-90 mg, to achieve the intended plasma glucose goal.
A primed constant infusion of 6,6-2d glucose was started at a rate of 0.444 (mol(min-1(kg-1 and run throughout the entire study period. Arterial and venous samples for glucose concentration and arterial samples for glucose enrichment were taken 10 min apart in triplicate at the end of each period.
Fat Study Design
A primed infusion of U-13C palmitate was started 2 hours after the glucose at a rate of 0.08(mol(min-1(kg-1, and 1 hour was allowed to reach steady state before samples were taken for assessing palmitate turnover. Four arterial, venous and breath samples were taken 10 min apart. A metabolic cart was used during the sampling period to measure the volume of CO2 expired. The same primed infusion of U-13C Palmitate was again started 2 hours after the start of the clamp with an identical sampling schedule.
Other measurements:
Blood flow
Leg blood flow was assessed during each period by the indocyanine green (ICG) dilution method ADDIN EN.CITE Wolfe2005505elderlypaper2-Converted.enlEndNote5056(Wolfe 2005). ICG was infused into the femoral artery at a rate of 60 mL/hr for 10 minutes prior to sampling. Three sets of simultaneous samples were drawn from the femoral vein in the same leg as the infusion, and from another venous site- either a peripheral line in the arm, the femoral vein of the other leg, or a subclavian venous line, assess the dilution of the dye. A blood sample from the second venous site was also sent to the clinical lab during the ICG infusion for a measurement of the hematocrit.
Resting energy expenditure
Resting energy expenditure was measured with a metabolic cart twice during each tracer study. The basal state measurement was done 3 hours after the start of the basal period and the clamp measurements were conducted 3 hours after the start of the clamp.
Intracellular Fat
Liver and soleus fat were assessed by magnetic resonance spectroscopy (MRS). MRS procedures were performed at 5am the morning following the infusion study.
Body composition
Whole body fat was measured using Dual X-Ray absorbiometry(DEXA) the day following the infusion study.
Muscle biopsy measurements:
Four muscle biopsies were obtained during each study. Three muscle biopsies were taken during the tracer study from the vastus lateralus muscle in the leg using a Bergstrom needle. These were taken 1 hour into the protocol, at the end of the basal period, and at the end of the clamp period. The fourth muscle biopsy was taken in the operating room during surgery following the tracer study.
The first muscle biopsy sample was used for baseline insulin signaling measurement and to assess fasting mitochondrial function. Twenty five to forty mg of the muscle sample was rinsed with cold saline and blotted, and then frozen at -80C until analysis for intracellular signaling components, including but not limited to the insulin receptor levels and phosphorylation, insulin receptor substrate 1 levels and phosphorylation, and protein kinase C. Thirty to forty mg of the muscle sample was placed into an ice-cold buffer and homogenized in a stabilizing buffer and then frozen at -80C until analysis.
The second muscle biopsy was used to measure the enrichment of either DAG, fatty acyl CoA or fatty acyl carnitine. It was also used to measure the concentration of some of these fat metabolites.
The third biopsy was taken at the end of the clamp period and used to measure the effect of insulin on the insulin receptor intracellular signaling cascade. The samples were processed as explained above.
The fourth muscle biopsy was taken during the second operation. This muscle was used to measure the ratio of the fatty acyl CoA: fatty acyl carnitine concentrations. This muscle sample was quickly rinsed with cold saline and snap frozen in liquid nitrogen before being transferred to a -80C freezer.
Study 4: Mitochondrial Function in Healthy Children
The majority of the questions posed in study three can be answered by comparing the treatment group to the PLA. References for the normal pediatric variables measured are generally available in the literature, with the exception of mitochondrial function. Thus, in order to quantitate the extent of mitochondrial damage in the burn patients, it is important to know the level in normal children. Children undergoing open abdominal surgeries for non-metabolic illness were chosen as our control population. A small piece of the rectus abdominis or external oblique muscle was removed from the margin of the site of the surgical incision site for mitochondrial oxidation analysis.
Chapter 3
Methods
Sample analysis for All studies
Blood samples
Plasma glucose concentration
After collection in sodium fluoride/potassium oxalate tubes, samples were spun and the plasma isolated. Venous and arterial plasma glucose concentration was measured on an YSI 2300 Stat glucose/lactate analyzer (YSI, Inc. Yellow Springs, OH). Samples were run twice and the instuments accuracy was checked with a standard each day before use.
Serum Insulin Concentrations
Blood was collected in serum separator test tubes and the serum was separated and stored at -80(C until analysis. After thawing, insulin levels were measured by RIA (Diagnostic Laboratories, Los Angeles, CA). The coefficient of variation for these measurements in our lab is <10%.
