Overexpression of Long-Chain Acyl-CoA Synthetase 5 Increases Fatty Acid Oxidation and Free Radical Formation While Attenuating Insulin Signaling in Primary Human Skeletal Myotubes

In rodent skeletal muscle, acyl-coenzyme A (CoA) synthetase 5 (ACSL-5) is suggested to localize to the mitochondria but its precise function in human skeletal muscle is unknown. The purpose of these studies was to define the role of ACSL-5 in mitochondrial fatty acid metabolism and the potential effects on insulin action in human skeletal muscle cells (HSKMC). Primary myoblasts isolated from vastus lateralis (obese women (body mass index (BMI) = 34.7 ± 3.1 kg/m2)) were transfected with ACSL-5 plasmid DNA or green fluorescent protein (GFP) vector (control), differentiated into myotubes, and harvested (7 days). HSKMC were assayed for complete and incomplete fatty acid oxidation ([1-14C] palmitate) or permeabilized to determine mitochondrial respiratory capacity (basal (non-ADP stimulated state 4), maximal uncoupled (carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP)-linked) respiration, and free radical (superoxide) emitting potential). Protein levels of ACSL-5 were 2-fold higher in ACSL-5 overexpressed HSKMC. Both complete and incomplete fatty acid oxidation increased by 2-fold (p < 0.05). In permeabilized HSKMC, ACSL-5 overexpression significantly increased basal and maximal uncoupled respiration (p < 0.05). Unexpectedly, however, elevated ACSL-5 expression increased mitochondrial superoxide production (+30%), which was associated with a significant reduction (p < 0.05) in insulin-stimulated p-Akt and p-AS160 protein levels. We concluded that ACSL-5 in human skeletal muscle functions to increase mitochondrial fatty acid oxidation, but contrary to conventional wisdom, is associated with increased free radical production and reduced insulin signaling

Metformin Improves Insulin Signaling in Obese Rats via Reduced IKK Action in a Fiber-Type Specific Manner

Metformin is a widely used insulin-sensitizing drug, though its mechanisms are not fully understood. Metformin has been shown to activate AMPK in skeletal muscle; however, its effects on the inhibitor of κB kinaseβ (IKKβ) in this same tissue are unknown. The aim of this study was to (1) determine the ability of metformin to attenuate IKKβ action, (2) determine whether changes in AMPK activity are associated with changes in IKKβ action in skeletal muscle, and (3) examine whether changes in AMPK and IKKβ function are consistent with improved insulin signaling. Lean and obese male Zuckers received either vehicle or metformin by oral gavage daily for four weeks (four groups of eight). Proteins were measured in white gastrocnemius (WG), red gastrocnemius (RG), and soleus. AMPK phosphorylation increased (P < .05) in WG in both lean (57%) and obese (106%), and this was supported by an increase in phospho-ACC in WG. Further, metformin increased IκBα levels in both WG (150%) and RG (67%) of obese rats, indicative of reduced IKKβ activity (P < .05), and was associated with reduced IRS1-pSer307 (30%) in the WG of obese rats (P < .02). From these data we conclude that metformin treatment appears to exert an inhibitory influence on skeletal muscle IKKβ activity, as evidenced by elevated IκBα levels and reduced IRS1-Ser307 phosphorylation in a fiber-type specific manner

Mitochondrial Hâ‚‚Oâ‚‚ emission and cellular redox state link excess fat intake to insulin resistance in both rodents and humans

