Jill M. Schimke

Effect of insulin on human skeletal muscle mitochondrial ATP production, protein synthesis, and mRNA transcripts

Mitochondria are the primary site of skeletal muscle fuel metabolism and ATP production. Although insulin is a major regulator of fuel metabolism, its effect on mitochondrial ATP production is not known. Here we report increases in vastus lateralis muscle mitochondrial ATP production capacity (32–42%) in healthy humans (P < 0.01) i.v. infused with insulin (1.5 milliunits/kg of fat-free mass per min) while clamping glucose, amino acids, glucagon, and growth hormone. Increased ATP production occurred in association with increased mRNA levels from both mitochondrial (NADH dehydrogenase subunit IV) and nuclear [cytochrome c oxidase (COX) subunit IV] genes (164–180%) encoding mitochondrial proteins (P < 0.05). In addition, muscle mitochondrial protein synthesis, and COX and citrate synthase enzyme activities were increased by insulin (P < 0.05). Further studies demonstrated no effect of low to high insulin levels on muscle mitochondrial ATP production for people with type 2 diabetes mellitus, whereas matched nondiabetic controls increased 16–26% (P < 0.02) when four different substrate combinations were used. In conclusion, insulin stimulates mitochondrial oxidative phosphorylation in skeletal muscle along with synthesis of gene transcripts and mitochondrial protein in human subjects. Skeletal muscle of type 2 diabetic patients has a reduced capacity to increase ATP production with high insulin levels.

Influence of fish oil on skeletal muscle mitochondrial energetics and lipid metabolites during high-fat diet

Omega-3 polyunsaturated fatty acids (n-3 PUFAs) enhance insulin sensitivity and glucose homeostasis in rodent models of insulin resistance. These beneficial effects have been linked with anti-inflammatory properties, but emerging data suggest that the mechanisms may also converge on mitochondria. We evaluated the influence of dietary n-3 PUFAs on mitochondrial physiology and muscle lipid metabolites in the context of high-fat diet (HFD) in mice. Mice were fed control diets (10% fat), HFD (60% fat), or HFD with fish oil (HFD+FO, 3.4% kcal from n-3 PUFAs) for 10 wk. Body mass and fat mass increased similarly in HFD and HFD+FO, but n-3 PUFAs attenuated the glucose intolerance that developed with HFD and increased expression of genes that regulate glucose metabolism in skeletal muscle. Despite similar muscle triglyceride levels in HFD and HFD+FO, long-chain acyl-CoAs and ceramides were lower in the presence of fish oil. Mitochondrial abundance and oxidative capacity were similarly increased in HFD and HFD+FO compared with controls. Hydrogen peroxide production was similarly elevated in HFD and HFD+FO in isolated mitochondria but not in permeabilized muscle fibers, likely due to increased activity and expression of catalase. These results support a hypothesis that n-3 PUFAs protect glucose tolerance, in part by preventing the accumulation of bioactive lipid mediators that interfere with insulin action. Furthermore, the respiratory function of skeletal muscle mitochondria does not appear to be a major factor in sphingolipid accumulation, glucose intolerance, or the protective effects of n-3 PUFAs.

Unique cellular and mitochondrial defects mediate FK506-induced islet β-cell dysfunction

Objective. To determine biological mechanisms involved in posttransplantation diabetes mellitus caused by the immunosuppressant tacrolimus (FK506). Methods. INS-1 cells and isolated rat islets were incubated with vehicle or FK506 and harvested at 24-hr intervals. Cells were assessed for viability, apoptosis, proliferation, cell insulin secretion, and content. Gene expression studies by microarray analysis, quantitative polymerase chain reaction, and motifADE analysis of the microarray data identified potential FK506-mediated pathways and regulatory motifs. Mitochondrial functions, including cell respiration, mitochondrial content, and bioenergetics were assessed. Results. Cell replication, viability, insulin secretion, oxygen consumption, and mitochondrial content were decreased (P<0.05) 1.2-, 1.27-, 1.77-, 1.32-, and 1.43-fold, respectively, after 48-hr FK506 treatment. Differences increased with time. FK506 (50 ng/mL) and cyclosporine A (800 ng/mL) had comparable effects. FK506 significantly decreased mitochondrial content and mitochondrial bioenergetics and showed a trend toward decreased oxygen consumption in isolated islets. Cell apoptosis and proliferation, mitochondrial DNA copy number, and ATP:ADP ratios were not significantly affected. Pathway analysis of microarray data showed FK506 modification of pathways involving ATP metabolism, membrane trafficking, and cytoskeleton remodeling. PGC1-&agr; mRNA was down-regulated by FK506. MotifADE identified nuclear factor of activated T-cells, an important mediator of &bgr;-cell survival and function, as a potential factor mediating both up- and down-regulation of gene expression. Conclusions. At pharmacologically relevant concentrations, FK506 decreases insulin secretion and reduces mitochondrial density and function without changing apoptosis rates, suggesting that posttransplantation diabetes induced by FK506 may be mediated by its effects on mitochondrial function.

