This document describes a study examining differences in skeletal muscle mitochondria between type 2 diabetes (T2D) patients and healthy individuals. Muscle samples were obtained and mitochondria were isolated from total homogenates. Proteins in the mitochondria were then identified using 2D gel electrophoresis and mass spectrometry. Results found both up-regulated and down-regulated enzymes in T2D patients, including increased levels of enzymes involved in fatty acid oxidation and decreased levels of enzymes in the tricarboxylic acid (TCA) cycle. This suggests altered regulation of specific fatty acid oxidation and TCA cycle enzymes in skeletal muscle mitochondria of T2D patients.

1. Proteomic and Ingenuity Pathway Analysis of Skeletal Muscle Mitochondria in Type 2 Diabetes
Patients Suggest Altered Regulation of Specific Fatty Acid Oxidation and TCA Cycle Enzymes
®
S. Kamath1, A. Monroy1, A. Chavez1, M. Wilson3, C. Carroll1, F. Casiraghi1, D. Tripathy1, S. Weintraub2, R. DeFronzo1 and F. Folli1
1Medicine-Diabetes, 2Biochemistry, and 3Institutional Research Core Facilities, University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States
INTRODUCTION MITOCHONDRIAL FRACTIONATION RESULTS SUMMARY
A B
Skeletal muscle is an important regulator of HUMAN MUSCLE MITOCHONDRIAL
COX IV content (% of total homogenate)
Validation by
NGT T2D p
Immunoblotting
glucose and lipids and impairment of this TOTAL HOMOGENATE 200
PROTEIN IDENTIFICATION IN 2D GEL Up-regulated in T2DM (~ 30 %, p<0.05)
metabolic function could possibly lead to the 150 Delta3,5-delta2,4-dienoyl-CoA isomerase (ECH1_HUMAN) 1.4 ± 0.5 1.9 ± 0.8 <0.02 Yes
Short chain 3-hydroxyacyl-CoA dehydrogenase (HCDH_HUMAN) 2.3 ± 0.8 3 ± 0.9 <0.04 Yes
onset of Type 2 Diabetes (T2D). 100 Enoyl-CoA hydratase (ECHM_HUMAN) 1.4 ± 0.4 2.2 ± 0.7 <0.02 Yes
Spin 700xg 10 min 4ºC
T2D subjects are characterized by an 50
3 pI 10 Adenylate kinase 3 (KAD3_HUMAN) 5.9 ± 1.6 8.9 ± 2.5 <0.001 Yes
Down-regulated in T2DM (~ 50%, p<0.05)
impaired ability to oxidize fat causing an Pyruvate dehydrogenase protein X component (ODPX_HUMAN) 6.9 ± 1.8 3.8 ± 0.9 <0.005 Yes
0
imbalance between fatty acid uptake and its Supernatant
10,000xg 30 min 4ºC
Total
homogenate
Mitochondria Cytosolic and
other fractions NDUS1
Dihydrolipolylysine-residue succinyltransferase component of 2-
oxoglutarate dehydrogenase complex (ODO2_HUMAN)
1.2 ± 0.2 0.6 ± 0.1 <0.07 Yes
ACON
IMMT ETFD
oxidation, which could lead to C MYH2 GRP75 ODP2
ALBU ALDH2
ALBU
ATPA SCOT DLDH
AL4A1
ATPA
KPYM
ATPA
ATP synthase alpha chain (ATPA_HUMAN) 2.9 ± 0.8 1.4 ± 0.3 <0.08 Yes
DHE3
intramyocellular lipid (IMCL) accumulation 140
ODO2
ODPX
ODPX EFTU FUMH
NUBM
NUBM UQCR2
ATPA Mitochondrial inner membrane protein (IMMT_HUMAN) 3.5 ± 0.9 2 ± 0.4 <0.002 Yes
Pellet 120
