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Diabetic cardiomyopathy (CM), occurring in the absence of hypertension, coronary artery disease, and valvular or congenital heart disease, is now recognized as a distinct, multifactorial disease leading to ventricular hypertrophy and abnormal myocardial contractility that correlates with an array of complex molecular and cellular changes. Animal models provide the unique opportunity to investigate mechanistic aspects of diabetic CM, but important caveats exist when extrapolating findings obtained from preclinical models of diabetes to humans. Indeed, animal models do not recapitulate the complexity of environmental factors, most notably the duration of the exposure to insulin resistance that may play a crucial role in the development of diabetic CM. Moreover, most preclinical studies are performed in animals with uncontrolled or poorly controlled diabetes, whereas patients tend to undergo therapeutic intervention. Finally, whilst type 2 diabetes mellitus prevalence trajectory mainly increases at 40- < 75 years (with a currently alarming increase at younger ages, however), it is a legitimate concern how closely rodent models employing young animals recapitulate the disease developing in old people. The aim of this review is to identify the current limitations of rodent models and to discuss how future mechanistic and preclinical studies should integrate key confounding factors to better mimic the diabetic CM phenotype.

International audience; Diabetic cardiomyopathy (DCM) is a leading complication in type 2 diabetes patients. Recently, we have shown that the reticulum-mitochondria Ca2+ uncoupling is an early and reversible trigger of the cardiac dysfunction in a diet-induced mouse model of DCM. Metformin is a first-line antidiabetic drug with recognized cardioprotective effect in myocardial infarction. Whether metformin could prevent the progression of DCM remains not well understood. We therefore investigated the effect of a chronic 6-week metformin treatment on the reticulum-mitochondria Ca2+ coupling and the cardiac function in our high-fat high-sucrose diet (HFHSD) mouse model of DCM. Although metformin rescued the glycemic regulation in the HFHSD mice, it did not preserve the reticulum-mitochondria Ca2+ coupling either structurally or functionally. Metformin also did not prevent the progression towards cardiac dysfunction, i.e., cardiac hypertrophy and strain dysfunction. In summary, despite its cardioprotective role, metformin is not sufficient to delay the progression to early DCM.

Diabetic cardiomyopathy (DCM) is a leading complication in type 2 diabetes patients. Recently, we have shown that the reticulum-mitochondria Ca

International audience; Hypobetalipoproteinemia is characterized by LDL-cholesterol and apolipoprotein B (apoB) plasma levels below the fifth percentile for age and sex. Familial hypobetalipoproteinemia (FHBL) is mostly caused by premature termination codons in the APOB gene, a condition associated with fatty liver and steatohepatitis. Nevertheless, many families with a FHBL phenotype carry APOB missense variants of uncertain significance (VUS). We here aimed to develop a proof-of-principle experiment to assess the pathogenicity of VUS using the genome editing of human liver cells. We identified a novel heterozygous APOB-VUS (p.Leu351Arg), in a FHBL family. We generated APOB knock-out (KO) and APOB-p.Leu351Arg knock-in Huh7 cells using CRISPR-Cas9 technology and studied the APOB expression, synthesis and secretion by digital droplet PCR and ELISA quantification. The APOB expression was decreased by 70% in the heterozygous APOB-KO cells and almost abolished in the homozygous-KO cells, with a consistent decrease in apoB production and secretion. The APOB-p.Leu351Arg homozygous cells presented with a 40% decreased APOB expression and undetectable apoB levels in cellular extracts and supernatant. Thus, the p.Leu351Arg affected the apoB secretion, which led us to classify this new variant as likely pathogenic and to set up a hepatic follow-up in this family. Therefore, the functional assessment of APOB-missense variants, using gene-editing technologies, will lead to improvements in the molecular diagnosis of FHBL and the personalized follow-up of these patients.