Hyperandrogenism sensitizes leukocytes to hyperglycemia to promote oxidative stress in lean reproductive-age women.

CONTEXT Hyperandrogenism and oxidative stress are related in polycystic ovary syndrome (PCOS), but it is unknown whether hyperandrogenemia can activate oxidative stress. OBJECTIVE The purpose of this study was to determine the effect of oral androgen administration on fasting and glucose-stimulated leukocytic reactive oxygen species (ROS) generation, reduced nicotinamide adenine dinucleotide phosphate oxidase p47(phox) subunit gene expression, and plasma thiobarbituric acid-reactive substances (TBARS) in lean healthy reproductive-age women. PARTICIPANTS, DESIGN, AND SETTING Sixteen lean healthy ovulatory reproductive-age women were treated with 130 mg dehydroepiandrosterone (DHEA) or placebo (n = 8 each) for 5 d in this randomized, controlled, double-blind study that was performed at an an academic medical center. MAIN OUTCOME MEASURES Leukocytic ROS generation, p47(phox) gene expression, and plasma TBARS were quantified in the fasting state and 2 h after glucose ingestion, before and after treatment. RESULTS Before treatment, subjects receiving DHEA or placebo exhibited no differences in androgens or any prooxidant markers while fasting and after glucose ingestion. Compared with placebo, DHEA administration raised levels of testosterone, androstenedione, and DHEA-sulfate, increased the percent change in glucose-challenged p47(phox) RNA content, and increased the percent change in fasting and glucose-challenged ROS generation from mononuclear cells and polymorphonuclear cells, p47(phox) protein content, and plasma TBARS. CONCLUSION Elevation of circulating androgens comparable to what is present in PCOS increases leukocytic ROS generation, p47(phox) gene expression, and plasma TBARS to promote oxidative stress in lean healthy reproductive-age women. Thus, hyperandrogenemia activates and sensitizes leukocytes to glucose in this population.

Hyperandrogenism sensitizes mononuclear cells to promote glucose-induced inflammation in lean reproductive-age women.

Hyperandrogenism and chronic low-grade inflammation are related in polycystic ovary syndrome (PCOS), but it is unknown whether hyperandrogenemia can activate inflammation. We determined the effect of oral androgen administration on fasting and glucose-stimulated nuclear factor-κB (NF-κB) activation and expression and related markers of inflammation in mononuclear cells (MNC) of lean reproductive-age women. Sixteen lean, ovulatory reproductive-age women were treated with 130 mg of DHEA or placebo (n = 8 each) for 5 days in a randomized, controlled, double-blind fashion. Nuclear activation of NF-κB, p65 and p105 NF-κB subunit RNA, TNFα and IL-1β mRNA, and NF-κB p65 and inhibitory-κB (IκB) protein were quantified from MNC obtained while fasting and 2 h after glucose ingestion, before and after DHEA or placebo administration. Before treatment, subjects receiving DHEA or placebo exhibited no differences in androgens or any inflammatory markers while fasting and after glucose ingestion. Compared with placebo, DHEA administration raised levels of testosterone, androstenedione, and DHEA-S, increased the percent change in fasting and glucose-challenged activated NF-κB, p65, p105, TNFα, and IL-1β RNA and p65 protein, and decreased the percent change in fasting and glucose-challenged IκB protein. We conclude that elevation of circulating androgens to the range observed in PCOS upregulates the NF-κB inflammation pathway in lean reproductive-age women. Thus, hyperandrogenemia activates and sensitizes MNC to glucose in this population.

