Sabina Paglialunga

Elevated sensitivity to diet-induced obesity and insulin resistance in mice lacking 4E-BP1 and 4E-BP2

The most common pathology associated with obesity is insulin resistance, which results in the onset of type 2 diabetes mellitus. Several studies have implicated the mammalian target of rapamycin (mTOR) signaling pathway in obesity. Eukaryotic translation initiation factor 4E-binding (eIF4E-binding) proteins (4E-BPs), which repress translation by binding to eIF4E, are downstream effectors of mTOR. We report that the combined disruption of 4E-BP1 and 4E-BP2 in mice increased their sensitivity to diet-induced obesity. Increased adiposity was explained at least in part by accelerated adipogenesis driven by increased expression of CCAAT/enhancer-binding protein delta (C/EBPdelta), C/EBPalpha, and PPARgamma coupled with reduced energy expenditure, reduced lipolysis, and greater fatty acid reesterification in the adipose tissue of 4E-BP1 and 4E-BP2 double KO mice. Increased insulin resistance in 4E-BP1 and 4E-BP2 double KO mice was associated with increased ribosomal protein S6 kinase (S6K) activity and impairment of Akt signaling in muscle, liver, and adipose tissue. These data clearly demonstrate the role of 4E-BPs as a metabolic brake in the development of obesity and reinforce the idea that deregulated mTOR signaling is associated with the development of the metabolic syndrome.

Omega‐3 supplementation alters mitochondrial membrane composition and respiration kinetics in human skeletal muscle

Following fish oil supplementation, omega‐3 fatty acids are incorporated into cellular membranes, which may affect lipid–protein interactions and therefore the function of embedded proteins. As the components of the electron transport chain required for oxidative phosphorylation are contained in the mitochondrial membrane, omega‐3 supplementation may alter metabolic function. We supplemented male participants for 12 weeks with fish oil [eicosapentaenoic acid (EPA) and docosahexanoic acid (DHA)] and analysed mitochondrial function and reactive oxygen species (ROS) emissions in permeabilized muscle fibres from the vastus lateralis muscle. Supplementation incorporated EPA and DHA into mitochondrial membranes, but did not result in changes in maximal mitochondrial respiratory function or pyruvate respiration kinetics. However, the apparent Km for ADP was decreased following supplementation, and was independent of creatine, changes in the protein content of ADP synthase or ANT transporters. The propensity for ROS emissions increased with omega‐3 supplementation, although there were no changes in markers of lipid or protein oxidative damage. These results demonstrate that omega‐3 supplementation improves mitochondrial ADP kinetics, suggesting post‐translational modification of existing proteins.

Reduced adipose tissue triglyceride synthesis and increased muscle fatty acid oxidation in C5L2 knockout mice

Activation of C5L2, a G-protein-coupled receptor, by acylation-stimulating protein/complement C3adesArg (ASP/C3adesArg) has been shown to stimulate triglyceride (TG) synthesis in both mature adipocytes and preadipocytes. ASP is an adipocyte-derived hormone that acts by increasing diacylglycerol acyltransferase activity and glucose transport. ASP-deficient mice (C3KO, precursor protein) are lean, display delayed postprandial TG clearance, increased food intake, and increased energy expenditure. The present study shows that C5L2KO mice on a low fat diet are hyperphagic (~60% increase in total food intake) yet maintain the same body weight and adipose tissue mass as wild-type (WT) controls. However, on a high fat diet, average adipocyte size and adipose tissue TG/DNA content were significantly reduced and postprandial TG clearance was delayed in C5L2KO. Adipose tissue TG synthesis (WT: 47.2 +/- 5.6 versus C5L2KO: 7.8 +/- 1.8 pmol/microg protein, P < 0.001), TG lipolysis (WT: 227.6 +/- 36.4 versus C5L2KO: 45.8 +/- 5.0 nmol/microg protein, P < 0.001), and fatty acid re-esterification (WT: 85.3 +/- 2.4% versus C5L2KO: 59.5 +/- 6.8%, P < 0.001) were significantly reduced in C5L2KO mice. Indirect calorimetry measurements revealed C5L2KO mice have unchanged oxygen consumption levels yet reduced respiratory quotient value, suggesting preferential fatty acid utilization over carbohydrate. In agreement, fatty acid oxidation was elevated in heart and skeletal muscle tissue in C5L2KO mice and skeletal muscle levels of uncoupling protein 3 (425.5 +/- 86.3%, P < 0.0001), CD36 (277.6 +/- 49.5%, P < 0.05), cytochrome c (252.6 +/- 33.9%, P < 0.05), and phospho-acetyl CoA carboxylase (118.4 +/- 9.3%, P < 0.05) were significantly increased in C5L2KO mice versus WT (100%). The study shows that in response to reduced TG storage in white adipose tissue, C5L2KO mice have developed a compensatory mechanism of increased muscle fat oxidation.