Obesity is associated with both chronic and acute respiratory illnesses, such as asthma, chronic obstructive pulmonary disease (COPD) or increased susceptibility to infectious diseases. Anatomical but also systemic and local metabolic alterations are proposed contributors to the pathophysiology of lung diseases in the context of obesity. To bring perspective to this discussion, we used NMR to compare the obesity-associated metabolomic profiles of the lung with those of the liver, heart, skeletal muscles, kidneys, brain and serum from male C57Bl/6J mice fed with a high-fat and high-sucrose (HFHSD) diet vs. standard (SD) chow for 14 weeks. Our results showed that the lung was the second most affected organ after the liver, and that the two organs shared reduced one-carbon (1C) metabolism and increased lipid accumulation. Altered 1C metabolism was found in all organs and in the serum, but serine levels were increased only in the lung of HFHSD compared to SD. Lastly, tricarboxylic acid (TCA)-derived metabolites were specifically and oppositely regulated in the serum and kidneys but not in other organs. Collectively, our data highlighted that HFHSD induced specific metabolic changes in all organs, the lung being the second most affected organ, the main alterations affecting metabolite concentrations of the 1C pathway and, to a minor extend, TCA. The absolute metabolite quantification performed in this study reveals some metabolic specificities affecting both the liver and the lung, that may reveal common metabolic determinants to the ongoing pathological process.

International audience; In the liver, contact sites between the endoplasmic reticulum (ER) and mitochondria (named MAMs) may be crucial hubs for the regulation of lipid metabolism, thus contributing to the exacerbation or prevention of fatty liver. We hypothesized that tether proteins located at MAMs could play a key role in preventing triglyceride accumulation in hepatocytes and nonalcoholic fatty liver disease (NAFLD) occurrence. To test this, we explored the role of two key partners in building MAM integrity and functionality, the glucose-regulated protein 75 (Grp75) and mitofusin 2 (Mfn2), which liver contents are altered in obesity and NAFLD. Grp75 or Mfn2 expression was either silenced using siRNA or overexpressed with adenoviruses in Huh7 cells.Silencing of Grp75 and Mfn2 resulted in decreased ER-mitochondria interactions, mitochondrial network fusion state and mitochondrial oxidative capacity, while overexpression of the two proteins induced mirror impacts on these parameters. Furthermore, Grp75 or Mfn2 silencing decreased cellular cholesterol content and enhanced triglyceride secretion in ApoB100 lipoproteins, while their overexpression led to reverse effects. Cellular phosphatidylcholine/phosphatidylethanolamine ratio was decreased only upon overexpression of the proteins, potentially contributing to altered ApoB100 assembly and secretion. Despite the opposite differences, both silencing and overexpression of Grp75 or Mfn2 induced triglyceride storage, although a fatty acid challenge was required to express the alteration upon protein silencing. Among the mechanisms potentially involved in this phenotype, ER stress was closely associated with altered triglyceride metabolism after Grp75 or Mfn2 overexpression, while blunted mitochondrial FA oxidation capacity may be the main defect causing triglyceride accumulation upon Grp75 or Mfn2 silencing. However, further studies are required to decipher the link between modulation of Grp75 or Mfn2 expression, change in MAM integrity and alteration of cholesterol content of the cell.In conclusion, Grp75 or Mfn2 silencing and overexpression in Huh7 cells contribute to altering MAM integrity and cholesterol storage in opposite directions, but all promote triglyceride accumulation through distinct cellular pathways. This study also highlights that besides Mfn2, Grp75 could play a central role in hepatic lipid and cholesterol metabolism in obesity and NAFLD.

We have determined the lipid, protein and miRNA composition of skeletal muscle (SkM)-released extracellular vesicles (ELVs) from Ob/ob (OB) vs wild-type (WT) mice. The results showed that atrophic insulin-resistant OB-SkM released less ELVs than WT-SkM, highlighted by a RAB35 decrease and an increase in intramuscular cholesterol content. Proteomic analyses of OB-ELVs revealed a group of 37 proteins functionally connected, involved in lipid oxidation and with catalytic activities. OB-ELVs had modified contents for phosphatidylcholine (PC 34-4, PC 40-3 and PC 34-0), sphingomyelin (Sm d18:1/18:1) and ceramides (Cer d18:1/18:0) and were enriched in cholesterol, likely to alleviated intracellular accumulation. Surprisingly many ELV miRNAs had a nuclear addressing sequence, and targeted genes encoding proteins with nuclear activities. Interestingly, SkM-ELV miRNA did not target mitochondria. The most significant function targeted by the 7 miRNAs altered in OB-ELVs was lipid metabolism. In agreement, OB-ELVs induced lipid storage in recipient adipocytes and increased lipid up-take and fatty acid oxidation in recipient muscle cells. In addition, OB-ELVs altered insulin-sensitivity and induced atrophy in muscle cells, reproducing the phenotype of the releasing OB muscles. These data suggest for the first time, a cross-talk between muscle cells and adipocytes, through the SkM-ELV route, in favor of adipose tissue expansion.