Plasma total FFA concentrations - Study 1 and 2 only
Total free fatty acids were measured using a chromatographic kit (Wako Diagnostics, Osaka, Japan)
Imaging
MRS soleus:
IMCL was measured with a 1H knee coil on a GE Advantage 1.5 Tesla whole body imager (General Electric, Milwaukee, WI). The calf of the dominant leg was secured within the center of the coil with the subject lying in a supine position and the ankle secured in a neutral position. A tube of 20 % Intralipid (IV high fat total parenteral feeding solution Baxter Healthcare Deerfield Park, IL) was placed inside the knee coil to obtain a standard external reference to normalize IMCL concentrations ADDIN EN.CITE Perseghin G199913EndNote017(Perseghin G 1999). The orientation of the leg in reference to the magnetic field (B0) and the coil position was selected from pilot studies to provide optimal splitting of the IMCL and extracellular fat (EMCL) resonances in the soleus muscle. After a preliminary localization image, 4 voxels (approximately 7 mm x 7 mm x 10 mm each) were chosen in soleus muscle free from fascia, gross fat marbling and vessels. The exact voxel volumes were recorded. A voxel was also chosen from the Intralipid( external reference. An optimized PRESS sequence with repetition time (TR) of 1188ms and echo time (TE) of 35 ms was run.
MRS Peak positions and areas of interest (extramuscular (CH2)n, intramuscular CH2)n, extramuscular (CH3), intramuscular CH3, total creatine (tCr), and tri-methylamines (TMA) were determined by time domain fitting using jMRUi ADDIN EN.CITE van den Boogaart A199698EndNote017van den Boogaart A199797EndNote06(van den Boogaart A 1996; van den Boogaart A 1997). It must be noted that there are compatibility problems between jMRUI and certain versions of GE software. GE software platforms 8x and 9x cannot be used with jMRUi versions 1.2 and 2.1 because the data generated a margin of error. Data from these GE platforms muay only be analyzed with jMRUi version 2.2, released January 2005. In brief, all water-suppressed FID (metabolite FID) were deconvoluted with the water unsuppressed FID (water FID) acquired from the same voxel in order to correct for zero-order phasing and removal of eddy-current induced artifacts ADDIN EN.CITE Klose U199052EndNote017(Klose U 1990). The resulting metabolite FIDs were analyzed with AMARES, a non-linear least square fitting algorithm operating in the time domain ADDIN EN.CITE Vanhamme L199795EndNote017(Vanhamme L 1997). The time domain model function was composed of four exponentially decaying sinusoids corresponding to the four Lorentzian peaks in the frequency domain assigned to the resonances of interest (i.e., EMCL and IMCL-(CH2)n- and -CH3 peaks). Additionally a Lorentzian decaying sinusoids were used to represent the additional lipid resonances, and two Gaussian decaying sinusoids were used to represent the tCr and TMA resonances. The prior knowledge information used for the AMARES fitting algorithm has been previously published by Rico-Sanz ADDIN EN.CITE Rico-Sanz J1999100EndNote017(Rico-Sanz J 1999). Spectra from voxels that did not have optimal shimming or clear intracellular and extracellular lipid peak resolution were not used in AMARES fitting analysis. This process was repeated for the Intralipid( phantom.
MRS liver
The LFAT was measured with a 1H whole body coil on the same system. Hepatic measurements were performed in the middle right lobe ADDIN EN.CITE Tarasow E20022EndNote017(Tarasow E 2002). A tube of Intralipid was again used for reference. After a preliminary localization scan, a voxel (approximately 30 mm x 30 mm x 20 mm) was chosen at a location free from large vessels. For study one, an optimized PRESS sequence with repetition time (TR) of 2000 ms and echo time (TE) of 40 ms was used to acquire 256 scans. For studies 2 and 3, an optimized PRESS sequence with repetition time (TR) of 1188 ms and echo time (TE) of 40 ms was used to acquire 128 scans. The settings used in study one are more sensitive, and are required as the healthy young have much less liver fat. Further, the time of the scans performed for studies two and three are dramatically reduced, making performance of these measures practical in the burned patients. These spectra represent an average LFAT measurement over the mid right lobe as respiratory gating was not conducted. By using light restraints across the chest and coaching shallow breathing, the movement induced by respiration was reduced. This was not possible in children who were intubated and receiving ambu bag ventilary assistance from a respiratory therapist during the scan. In these children, care was taken to scan the area of the liver close to the lumbar spine. The processing of the scans with jMRUi was similar to as explained above, except that the scans from the liver were not phased with the water, due to the inherent movement from respiration. Thus phasing would introduce error, rather than reduce variability, as it does for the soleus scans.
Dual X-Ray absorptometry (DEXA)
Whole body dexa scans were conducted on a Discovery QDR 4500A (Hologic Bedford, MA) following the MRI, in all studies. Healthy volunteers were dressed in metal free clothing, generally scrubs. In the patients, burn dressings were removed to the fullest extent allowed clinically. All scans were analyzed by the same person to reduce variability.
