Matthew W. Hulver

Control of mitochondrial metabolism and systemic energy homeostasis by microRNAs 378 and 378

Obesity and metabolic syndrome are associated with mitochondrial dysfunction and deranged regulation of metabolic genes. Peroxisome proliferator-activated receptor γ coactivator 1β (PGC-1β) is a transcriptional coactivator that regulates metabolism and mitochondrial biogenesis through stimulation of nuclear hormone receptors and other transcription factors. We report that the PGC-1β gene encodes two microRNAs (miRNAs), miR-378 and miR-378*, which counterbalance the metabolic actions of PGC-1β. Mice genetically lacking miR-378 and miR-378* are resistant to high-fat diet-induced obesity and exhibit enhanced mitochondrial fatty acid metabolism and elevated oxidative capacity of insulin-target tissues. Among the many targets of these miRNAs, carnitine O-acetyltransferase, a mitochondrial enzyme involved in fatty acid metabolism, and MED13, a component of the Mediator complex that controls nuclear hormone receptor activity, are repressed by miR-378 and miR-378*, respectively, and are elevated in the livers of miR-378/378* KO mice. Consistent with these targets as contributors to the metabolic actions of miR-378 and miR-378*, previous studies have implicated carnitine O-acetyltransferase and MED13 in metabolic syndrome and obesity. Our findings identify miR-378 and miR-378* as integral components of a regulatory circuit that functions under conditions of metabolic stress to control systemic energy homeostasis and the overall oxidative capacity of insulin target tissues. Thus, these miRNAs provide potential targets for pharmacologic intervention in obesity and metabolic syndrome.

Toll-like receptor 4 modulates skeletal muscle substrate metabolism

Toll-like receptor 4 (TLR4), a protein integral to innate immunity, is elevated in skeletal muscle of obese and type 2 diabetic humans and has been implicated in the development of lipid-induced insulin resistance. The purpose of this study was to examine the role of TLR4 as a modulator of basal (non-insulin-stimulated) substrate metabolism in skeletal muscle with the hypothesis that its activation would result in reduced fatty acid oxidation and increased partitioning of fatty acids toward neutral lipid storage. Human skeletal muscle, rodent skeletal muscle, and skeletal muscle cell cultures were employed to study the functional consequences of TLR4 activation on glucose and fatty acid metabolism. Herein, we demonstrate that activation of TLR4 with low (metabolic endotoxemia) and high (septic conditions) doses of LPS results in increased glucose utilization and reduced fatty acid oxidation in skeletal muscle and that these changes in metabolism in vivo occur in concert with increased circulating triglycerides. Moreover, animals with a loss of TLR4 function possess increased oxidative capacity in skeletal muscle and present with lower fasting levels of triglycerides and nonesterified free fatty acids. Evidence is also presented to suggest that these changes in substrate metabolism under metabolic endotoxemic conditions are independent of skeletal muscle-derived proinflammatory cytokine production. This report illustrates that skeletal muscle is a target for circulating endotoxin and may provide critical insight into the link between a proinflammatory state and dysregulated metabolism as observed with obesity, type 2 diabetes, and metabolic syndrome.

The pivotal role of pyruvate dehydrogenase kinases in metabolic flexibility

Metabolic flexibility is the capacity of a system to adjust fuel (primarily glucose and fatty acids) oxidation based on nutrient availability. The ability to alter substrate oxidation in response to nutritional state depends on the genetically influenced balance between oxidation and storage capacities. Competition between fatty acids and glucose for oxidation occurs at the level of the pyruvate dehydrogenase complex (PDC). The PDC is normally active in most tissues in the fed state, and suppressing PDC activity by pyruvate dehydrogenase (PDH) kinase (PDK) is crucial to maintain energy homeostasis under some extreme nutritional conditions in mammals. Conversely, inappropriate suppression of PDC activity might promote the development of metabolic diseases. This review summarizes PDKs’ pivotal role in control of metabolic flexibility under various nutrient conditions and in different tissues, with emphasis on the best characterized PDK4. Understanding the regulation of PDC and PDKs and their roles in energy homeostasis could be beneficial to alleviate metabolic inflexibility and to provide possible therapies for metabolic diseases, including type 2 diabetes (T2D).