Cellular senescence is a cell program induced by various stresses that leads to a stable proliferation arrest and to a senescence-associated secretory phenotype. Accumulation of senescent cells during age-related diseases participates in these pathologies and regulates healthy lifespan. Recent evidences point out a global dysregulated intracellular metabolism associated to senescence phenotype. Nonetheless, the functional contribution of metabolic homeostasis in regulating senescence is barely understood. In this work, we describe how the mevalonate pathway, an anabolic pathway leading to the endogenous biosynthesis of poly-isoprenoids, such as cholesterol, acts as a positive regulator of cellular senescence in normal human cells. Mechanistically, this mevalonate-induced senescence is partly mediated by the downstream cholesterol biosynthetic pathway. This pathway promotes transcriptional activity of ERRα leading to dysfunctional mitochondria, ROS production, DNA damage and a p53-dependent senescence. Supporting the relevance of these observations, increase of senescence in liver due to a high-fat diet regimen is abrogated in ERRα knockout mouse. Overall, this work unravels the role of cholesterol biosynthesis in the induction of an ERRα-dependent mitochondrial program leading to cellular senescence and related pathological alterations.

Over the course of diet-induced obesity (DIO), adipose tissue macrophage (ATM) populations transition from highly oxidative and protective to highly inflammatory and metabolically deleterious. Here, we demonstrate that the Interferon Regulatory Factor (IRF)-5 is a key molecular switch mediating repression of macrophage oxidative capacity early in DIO. ATMs from mice with a myeloid-specific deletion of IRF5 are characterised by increased mitochondrial activity and oxidative respiration compared to ATMs from wild-type mice. This hyper-oxidative phenotype is inducible and reversible in vitro by delivery of an inhibitory IRF5-decoy peptide and through IRF5 adenoviral over-expression, respectively. In a data-driven approach, using public IRF5-cistrome data and in-house RNA-sequencing, we identified a transcriptional mechanism by which cellular oxidative capacity is repressed in an IRF5-dependent manner. The hyper-oxidative phenotype of IRF5-deficient macrophages is mediated by the Growth Hormone Inducible Transmembrane Protein (GHITM), known for maintaining mitochondrial architecture for optimal oxidative respiration. Cas9-mediated simultaneous knock-down of GHITM and of IRF5 reverses the hyper-oxidative phenotype associated with IRF5-deficiency in vitro. IRF5-dependent regulation of GHITM expression and mitochondrial activity extends to human ATMs and monocytes from obese and diabetic patients.

Although the mechanism of action of the antidiabetic drug metformin is still a matter of discussions, it is well accepted that the gut plays an important role. To gain more insights into the mechanisms that occur in the different regions of the intestinal tract in response to metformin treatment, adult male mice were fed a high-fat-high sucrose (HFS) diet for 8 days. Metformin treatment by gavage (300 mg/day/kg body weight) during the HFS diet improved glucose tolerance. Metformin counteracted HFS diet-induced overexpression of a network of genes involved in the transport of glucose and fatty acids in the small intestine, and induced beneficial modification of secondary bile acid profile in the caecum, with reduction in DCA and LCA levels and increased abundance of UDCA and TUDCA, potentially leading to FRX inhibition. In parallel, metformin treatment was associated with specific changes in the microbiota composition in the lumen of the different regions of the gut, counteracting the effects of HFS diet on the abundances of some bacterial groups generally associated with metabolic disturbances (f-Lachnospiraceae, f-Petostreptococcaceae, g-Clostidium). In addition, metformin promotes a strong increase in the abundance of Akkermansia muciniphila in the intestinal lumen all along the gut. Therefore, the present work clearly emphasises the role of all the regions of the intestinal tract in the beneficial action of the antidiabetic drug metformin in a prediabetic mouse model.