Calculations
Matsuda Model-Study 1 and 2 only
The composite model for Insulin Sensitivity designed by Matsuda ADDIN EN.CITE Matsuda M199933EndNote017(Matsuda M 1999) was used to assess whole body insulin sensitivity following a 75 g oral glucose load.
Equation 1: M= _______10,000_______
Sqrt (FPG x FPI x Mean OGTT G x Mean OGTT I)
Where FPG is fasting plasma glucose and FPI is fasting plasma insulin. The value derived from this equation is an M value of glucose uptake in mg/kg/min which is approximated to results that would likely have been obtained if a more invasive hyperinsulinemic-euglycemic clamp had been performed ADDIN EN.CITE Matsuda M199933EndNote017(Matsuda M 1999). FPG stands for fasting plasma glucose, FPI for fasting plasma insulin, Mean OGTT G and I for the mean plasma glucose level and insulin level, respectively, during the 2-hour OGTT.
The range of values is from 0-14, with 14 being the highest level of insulin sensitivity, and 0 the lowest.
sample analysis for study 3 only
Whole blood and plasma analysis
Glucose enrichment
After collection in lithium heparin tubes, arterial samples were centrifuged and the plasma was removed and frozen at -80(C until analysis. The thawed plasma was mixed with barium hydroxide and zinc sulfate, vortexed, and placed on ice for 30 min. The samples were then centrifuged, and the supernatant was added to 200-400 H cation CL anion columns. The glucose was eluted through the columns with water, and placed on the speed vacuum without heat to dry. The samples were then mixed with 2:1 acetic anhydride:pyridine to be run on the GCMS. The penta-acetate derivative was examined at the 200/202 mass fragments.
Free Fatty Acid Concentration and Enrichment
The blood was collected in EDTA tubes and the isolated plasma frozen at -80(C. 200(L of plasma was added to an internal standard of heptadecanoic acid and 5 mL of extraction solution of HCl, n-Heptane and 2-Propanol. The mixture was vortexed for 30 minutes and then 3 mL of n-Heptane and 2 mL of water were added. The samples were vortexed again for 20 minutes and shaken for 15 minutes and then the upper organic phase was removed and evaporated under nitrogen. The solid was reconstituted with 50(L chloroform. Then the solution was pipetted onto a thin layer chromatography plate. After all of the sample has been loaded to individual columns, along with standards, the plate was run in a tank with heptane, ethyl ether and glacial acetic acid (70:30:1). The solution was allowed to run to within 1 cm of the top of the plate to maximize separation. The plate was allowed to dry and then sprayed with a primuline solution to force the visualization of the lipid bands on the plate. The FFA band is the third band from the top, below the cholesterol and TG band. The FFA bands were cut off the plate and purified in the same manner as the DAG samples (see below). The solid was reconstituted in heptane for GC and MS. For the enrichment of the methyl palmitate, an HP/Agilent 5973 Mass Spectrometer (Wilmington, DE) is used. The ions of interest are at M+0, M+15 and M+16, which yield fragments of 270, 285 and 286. For the concentration, the Hewlet Packard 5890 Gas Chromatography machine with the 3392A Hewlett Packard integrator was used for the first half of the study. The concentrations for the second half of the children enrolled were measured on a 6890n gas chromatogram with a 7683B auto injector (Agilent technologies, Palo Alto, CA)
Carbon Dioxide Concentration and Enrichment
Blood CO2 samples were collected in 15 ml vacutainer tubes containing 7 drops of 85% phosphoric acid. The blood was collected in blood gas syringes, and injected directly into the vacutainer tubes so as not to allow outside air to enter the tubes. The samples were loaded onto the IRMS (VG ISOGAS, Cheshire, England) for analysis. A standard curve prepared from the phosphoric acid and 45CO2 standard (Bayer, East Walpole, MA) was used to calculate the concentration in each of the blood sample. When the sample entered the mass spectrometer chamber, the concentration was measured by the ion chamber beam. Then the IRMS calculates the enrichment of mass ratios 44/45 compared to that of a standard CO2 gas. Samples were analyzed within 14 days of collection to avoid a decrease in the concentration and enrichment, possibly through condensation of the CO2 molecules.
Plasma Glycerol
The blood was collected in EDTA tubes and the separated plasma was frozen at -80(C. Ten l of the sample was used to determine the glycerol concentration using an enzymatic free glycerol determination kit (Sigma, St Louis, MO).
Muscle Tissue
Diacyglycerol (DAG)
Muscle samples were quickly cleaned with saline and frozen at -80(C until analysis. Frozen muscle tissue was pulverized with a Bessman medium tissue pulverizer (Spectrum, Houston, TX) at -80 C and a weighed sample put into a test tube with 3 ml of extraction solution (1:2 (v/v) Methanol:Chloroform containing 0.05 mg/ml BHT). Internal standards for triglycerides (1mg Tri-C17:0/ml chloroform) and DAG (0.1mg 1, 3-Dipentadecanoyl-glycerol /ml choloroform) were added, and the sample was vortexed and then refrigerated for 24 hours to allow for extraction of the lipids. Glass pipettes were used for all mixtures containing chloroform.