Evidence for a Direct Effect of the NAD+ Precursor Acipimox on Muscle Mitochondrial Function in Humans

Recent preclinical studies showed the potential of nicotinamide adenine dinucleotide (NAD+) precursors to increase oxidative phosphorylation and improve metabolic health, but human data are lacking. We hypothesize that the nicotinic acid derivative acipimox, an NAD+ precursor, would directly affect mitochondrial function independent of reductions in nonesterified fatty acid (NEFA) concentrations. In a multicenter randomized crossover trial, 21 patients with type 2 diabetes (age 57.7 ± 1.1 years, BMI 33.4 ± 0.8 kg/m2) received either placebo or acipimox 250 mg three times daily dosage for 2 weeks. Acipimox treatment increased plasma NEFA levels (759 ± 44 vs. 1,135 ± 97 μmol/L for placebo vs. acipimox, P < 0.01) owing to a previously described rebound effect. As a result, skeletal muscle lipid content increased and insulin sensitivity decreased. Despite the elevated plasma NEFA levels, ex vivo mitochondrial respiration in skeletal muscle increased. Subsequently, we showed that acipimox treatment resulted in a robust elevation in expression of nuclear-encoded mitochondrial gene sets and a mitonuclear protein imbalance, which may indicate activation of the mitochondrial unfolded protein response. Further studies in C2C12 myotubes confirmed a direct effect of acipimox on NAD+ levels, mitonuclear protein imbalance, and mitochondrial oxidative capacity. To the best of our knowledge, this study is the first to demonstrate that NAD+ boosters can also directly affect skeletal muscle mitochondrial function in humans.

High-fat diet-induced mitochondrial biogenesis is regulated by mitochondrial derived reactive oxygen species activation of CaMKII

Calcium/calmodulin-dependent protein kinase (CaMK) activation induces mitochondrial biogenesis in response to increasing cytosolic calcium concentrations. Calcium leak from the ryanodine receptor (RyR) is regulated by reactive oxygen species (ROS), which is increased with high-fat feeding. We examined whether ROS-induced CaMKII-mediated signaling induced skeletal muscle mitochondrial biogenesis in selected models of lipid oversupply. In obese Zucker rats and high-fat–fed rodents, in which muscle mitochondrial content was upregulated, CaMKII phosphorylation was increased independent of changes in calcium uptake because sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) protein expression or activity was not altered, implicating altered sarcoplasmic reticulum (SR) calcium leak in the activation of CaMKII. In support of this, we found that high-fat feeding increased mitochondrial ROS emission and S-nitrosylation of the RyR, whereas hydrogen peroxide induced SR calcium leak from the RyR and activation of CaMKII. Moreover, administration of a mitochondrial-specific antioxidant, SkQ, prevented high-fat diet–induced phosphorylation of CaMKII and the induction of mitochondrial biogenesis. Altogether, these data suggest that increased mitochondrial ROS emission is required for the induction of SR calcium leak, activation of CaMKII, and induction of mitochondrial biogenesis in response to excess lipid availability.