Mitochondrial diseases are genetic disorders that lead to impaired mitochondrial function, resulting in exercise intolerance and muscle weakness. In patients, muscle fatigue due to defects in mitochondrial oxidative capacities commonly precedes muscle weakness. In mice, deletion of the fast-twitch skeletal muscle-specific Tfam gene (Tfam KO) leads to a deficit in respiratory chain activity, severe muscle weakness and early death. Here, we performed a time-course study of mitochondrial and muscular dysfunctions in 11- and 14-week-old Tfam KO mice, i.e. before and when mice are about to enter the terminal stage, respectively. Although force in the unfatigued state was reduced in Tfam KO mice compared to control littermates (wild type) only at 14 weeks, during repeated submaximal contractions fatigue was faster at both ages. During fatiguing stimulation, total phosphocreatine breakdown was larger in Tfam KO muscle than in wild-type muscle at both ages, whereas phosphocreatine consumption was faster only at 14 weeks. In conclusion, the Tfam KO mouse model represents a reliable model of lethal mitochondrial myopathy in which impaired mitochondrial energy production and premature fatigue occur before muscle weakness and early death.

International audience; In humans, insulin resistance has been linked to an impaired metabolic transition from fasting to feeding (metabolic flexibility; MetFlex). Previous studies suggest that mitochondrial dynamics response is a putative determinant of MetFlex; however, this has not been studied in humans. Thus, the aim of this study was to investigate the mitochondrial dynamics response in the metabolic transition from fasting to feeding in human peripheral blood mononuclear cells (PBMCs). Six male subjects fasted for 16 h (fasting), immediately after which they consumed a 75-g oral glucose load (glucose). In both fasting and glucose conditions, blood samples were taken to obtain PBMCs. Mitochondrial dynamics were assessed by electron microscopy images. We exposed in vitro acetoacetate-treated PBMCs to the specific IP3R inhibitor Xestospongin B (XeB) to reduce IP3R-mediated mitochondrial Ca(2+) accumulation. This allowed us to evaluate the role of ER-mitochondria Ca(2+) exchange in the mitochondrial dynamic response to substrate availability. To determine whether PBMCs could be used in obesity context (low MetFlex), we measured mitochondrial dynamics in mouse spleen-derived lymphocytes from WT and ob/ob mice. We demonstrated that the transition from fasting to feeding reduces mitochondria-ER interactions, induces mitochondrial fission and reduces mitochondrial cristae density in human PBMCs. In addition, we demonstrated that IP3R activity is key in the mitochondrial dynamics response when PBMCs are treated with a fasting-substrate in vitro. In murine mononuclear-cells, we confirmed that mitochondria-ER interactions are regulated in the fasted-fed transition and we further highlight mitochondria-ER miscommunication in PBMCs of diabetic mice. In conclusion, our results demonstrate that the fasting/feeding transition reduces mitochondria-ER interactions, induces mitochondrial fission and reduces mitochondrial cristae density in human PBMCs, and that IP3R activity may potentially play a central role.

International audience; The liver is a major organ that coordinates the metabolic flexibility of the whole body, which is characterized by the ability to adapt dynamically in response to fluctuations in energy needs and supplies. In this context, hepatocyte mitochondria are key partners in fine-tuning metabolic flexibility. Here we review the metabolic and signalling pathways carried by mitochondria in the liver, the major pathways that regulate mitochondrial function and how they function in health and metabolic disorders associated to obesity, i.e. insulin resistance, non-alcoholic steatosis and steatohepatitis and hepatocellular carcinoma. Finally, strategies targeting mitochondria to counteract liver disorders are discussed.