Following extraction the solution was centrifuged at 3,500 rpm for 30 min at 4(C. The supernatant was transferred and dried under gent l e n i t r o g e n f l o w . W h e n d r y , 5 0 l o f c h l o r o f o r m w a s a d d e d , a n d t h e n t h e s o l u t i o n w a s l o a d e d o n t o t h i n l a y e r c h r o m a t o g r a p h y p l a t e s w i t h c a p i l l a r y t u b e s , a l o n g w i t h e x t e r n a l s t a n d a r d s i n s e p a r a t e l a n e s . T h e p l a t e w a s p u t i n t o a t a n k c o n t a i n i n g H e x a n e : E t h y l E ther:Acetic Acid 70:30:1 (v/v) and the fluid allowed to rise to approx 1 cm from the top of the plate. The plate was then dried, and sprayed with primuline solution and the DAG and TAG band were visualized under a UV lamp at a wavelength of 365 nm.
The TAG and DAG bands were scraped into 13 x 100 screw top tubes and 50 l h e x a n e a n d 1 m l 1 4 % B F 3 - C H 3 O H w e r e a d d e d . T h e l i p i d d i s s o l v e s i n t h e h e x a n e , w h e r e a s t h e B F 3 - C H 3 O H d i s s o l v e s t h e s i l i c o n e f r o m t h e c h r o m a t o g r a p h y p l a t e . T h e s a m p l e s w e r e t h e n v o r t e x e d f o r 3 m i n b y h a n d , h e a t e d a t 1 0 0 C f o r 4 m i n a n d c o o l e d i n a n i c e b a t h . O n e m l o f d d - H 2 O a n d 2 m l h e x a n e w e r e a d d e d , a n d t h e s o l u t i o n s h a k e n f o r 1 5 m i n , t h e n c e n t r i f u g e d f o r 1 5 m i n a t 2 , 0 0 0 r p m . T h e u p p e r p h a s e c o n t a i n i n g t h e h e x a n e a n d s a m p l e w a s r e m o v e d , d r i e d , a n d t h e n r e c o n s t i t u t e d i n 1 0 0 l h e x a n e f o r D A G a n d 2 0 0 l f o r TAG in preparation for analysis by GC on a 6890n gas chromatogram with a 7683B auto injector (Agilent technologies, Palo Alto, CA)
Fatty Acyl CoA
Muscle samples were quickly rinsed with saline and frozen at -80C until analysis. Up to 40 mg of muscle was pulverized at -80C, and then 500L of a 1:4 solution of 2-propranol and 100mM KH2PO4 buffer (pH=4.7) solution was added. An internal standard of heptadecanoyl CoA(50L of 1ng/ml) was added and the sample was vortexed for 5 min. The samples were centrifuged f o r 2 0 m i n a t 1 4 , 0 0 0 g a t 4 C , a n d t h e n 8 0 0 l a c e t o n i t r i l e w a s a d d e d t o t h e p e l l e t f r o m t h e t i s s u e s u s p e n s i o n . T h e s a m p l e s w e r e a g a i n v o r t e x e d f o r 5 m i n a n d c e n t r i f u g e d a t 1 4 , 0 0 0 g f o r 2 0 m i n a t 4 C . T h e s u p e r n a t a n t w a s t h e n d r i e d u n d e r N 2 . W h e n d r y , t h e r e s i d u e w a s r e - s u s p e n d e d i n 2 0 0 l 8 0 : 2 0 o f 2 - p r o p a n o l : 1 m M a c e t i c a c i d ( i n d d - H 2 O , v / v = 6 0 l / m l ) . T h e s o l u t i o n w a s s o n i c a t e d f o r 1 5 m i n a n d v o r t e x e d f o r 2 m i n t o f u l l y m i x t h e C o A i n t o t h e s o l u t i o n . T h e s a m p l e w a s t h e n c e n t r i f u g e d a t 1 4 , 0 0 0 g f o r 2 0 m i n a t 4C to separate out any salts. The supernatant was then transferred to a glass vial for LCMS analysis.
The HPLC/MS analysis was performed using an Agilent 1100 series liquid chromatograph 1956B SL single quadrupole mass spectrometer (Agilent Technologies, Palo Alto, CA, USA) with a binary gradient pump, heated column compartment, auto-sampler, and electrospray ionization source. The system was controlled by LC/MSD Chem-Station software Rev. A.10.02 (Agilent Technologies). The analytical column was a reversed phase Zorbax Extend-C18 2.1 ( 150 mm, 5(m with corresponding guard cartridge.