UQCR1 UQCR1 ECHB Down-regulated in T2DM (~ 20%, p<0.05)
within the skeletal muscle and induce insulin
(% of total homogenate)
ACTB ACADM IDHP
Resuspend in homogenization
Adenylate kinase 1
100 ACADS ACADS KCRM KCRS THIL MDHM
3,2-trans-enoyl-CoA isomerase (D3D2_HUMAN) 4.2 ± 0.1 3.3 ± 0.8 <0.02 No
TPM2 ALDOA
Buffer 80,000xg 1 h 4ºC
resistance. 80
ODP2 ECH1 ECH1
VADC2
ETFA
VDAC2 HCDH HCDH
Dihydrolipoamide dehydrogenase (DLDH_HUMAN) 2.5 ± 0.8 2 ± 0.6 <0.005 Yes
TPM2 3HIDH
VDAC1
Cytochrome c oxidase subunit VIb isoform 1 (CX6B1_HUMAN) 1.6 ± 0.3 1 ± 0.2 <0.002 Yes
PURPOSE
60 D3D2 VDAC1 ADT1
CAH1 ECHB
ECHM TRIS
HSPB1 TRIS Myosin regulatory light chain 2 (MLRV_HUMAN) 7.7 ± 2.6 5.8 ± 2.2 <0.002 Yes
40 PRDX3 VDAC1
CAH3
MITOCHONDRIA MLE1 NUGM
ES1
CHCH3 KAD3
ATPO
MLE1 ES1 HCD2
ENRICHED FRACTION 20 HUHM HUHM UCRI NIDM
NUIM HUHM
SODM
PEPB Table 2. List of mitochondrial proteins differentially expressed
0
in T2D. Numbers are expressed as spot intensities ( × 10-6).
Total Mitochondria Cytosolic and ATP5H ATP5H
To identify protein markers of T2D homogenate other fractions CASQ1 MLRV MLRV CRYAB
NDUS4
Data are expressed as means ± SEM, (n = 5)
mitochondrial dysfunction using a proteomic Figure 1A. Human muscle mitochondrial enriched fraction
NUPM
NUYM
strategy to compare protein expression in was obtained by differential centrifugation from vastus ATPD
skeletal muscle biopsies from normal
lateralis percutaneous biopsy. Figure 1B and 1C.
MLRV
COX5A LEG1
COX5B
HBB
MYG
N5BM
PATHWAYS ALTERED IN
Immunoblot analysis of human skeletal muscle sub- MLE1
glucose tolerant (NGT) and T2D subjects. cellular fractionation using mitochondrial (COX IV) and
NUFM
CX6B1
HBA UCR6 ATP5I
T2D MITOCHONDRIA
cytoplasmic (AK1) markers respectively.
Figure 3. Human skeletal muscle mitochondrial 2D-gel
METHODS PROTEIN IDENTIFICATION protein map were created with 100 proteins identified.
Canonical pathways
Lysine degradation (Ratio 4/136, p = 9.04 x 10-8)
Fatty acid elongation in mitochondria (Ratio 3/45, p = 2.64 x 10-7)
SUBJECT CHARACTERISTICS DIFFERENTIAL PROTEIN EXPRESSION Fatty acid metabolism (Ratio 4/196, p = 2.84 x 10-6)
Gene Ontology Biological Processes
2D GELS AND STAINING
IN T2D MITOCHONDRIA Carboxylic acid metabolic process (p = 6.2 x 10-7)
Fatty acid catabolic process (p = 3 x 10-4)
Image Analysis
Table 3. Network and pathway analysis of mitochondrial
Normal Type 2
NGT T2DM proteins with altered expression using Ingenuity software
Glucose Diabetic Cut spots of interest
Tolerant (T2D)
(NGT)
Digestion IMMT DLDH ATPA IMMT DLDH ATPA CONCLUSIONS
ODO2 ODPX ODO2 ODPX
Male/ Female 4/4 4/4 HPLC-ESI-MS/MS
ECH1 HCDH ECH1 HCDH This study demonstrates selective
Age (Years) 36 ± 5 50 ± 3 † Database Search ECHM D3D2 alterations in the stoichiometry of
ECHM D3D2
BMI (kg/m2) 26 ± 2 36 ± 2 * KAD3 KAD3 mitochondrial proteins in T2D subjects.
