Dynamin-related protein 1 regulates substrate oxidation in skeletal muscle by stabilizing cellular and mitochondrial calcium dynamics.

Mitochondria undergo continuous cycles of fission and fusion to promote inheritance, regulate quality control, and mitigate organelle stress. More recently, this process of mitochondrial dynamics has been demonstrated to be highly sensitive to nutrient supply, ultimately conferring bioenergetic plasticity to the organelle. However, whether regulators of mitochondrial dynamics play a causative role in nutrient regulation remains unclear. In this study, we generated a cellular loss-of-function model for dynamin-related protein 1 (DRP1), the primary regulator of outer membrane mitochondrial fission. Loss of DRP1 (shDRP1) resulted in extensive ultrastructural and functional remodeling of mitochondria, characterized by pleomorphic enlargement, increased electron density of the matrix, and defective NADH and succinate oxidation. Despite increased mitochondrial size and volume, shDRP1 cells exhibited reduced cellular glucose uptake and mitochondrial fatty acid oxidation. Untargeted transcriptomic profiling revealed severe downregulation of genes required for cellular and mitochondrial calcium homeostasis, which was coupled to loss of ATP-stimulated calcium flux and impaired substrate oxidation stimulated by exogenous calcium. The insights obtained herein suggest that DRP1 regulates substrate oxidation by altering whole-cell and mitochondrial calcium dynamics. These findings are relevant to the targetability of mitochondrial fission and have clinical relevance in the identification of treatments for fission-related pathologies such as hereditary neuropathies, inborn errors in metabolism, cancer, and chronic diseases.

Rickettsia conorii survival in THP-1 macrophages involves host lipid droplet alterations and active rickettsial protein production.

Rickettsia conorii is a Gram-negative, cytosolic intracellular bacterium that has classically been investigated in terms of endothelial cell infection. However, R. conorii and other human pathogenic Rickettsia species have evolved mechanisms to grow in various cell types, including macrophages, during mammalian infection. During infection of these phagocytes, R. conorii shifts the host cell's overall metabolism towards an anti-inflammatory M2 response, metabolically defined by an increase in host lipid metabolism and oxidative phosphorylation. Lipid metabolism has more recently been identified as a key regulator of host homeostasis through modulation of immune signalling and metabolism. Intracellular pathogens have adapted mechanisms of hijacking host metabolic pathways including host lipid catabolic pathways for various functions required for growth and survival. In the present study, we hypothesised that alterations of host lipid droplets initiated by lipid catabolic pathways during R. conorii infection is important for bacterial survival in macrophages. Herein, we determined that host lipid droplet modulation is initiated early during R. conorii infection, and these alterations rely on active bacteria and lipid catabolic pathways. We also find that these lipid catabolic pathways are essential for efficient bacterial survival. Unlike the mechanisms used by other intracellular pathogens, the catabolism of lipid droplets induced by R. conorii infection is independent of upstream host peroxisome proliferator-activated receptor-alpha (PPARα) signalling. Inhibition of PPARÉ£ signalling and lipid droplet accumulation in host cells cause a significant decrease in R. conorii survival suggesting a negative correlation with lipid droplet production and R. conorii survival. Together, these results strongly suggest that the modulation of lipid droplets in macrophage cells infected by R. conorii is an important and underappreciated aspect of the infection process. TAKE AWAYS: Host lipid droplets are differentially altered in early and replicative stages of THP-1 macrophage infection with R. conorii. Lipid droplet alterations are initiated in a bacterial-dependent manner and do not require host peroxisome proliferator-activated receptors α or É£ activation. Pharmacological inhibition of host lipid catabolic processes during R. conorii infection indicates a requirement of lipid catabolism for bacterial survival and initiation of lipid droplet modulation. A significant increase in host lipid droplets during infection has a negative impact on R. conorii survival in THP-1 macrophages.

Extensive metabolic remodeling after limiting mitochondrial lipid burden is consistent with an improved metabolic health profile.

