Chad R. Hancock

High-fat diets cause insulin resistance despite an increase in muscle mitochondria

It has been hypothesized that insulin resistance is mediated by a deficiency of mitochondria in skeletal muscle. In keeping with this hypothesis, high-fat diets that cause insulin resistance have been reported to result in a decrease in muscle mitochondria. In contrast, we found that feeding rats high-fat diets that cause muscle insulin resistance results in a concomitant gradual increase in muscle mitochondria. This adaptation appears to be mediated by activation of peroxisome proliferator-activated receptor (PPAR)δ by fatty acids, which results in a gradual, posttranscriptionally regulated increase in PPAR γ coactivator 1α (PGC-1α) protein expression. Similarly, overexpression of PPARδ results in a large increase in PGC-1α protein in the absence of any increase in PGC-1α mRNA. We interpret our findings as evidence that raising free fatty acids results in an increase in mitochondria by activating PPARδ, which mediates a posttranscriptional increase in PGC-1α. Our findings argue against the concept that insulin resistance is mediated by a deficiency of muscle mitochondria.

A Role for the Transcriptional Coactivator PGC-1α in Muscle Refueling

The transcriptional coactivator peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) has been identified as an inducible regulator of mitochondrial function. Skeletal muscle PGC-1α expression is induced post-exercise. Therefore, we sought to determine its role in the regulation of muscle fuel metabolism. Studies were performed using conditional, muscle-specific, PGC-1α gain-of-function and constitutive, generalized, loss-of-function mice. Forced expression of PGC-1α increased muscle glucose uptake concomitant with augmentation of glycogen stores, a metabolic response similar to post-exercise recovery. Induction of muscle PGC-1α expression prevented muscle glycogen depletion during exercise. Conversely, PGC-1α-deficient animals exhibited reduced rates of muscle glycogen repletion post-exercise. PGC-1α was shown to increase muscle glycogen stores via several mechanisms including stimulation of glucose import, suppression of glycolytic flux, and by down-regulation of the expression of glycogen phosphorylase and its activating kinase, phosphorylase kinase α. These findings identify PGC-1α as a critical regulator of skeletal muscle fuel stores.

Raising plasma fatty acid concentration induces increased biogenesis of mitochondria in skeletal muscle

A number of studies have reported that a high-fat diet induces increases in mitochondrial fatty acid oxidation enzymes in muscle. In contrast, in two recent studies raising plasma free fatty acids (FFA) resulted in a decrease in mitochondria. In this work, we reevaluated the effects of raising FFA on muscle mitochondrial biogenesis and capacity for fat oxidation. Rats were fed a high-fat diet and given daily injections of heparin to raise FFA. This treatment induced an increase in mitochondrial biogenesis in muscle, as evidenced by increases in mitochondrial enzymes of the fatty acid oxidation pathway, citrate cycle, and respiratory chain, with an increase in the capacity to oxidize fat, as well as an increase in mitochondrial DNA copy number. Raising FFA also resulted in an increase in binding of peroxisome proliferator-activated receptor (PPAR) δ to the PPAR response element on the carnitine palmitoyltransferase 1 promoter. We interpret our results as evidence that raising FFA induces an increase in mitochondrial biogenesis in muscle by activating PPARδ.

Does calorie restriction induce mitochondrial biogenesis? A reevaluation

It has been reported that 30% calorie restriction (CR) for 3 mo results in large increases in mitochondrial biogenesis in heart, brain, liver, and adipose tissue, with concomitant increases in respiration and ATP synthesis. We found these results surprising, and performed this study to determine whether 30% CR does induce an increase in mitochondria in heart, brain, liver, adipose tissue, and/or skeletal muscle. To this end, we measured the levels of a range of mitochondrial proteins, and mRNAs. With the exception of long‐chain acyl‐CoA dehydrogenase protein level, which was increased ~60% in adipose tissue, none of the mitochondrial proteins or mRNAs that we measured were increased in rats subjected to 30% CR for 14 wk. There was also no increase in citrate synthase activity. Because it is not possible to have an increase in mitochondria without any increase in key mitochondrial proteins, we conclude that 30% CR does not induce an increase in mitochondria in heart, brain, liver, adipose tissue, or skeletal muscle in laboratory rodents.—Hancock, C. R., Han, D.‐H., Higashida, K., Kim, S. H., Holloszy, J. O. Does calorie restriction induce mito‐chondrial biogenesis? A reevaluation. FASEB J. 25, 785–791 (2011). www.fasebj.org

Deficiency of the Mitochondrial Electron Transport Chain in Muscle Does Not Cause Insulin Resistance