PROTEIN ID
HbA1c (%) 5.1 ± 0.1 9 ± 0.7 * MLRV MLRV Mitochondrial dysregulation in human
Rd (mg/kg.min-1) 10 ± 1 3±1* Figure 2. Human muscle proteins were separated by 2D-gel
skeletal muscle could provide direct
electrophoresis, gels were stained using SYPRO Ruby. insights into the molecular pathogenesis
TG (mg/dl) 122.4 ± 281.6 ± of lipotoxicity in T2D.
Spots were excised, digested with trypsin and analysed by
44.6 82.2 † CX6B1 CX6B1
HPLC-ESI-MS/MS. Protein identifications were based on
Table 1. Clinical, laboratory and metabolic characteristics Mascot searches of SwissProt followed by post-processing
Figure 4. Gels were stained for total protein using SYPRO Ruby.
ACKNOWLEDGEMENTS
of subjects. Data are expressed as means ± SEM. with Scaffold. All reported peptide and protein assignments
HbA1c = Glycated haemoglobin, Rd = Insulin-stimulated Images were analysed, spots matched, edited and statistically This study was supported by an NIH-DDK grant to Dr. Franco Folli, M.D.,
were >95% confidence (Scaffold).
rate of glucose disposal, TG = Serum triglycerides. compared using PDQuest (Bio-Rad). Ph.D under grant number R01 DK080148 from the National Institutes of
* p < 0.001, † p < 0.05
Health–Diabetes/ Digestive/ Kidney Diseases.

2. Insulin Signaling Defects in the Liver of a Non-Human Primate Model of NAFLD
S. Kamath1, A.O. Chavez1, A. Davalli2, G.B. Hubbard5,6, E.J. Dick5, M. Palomo1, G. Halff3,4, G. Abrahamian3,4, R. Bastarrachea5,
® A.G. Comuzzie5,6, D. Tripathy1, A. Gastaldelli1, R.A. DeFronzo1, F. Folli1
1Medicine/Diabetes,UTHSCSA, San Antonio, TX, United States, 2Istituto Scientifico San Raffaelle, Milano, Italy, 3Surgery, UTHSCSA, San Antonio, TX, United States, 4Transplant Center, UTHSCSA,
San Antonio, TX, United States, 5Genetics, SFBR, San Antonio, TX, United States, 6SNPRC, San Antonio, TX, United States.
SUMMARY
Non-Alcoholic Fatty Liver Disease (NAFLD) represents SUBJECT CHARACTERISTICS • Histological analysis demonstrate a progressive
a broad spectrum of liver pathologies ranging from increase in macrovesicular steatosis in IR baboons.
Insulin Sensitive (IS) Insulin Resistant (IR)
simple fatty liver steatosis and non-alcoholic • Elevated levels of triglycerides are present in the liver
(n=8) (n=8)
steatohepatitis (NASH) to fibrosis and cirrhosis. of IR baboons and they correlate strongly with insulin
Male/Female 5/3 3/5
Age (years) 19 ± 0.8 21 ± 0.9
resistance in vivo.
Hepatic steatosis occurs when the rate of free fatty • Insulin signaling is significantly impaired in the Akt-
Body Mass Index (BMI) 22 ± 0.6 28 ± 0.8 *
acid (FFA) input marked by uptake of fatty acids from the GSK3-beta component of the pathway.
Rd90-120 min (mg/kg. min-1) 11 ± 0.7 2 ± 0.3 *
plasma and esterifcation is greater than fatty acid
Body fat (%) 5 ± 0.3 15 ± 1 *
oxidation and its export as triglyceride within very low-
HOMA-IR 2.4 ± 0.5 7.4 ± 0.8 *
density lipoprotein (VLDL).
FPG (mg/dL) 98 ± 2 114 ± 2
Figure 3A-F. Hepatic triglyceride levels in IR baboons and its correlation with anthropometric and
Hepatic Insulin metabolic characteristics. Triglycerides were measured with a Triglyceride quantification kit. Data are
Numerous studies have linked hepatic steatosis with 322 ± 4 240 ± 2 *
clearance (mL/m2.min-1) expressed as means ± SEM. * p < 0.05
Papio Hamadryas
factors of the metabolic syndrome, particularly obesity,
Table 1. Clinical anthropometric, laboratory, and glucose metabolic characteristics of the baboons.