Mitochondrial lipid overload in skeletal muscle contributes to insulin resistance, and strategies limiting this lipid pressure improve glucose homeostasis; however, comprehensive cellular adaptations that occur in response to such an intervention have not been reported. Herein, mice with skeletal muscle-specific deletion of carnitine palmitoyltransferase 1b (Cpt1bM-/-), which limits mitochondrial lipid entry, were fed a moderate fat (25%) diet, and samples were subjected to a multimodal analysis merging transcriptomics, proteomics, and nontargeted metabolomics to characterize the coordinated multilevel cellular responses that occur when mitochondrial lipid burden is mitigated. Limiting mitochondrial fat entry predictably improves glucose homeostasis; however, remodeling of glucose metabolism pathways pales compared with adaptations in amino acid and lipid metabolism pathways, shifts in nucleotide metabolites, and biogenesis of mitochondria and peroxisomes. Despite impaired fat utilization, Cpt1bM-/- mice have increased acetyl-CoA (14-fold) and NADH (2-fold), indicating metabolic shifts yield sufficient precursors to meet energy demand; however, this does not translate to enhance energy status as Cpt1bM-/- mice have low ATP and high AMP levels, signifying energy deficit. Comparative analysis of transcriptomic data with disease-associated gene-sets not only predicted reduced risk of glucose metabolism disorders but was also consistent with lower risk for hepatic steatosis, cardiac hypertrophy, and premature death. Collectively, these results suggest induction of metabolic inefficiency under conditions of energy surfeit likely contributes to improvements in metabolic health when mitochondrial lipid burden is mitigated. Moreover, the breadth of disease states to which mechanisms induced by muscle-specific Cpt1b inhibition may mediate health benefits could be more extensive than previously predicted.

The RNA binding protein HuR influences skeletal muscle metabolic flexibility in rodents and humans.

BACKGROUND: Metabolic flexibility can be assessed by changes in respiratory exchange ratio (RER) following feeding. Though metabolic flexibility (difference in RER between fasted and fed state) is often impaired in individuals with obesity or type 2 diabetes, the cellular processes contributing to this impairment are unclear. MATERIALS AND METHODS: From several clinical studies we identified the 16 most and 14 least metabolically flexible male and female subjects out of >100 participants based on differences between 24-hour and sleep RER measured in a whole-room indirect calorimeter. Global skeletal muscle gene expression profiles revealed that, in metabolically flexible subjects, transcripts regulated by the RNA binding protein, HuR, are enriched. We generated and characterized mice with a skeletal muscle-specific knockout of the HuR encoding gene, Elavl1 (HuRm-/-). RESULTS: Male, but not female, HuRm-/- mice exhibit metabolic inflexibility, with mild obesity, impaired glucose tolerance, impaired fat oxidation and decreased in vitro palmitate oxidation compared to HuRfl/fl littermates. Expression levels of genes involved in mitochondrial fatty acid oxidation and oxidative phosphorylation are decreased in both mouse and human muscle when HuR is inhibited. CONCLUSIONS: HuR inhibition results in impaired metabolic flexibility and decreased lipid oxidation, suggesting a role for HuR as an important regulator of skeletal muscle metabolism.

An Extract of Russian Tarragon Prevents Obesity-Related Ectopic Lipid Accumulation.

SCOPE: The primary disorder underlying metabolic syndrome is insulin resistance due to excess body weight and abdominal visceral fat accumulation. In this study, it is asked if dietary intake of an ethanolic extract from Russian tarragon (Artemisia dracunculus L., termed PMI5011), shown to improve glucose utilization by enhancing insulin signaling in skeletal muscle, could prevent obesity-induced insulin resistance, skeletal muscle metabolic inflexibility, and ectopic lipid accumulation in the skeletal muscle and liver. METHODS AND RESULTS: Male wild-type mice are fed a high-fat diet alone or supplemented with PMI5011 (1% w/w) over 3 months. Dietary intake of PMI5011 improved fatty acid oxidation and metabolic flexibility in the skeletal muscle, reduced insulin levels, and enhanced insulin signaling in the skeletal muscle and liver independent of robust changes in expression of factors that control fatty acid oxidation. This corresponds with significantly reduced lipid accumulation in the skeletal muscle and liver, although body weight gain is comparable to a high-fat diet alone. CONCLUSION: Previous studies showed that PMI5011 enhances insulin sensitivity in the setting of established obesity-induced insulin resistance. The current study demonstrates that dietary intake of PMI5011 prevents high-fat diet-induced insulin resistance, metabolic dysfunction, and ectopic lipid accumulation in the skeletal muscle and liver without reducing body weight.

