OBJECTIVEInsulin resistance in skeletal muscle is an early phenomenon in the pathogenesis of type 2 diabetes. Studies of insulin resistance usually are highly focused. However, approaches that give a more global picture of abnormalities in insulin resistance are useful in pointing out new directions for research. In previous studies, gene expression analyses show a coordinated pattern of reduction in nuclear-encoded mitochondrial gene expression in insulin resistance. However, changes in mRNA levels may not predict changes in protein abundance. An approach to identify global protein abundance changes involving the use of proteomics was used here.RESEARCH DESIGN AND METHODSMuscle biopsies were obtained basally from lean, obese, and type 2 diabetic volunteers (n = 8 each); glucose clamps were used to assess insulin sensitivity. Muscle protein was subjected to mass spectrometry–based quantification using normalized spectral abundance factors.RESULTSOf 1,218 proteins assigned, 400 were present in at least half of all subjects. Of these, 92 were altered by a factor of 2 in insulin resistance, and of those, 15 were significantly increased or decreased by ANOVA (P < 0.05). Analysis of protein sets revealed patterns of decreased abundance in mitochondrial proteins and altered abundance of proteins involved with cytoskeletal structure (desmin and alpha actinin-2 both decreased), chaperone function (TCP-1 subunits increased), and proteasome subunits (increased).CONCLUSIONSThe results confirm the reduction in mitochondrial proteins in insulin-resistant muscle and suggest that changes in muscle structure, protein degradation, and folding also characterize insulin resistance.

Changes in protein abundance in skeletal muscle are central to a large number of metabolic and other disorders, including, and perhaps most commonly, insulin resistance. Proteomics analysis of human muscle is an important approach for gaining insight into the biochemical basis for normal and pathophysiological conditions. However, to date, the number of proteins identified by this approach has been limited, with 107 different proteins being the maximum reported so far. Using a combination of one-dimensional gel electrophoresis and high performance liquid chromatography electrospray ionization tandem mass spectrometry, we identified 954 different proteins in human vastus lateralis muscle obtained from three healthy, nonobese subjects. In addition to a large number of isoforms of contractile proteins, we detected all proteins involved in the major pathways of glucose and lipid metabolism in skeletal muscle. Mitochondrial proteins accounted for 22% of all proteins identified, including 55 subunits of the respiratory complexes I-V. Moreover, a number of enzymes involved in endocrine and metabolic signaling pathways as well as calcium homeostasis were identified. These results provide the most comprehensive characterization of the human skeletal muscle proteome to date. These data hold promise for future global assessment of quantitative changes in the muscle proteome of patients affected by disorders involving skeletal muscle.

OBJECTIVEThe contribution of mitochondrial dysfunction to skeletal muscle insulin resistance remains elusive. Comparative proteomics are being applied to generate new hypotheses in human biology and were applied here to isolated mitochondria to identify novel changes in mitochondrial protein abundance present in insulin-resistant muscle.RESEARCH DESIGN AND METHODSMitochondria were isolated from vastus lateralis muscle from lean and insulin-sensitive individuals and from obese and insulin-resistant individuals who were otherwise healthy. Respiration and reactive oxygen species (ROS) production rates were measured in vitro. Relative abundances of proteins detected by mass spectrometry were determined using a normalized spectral abundance factor method.RESULTSNADH- and FADH2-linked maximal respiration rates were similar between lean and obese individuals. Rates of pyruvate and palmitoyl-dl-carnitine (both including malate) ROS production were significantly higher in obesity. Mitochondria from obese individuals maintained higher (more negative) extramitochondrial ATP free energy at low metabolic flux, suggesting that stronger mitochondrial thermodynamic driving forces may underlie the higher ROS production. Tandem mass spectrometry identified protein abundance differences per mitochondrial mass in insulin resistance, including lower abundance of complex I subunits and enzymes involved in the oxidation of branched-chain amino acids (BCAA) and fatty acids (e.g., carnitine palmitoyltransferase 1B).CONCLUSIONSWe provide data suggesting normal oxidative capacity of mitochondria in insulin-resistant skeletal muscle in parallel with high rates of ROS production. Furthermore, we show specific abundance differences in proteins involved in fat and BCAA oxidation that might contribute to the accumulation of lipid and BCAA frequently associated with the pathogenesis of insulin resistance.