High dietary fat intake leads to insulin resistance in skeletal muscle, and this represents a major risk factor for type 2 diabetes and cardiovascular disease. Mitochondrial dysfunction and oxidative stress have been implicated in the disease process, but the underlying mechanisms are still unknown. Here we show that in skeletal muscle of both rodents and humans, a diet high in fat increases the Hâ‚‚Oâ‚‚-emitting potential of mitochondria, shifts the cellular redox environment to a more oxidized state, and decreases the redox-buffering capacity in the absence of any change in mitochondrial respiratory function. Furthermore, we show that attenuating mitochondrial Hâ‚‚Oâ‚‚ emission, either by treating rats with a mitochondrial-targeted antioxidant or by genetically engineering the overexpression of catalase in mitochondria of muscle in mice, completely preserves insulin sensitivity despite a high-fat diet. These findings place the etiology of insulin resistance in the context of mitochondrial bioenergetics by demonstrating that mitochondrial Hâ‚‚Oâ‚‚ emission serves as both a gauge of energy balance and a regulator of cellular redox environment, linking intracellular metabolic balance to the control of insulin sensitivity. Original version available at http://www.jci.org/articles/view/3704

Mitochondrial Phenotype as a Driver of the Racial Dichotomy in Obesity and Insulin Resistance

African Americans (AA) are disproportionately burdened by metabolic diseases. While largely unexplored between Caucasian (C) and AA, differences in mitochondrial bioenergetics may provide crucial insight to mechanisms for increased susceptibility to metabolic diseases. AA display lower total energy expenditure and resting metabolic rate compared to C, but paradoxically have a higher amount of skeletal muscle mass, suggestive of inherent energetic efficiency differences between these races. Such adaptations would increase the chances of overnutrition in AA; however, these disparities would not explain the racial difference in insulin resistance (IR) in healthy subjects. Hallmarks associated with insulin resistance (IR), such as reduced mitochondrial oxidative capacity and metabolic inflexibility are present even in healthy AA without a metabolic disease. These adaptations might be influential of mitochondrial “substrate preference” and could play a role in disproportionate IR rates among races. A higher glycolytic flux and provision of shuttles transferring electrons from cytosol to mitochondrial matrix could be a contributing factor in development of IR via heightened reactive oxygen species (ROS) production. This review highlights the above concepts and provides suggestions for future studies that could help delineate molecular premises behind potential impairments in insulin signaling and metabolic disease susceptibility in AA

Peroxisomal gene and protein expression increase in response to a high-lipid challenge in human skeletal muscle

Peroxisomes are essential for lipid metabolism and disruption of liver peroxisomal function results in neonatal death. Little is known about how peroxisomal content and activity respond to changes in the lipid environment in human skeletal muscle (HSkM).Aims:We hypothesized and tested that increased peroxisomal gene/protein expression and functionality occur in HSkM as an adaptive response to lipid oversupply.Materials and methods:HSkM biopsies, derived from a total of sixty-two subjects, were collected for 1) examining correlations between peroxisomal proteins and intramyocellular lipid content (IMLC) as well as between peroxisomal functionality and IMLC, 2) assessing peroxisomal gene expression in response to acute- or 7-day high fat meal (HFM), and in human tissue derived primary myotubes for 3) treating with high fatty acids to induce peroxisomal adaptions. IMLC were measured by both biochemical analyses and fluorescent staining. Peroxisomal membrane protein PMP70 and biogenesis gene ( PEX ) expression were assessed using western blotting and realtime qRT-PCR respectively. 1- 14 C radiolabeled lignocerate and palmitate oxidation assays were performed for peroxisomal and mitochondrial functionality respectively.Results:1) Under fasting conditions, HSkM tissue demonstrated a significant correlation ( P ≪-¯0.05) between IMCL and the peroxisomal biogenesis factor 19 (PEX19) protein as well as between lipid content and palmitate and lignocerate complete oxidation. 2) Similarly, post-HFM, additional PEX genes ( Pex19 , PEX11A , and PEX5 ) were significantly ( P ≪-¯0.05) upregulated. 3) Increments in PMP70 , carnitine octanoyl transferase (CrOT), PGC-1a , and ERRa mRNA were observed post-fatty acid incubation in HSkM cells. PMP70 protein was significantly ( P ≪-¯0.05) elevated 48-h post lipid treatment.Conclusions:These results are the first to associate IMLC with peroxisomal gene/protein expression and function in HSkM suggesting an adaptive role for peroxisomes in lipid metabolism in this tissue