Data are expressed as means ± SEM. FPG = fasting plasma glucose; Rd = insulin-stimulated rate of A B C
type 2 diabetes and hyperlipidemia. glucose disposal. * p<0.05
Insulin resistance, a prominent feature of obesity and
type 2 diabetes mellitus, is a key factor in the
development of steatosis.
Figure 4A-C. Phosphorylation of Insulin receptor-beta in baboon liver at baseline, 30, and 120 mins during
the euglycemic insulin clamp using ELISA. Data are expressed as means ± SEM. * p < 0.05, ** p < 0.05
Figure 1. Schematic representation of the euglycemic clamp. Insulin sensitivity was assessed using a
60 mU.m2.min-1 euglycemic insulin clamp and liver biopsies performed at baseline, and repeated at
30' and 120' after insulin. A B C We show that the baboon is a non-human primate model
of NAFLD, with marked impairment of insulin signaling in
the liver, which could provide novel insights for prevention
and treatment of human NAFLD and associated co-
NAFLD is emerging as the most common liver
morbidities.
disorder perhaps due to the rise in incidence of obesity
and type 2 diabetes; its prevalence is estimated to be
around 20% of the general population, and up to 70% for Figure 5A-C. Phosphorylation of Akt in baboon liver at baseline, 30, and 120 mins during the euglycemic
insulin clamp. Data are expressed as means ± SEM. * p < 0.05, ** p < 0.05, # p < 0.05, # # p < 0.05
those with obesity.
Prevalence of and risk factors for hepatic steatosis in
A B C
Northern Italy.
It is important to identify patients with NAFLD in the Bellentani S, Saccoccio G, Masutti F, Crocè LS, Brandi G,
stage of preclinical diabetes or glucose intolerance Sasso F, Cristanini G, Tiribelli C. Annals of Internal Medicine
because they are at a high risk of developing type 2 132:112-7, 2000.
diabetes mellitus which could lead to progressive Nonalcoholic fatty liver disease: Pathogenesis and the role of
fibrosis. antioxidants.
Figure 6A-C. Phosphorylation of GSK3-beta in baboon liver at baseline, 30, and 120 mins during the
Mehta K, Van Thiel DH, Shah N, Mobarhan S. Nutrition
In the current investigation, we present a non- euglycemic insulin clamp. Data are expressed as means ± SEM. * p < 0.05, ** p < 0.05, # p< 0.05 Reviews 60:289-93, 2002.
genetically modified non-human primate model, the Physiological and molecular determinants of insulin action in
A B C
baboon (Papio hamadryas), which is obese, non-diabetic the baboon.
with proven characteristics of insulin resistance in Chavez AO, Lopez-Alvarenga JC, Tejero ME, Triplitt C,
skeletal muscle and adipose tissue, and most Bastarrachea RA, Sriwijitkamol A, Tantiwong P, Voruganti VS,
importantly, by virtue of its close resemblance to Musi N, Comuzzie AG, DeFronzo RA, Folli F. Diabetes 57:899-
908, 2008.
humans, represents a model to study NAFLD, hepatic
Obesity and nonalcoholic fatty liver disease: biochemical,
insulin resistance, and possibly overt NASH. Figure 7A-C. Phosphorylation of MAPK in baboon liver at baseline, 30, and 120 mins during the
euglycemic insulin clamp. Data are expressed as means ± SEM. * p < 0.05 metabolic, and clinical implications. Fabbrini E, Sullivan S,
Figure 2A-L. Representative sections of liver from baboons with various degrees of insulin sensitivity showing
Klein S.
progressive increase in hepatic steatosis and disruption of the portal triad architecture. Figure 2A, C, E, and G stained Hepatology 51:679-89,2010.
with H&E, Figure 2B, D, F and H stained with Masson’s Trichrome. Figure 2I-L (H&E, 40X) showing regions of steatosis,
focal steatosis and ballooning consistent with the presence of NAFLD.