C5L2 receptor disruption enhances the development of diet-induced insulin resistance in mice

INTRODUCTION Acylation stimulating protein (ASP) is a hormone secreted by the adipose tissue that has been shown to increase triglyceride storage and glucose transport in adipocytes. These effects are mediated by C5L2 receptor, which has also been associated with inflammatory effects. C5L2 deficient mice on a low-fat diet are hyperphagic yet lean due to increased energy expenditure. The present study assessed insulin sensitivity and metabolic and inflammatory changes in C5L2KO mice vs WT in diet-induced obesity. METHODS We placed C5L2KO and WT mice on a diabetogenic diet for 12 weeks and examined in vivo and ex vivo metabolism. RESULTS C5L2KO mice on a diabetogenic diet exhibit decreased insulin sensitivity. Whole body substrate partitioning is evidenced through increased glucose uptake by the liver and decreased uptake by adipose tissue and skeletal muscle. Lipid content of both liver and skeletal muscle was higher in C5L2KO mice vs WT. Furthermore, elevated levels of macrophage markers were found in adipose tissue, liver and skeletal muscle of C5L2KO mice vs WT. Several inflammatory cytokines such as IL-6, MIP-1α and KC were also elevated in plasma of C5L2KO mice vs WT. CONCLUSIONS Overall, we demonstrated that C5L2KO mice fed a diabetogenic diet develop more severe insulin resistance than WT mice through altered substrate partitioning, ectopic fat deposition and a pro-inflammatory phenotype.

Shift in metabolic fuel in acylation-stimulating protein-deficient mice following a high-fat diet.

ASP-deficient mice (C3 KO) have delayed postprandial TG clearance, are hyperphagic, and display increased energy expenditure. Markers of carbohydrate and fatty acid metabolism in the skeletal muscle and heart were examined to evaluate the mechanism. On a high-fat diet, compared with wild-type mice, C3 KO mice have increased energy expenditure, decreased RQ, lower ex vivo glucose oxidation (-39%, P = 0.018), and higher ex vivo fatty acid oxidation (+68%, P = 0.019). They have lower muscle glycogen content (-25%, P < 0.05) and lower activities for the glycolytic enzymes glycogen phosphorylase (-31%, P = 0.005), hexokinase (-43%, P = 0.007), phosphofructokinase (-51%, P < 0.0001), and GAPDH (-15%, P = 0.04). Analysis of mitochondrial enzyme activities revealed that hydroxyacyl-coenzyme A dehydrogenase was higher (+25%, P = 0.004) in C3 KO mice. Furthermore, Western blot analysis of muscle revealed significantly higher fatty acid transporter CD36 (+40%, P = 0.006) and cytochrome c (a marker of mitochondrial content; +69%, P = 0.034) levels in C3 KO mice, whereas the activity of AMP kinase was lower (-48%, P = 0.003). Overall, these results demonstrate a shift in the metabolic potential of skeletal muscle toward increased fatty acid utilization. Whether this is 1) a consequence of decreased adipose tissue storage with repartitioning toward muscle or 2) a direct result of the absence of ASP interaction with the receptor C5L2 in muscle remains to be determined. However, these in vivo data suggest that ASP inhibition could be a potentially viable approach in correcting muscle metabolic dysfunction in obesity.

Effects of Bezafibrate Treatment in a Patient and a Carrier With Mutations in the PNPLA2 Gene, Causing Neutral Lipid Storage Disease With Myopathy