HPLC separation was conducted using a binary gradient with a 0.05% volatile reagent tri-ethylamine in water (A) and acetonitrile (B) at a flow rate of 0.3 ml/min. The gradient started at 15% B, increased to 35% B over 20 min, then rapidly increased to 90% B over 1 min and stayed at 90% B for 2 min. The post-run re-equilibrium time was 5 min for a total run time of 30 min. The column temperature was 30(C during the run and 4(C for the auto-sampler. Sample injection volume was 20~60 (l.
Electrospray mass spectrometry was performed under negative ion mode. Electrospray ion source parameters were: drying gas flow, 10 L/min; neubulizer pressure:, 20 psi; drying gas temperature, 350(C; capillary voltage, 4000 V; fragmentor voltage, 300 eV. The detector was turned on from 10 min tunill 25 min. All standard and tissue samples were analyzed in duplicate by HPLC/MS and the average values were used for the calculations.
Fatty Acyl Car n i t i n e
M u s c l e s a m p l e s w e r e q u i c k l y r i n s e d w i t h s a l i n e a n d f r o z e n a t - 8 0 C u n t i l a n a l y s i s . U p t o 1 0 0 m g o f t i s s u e w a s p u l v e r i z e d a n d t r a n s f e r r e d t o 1 . 5 m l p l a s t i c c e n t r i f u g e t u b e a n d t h e w e i g h e d . F i f t y l f r e s h l y m a d e 1 M K H 2 P O 4 w a s a d d e d t o t h e s a m p l e a l o n g w i t h 1 0 l i n t e r n a l s t a n d a r d ( I n t e r n a l s t a n d a r d d 3 - C 1 6 : 0 a c y l - c a r n i t i n e : 1 n g / l i n 3 : 1 A c n / M e O H ) a n d 1 m l f r e s h l y m a d e 3 : 1 A c n / M e O H ( v / v ) . T h e s o l u t i o n w a s v o r t e x e d f o r 2 m i n a n d t h e p e l l e t g r o u n d w i t h a m e c h a n i c a l g r i n d e r f o r 2 m i n . T h e m i x t u r e w a s t h e n centrifuged at 14,000g for 20 min at 4C, and the supernatant dried under N2. When dry, 100 l of 3:1 Acn/MeOH was added, and the sample then vortexed for 5 min. The solution was further sonicated for 15 min, and then centrifuged at 14,000g for 20 min at 4(C. The clear solution was transferred into an analytical vial with a sharp insert and kept at 20(C until HPLC-MS analysis.
The HPLC/MS analysis was performed using an Agilent 1100 series liquid chromatograph 1956B SL single quadrupole mass spectrometer (Agilent Technologies, Palo Alto, CA, USA) with a binary gradient pump, a heated column compartment, an autosampler, and was equipped with an electrospray ionization source. The system was controlled by LC/MSD Chem-Station software Rev. A.10.02 (Agilent Technologies). The analytical column was a reversed phase Zorbax Eclipse XDB-C8 4.6 ( 150 mm, 5um with corresponding guard cartridge (Agilent Technologies).
HPLC separation was achieved by means of a binary gradient with a volatile ion-pairing reagent that consisted of 0.05% heptafluorobutyric acid in water (A) and acetonitrile (B) at a flow rate of 1 ml/min. The gradient started at 10% B, increased to 80% B over 16 min and stayed at 80% for 9 min. The post-run re-equilibrium time was 5 min for a total run time of 30 min. The column temperature was 30(C during the run and 4(C for the auto-sampler. The sample injection volume was 1~10 l.
Electrospray mass spectrometry was performed in the positive ion mode. Electrospray ion source parameters were: drying gas flow, 13 L/min; neubulizer pressure, 60 psi; drying gas temperature, 350(C; capillary voltage, 400 V; fregmentor voltage, 200 eV. The detector was turned on from 15 min till 25 min. All standard and tissue samples were analyzed in duplicate by HPLC/MS and the average values were used for the calculations.
Mitochondrial enzyme activity -Cytochrome C oxidase, (-HAD, Citrate synthase
25-40 mg of vastus lateralis biopsies were immediately homogenized in a sucrose/EDTA/Tris buffer, and frozen at -80(C. The samples were sent to a collaborator Dr. Beatrice Moriro. She has measured the substrate capacities of several key mitochondrial enzymes using their immediate substrates: cytochrome c-oxidase with cytochrome C (COX), citrate synthase (CS) with coenzyme A and (-HAD with NADH.
Mitochondrial oxidation rates
Oxygen consumption in saponin-skinned muscle fibers was measured polarographically using a Clark-type electrode (Hansatech, Norfolk, UK) in a water jacketed glass chamber at 30 0C. The muscle biopsies, 15 to 30 mg wet weight, were first washed and minced into small bundles in relaxing solution. And then the small muscle fibers were incubated in saponin solution to permeabilize sarcolemma membrane. The glucose oxidation capacity was measured with 2 mM malate and 10 mM pyruvate as substrates plus 0.5 mM ADP. To measure mitochondrial FFA oxidation, pyruvate was replaced by 1 mM palmitoyl-L-carnitine. The final respiration activity was normalized by oxygen consumed per min over CS activity per mg protein.