Background It has been proposed that muscle insulin resistance in type 2 diabetes is due to a selective decrease in the components of the mitochondrial electron transport chain and results from accumulation of toxic products of incomplete fat oxidation. The purpose of the present study was to test this hypothesis. Methodology/Principal Findings Rats were made severely iron deficient, by means of an iron-deficient diet. Iron deficiency results in decreases of the iron containing mitochondrial respiratory chain proteins without affecting the enzymes of the fatty acid oxidation pathway. Insulin resistance was induced by feeding iron-deficient and control rats a high fat diet. Skeletal muscle insulin resistance was evaluated by measuring glucose transport activity in soleus muscle strips. Mitochondrial proteins were measured by Western blot. Iron deficiency resulted in a decrease in expression of iron containing proteins of the mitochondrial respiratory chain in muscle. Citrate synthase, a non-iron containing citrate cycle enzyme, and long chain acyl-CoA dehydrogenase (LCAD), used as a marker for the fatty acid oxidation pathway, were unaffected by the iron deficiency. Oleate oxidation by muscle homogenates was increased by high fat feeding and decreased by iron deficiency despite high fat feeding. The high fat diet caused severe insulin resistance of muscle glucose transport. Iron deficiency completely protected against the high fat diet-induced muscle insulin resistance. Conclusions/Significance The results of the study argue against the hypothesis that a deficiency of the electron transport chain (ETC), and imbalance between the ETC and β-oxidation pathways, causes muscle insulin resistance.

Chronic AMP-activated protein kinase activation and a high-fat diet have an additive effect on mitochondria in rat skeletal muscle

Factors that stimulate mitochondrial biogenesis in skeletal muscle include AMP-activated protein kinase (AMPK), calcium, and circulating free fatty acids (FFAs). Chronic treatment with either 5-aminoimidazole-4-carboxamide riboside (AICAR), a chemical activator of AMPK, or increasing circulating FFAs with a high-fat diet increases mitochondria in rat skeletal muscle. The purpose of this study was to determine whether the combination of chronic chemical activation of AMPK and high-fat feeding would have an additive effect on skeletal muscle mitochondria levels. We treated Wistar male rats with a high-fat diet (HF), AICAR injections (AICAR), or a high-fat diet and AICAR injections (HF + AICAR) for 6 wk. At the end of the treatment period, markers of mitochondrial content were examined in white quadriceps, red quadriceps, and soleus muscles, predominantly composed of unique muscle-fiber types. In white quadriceps, there was a cumulative effect of treatments on long-chain acyl-CoA dehydrogenase, cytochrome c, and peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) protein, as well as on citrate synthase and beta-hydroxyacyl-CoA dehydrogenase (beta-HAD) activity. In contrast, no additive effect was noted in the soleus, and in the red quadriceps only beta-HAD activity increased additively. The additive increase of mitochondrial markers observed in the white quadriceps may be explained by a combined effect of two separate mechanisms: high-fat diet-induced posttranscriptional increase in PGC-1alpha protein and AMPK-mediated increase in PGC-1alpha protein via a transcriptional mechanism. These data show that chronic chemical activation of AMPK and a high-fat diet have a muscle type specific additive effect on markers of fatty acid oxidation, the citric acid cycle, the electron transport chain, and transcriptional regulation.

Skeletal muscle dysfunction in muscle-specific LKB1 knockout mice

Liver kinase B1 (LKB1) is a tumor-suppressing protein that is involved in the regulation of muscle metabolism and growth by phosphorylating and activating AMP-activated protein kinase (AMPK) family members. Here we report the development of a myopathic phenotype in skeletal and cardiac muscle-specific LKB1 knockout (mLKB1-KO) mice. The myopathic phenotype becomes overtly apparent at 30-50 wk of age and is characterized by decreased body weight and a proportional reduction in fast-twitch skeletal muscle weight. The ability to ambulate is compromised with an often complete loss of hindlimb function. Skeletal muscle atrophy is associated with a 50-75% reduction in mammalian target of rapamycin pathway phosphorylation, as well as lower peroxisome proliferator-activated receptor-alpha coactivator-1 content and cAMP response element binding protein phosphorylation (43 and 40% lower in mLKB1-KO mice, respectively). Maximum in situ specific force production is not affected, but fatigue is exaggerated, and relaxation kinetics are slowed in the myopathic mice. The increased fatigue is associated with a 30-78% decrease in mitochondrial protein content, a shift away from type IIA/D toward type IIB muscle fibers, and a tendency (P=0.07) for decreased capillarity in mLKB1-KO muscles. Hearts from myopathic mLKB1-KO mice exhibit grossly dilated atria, suggesting cardiac insufficiency and heart failure, which likely contributes to the phenotype. These findings indicate that LKB1 plays a critical role in the maintenance of both skeletal and cardiac function.

Reductions in RIP140 are not required for exercise- and AICAR-mediated increases in skeletal muscle mitochondrial content.