OBJECTIVEAging increases the risk of developing impaired glucose tolerance (IGT) and type 2 diabetes. It has been proposed that increased reactive oxygen species (ROS) generation by dysfunctional mitochondria could play a role in the pathogenesis of these metabolic abnormalities. We examined whether aging per se (in subjects with normal glucose tolerance [NGT]) impairs mitochondrial function and how this relates to ROS generation, whether older subjects with IGT have a further worsening of mitochondrial function (lower ATP production and elevated ROS generation), and whether exercise reverses age-related changes in mitochondrial function.RESEARCH DESIGN AND METHODSMitochondrial ATP and ROS production were measured in muscle from younger individuals with NGT, older individuals with NGT, and older individuals with IGT. Measurements were performed before and after 16 weeks of aerobic exercise.RESULTSATP synthesis was lower in older subjects with NGT and older subjects with IGT versus younger subjects. Notably, mitochondria from older subjects (with NGT and IGT) displayed reduced ROS production versus the younger group. ATP and ROS production were similar between older groups. Exercise increased ATP synthesis in the three groups. Mitochondrial ROS production also increased after training. Proteomic analysis revealed downregulation of several electron transport chain proteins with aging, and this was reversed by exercise.CONCLUSIONSOld mitochondria from subjects with NGT and IGT display mitochondrial dysfunction as manifested by reduced ATP production but not with respect to increased ROS production. When adjusted to age, the development of IGT in elderly individuals does not involve changes in mitochondrial ATP and ROS production. Lastly, exercise reverses the mitochondrial phenotype (proteome and function) of old mitochondria.

Mitochondria can be isolated from skeletal muscle in a manner that preserves tightly coupled bioenergetic function in vitro. The purpose of this study was to characterize the composition of such preparations using a proteomics approach. Mitochondria isolated from human vastus lateralis biopsies were functional as evidenced by their response to carbohydrate and fat-derived fuels. Using one-dimensional gel electrophoresis and HPLC-ESI-MS/MS, 823 unique proteins were detected, and 487 of these were assigned to the mitochondrion, including the newly characterized SIRT5, MitoNEET and RDH13. Proteins detected included 9 of the 13 mitochondrial DNA-encoded proteins and 86 of 104 electron transport chain (ETC) and ETC-related proteins. In addition, 59 of 78 proteins of the 55S mitoribosome, several TIM and TOM proteins and cell death proteins were present. This study presents an efficient method for future qualitative assessments of proteins from functional isolated mitochondria from small samples of healthy and diseased skeletal muscle.

SUMMARY Type 2 diabetes Mellitus (T2DM) and aging are characterized by insulin resistance, lower mitochondrial density and function and increased production of reactive oxygen species (ROS). In lower organisms continuous remodeling critically maintains the function and life cycle of mitochondria, in part by the protease pcp1 (PARL ortholog). We therefore examined whether variation in PARL protein content is associated with mitochondrial abnormalities and insulin resistance. Relative to healthy, young individuals (23±1y), PARL mRNA and mitochondrial mass were both reduced in elderly subjects (64.4±1.2 y; 51% and 44% respectively) and in subjects with T2DM (51.8±3 y; 31% and 41% respectively; all p<0.05). Muscle knock-down of PARL in mice resulted in lower mitochondrial content (−31±3%, p<0.05), lower OPA1 and PGC1α protein levels and impaired insulin signaling. Furthermore, mitochondrial cristae were malformed and resulted in elevated in vivo oxidative stress. Adenoviral suppression of PARL protein in healthy myotubes lowered mitochondrial mass (−33±8%), insulin stimulated glycogen synthesis (−33±9%) and increased ROS production (2-fold) (all p<0.05). We propose that lower PARL expression may contribute to the mitochondrial abnormalities seen in aging and T2DM.

