Citations

The forkhead box O3a (FOXO3a) transcription factor has been shown to regulate glucose metabolism, muscle atrophy, and cell death in postmitotic cells. Its role in regulation of mitochondrial and myocardial function is not well studied. Based on previous work, we hypothesized that FOXO3a, through BCL2/adenovirus E1B 19-kDa protein-interacting protein 3 (BNIP3), modulates mitochondrial morphology and function in heart failure (HF). We modulated the FOXO3a-BNIP3 pathway in normal and phenylephrine (PE)-stressed adult cardiomyocytes (ACM) in vitro and developed a cardiotropic adeno-associated virus serotype 9 encoding dominant-negative FOXO3a (AAV9.dn-FX3a) for gene delivery in a rat model of HF with preserved ejection fraction (HFpEF). We found that FOXO3a upregulates BNIP3 expression in normal and PE-stressed ACM, with subsequent increases in mitochondrial Ca2+, leading to decreased mitochondrial membrane potential, mitochondrial fragmentation, and apoptosis. Whereas dn-FX3a attenuated the increase in BNIP3 expression and its consequences in PE-stressed ACM, AAV9.dn-FX3a delivery in an experimental model of HFpEF decreased BNIP3 expression, reversed adverse left ventricular remodeling, and improved left ventricular systolic and, particularly, diastolic function, with improvements in mitochondrial structure and function. Moreover, AAV9.dn-FX3a restored phospholamban phosphorylation at S16 and enhanced dynamin-related protein 1 phosphorylation at S637. Furthermore, FOXO3a upregulates maladaptive genes involved in mitochondrial apoptosis, autophagy, and cardiac atrophy. We conclude that FOXO3a activation in cardiac stress is maladaptive, in that it modulates Ca2+ cycling, Ca2+ homeostasis, and mitochondrial dynamics and function. Our results suggest an important role of FOXO3a in HF, making it an attractive potential therapeutic target. Listen to this article's corresponding podcast at http://ajpheart.podbean.com/e/role-of-foxo3a-in-heart-failure/ .

Insulin regulates skeletal muscle protein degradation, but the types of proteins being degraded in vivo remain to be determined due to methodological limitations. We present a method to assess the types of skeletal muscle proteins that are degraded by extracting their degradation products as low-molecular weight (LMW) peptides from muscle samples. High-resolution mass spectrometry was used to identify the original intact proteins that generated the LMW peptides, which we validated in rodents and then applied to humans. We deprived insulin from insulin-treated streptozotocin (STZ) diabetic mice for 6 and 96 h and for 8 h in type 1 diabetic humans (T1D) for comparison with insulin-treated conditions. Protein degradation was measured using activation of autophagy and proteasome pathways, stable isotope tracers, and LMW approaches. In mice, insulin deprivation activated proteasome pathways and autophagy in muscle homogenates and isolated mitochondria. Reproducibility analysis of LMW extracts revealed that ∼80% of proteins were detected consistently. As expected, insulin deprivation increased whole body protein turnover in T1D. Individual protein degradation increased with insulin deprivation, including those involved in mitochondrial function, proteome homeostasis, nDNA support, and contractile/cytoskeleton. Individual mitochondrial proteins that generated more LMW fragment with insulin deprivation included ATP synthase subunit-γ (+0.5-fold, P = 0.007) and cytochrome c oxidase subunit 6 (+0.305-fold, P = 0.03). In conclusion, identifying LMW peptide fragments offers an approach to determine the degradation of individual proteins. Insulin deprivation increases degradation of select proteins and provides insight into the regulatory role of insulin in maintaining proteome homeostasis, especially of mitochondria.

Insulin plays pivotal role in cellular fuel metabolism in skeletal muscle. Despite being the primary site of energy metabolism, the underlying mechanism on how insulin deficiency deranges skeletal muscle mitochondrial physiology remains to be fully understood. Here we report an important link between altered skeletal muscle proteome homeostasis and mitochondrial physiology during insulin deficiency. Deprivation of insulin in streptozotocin-induced diabetic mice decreased mitochondrial ATP production, reduced coupling and phosphorylation efficiency, and increased oxidant emission in skeletal muscle. Proteomic survey revealed that the mitochondrial derangements during insulin deficiency were related to increased mitochondrial protein degradation and decreased protein synthesis, resulting in reduced abundance of proteins involved in mitochondrial respiration and β-oxidation. However, a paradoxical upregulation of proteins involved in cellular uptake of fatty acids triggered an accumulation of incomplete fatty acid oxidation products in skeletal muscle. These data implicate a mismatch of β-oxidation and fatty acid uptake as a mechanism leading to increased oxidative stress in diabetes. This notion was supported by elevated oxidative stress in cultured myotubes exposed to palmitate in the presence of a β-oxidation inhibitor. Together, these results indicate that insulin deficiency alters the balance of proteins involved in fatty acid transport and oxidation in skeletal muscle, leading to impaired mitochondrial function and increased oxidative stress.