Differential Effect of Endurance Training on Mitochondrial Protein Damage, Degradation, and Acetylation in the Context of Aging

Acute aerobic exercise increases reactive oxygen species and could potentially damage proteins, but exercise training (ET) enhances mitochondrial respiration irrespective of age. Here, we report a differential impact of ET on protein quality in young and older participants. Using mass spectrometry we measured oxidative damage to skeletal muscle proteins before and after 8 weeks of ET and find that young but not older participants reduced oxidative damage to both total skeletal muscle and mitochondrial proteins. Young participants showed higher total and mitochondrial derived semitryptic peptides and 26S proteasome activity indicating increased protein degradation. ET however, increased the activity of the endogenous antioxidants in older participants. ET also increased skeletal muscle content of the mitochondrial deacetylase SIRT3 in both groups. A reduction in the acetylation of isocitrate dehydrogenase 2 was observed following ET that may counteract the effect of acute oxidative stress. In conclusion aging is associated with an inability to improve skeletal muscle and mitochondrial protein quality in response to ET by increasing degradation of damaged proteins. ET does however increase muscle and mitochondrial antioxidant capacity in older individuals, which provides increased buffering from the acute oxidative effects of exercise.

High Insulin Combined With Essential Amino Acids Stimulates Skeletal Muscle Mitochondrial Protein Synthesis While Decreasing Insulin Sensitivity in Healthy Humans

CONTEXT Insulin and essential amino acids (EAAs) regulate skeletal muscle protein synthesis, yet their independent effects on mitochondrial protein synthesis (MiPS) and oxidative function remain to be clearly defined. OBJECTIVE The purpose of this study was to determine the effects of high or low insulin with or without EAAs on MiPS. DESIGN Thirty participants were randomized to 3 groups of 10 each with each participant studied twice. Study groups comprised (1) low and high insulin, (2) low insulin with and without EAAs, and (3) high insulin with and without EAAs. SETTING The study was conducted in an in-patient clinical research unit. PARTICIPANTS Eligible participants were 18 to 45 years old, had a body mass index of <25 kg/m(2), and were free of diseases and medications that might impair mitochondrial function. INTERVENTION Low (∼ 6 μU/mL) and high (∼ 40 μU/mL) insulin levels were maintained by iv insulin infusion during a somatostatin clamp while maintaining euglycemia (4.7-5.2 mM) and replacing GH and glucagon. The EAA infusion was 5.4% NephrAmine. l-[ring-(13)C6]Phenylalanine was infused, and muscle needle biopsies were performed. MAIN OUTCOMES Muscle MiPS, oxidative enzymes, and plasma amino acid metabolites were measured. RESULTS MiPS and oxidative enzyme activities did not differ between low and high insulin (MiPS: 0.07 ± 0.009 vs 0.07 ± 0.006%/h, P = .86) or between EAAs and saline during low insulin (MiPS: 0.05 ± 0.01 vs 0.07 ± 0.01, P = .5). During high insulin, EAAs in comparison with saline increased MiPS (0.1 ± 0.01 vs 0.06 ± 0.01, P < .05) and cytochrome c oxidase activity (P < .05) but not citrate synthase (P = .27). EAA infusion decreased (P < .05) the glucose infusion rates needed to maintain euglycemia during low (∼ 40%) and high insulin (∼ 24%). CONCLUSION EAAs increased MiPS and oxidative enzyme activity only with high insulin concentrations.

Citrulline stimulates muscle protein synthesis in the post-absorptive state in healthy people fed a low-protein diet – A pilot study

BACKGROUND & AIMS Amino acid (AA) availability is critical to maintain protein homeostasis and reduced protein intake causes a decline in protein synthesis. Citrulline, an amino acid metabolite, has been reported to stimulate muscle protein synthesis in malnourished rats. METHODS To determine whether citrulline stimulates muscle protein synthesis in healthy adults while on a low-protein diet, we studied 8 healthy participants twice in a cross-over study design. Following a 3-days of low-protein intake, either citrulline or a non-essential AA mixture (NEAA) was given orally as small boluses over the course of 8 h. [ring-(13)C6] phenylalanine and [(15)N] tyrosine were administered as tracers to assess protein metabolism. Fractional synthesis rates (FSR) of muscle proteins were measured using phenylalanine enrichment in muscle tissue fluid as the precursor pool. RESULTS FSR of mixed muscle protein was higher during the administration of citrulline than during NEAA (NEAA: 0.049 ± 0.005; citrulline: 0.060 ± 0.006; P = 0.03), while muscle mitochondrial protein FSR and whole-body protein turnover were not different between the studies. Citrulline administration increased arginine and ornithine plasma concentrations without any effect on glucose, insulin, C-peptide, and IGF-1 levels. Citrulline administration did not promote mitochondria protein synthesis, transcripts, or citrate synthesis. CONCLUSIONS Citrulline ingestion enhances mixed muscle protein synthesis in healthy participants on 3-day low-protein intake. This anabolic action of citrulline appears to be independent of insulin action and may offer potential clinical application in conditions involving low amino acid intake.