Mice lacking microRNA 133a develop dynamin 2–dependent centronuclear myopathy

MicroRNAs modulate cellular phenotypes by inhibiting expression of mRNA targets. In this study, we have shown that the muscle-specific microRNAs miR-133a-1 and miR-133a-2 are essential for multiple facets of skeletal muscle function and homeostasis in mice. Mice with genetic deletions of miR-133a-1 and miR-133a-2 developed adult-onset centronuclear myopathy in type II (fast-twitch) myofibers, accompanied by impaired mitochondrial function, fast-to-slow myofiber conversion, and disarray of muscle triads (sites of excitation- contraction coupling). These abnormalities mimicked human centronuclear myopathies and could be ascribed, at least in part, to dysregulation of the miR-133a target mRNA that encodes dynamin 2, a GTPase implicated in human centronuclear myopathy. Our findings reveal an essential role for miR-133a in the maintenance of adult skeletal muscle structure, function, bioenergetics, and myofiber identity; they also identify a potential modulator of centronuclear myopathies.

Oligomeric Cocoa Procyanidins Possess Enhanced Bioactivity Compared to Monomeric and Polymeric Cocoa Procyanidins for Preventing the Development of Obesity, Insulin Resistance, and Impaired Glucose Tolerance during High-Fat Feeding

There is interest in the potential of cocoa flavanols, including monomers and procyanidins, to prevent obesity and type-2 diabetes. Fermentation and processing of cocoa beans influence the qualitative and quantitative profiles of individual cocoa constituents. Little is known regarding how different cocoa flavanols contribute to inhibition of obesity and type-2 diabetes. The objective of this study was to compare the impacts of long-term dietary exposure to cocoa flavanol monomers, oligomers, and polymers on the effects of high-fat feeding. Mice were fed a high-fat diet supplemented with either a cocoa flavanol extract or a flavanol fraction enriched with monomeric, oligomeric, or polymeric procyanidins for 12 weeks. The oligomer-rich fraction proved to be most effective in preventing weight gain, fat mass, impaired glucose tolerance, and insulin resistance in this model. This is the first long-term feeding study to examine the relative activities of cocoa constituents on diet-induced obesity and insulin resistance.

Endurance capacity in maturing mdx mice is markedly enhanced by combined voluntary wheel running and green tea extract

Duchenne muscular dystrophy is characterized by the absence of dystrophin from muscle cells. Dystrophic muscle cells are susceptible to oxidative stress. We tested the hypothesis that 3 wk of endurance exercise starting at age 21 days in young male mdx mice would blunt oxidative stress and improve dystrophic skeletal muscle function, and these effects would be enhanced by the antioxidant green tea extract (GTE). In mice fed normal diet, average daily running distance increased 300% from week 1 to week 3, and total distance over 3 wk was improved by 128% in mice fed GTE. Running, independent of diet, increased serum antioxidant capacity, extensor digitorum longus tetanic stress, and total contractile protein content, heart citrate synthase, and heart and quadriceps beta-hydroxyacyl-CoA dehydrogenase activities. GTE, independent of running, decreased serum creatine kinase and heart and gastrocnemius lipid peroxidation and increased gastrocnemius citrate synthase activity. These data suggest that both endurance exercise and GTE may be beneficial as therapeutic strategies to improve muscle function in mdx mice.

The molecular mechanism linking muscle fat accumulation to insulin resistance.

Skeletal muscle insulin resistance is a co-morbidity of obesity and a risk factor for the development of type 2 diabetes mellitus. Insulin resistance is associated with the accumulation of intramyocellular lipids. Intramyocellular triacylglycerols do not appear to be the cause of insulin resistance but are more likely to be a marker of other lipid intermediates such as fatty acyl-CoA, ceramides or diacylglycerols. Fatty acyl-CoA, ceramides and diacylglycerols are known to directly alter various aspects of the insulin signalling cascade. Insulin signalling is inhibited by the phosphorylation of serine and threonine residues at the levels of the insulin receptor and insulin receptor substrate 1. Protein kinase C is responsible for the phosphorylation of the serine and threonine residues. Fatty acyl-CoA and diacylglycerols are known to activate protein kinase C. The cause of the intramyocellular accumulation of fatty acyl-CoA and diacylglycerols is unclear at this time. Reduced fatty acid oxidation does not appear to be responsible, as fatty acyl-CoA accumulates in skeletal muscle with a normal fatty acid oxidative capacity. Other potential mechanisms include oversupply of lipids to muscle and/or up regulated fatty acid transport.