Context:Numerous factors are purported to influence insulin sensitivity including age, adiposity, mitochondrial function, and physical fitness. Univariate associations cannot address the complexity of insulin resistance or the interrelationship among potential determinants.Objective:The objective of the study was to identify significant independent predictors of insulin sensitivity across a range of age and adiposity in humans.Design, Setting, and Participants:Peripheral and hepatic insulin sensitivity were measured by two stage hyperinsulinemic-euglycemic clamps in 116 men and women (aged 19–78 y). Insulin-stimulated glucose disposal, the suppression of endogenous glucose production during hyperinsulinemia, and homeostatic model assessment of insulin resistance were tested for associations with 11 potential predictors. Abdominal subcutaneous fat, visceral fat (AFVISC), intrahepatic lipid, and intramyocellular lipid (IMCL) were quantified by magnetic resonance imaging and spectroscopy. Skeletal muscle mitochondrial respiratory capacity (state 3), coupling efficiency, and reactive oxygen species production were evaluated from muscle biopsies. Aerobic fitness was measured from whole-body maximum oxygen uptake (VO2 peak), and metabolic flexibility was determined using indirect calorimetry.Results:Multiple regression analysis revealed that AFVISC (P < .0001) and intrahepatic lipid (P = .002) were independent negative predictors of peripheral insulin sensitivity, whereas VO2 peak (P = .0007) and IMCL (P = .023) were positive predictors. Mitochondrial capacity and efficiency were not independent determinants of peripheral insulin sensitivity. The suppression of endogenous glucose production during hyperinsulinemia model of hepatic insulin sensitivity revealed percentage fat (P < .0001) and AFVISC (P = .001) as significant negative predictors. Modeling homeostatic model assessment of insulin resistance identified AFVISC (P < .0001), VO2 peak (P = .001), and IMCL (P = .01) as independent predictors.Conclusion:The reduction in insulin sensitivity observed with aging is driven primarily by age-related changes in the content and distribution of adipose tissue and is independent of muscle mitochondrial function or chronological age.

Caloric restriction (CR) improves insulin sensitivity and reduces the incidence of diabetes in obese individuals. The underlying mechanisms whereby CR improves insulin sensitivity are not clear. We evaluated the effect of 16 weeks of CR on whole-body insulin sensitivity by pancreatic clamp before and after CR in 11 obese participants (BMI = 35 kg/m2) compared with 9 matched control subjects (BMI = 34 kg/m2). Compared with the control subjects, CR increased the glucose infusion rate needed to maintain euglycemia during hyperinsulinemia, indicating enhancement of peripheral insulin sensitivity. This improvement in insulin sensitivity was not accompanied by changes in skeletal muscle mitochondrial oxidative capacity or oxidant emissions, nor were there changes in skeletal muscle ceramide, diacylglycerol, or amino acid metabolite levels. However, CR lowered insulin-stimulated thioredoxin-interacting protein (TXNIP) levels and enhanced nonoxidative glucose disposal. These results support a role for TXNIP in mediating the improvement in peripheral insulin sensitivity after CR.

ObjectiveTo examine the association of gene variants of uncoupling proteins (UCP)‐2 and −3 with obesity and gastrointestinal (GI) traits.MethodsIn 255 overweight or obese adults, the associations of gene variants in UCP‐2 (−3474, rs659366) and UCP‐3 (rs1626521, rs2075577, rs15763) with body weight (BW) and GI traits were studied. Gene variants were genotyped by TaqMan® assay. The associations of genotypes with BW and GI traits (gastric emptying, gastric volume, satiety by buffet meal, satiation by nutrient drink test and GI hormones) were assessed using ANOVA corrected for false detection rate (FDR).ResultsA novel UCP‐3 gene variant, rs1626521, was identified; it was associated with BW (P = 0.039), waist circumference (P = 0.035), and significantly higher postprandial gastric volume (P = 0.003) and calories ingested at buffet meal (P = 0.006, both significant with FDR). In a subgroup of 11 participants, rs1626521 was also associated with reduced mitochondrial bioenergetics efficiency in skeletal muscle (P = 0.051). In an in vitro study in HEK293 cells, rs1626521 reduced UCP‐3 protein expression (P = 0.049). Associations detected between other genotypes and GI traits were nonsignificant with FDR.ConclusionsA newly identified functional variant (rs1626521) in UCP‐3 affects postprandial gastric functions and satiety and may contribute to weight gain and alter human mitochondrial function.