Intracellular signaling
25-40 mg of astus lateralis muscle tissue samples (25-40 mg) collected during the basal state and at the end of the hyper-insulinemic euglycemic clamp were used to measure insulin signaling. All work was performed by Jennifer Zwetsloot at Eastern Carolina University.
Tissue homogenization
26-40 mg of tissue was weighed and homogenized in 1 ml of buffer containing Herpes, EDTA, NaF, Na pyrophosphate, Na orthovanadate, leupeptin, aprotinin, protease inhibitor cocktail, phosphatase cocktail I, and phosphatase cocktail II. The cytosol was separated from the membrane fraction by centrifugation, and the membranes further purified with Triton. The protein concentration in each was then measured with BCA.
p-Ty Immunoprecipitation
100ug of protein was added to 700ul homogenizing buffer and tyrosine antibody and incubated overnight at 4C. The proteins were then washed, centrifuged and 35ul 1X Bio Rad lamemil buffer +10% Pierce Bond breaker were added. The samples were heated at 100( C for 10 minutes then all of the sample was loaded onto 4-15% Tris Criterion Gel and run according to Criterion Protocol (200V run 1 hr, 100V transfer 1 hr). The gel was blocked with 5% milk for 1 hour at room temp with the following antibodies: 1AB SC IR(mem) or SC IRS1(Cyt) rabbit polyclonal 1:500 in 5% milk. After blocking, the gel was washed with TBST 2x5min and 2x10min, Amersham anti-rabbit 2AB in 5% milk 1 hr and Amersham ECL. After blocking, the gel was stripped to remove the antibodies with Cyt pTy Blot 2x15min and then washed 4x5min in TBS. The gel was then reprobed with Cyt blot for PI3K p85 content. The gel was blocked for 1 hour in 5% milk with 1AB Cell Signaling PI3K p85 1:1000 in 5% BSA overnight 4(C then washed with TBST 2x5min and 2x10min then incubated with 2AB cell Signaling in 2% BSA for 1 hour and finished with Cell Signaling ECL.
IRS1 Immunoprecipitation
Eight hundred ug of protein was added to 700ul of homogenizing buffer and incubated overnight with 40ul of SC-IRS1 mouse monoclonal AB. The next day, 160ul of 50% Sepharose A slurry (washed 2x with homogenizing buffer) was added and the mixture was rotated for 2 hours at 4(C. It was then washed with homogenizing buffer that had been diluted 5 fold. Then 160ul 1X Bio Rad laemmli buffer +10% Pierce Bond breaker were added, and the mixture was heated at 100 (C for 10 minutes and then centrifuged. All of the sample was loaded onto 4 different 4-15% Tris Criterion Gels and run according to Criterion Protocol (200V run 1 hr, 100V transfer 1 hr). The gels were blocked with 5% milk for 1 hour at room temperature, washed with TBST for 2x5min, and then 1AB Cell signaling IRS1ser 1:1000 in 5% BSA was added and incubated on the gel at 4(C for 2x10min. The gel was washed with TBST 2x5min, and 2x10min, and then incubated with 2AB cell signaling in 2% BSA for 1 hour. The bands were read with finish with Cell Signaling ECL. After reading, the gel was stripped for 2x15min then washed 4x5min The gel was then re-probed with TBS for IRS1 content by blocking for 1 hour in 5% milk with 1AB Santa Cruz polyclonal rabbit IRS1 1:500 in 5% milk 4(C. The gel was washed with TBST 2x5min, 2x10min and then incubated with Amersham anti-rabbit 2AB in 5% milk four 1 hour prior to measurement with Amersham ECL.
PKC Content
Buffer was mixed with 30-50ug of protein and heated at 100 (C for 10 minutes and then centrifuged. The sample was loaded onto to 7.5% Tris Criterion Gels.Run according to Criterion Protocol (200V run 1 hr, 100V transfer 1 hr). The gels were then blocked for 1 hour with 5% milk and 1AB Santa Cruz polyclonal rabbit PKC 1:250 in 5% milk at 4(C. After incubation the gel was washed with TBST 2x5min, 2x10min, then incubated with Amersham anti-rabbit 2AB in 5% milk for 1 hour prior to reading with and Amersham ECL. The gel was stripped 2x15min and washed 4x5min TBS and then reproved in a similar manner for other PKC isoforms.
Resting energy expenditure
Resting energy expenditure and the volume of CO2 expired was measured on a Vmax 29 metabolic cart (Sensormedics, Yorba Linda, CA) twice per study. Measurements were taken for 20 minutes prior to blood sampling in the third hours of both the basal and clamp periods. The steady state average for ten minutes within the period sampled was used to calculated the mean REE and v CO2 for the period. The children were not given sedation during the study, and the room was dark and quiet during the study to promote the rested state.