Impaired Mitochondrial Fat Oxidation Induces FGF21 in Muscle.

Fatty acids are the primary fuel source for skeletal muscle during most of our daily activities, and impaired fatty acid oxidation (FAO) is associated with insulin resistance. We have developed a mouse model of impaired FAO by deleting carnitine palmitoyltransferase-1b specifically in skeletal muscle (Cpt1b(m-/-)). Cpt1b(m-/-) mice have increased glucose utilization and are resistant to diet-induced obesity. Here, we show that inhibition of mitochondrial FAO induces FGF21 expression specifically in skeletal muscle. The induction of FGF21 in Cpt1b-deficient muscle is dependent on AMPK and Akt1 signaling but independent of the stress signaling pathways. FGF21 appears to act in a paracrine manner to increase glucose uptake under low insulin conditions, but it does not contribute to the resistance to diet-induced obesity.

Myokine Expression in Muscle and Myotubes in Response to Exercise Stimulation.

PURPOSE: Myokines have been shown to affect muscle physiology and exert systemic effects. We endeavored to investigate a panel of myokine mRNA expression after a single exercise bout (studies 1 and 2) to measure myokine mRNA in primary human myotubes in an in vitro exercise model (study 2). METHODS: Vastus lateralis muscle biopsies were obtained from 20 healthy males (age, 24.0 ± 4.5 yr; BMI, 23.6 ± 1.8 kg·m)(-2) before and after a single exercise bout (650 kcal at 50% V˙O2max). Primary myotubes from active and sedentary male donors were treated with a pharmacological cocktail (palmitate, forskolin, and ionomycin (PFI)) to mimic exercise-stimulated contractions in vitro. RESULTS: Interleukin 6 and 8 (IL-6 and IL-8), leukocyte-inducing factor, and connective tissue growth factor (CTGF) mRNA levels increased approximately 10-fold after a single exercise bout (all P < 0.001), whereas myostatin levels decreased (P < 0.05). Key correlations between myokine expression and parameters of muscle and whole-body physiology were found: myostatin versus skeletal muscle citrate synthase activity (r = -0.69, P < 0.001), V˙O2max (r = -0.64, P = 0.002) and the percentage of Type I fibers (r = -0.55, P = 0.01); IL-6 versus the RER (r = 0.45, P = 0.04), homeostatic model assessment of insulin resistance (r = 0.44, P = 0.05), and serum lactate (r = 0.50, P = 0.02). Myokine expressions in myotubes from sedentary donors for CTGF and myostatin decreased, whereas IL-6 and IL-8 increased after PFI treatment. In myotubes from active donors, myokine expression increased for IL-6, CTGF, and myostatin but decreased for IL-8 after PFI treatment. CONCLUSION: These data offer insight into the differences in regulation of myokine expression and their possible physiologic relationships.

Thiobenzothiazole-modified Hydrocortisones Display Anti-inflammatory Activity with Reduced Impact on Islet ß-Cell Function.

Glucocorticoids signal through the glucocorticoid receptor (GR) and are administered clinically for a variety of situations, including inflammatory disorders, specific cancers, rheumatoid arthritis, and organ/tissue transplantation. However, glucocorticoid therapy is also associated with additional complications, including steroid-induced diabetes. We hypothesized that modification of the steroid backbone is one strategy to enhance the therapeutic potential of GR activation. Toward this goal, two commercially unavailable, thiobenzothiazole-containing derivatives of hydrocortisone (termed MS4 and MS6) were examined using 832/13 rat insulinoma cells as well as rodent and human islets. We found that MS4 had transrepression properties but lacked transactivation ability, whereas MS6 retained both transactivation and transrepression activities. In addition, MS4 and MS6 both displayed anti-inflammatory activity. Furthermore, MS4 displayed reduced impact on islet ß-cell function in both rodent and human islets. Similar to dexamethasone, MS6 promoted adipocyte development in vitro, whereas MS4 did not. Moreover, neither MS4 nor MS6 activated the Pck1 (Pepck) gene in primary rat hepatocytes. We conclude that modification of the functional groups attached to the D-ring of the hydrocortisone steroid molecule produces compounds with altered structure-function GR agonist activity with decreased impact on insulin secretion and reduced adipogenic potential but with preservation of anti-inflammatory activity.