International audience; Aims/hypothesis Disrupted intracellular Ca2+ handling is known to play a role in diabetic cardiomyopathy but it has also been postulated to contribute to obesity- and type 2 diabetes-associated skeletal muscle dysfunction. Still, there is so far very limited functional insight into whether, and if so to what extent, muscular Ca2+ homeostasis is affected in this situation, so as to potentially determine or contribute to muscle weakness. In differentiated muscle, force production is under the control of the excitation-contraction coupling process: upon plasma membrane electrical activity, the Ca(V)1.1 voltage sensor/Ca2+ channel in the plasma membrane triggers opening of the ryanodine receptor Ca2+ release channel in the sarcoplasmic reticulum (SR) membrane. Opening of the ryanodine receptor triggers the rise in cytosolic Ca2+, which activates contraction while Ca2+ uptake by the SR ATPase Ca(2+-)pump promotes relaxation. These are the core mechanisms underlying the tight control of muscle force by neuronal electrical activity. This study aimed at characterising their inherent physiological function in a diet-induced mouse model of obesity and type 2 diabetes.Methods Intact muscle fibres were isolated from mice fed either with a standard chow diet or with a high-fat, high-sucrose diet generating obesity, insulin resistance and glucose intolerance. Properties of muscle fibres were investigated with a combination of whole-cell voltage-clamp electrophysiology and confocal fluorescence imaging. The integrity and density of the plasma membrane network (transverse tubules) that carries the membrane excitation throughout the muscle fibres was assessed with the dye Di-8-ANEPPS. Ca(V)1.1 Ca2+ channel activity was studied by measuring the changes in current across the plasma membrane elicited by voltage-clamp depolarising pulses of increasing amplitude. SR Ca2+ release through ryanodine receptors was simultaneously detected with the Ca2+-sensitive dye Rhod-2 in the cytosol. CaV1.1 voltage-sensing activity was separately characterised from the properties of intra-plasma-membrane charge movement produced by short voltage-clamp depolarising pulses. Spontaneous Ca2+ release at rest was assessed with the Ca2+-sensitive dye Fluo-4. The rate of SR Ca2+ uptake was assessed from the time course of cytosolic Ca2+ recovery after the end of voltage excitation using the Ca2+-sensitive dye Fluo-4FF. The response to a fatigue-stimulation protocol was determined from the time course of decline of the peak Fluo-4FF Ca2+ transients elicited by 30 trains of 5-ms-long depolarising pulses delivered at 100 Hz.Results The transverse tubule network architecture and density were well preserved in the fibres from the obese mice. The Ca(V)1.1 Ca2+ current and voltage-sensing properties were also largely unaffected with mean values for maximum conductance and maximum amount of charge of 234 +/- 12 S/F and 30.7 +/- 1.6 nC/mu F compared with 196 +/- 13 S/F and 32.9 +/- 2.0 nC/mu F in fibres from mice fed with the standard diet, respectively. Voltage-activated SR Ca2+ release through ryanodine receptors also exhibited very similar properties in the two groups with mean values for maximum rate of Ca2+ release of 76.0 +/- 6.5 and 78.1 +/- 4.4 mu mol l(-1) ms(-1), in fibres from control and obese mice, respectively. The response to a fatigue protocol was also largely unaffected in fibres from the obese mice, and so were the rate of cytosolic Ca2+ removal and the spontaneous Ca2+ release activity at rest.Conclusions/interpretation The functional properties of the main mechanisms involved in the control of muscle Ca2+ homeostasis are well preserved in muscle fibres from obese mice, at the level of both the plasma membrane and of the SR. We conclude that intracellular Ca2+ handling and excitation-contraction coupling in skeletal muscle fibres are not primary targets of obesity and type 2 diabetes.