CALCULATIONS
Plasma
Glucose Kinetics
Glucose Infusion rate (M-value) during the hyperinsulinemic-euglycemic clamp
Glucose uptake in mg/kg/min was calculated by measuring the amount of D20 infused over an hour to maintain a steady state of plasma glucose within the desired range. 60 minutes of steady state of both the infusion and the plasma glucose were chosen, and the majority of steady states occurred within the 3rd hour of the clamp. A sample of D20 was collected from each study, and the actual concentration of the glucose was measured, and the real concentration was used. The mean measured concentration was 17.5g in 500mL, not 20g per 500mL as expected. The calculation is:
Equation 1: M-value =(mg glucose infused per min /weight in kg)
The results are expressed at glucose infusion in mg/kg/min.
Glucose clearance during the clamp
Glucose clearance represents the disappearance of the glucose infused into the blood. The only difference between this and the previous calculation is the division of the rate infused by the plasma concentration of glucose.
Equation 2:
Glucose clearance = (mg glucose infused/min /weight in kg)
ave plasma [glucose]
The results are expressed as dl/kg/min cleared.
Endogenous Glucose Production
Glucose enrichment was used for these calculations. The glucose enrichment as measured on the mass spec was corrected for the overlap between M+1 and M+2, and also corrected for the skew. The rate of endogenous release of glucose by the liver, representing both gluconeogenous and glycogenolysis was calculated by measuring the dilution of the infused tracer by unlabeled glucose. Thus during the basal period the calculation was:
Equation 3: Ra = rate of the glucose infusion
arterial enrichment
However, during the hyperinsulinemic-euglycemic clamp, a large amount of unlabelled glucose was infused, so the Ra must be corrected for by subtracting the amount of unlabelled glucose infused to maintain euglycemia. The equation was:
Equation 4:
Ra =(rate of the labeled glucose infusion)- unlabelled glucose infused
arterial enrichment
The resultant units for both of these equations are glucose release in mg/kg/min.
Glucose Net Balance
The net glucose uptake across the leg was calculated by the following equation:
Equation 5:
Glucose uptake = ([Artery] [Vein]) x plasma flow
There is a 5% difference in glucose concentration between whole blood and plasma, so since plasma glucose was measured, the plasma flow rather than blood flow was used for the above calculation. The units of leg glucose uptake are mg/100ml leg/min.
FFA Kinetics
Whole Body Oxidation
The data from the IRMS was used to calculate the tracer/tracee ratio (TTR) of breath CO2. First the background sample was analyzed, and the enrichment of the background was subtracted from the standard to obtain the background enrichment of CO2 in the subject prior to the infusion. Then each of the 4 sets of samples was also subtracted from the standard CO2. Then the averages of the backgrounds were subtracted from the adjusted average of the samples to calculate the increase in enrichment in the breath during the plateau period denoted R. To calculate the enrichment in the venous plasma palmitate, the contribution of M+15 to M+16 must be accounted. For the following equations were used to do this:
Equation 6: TTR(M+15)=R(1-A)n
Equation 7: TTR(M+16) = (R-Eqn 2)*(1-A)n
Where R =M+X/M+0 ratio, A = 0.011 and n=mass number. The (1-A)n is the skew factor, which counts for the natural abundance of the isotope in the molecule, and A in this case is 0.011, for carbon. The precursor enrichment was then the sum of the two equations above. To calculate the % of M+15 or M+16, the outcome of either equation 6 or 7 is divided by the precursor enrichment. This could then be multiplied by the rate of infusion, to calculate the rate of appearance of label in the bloodstream. To calculate the amount of palmitate oxidation, the following calculation was used:
Equation 8:
Palmitate Oxidation = Enrichment CO2 * Volume CO2
Precursor Enrich*Acetate Corr factor*(%M+16*16 + %M+15*15)
The acetate correction factor used for these calculations was 0.90, as determined by studies in a similar patient population of pediatric burns.
The percent of palmitate uptake oxidized was:
Equation 9:
Uptake oxidized = (palmitate oxidation*100%)/Rd of palmitate
The rate of total fat oxidation was also calculated from the data collected with indirect calorimetry ADDIN EN.CITE Wolfe2005505elderlypaper2-Converted.enlEndNote5056(Wolfe 2005). The formula for fat oxidation is:
Equation 10:
Total fat oxidation= (1.67*VO2) (1.67*VCO2) (1.94n)
Where n is the rate of urinary nitrogen excretion and VO2 and VCO2 are expressed in L/min. The results are expressed as fat oxidized in g/min. The n value for this study was assumed to be 0.007 g/min, based on previous research in burned children ADDIN EN.CITE Patterson1997197elderlypaper2-Converted.enlEndNote19717(Patterson 1997).