Albumin-bound fatty acids but not albumin itself alter redox balance in tubular epithelial cells and induce a peroxide-mediated redox-sensitive apoptosis.

Albuminuria is associated with metabolic syndrome and diabetes. It correlates with the progression of chronic kidney disease, particularly with tubular atrophy. The fatty acid load on albumin significantly increases in obesity, presenting a proinflammatory environment to the proximal tubules. However, little is known about changes in the redox milieu during fatty acid overload and how redox-sensitive mechanisms mediate cell death. Here, we show that albumin with fatty acid impurities or conjugated with palmitate but not albumin itself compromised mitochondrial and cell viability, membrane potential and respiration. Fatty acid overload led to a redox imbalance which deactivated the antioxidant protein peroxiredoxin 2 and caused a peroxide-mediated apoptosis through the redox-sensitive pJNK/caspase-3 pathway. Transfection of tubular cells with peroxiredoxin 2 was protective and mitigated apoptosis. Mitochondrial fatty acid entry and ceramide synthesis modulators suggested that mitochondrial ß oxidation but not ceramide synthesis may modulate lipotoxic effects on tubular cell survival. These results suggest that albumin overloaded with fatty acids but not albumin itself changes the redox environment in the tubules, inducing a peroxide-mediated redox-sensitive apoptosis. Thus, mitigating circulating fatty acid levels may be an important factor in both preserving redox balance and preventing tubular cell damage in proteinuric diseases.

Regulation of islet beta-cell pyruvate metabolism: interactions of prolactin, glucose, and dexamethasone.

Prolactin (PRL) induces beta-cell proliferation and glucose-stimulated insulin secretion (GSIS) and counteracts the effects of glucocorticoids on insulin production. The mechanisms by which PRL up-regulates GSIS are unknown. We used rat islets and insulinoma (INS-1) cells to explore the interactions of PRL, glucose, and dexamethasone (DEX) in the regulation of beta-cell pyruvate carboxylase (PC), pyruvate dehydrogenase (PDH), and the pyruvate dehydrogenase kinases (PDKs), which catalyze the phosphorylation and inactivation of PDH. PRL increased GSIS by 37% (P < 0.001) in rat islets. Glucose at supraphysiological concentrations (11 mm) increased PC mRNA in islets; in contrast, PRL suppressed PC mRNA levels in islets and INS-1 cells, whereas DEX was without effect. Neither PRL nor DEX altered PC protein or activity levels. In INS-1 cells, PRL increased PDH activity 1.4- to 2-fold (P < 0.05-0.001) at glucose concentrations ranging from 2.5-11 mm. DEX reduced PDH activity; this effect was reversed by PRL. PDK1, -2, -3, and -4 mRNAs were detected in both islets and insulinoma cells, but the latter expressed trivial amounts of PDK4. PRL reduced PDK2 mRNA and protein levels in rat islets and INS-1 cells and PDK4 mRNA in islets; DEX increased PDK2 mRNA in islets and INS-1 cells; this effect was reversed by PRL. Our findings suggest that PRL induction of GSIS is mediated by increases in beta-cell PDH activity; this is facilitated by suppression of PDKs. PRL counteracts the effects of DEX on PDH and PDK expression, suggesting novel roles for the lactogens in the defense against diabetes.

Metabolic inflexibility in skeletal muscle: a prelude to the cardiometabolic syndrome?

Peripheral insulin resistance, which is largely dependent on skeletal muscle, is closely linked to the development of the cardiometabolic syndrome. Metabolic flexibility is the capacity for skeletal muscle to acutely shift its reliance between lipids or glucose during fasting or postprandial conditions. Obese and insulin-resistant individuals display elevated intramuscular lipids, impaired vasculature function, decreased fatty add oxidation during fasting, and reduced postprandial glucose metabolism. Impairments in metabolic flexibility are linked to physical inactivity, excess energy intake and obesity, and genetic predisposition. Each of these factors precludes the development of insulin resistance and the cardiometabolic syndrome by mechanistic links that are not fully understood.