Lefort N, St-Amand E, Morasse S, Côté CH, Marette A. The ␣-subunit of AMPK is essential for submaximal contraction-mediated glucose transport in skeletal muscle in vitro. Am J Physiol Endocrinol Metab 295: E1447-E1454, 2008. First published September 23, 2008 doi:10.1152/ajpendo.90362.2008.-AMP-activated protein kinase (AMPK) is a key signaling protein in the regulation of skeletal muscle glucose uptake, but its role in mediating contraction-induced glucose transport is still debated. The effect of contraction on glucose transport is impaired in EDL muscle of transgenic mice expressing a kinase-dead, dominant negative form of the AMPK␣ 2 subunit (KD-AMPK␣2 mice). However, maximal force production is reduced in this muscle, raising the possibility that the defect in glucose transport was due to a secondary decrease in force production and not impaired AMPK␣ 2 activity. Generation of force-frequency curves revealed that muscle force production is matched between wild-type (WT) and KD-AMPK␣2 mice at frequencies Յ50 Hz. Moreover, AMPK activation is already maximal at 50 Hz in muscles of WT mice. When EDL muscles from WT mice were stimulated at a frequency of 50 Hz for 2 min (200-ms train, 1/s, 30 volts), contraction caused an ϳ3.5-fold activation of AMPK␣2 activity and an ϳ2-fold stimulation of glucose uptake. Conversely, whereas force production was similar in EDL of KD-AMPK␣2 animals, no effect of contraction was observed on AMPK␣2 activity, and glucose uptake stimulation was reduced by 50% (P Ͻ 0.01) As expected, 5-aminoimidazole-4-carboxamide-1-␤-D-ribofuranosyl 5Ј-monophosphate (AICAR) caused a 2.3-fold stimulation of AMPK␣ 2 activity and a 1.7-fold increase in glucose uptake in EDL from WT mice, whereas no effect was detected in muscle from KD-AMPK␣ 2 mice. These data demonstrate that AMPK activation is essential for both AICAR and submaximal contractioninduced glucose transport in skeletal muscle but that AMPK-independent mechanisms are also involved. adenosine 5Ј-monophosphate-activated protein kinase 5Ј-AMP-ACTIVATED PROTEIN KINASE (AMPK) is a member of a metabolite-sensing protein kinase family that responds to modulations in cellular energy levels (6, 14). When AMPK "senses" decreased energy stores, it acts to switch off ATPconsuming pathways and switch on alternative pathways for ATP regeneration. AMPK is a ubiquitous heterotrimer comprised of a catalytic ␣-and regulatory ␤-and ␥-subunits. It is thought to be a key actor in the alternative, non-insulinmediated pathways leading to glucose uptake in skeletal muscle (13). Because AMPK is potently activated by muscular contraction, it is also believed to represent a key signaling molecule for exercise-induced glucose transport (15,28,29,35,38). In rats, exercise predominantly increases the activity of the ␣ 2 -subunit of AMPK while tetanic contractions ex vivo increase both the ␣ 1 and ␣ 2 AMPK subunits (26). Exercise performed at 65-70% V O 2max preferentially stimulates AMPK␣ 2 in human muscle, whereas activation of AMPK␣ 1 at this intensity is still cont...

Aims/hypothesis Insulin resistance in skeletal muscle is linked to mitochondrial dysfunction in obesity and type 2 diabetes. Emerging evidence indicates that reversible phosphorylation regulates oxidative phosphorylation (OxPhos) proteins. The aim of this study was to identify and quantify site-specific phosphorylation of the catalytic beta subunit of ATP synthase (ATPsyn-β) and determine protein abundance of ATPsyn-β and other OxPhos components in skeletal muscle from healthy and insulin-resistant individuals. Methods Skeletal muscle biopsies were obtained from lean, healthy, obese, non-diabetic and type 2 diabetic volunteers (each group n=10) for immunoblotting of proteins, and hypothesis-driven identification and quantification of phosphorylation sites on ATPsyn-β using targeted nanospray tandem mass spectrometry. Volunteers were metabolically characterised by euglycaemic-hyperinsulinaemic clamps. Results Seven phosphorylation sites were identified on ATPsyn-β purified from human skeletal muscle. Obese individuals with and without type 2 diabetes were characterised by impaired insulin-stimulated glucose disposal rates, and showed a ∼30% higher phosphorylation of ATPsyn-β at Tyr361 and Thr213 (within the nucleotidebinding region of ATP synthase) as well as a coordinated downregulation of ATPsyn-β protein and other OxPhos components. Insulin increased Tyr361 phosphorylation of ATPsyn-β by ∼50% in lean and healthy, but not insulinresistant, individuals. Conclusions/interpretation These data demonstrate that ATPsyn-β is phosphorylated at multiple sites in human skeletal muscle, and suggest that abnormal site-specific phosphorylation of ATPsyn-β together with reduced content of OxPhos proteins contributes to mitochondrial dysfunction in insulin resistance. Further characterisation of phosphorylation of ATPsyn-β may offer novel targets of treatment in human diseases with mitochondrial dysfunction, such as diabetes.