Neutral lipid storage disease with myopathy (NLSDM) is a rare but severe genetic disorder characterized by excessive lipid accumulation in tissues including skin, bone marrow, heart, liver, and muscles. Clinically, NLSDM patients present with severe dilated cardiomyopathy, skeletal muscle myopathy, and insulin resistance.1 NLSDM is caused by a defect in the PNPLA2 gene encoding the enzyme adipose triglyceride lipase (ATGL),2 which catalyzes the breakdown of triglycerides in multiple tissues and is the rate-limiting step of lipolysis. Although heterozygous carriers and homozygous patients both present with similar clinical symptoms, the severity of these symptoms in homozygous patients is more dramatic, leading to premature death attributed to dilated cardiomyopathy in some patients.1,2 To date, the only available treatment is strict dietary guidelines and is focused on treating the comorbidities rather than targeting the primary defect. To investigate cardiac lipotoxicity in NLSDM, ATGL-deficient mice have been investigated. Just like NLSDM patients, ATGL-deficient mice are also characterized by excessive lipid storage in skeletal muscle, liver, and heart, and they develop cardiomyopathy at a young age, resulting in premature death.3 Interestingly, we reported recently that a lack of ATGL resulted in a diminished cardiac peroxisome proliferator-activated receptor (PPAR) activity and mitochondrial function, suggesting that lipolysis-derived fatty acids or fatty acid byproducts serve as transcriptional activators of PPARs.4 Subsequently, pharmacological treatment of ATGL-deficient mice with PPAR agonists completely reversed the mitochondrial defects, restored cardiac function, and prevented premature death. These promising findings inspired us to investigate whether PPAR agonist treatment in patients and carriers of a PNPLA2 gene defect could also have beneficial effects. Although NSLDM is a very rare disease, we had the opportunity to study 2 sisters with PNPLA2 gene mutations. Patient 1 is a 37-year-old woman with a body mass index of 21.4 kg/m2. …

Rapid repression of ADP transport by palmitoyl-CoA is attenuated by exercise training in humans; a potential mechanism to decrease oxidative stress and improve skeletal muscle insulin signaling

Mitochondrial ADP transport may represent a convergence point unifying two prominent working models for the development of insulin resistance, as reactive lipids (specifically palmitoyl-CoA [P-CoA]) can inhibit ADP transport and subsequently increase mitochondrial reactive oxygen species emissions. In the current study, we aimed to determine if exercise training in humans diminished P-CoA attenuation of mitochondrial ADP respiratory sensitivity. Six weeks of exercise training increased whole-body glucose homeostasis and skeletal muscle Akt signaling and reduced markers of oxidative stress without reducing maximal mitochondrial H2O2 emissions. To ascertain if enhanced mitochondrial ADP transport contributed to the improvement in the in vivo oxidative state, we determined mitochondrial ADP sensitivity in the presence and absence of P-CoA. In the absence of P-CoA, exercise training reduced mitochondrial ADP sensitivity. In contrast, exercise training increased mitochondrial ADP sensitivity with P-CoA present. We further show that P-CoA noncompetitively inhibits mitochondrial ADP transport and the ability of ADP to attenuate mitochondrial H2O2 emission. Altogether, the current data provide a potential mechanism for how P-CoA contributes to insulin resistance and highlight the ability of exercise training to diminish P-CoA attenuation in mitochondrial ADP transport.

Ablating the protein TBC1D1 impairs contraction-induced sarcolemmal glucose transporter 4 redistribution but not insulin-mediated responses in rats

TBC1 domain family member 1 (TBC1D1), a Rab GTPase-activating protein and paralogue of Akt substrate of 160 kDa (AS160), has been implicated in both insulin- and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase-mediated glucose transporter type 4 (GLUT4) translocation. However, the role of TBC1D1 in contracting muscle remains ambiguous. We therefore explored the metabolic consequence of ablating TBC1D1 in both resting and contracting skeletal muscles, utilizing a rat TBC1D1 KO model. Although insulin administration rapidly increased (p < 0.05) plasma membrane GLUT4 content in both red and white gastrocnemius muscles, the TBC1D1 ablation did not alter this response nor did it affect whole-body insulin tolerance, suggesting that TBC1D1 is not required for insulin-induced GLUT4 trafficking events. Consistent with findings in other models of altered TBC1D1 protein levels, whole-animal and ex vivo skeletal muscle fat oxidation was increased in the TBC1D1 KO rats. Although there was no change in mitochondrial content in the KO rats, maximal ADP-stimulated respiration was higher in permeabilized muscle fibers, which may contribute to the increased reliance on fatty acids in resting KO animals. Despite this increase in mitochondrial oxidative capacity, run time to exhaustion at various intensities was impaired in the KO rats. Moreover, contraction-induced increases in sarcolemmal GLUT4 content and glucose uptake were lower in the white gastrocnemius of the KO animals. Altogether, our results highlight a critical role for TBC1D1 in exercise tolerance and contraction-mediated translocation of GLUT4 to the plasma membrane in skeletal muscle.