Impact of insulin deprivation and treatment on sphingolipid distribution in different muscle subcellular compartments of streptozotocin-diabetic C57Bl/6 mice

Insulin deprivation in type 1 diabetes (T1D) individuals increases lipolysis and plasma free fatty acids (FFA) concentration, which can stimulate synthesis of intramyocellular bioactive lipids such as ceramides (Cer) and long-chain fatty acid-CoAs (LCFa-CoAs). Ceramide was shown to decrease muscle insulin sensitivity, and at mitochondrial levels it stimulates reactive oxygen species production. Here, we show that insulin deprivation in streptozotocin diabetic C57BL/6 mice increases quadriceps muscle Cer content, which was correlated with a concomitant decrease in the body fat and increased plasma FFA, glycosylated hemoglobin level (%Hb A1c), and muscular LCFa-CoA content. The alternations were accompanied by an increase in protein expression in LCFa-CoA and Cer synthesis (FATP1/ACSVL5, CerS1, CerS5), a decrease in the expression of genes implicated in muscle insulin sensitivity (GLUT4, GYS1), and inhibition of insulin signaling cascade by Aktα and GYS3β phosphorylation under acute insulin stimulation. Both the content and composition of sarcoplasmic fraction sphingolipids were most affected by insulin deprivation, whereas mitochondrial fraction sphingolipids remained stable. The observed effects of insulin deprivation were reversed, except for content and composition of LCFa-CoA, CerS protein expression, GYS1 gene expression, and phosphorylation status of Akt and GYS3β when exogenous insulin was provided by subcutaneous insulin implants. Principal component analysis and Pearsons correlation analysis revealed close relationships between the features of the diabetic phenotype, the content of LCFa-CoAs and Cers containing C18-fatty acids in sarcoplasm, but not in mitochondria. Insulin replacement did not completely rescue the phenotype, especially regarding the content of LCFa-CoA, or proteins implicated in Cer synthesis and muscle insulin sensitivity. These persistent changes might contribute to muscle insulin resistance observed in T1D individuals.

Acute Free Fatty Acid Elevation Eliminates Endurance Training Effect on Insulin Sensitivity

CONTEXT Both training and normal body mass index are associated with high insulin sensitivity, but the mechanism may be different. OBJECTIVE The aim of the study was to examine whether lean trained humans may be protected from acute free fatty acid (FFA)-induced insulin resistance compared with lean sedentary humans. DESIGN AND SETTING We conducted an interventional trial using either a 6-h lipid (20% Intralipid at 90 ml/h) or glycerol (2.25 g/100 ml at 90 ml/h) infusion along with a concurrent hyperinsulinemic-euglycemic clamp and serial muscle biopsies (0, 120, 360 min) at a clinical research unit at the University of Minnesota. PATIENTS OR PARTICIPANTS The study included lean endurance-trained (n = 14) and sedentary (n = 14) individuals matched for age, gender, and body mass index. MAIN OUTCOME MEASURES We measured the decline in glucose infusion rate (GIR) during the hyperinsulinemic-euglycemic clamp. RESULTS The trained group had higher baseline mitochondrial DNA copy number, mRNA of cytochrome C oxidase subunit 3, and insulin sensitivity (as measured by GIR) compared with the sedentary group. When FFA was acutely elevated to the upper physiological range (0.6-0.7 mEq/liter) by lipid infusion, the GIR in both activity groups declined similarly compared with their respective glycerol controls, although insulin signaling, as measured by Ser 473 pAKT/AKT, remained comparable. Specific to the trained group, the stimulatory effect of hyperinsulinemia on mitochondrial mRNA levels during the glycerol infusion was absent during the lipid infusion. CONCLUSIONS Elevated FFA had similar effects in reducing insulin sensitivity in trained and sedentary humans. In trained participants, this decline was associated with alterations in the skeletal muscle mitochondrial mRNA response to hyperinsulinemia.