Plasma leptin and exercise: recent findings.

It is established that plasma leptin is associated with satiety and that leptin stimulates lipid metabolism, and increases energy expenditure. These effects implicate leptin as a major regulator of energy homeostasis, which may serve to limit excess energy storage. As plasma leptin concentrations are tightly coupled with fat mass in humans, decreases in adipose mass with weight loss coincide with decreased concentrations of circulating leptin. However, due to many confounding factors, the effects of exercise on circulating leptin are less clear. The data from investigations examining single exercise bouts suggest that serum leptin concentrations are unaltered by short duration (41 minutes or less), non-exhaustive exercise, but may be affected by short duration, exhaustive exercise. More convincingly, studies investigating long duration exercise bouts indicate that serum leptin concentrations are reduced with exercise durations ranging from one to multiple hours. These findings raise speculation that exercise-associated reductions in leptin may be due to alterations in nutrient availability or nutrient flux at the level of the adipocytes, the primary site of leptin production and secretion. Thus, one purpose of this review is to discuss the effects of exercise on circulating leptin concentrations with special emphasis on studies that have examined single exercise bouts that are associated with high levels of energy expenditure and energy deficit. In addition, a ‘nutrient sensing pathway’ (the hexosamine biosynthetic pathway), which regulates leptin gene expression, will be discussed as a possible mechanism by which exercise-induced energy deficit may modulate serum leptin concentrations.

Lipid Partitioning, Incomplete Fatty Acid Oxidation, and Insulin Signal Transduction in Primary Human Muscle Cells: Effects of Severe Obesity, Fatty Acid Incubation, and Fatty Acid Translocase/CD36 Overexpression

CONTEXT Intracellular lipid partitioning toward storage and the incomplete oxidation of fatty acids (FA) have been linked to insulin resistance. OBJECTIVE To gain insight into how intracellular lipid metabolism is related to insulin signal transduction, we examined the effects of severe obesity, excess FA, and overexpression of the FA transporter, FA translocase (FAT)/CD36, in primary human skeletal myocytes. DESIGN, SETTING, AND PATIENTS Insulin signal transduction, FA oxidation, and metabolism were measured in skeletal muscle cells harvested from lean and severely obese women. To emulate the obesity phenotype in our cell culture system, we incubated cells from lean individuals with excess FA or overexpressed FAT/CD36 using recombinant adenoviral technology. RESULTS Complete oxidation of FA was significantly reduced, whereas total lipid accumulation, FA esterification into lipid intermediates, and incomplete oxidation were up-regulated in the muscle cells of severely obese subjects. Insulin signal transduction was reduced in the muscle cells from severely obese subjects compared to lean controls. Incubation of muscle cells from lean subjects with lipids reduced insulin signal transduction and increased lipid storage and incomplete FA oxidation. CD36 overexpression increased FA transport capacity, but did not impair complete FA oxidation and insulin signal transduction in muscle cells from lean subjects. CONCLUSIONS Cultured myocytes from severely obese women express perturbations in FA metabolism and insulin signaling reminiscent of those observed in vivo. The obesity phenotype can be recapitulated in muscle cells from lean subjects via exposure to excess lipid, but not by overexpressing the FAT/CD36 FA transporter.

Regulation of skeletal muscle oxidative capacity and insulin signaling by the mitochondrial rhomboid protease PARL.

Type 2 diabetes mellitus (T2DM) and aging are characterized by insulin resistance and impaired mitochondrial energetics. In lower organisms, remodeling by the protease pcp1 (PARL ortholog) maintains the function and lifecycle of mitochondria. We examined whether variation in PARL protein content is associated with mitochondrial abnormalities and insulin resistance. PARL mRNA and mitochondrial mass were both reduced in elderly subjects and in subjects with T2DM. Muscle knockdown of PARL in mice resulted in malformed mitochondrial cristae, lower mitochondrial content, decreased PGC1alpha protein levels, and impaired insulin signaling. Suppression of PARL protein in healthy myotubes lowered mitochondrial mass and insulin-stimulated glycogen synthesis and increased reactive oxygen species production. We propose that lower PARL expression may contribute to the mitochondrial abnormalities seen in aging and T2DM.