Palmitate Rate of Appearance
The IRMS data on CO2 enrichment in the arterial and venous blood was analyzed in the same manner as was described for the whole body oxidation. The tracer and the tracee should be taken up in the same amounts, so if there was no palmitate being released, then the TTR between the artery and the vein would be the same. However, any dilution in the TTR between the artery and the vein represents release of unlabelled palmitate. The following calculations were used for the palmitate kinetics of the leg:
Equation 11:
Rate of Palmitate =Arterial Concentration* Plasma Flow
Equation 12:
Rate of Palmitate out= Venous Concentration* Plasma Flow
Equation 13:
Fractional Extraction= (Art Enrich *[Art]-Ven enrich[Ven ])/ Art Enrich [Art]
Equation 14:
Palmitate Uptake by the leg:= Fractional Extraction* Rate of Palmitate in
Palmitate Leg Oxidation
The IRMS data on CO2 enrichment in the arterial and venous blood was analyzed in the same manner as was described for the whole body oxidation.
Equation 15:
Leg ox= Blood flow * ((Ven CO2 Con *Erich) (Art CO2 Con. *Enrich) )
Precursor Enrich*Acetate Corr factor*(%M+16*16 + %M+15*15)
Equation 16:
Percent Palmitate Uptake Oxidized= Palmitate oxidized/palmitate uptake
Palmitate rates were converted to FFA rate by dividing by the % FFA that are palmitate, which was approximately 35% in this patient poulaion.
Muscle
DAG, TAG, Fatty acyl CoA, and Fatty acyl carnitine concentrations
The concentrations of all of these were determined using the internal standard method. The internal standards were C 15:0 DAG, C17:0 Fatty acyl CoA and D3-palmitate fatty acyl carnitine. The concentration of the palmitate was determined by multiplying the area under the curve of the palmitate by the area under the curve of the internal standard divided by the molar concentration of IS. Total long chain fatty acid conctration was determined similarly.
Imaging
MRS of soleus and liver
TG levels were computed as a ratio relative to the Intralipid standard using the following formula:
Equation 17: TG = [(PM / VM) / (PI / VI)]
where PM is the tissue lipid methylene peak area, VM is the total measured tissue voxel volume, PI is the Intralipid peak area, and VI is the Intralipid voxel volume. The results of this calculation is a ratio and thus unitless.
DEXA
The fitting program included with the hologic DEXA machine calculates the percent body fat based on the density of the tissue.
STATISTICS
Study 1
All results were reported as mean ( SEM. Differences between young and elderly were evaluated using a 2-tailed Student's t test. Differences in glucose and insulin over time between young and elderly were evaluated using a two-way ANOVA with factors time and age, followed by Tukey's test when appropriate. Pearson correlations between physiological factors were examined. The effect of percent body fat was factored out of the correlation by dividing the relevant factor by the percent body fat.
Study 2
All results were reported as meanSEM. Differences between the groups and across time were analyzed using a 2 way ANOVA, with a Tukeys test to delineate any detected differences.
Study 3
As baseline measures for individuals vary greatly, the change of each individual was measured and compared instead of measuring group means. The type of statistical test varied, depending on the model being used. For glucose metabolism, we were interested in the response to insulin before and after treatment. Thus the change from the basal period to the clamp period for each study was calculated and compared from week 1 to week 3 using a paired t-test. An unpaired t-test was used to detect difference between the PLA and FEN groups after treatment. As change was predicted a priori, the alpha value is 0.10 for all statistics. The comparisons of interest were between the basal state at week1 and 3, and the clamp state at week 1 and 3.
All raw data is shown with standard errors. Statistics were run using Sigma Stat software package, version 2.03 (SPSS, Chicago, IL)
Study 4
The data from study four are compared to the data from study three using an un-paired t-test.
Chapter 4
Patient Demographics and clinical labs
Study 1: Young vs. Elderly
Twenty-five volunteers, nine young (5 female, 4 male; ages 271; range 20-32) and sixteen elderly (11 female, 5 male; ages 691; range 65 to 74) were enrolled in the study. The groups were racially balanced, with 6 Caucasians, 2 Hispanics and 1 African American in the young group and 13 Caucasians, 3 Hispanics and 1 African American in the elderly group. 1 young and two elderly had an immediate family member who had been diagnosed with diabetes, and all others had no family history of diabetes. All subjects read and signed an informed consent. The project was approved by the Institutional Review Board at the University of Texas Medical Branch, Galveston, Texas. The mean baseline clinical data are shown in Table 4.1.
Several baseline parameters were significantly different between the young and elderly (Table 4.1). The elderly had a higher average HbA1C, fasting insulin and fasting glucose, although these were not outside the normal range, and no individual was in the diabetic range. The elderly had a higher total cholesterol and LDL fraction.
Table 4.1
Study 1 Volunteer Baseline Laboratory MeasurementsNormal RangeYoungElderlyFasting Glucose (mg/dL)70-1008831004*Fas t i n g I n s u l i n ( U / m L ) 5 - 2 7 4 . 4 0 . 9 8 . 5 1 . 3 * H b A 1 C ( % ) <