Thyroid hormone excess is associated with increased energy expenditure. The contributions of increases in spontaneous physical activity and nonexercise activity thermogenesis (NEAT) to this effect have not been defined. To address the hypothesis that hyperthyroidism is associated with increased spontaneous physical activity and NEAT, we rendered rats hyperthyroid by using continuous infusion of high-dose triiodothyronine for 14 days and measured the effects on physical activity and NEAT. On day 14, in the hyperthyroid group the mean ± SD triiodothyronine concentration was 755 ± 137 (range 574–919) ng/dl and in the control group 59 ± 0.5 (58–59) ng/dl. Over the 14-day treatment period, mean spontaneous physical activity increased in the hyperthyroid rats from 24 ± 7 to 36 ± 6 activity units (AU)/min; P < 0.001 but did not increase in the controls (23 ± 7 vs. 22 ± 4 AU/min). Also, over the 14-day period, daily NEAT increased in the hyperthyroid rats from 8.1 ± 2.8 to 19.7 ± 5.0 kcal/day ( P < 0.001) but did not increase in the controls (8.7 ± 3.5 cf 9.4 ± 1.7 kcal/day; not significant). In conclusion, hyperthyroidism is associated with increased spontaneous physical activity and NEAT.

Rodent skeletal muscle mitochondrial DNA has been shown to be a potential site of oxidative damage during aging. Caloric restriction (CR) is reported to reduce oxidative stress and prolong life expectancy in rodents. Gene expression profiling and measurement of mitochondrial ATP production capacity were performed in skeletal muscle of male rats after feeding them either a control diet or calorie-restricted diet (60% of control diet) for 36 wk to determine the potential mechanism of the beneficial effects of CR. CR enhanced the transcripts of genes involved in reactive oxygen free radical scavenging function, tissue development, and energy metabolism while decreasing expression of those genes involved in signal transduction, stress response, and structural and contractile proteins. Real-time PCR measurments confirmed the changes in transcript levels of cytochrome- c oxidase III, superoxide dismutase (SOD)1, and SOD2 that were noted by the microarray approach. Mitochondrial ATP production and citrate synthase were unaltered by the dietary changes. We conclude that CR alters transcript levels of several genes in skeletal muscle and that mitochondrial function in skeletal muscle remains unaltered by the dietary intervention. Alterations in transcripts of many genes involved in reactive oxygen scavenging function may contribute to the increase in longevity reported with CR.

High-fat diets are reported to increase oxidative stress in a variety of tissues, whereas antioxidant supplementation prevents many diseases attributed to high-fat diet. Rodent skeletal muscle mitochondrial DNA has been shown to be a potential site of oxidative damage. We hypothesized that the effects of a high-fat diet on skeletal muscle DNA functions would be attenuated or partially reversed by antioxidant supplementation. Gene expression profiling and measurement of mitochondrial ATP production capacity were performed in skeletal muscle from male rats after feeding one of three diets (control, high-fat diet with or without antioxidants) for 36 wk. The high-fat diet altered transcript levels of 18 genes of 800 surveyed compared with the control-fed rats. Alterations included reduced expression of genes involved in free-radical scavenging and tissue development and increased expression of stress response and signal transduction genes. The magnitude of these alterations due to high-fat diet was reduced by antioxidant supplementation. Real-time PCR measurements confirmed the changes in transcript levels of cytochrome c oxidase subunit III and superoxide dismutase-1 and -2 noted by microarray approach. Mitochondrial ATP production was unaltered by dietary changes or antioxidant supplemention. It is concluded that the high-fat diet increases the transcription of genes involved in stress response but reduces those of free-radical scavenger enzymes, resulting in reduced DNA repair/metabolism (increased DNA damage). Antioxidants partially prevent these changes. Mitochondrial functions in skeletal muscle remain unaltered by the dietary intervention due to many adaptive changes in gene transcription.