Receptor interacting protein 1 (RIP140) has recently been demonstrated to be a key player in the regulation of skeletal muscle mitochondrial content. We have shown that β-guanadinopropionic acid (β-GPA) feeding reduces RIP140 protein content and mRNA levels concomitant with increases in mitochondrial content (Williams DB, Sutherland LN, Bomhof MR, Basaraba SA, Thrush AB, Dyck DJ, Field CJ, Wright DC. Am J Physiol Endocrinol Metab 296: E1400-E1408, 2009). Since β-GPA feeding reduces high-energy phosphate levels and activates AMPK, alterations reminiscent of exercise, we hypothesized that exercise training would reduce RIP140 protein content. We further postulated that an acute bout of exercise, or interventions known to induce the expression of mitochondrial enzymes or genes involved in mitochondrial biogenesis, would result in decreases in nuclear RIP140 content. Two weeks of daily swim training increased markers of mitochondrial content in rat skeletal muscle independent of reductions in RIP140 protein. Similarly, high-intensity exercise training in humans failed to reduce RIP140 content despite increasing skeletal muscle mitochondrial enzymes. We found that 6 wk of daily 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) injections had no effect on RIP140 protein content in rat skeletal muscle while RIP140 content from LKB1 knockout mice was unaltered despite reductions in mitochondria. An acute bout of exercise, AICAR treatment, and epinephrine injections increased the mRNA levels of PGC-1α, COXIV, and lipin1 independent of decreases in nuclear RIP140 protein. Surprisingly these interventions increased RIP140 mRNA expression. In conclusion our results demonstrate that decreases in RIP140 protein content are not required for exercise and AMPK-dependent increases in skeletal muscle mitochondrial content, nor do acute perturbations alter the cellular localization of RIP140 in parallel with the induction of genes involved in mitochondrial biogenesis.

AICAR inhibits ceramide biosynthesis in skeletal muscle

BackgroundThe worldwide prevalence of obesity has lead to increased efforts to find therapies to treat obesity-related pathologies. Ceramide is a well-established mediator of several health problems that arise from adipose tissue expansion. The purpose of this study was to determine whether AICAR, an AMPK-activating drug, selectively reduces skeletal muscle ceramide synthesis.MethodsMurine myotubes and rats were challenged with palmitate and high-fat diet, respectively, to induce ceramide accrual, in the absence or presence of AICAR. Transcript levels of the rate-limiting enzyme in ceramide biosynthesis, serine palmitoyltransferase 2 (SPT2) were measured, in addition to lipid analysis. Student’s t-test and ANOVA were used to assess the association between outcomes and groups.ResultsPalmitate alone induced an increase in serine palmitoyltransferase 2 (SPT2) expression and an elevation of ceramide levels in myotubes. Co-incubation with palmitate and AICAR prevented both effects. However, ceramide and SPT2 increased with the addition of compound C, an AMPK inhibitor. In rats fed a high-fat diet (HFD), soleus SPT2 expression increased compared with normal chow-fed littermates. Moreover, rats on HFD that received daily AICAR injections had lower SPT2 levels and reduced muscle ceramide content compared with those on HFD only.ConclusionsThese results suggest that AICAR reduces ceramide synthesis by targeting SPT2 transcription, likely via AMPK activation as AMPK inhibition prevented the AICAR-induced improvements. Given the role of skeletal muscle ceramide in insulin resistance, it is tempting to speculate that interventions that activate AMPK may lead to long-term ceramide reduction and improved metabolic function.

Iron deficiency causes a shift in AMP-activated protein kinase (AMPK) subunit composition in rat skeletal muscle

BackgroundAs a cellular energy sensor, the 5’AMP-activated protein kinase (AMPK) is activated in response to energy stresses such as hypoxia and muscle contraction. To determine effects of iron deficiency on AMPK activation and signaling, as well as the AMPK subunit composition in skeletal muscle, rats were fed a control (C=50-58 mg/kg Fe) or iron deficient (ID=2-6 mg/kg Fe) diet for 6–8 wks.ResultsTheir respective hematocrits were 47.5% ± 1.0 and 16.5% ± 0.6. Iron deficiency resulted in 28.3% greater muscle fatigue (p<0.01) in response to 10 min of stimulation (1 twitch/sec) and was associated with a greater reduction in phosphocreatine (C: Resting 24.1 ± 0.9 μmol/g, Stim 13.1 ± 1.5 μmol/g; ID: Resting 22.7 ± 1.0 μmol/g, Stim 3.2 ± 0.7 μmol/g; p<0.01) and ATP levels (C: Resting 5.89 ± 0.48 μmol/g, Stim 6.03 ± 0.35 μmol/g; ID: Resting 5.51 ± 0.20 μmol/g, Stim 4.19 ± 0.47 μmol/g; p<0.05). AMPK activation increased with stimulation in muscles of C and ID animals. A reduction in Cytochrome c and other iron-dependent mitochondrial proteins was observed in ID animals (p<0.01). The AMPK catalytic subunit (α) was examined because both isoforms are known to play different roles in responding to energy challenges. In ID animals, AMPKα2 subunit protein content was reduced to 71.6% of C (p<0.05), however this did not result in a significant difference in resting AMPKα2 activity. AMPKα1 protein was unchanged, however an overall increase in AMPKα1 activity was observed (C: 0.91 pmol/mg/min; ID: 1.63 pmol/mg/min; p<0.05). Resting phospho Acetyl CoA Carboxylase (pACC) was unchanged. In addition, we observed significant reductions in the β2 and γ3 subunits of AMPK in response to iron deficiency.ConclusionsThis study indicates that chronic iron deficiency causes a shift in the expression of AMPKα, β, and γ subunit composition. Iron deficiency also causes chronic activation of AMPK as well as an increase in AMPKα1 activity in exercised skeletal muscle.