Disrupted intracellular CaIntact muscle fibres were isolated from mice fed either with a standard chow diet or with a high-fat, high-sucrose diet generating obesity, insulin resistance and glucose intolerance. Properties of muscle fibres were investigated with a combination of whole-cell voltage-clamp electrophysiology and confocal fluorescence imaging. The integrity and density of the plasma membrane network (transverse tubules) that carries the membrane excitation throughout the muscle fibres was assessed with the dye Di-8-ANEPPS. CaThe transverse tubule network architecture and density were well preserved in the fibres from the obese mice. The CaThe functional properties of the main mechanisms involved in the control of muscle Ca

IntroductionDeterminants of health and diseases in humans involve complex interactions between diet, gut microbiota and host metabolism. The liver is a major organ that coordinates host adaptations to environmental factors. Mitochondria and endoplasmic reticulum tightly regulate liver nutrient sensing and metabolic adaptations, shaping its metabolic flexibility. Both organelles interact through contact points (named MAMs) in order to exchange calcium and lipids, and MAMs’ integrity settles metabolic flexibility of the liver. Disrupted MAMs’ integrity in obesity is linked to liver steatosis and insulin resistance. This occurs in association with alterations circulating amino acid profiles, among which glycine. Meta-analyses showed that the low plasma glycine concentration observed in obesity is a predictive factor for developing type 2 diabetes. We thus aimed at exploring whether glycine could participate to the regulation of the hepatic response to insulin and whether mechanisms may involve regulation of mitochondrial-ER (MAMs) interactions.Materials and MethodsThe study was carried out in 12 week-old male C57B16J mice fed a standard chow (n = 12/group, 36 total) in accordance with the French guidelines for the care and use of animals. Glycine (1.2g/kg) was provided for 3 days in drinking water vs. water vs. isonitrogenous placebo amino acids. Liver was collected after an overnight fast, 15 min after ip injection of saline (n = 6/group) or insulin (0.75U/kg, n = 6/group). Liver samples were fixed in glutaraldehyde/cacodylate for quantifying MAMs by transmission electronic microscopy. Fractions enriched in MAMs were isolated by differential ultracentrifugation on fresh tissue for Western Blot explorations. Insulin response was assessed through Akt phosphorylation at Ser473.ResultsGlycine supplementation increased the percentage of interaction between ER and mitochondria (compare to mitochondrial surface) for all spacing from 10 to 50 nm. Thus, glycine supplementation induced a 45% increase in mitochondrial-ER (MAMs) interactions compared to other groups (p < 0.001). In agreement to the improved MAMs’ integrity, insulin response was enhanced by 50% after glycine supplementation compared to other groups (p < 0.05). Finally, protein analysis of the MAMs' fraction corroborates the importance of PP2A as a key enzyme regulating MAMs’ integrity.ConclusionGlycine is therefore an interesting nutritional actor that can play a crucial role in regulating the integrity of MAMs in the liver, thus contributing to the control of the insulin response.

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Whereas reactive oxygen species (ROS) can have opposite impacts on insulin signaling, they have mainly been associated with mitochondrial dysfunction in skeletal muscle. We analyzed the relationship between these three features in skeletal muscle of senescence accelerated mice (SAM) prone (P8), which are characterized by enhanced oxidative stress compared to SAM resistant (R1). Oxidative stress, ROS production, antioxidant system, mitochondrial content and functioning, as well as in vitro and in vivo insulin signaling were investigated in gastrocnemius and quadriceps muscles. In SAMP8 compared to SAMR1, muscle content in carbonylated proteins was two-fold (p < 0.01) and ROS production by xanthine oxidase 70% (p < 0.05) higher. Furthermore, insulin-induced Akt phosphorylation measured in vivo and ex vivo as well as muscle glucose uptake measured ex vivo were significantly higher (p < 0.05). Mitochondrial respiration evidenced uncoupling and higher respiration rates with substrates of complexes II and IV, in agreement with higher maximal activity of complexes II and IV (+ 18% and 62%, respectively, p < 0.05). By contrast, maximal activity of complex I was 22% lower (p < 0.05). All strain differences were corrected after 6 months of N-acetylcysteine (NAC) treatment, thus supporting the involvement of high ROS production in these differences. In conclusion in muscle of SAMP8 compared to SAMR1, high ROS production is associated to higher insulin sensitivity and glucose uptake but to lower mitochondrial complex I activity. These conflicting adaptations, with regards to the resulting imbalance between NADH production and use, were associated with intrinsic adjustments in the mitochondrial respiration chain (mitochondrial uncoupling, enhanced complexes II and IV activity). We propose that these bioenergetics adaptations may help at preserving muscle metabolic flexibility of SAMP8.

Introduction Type 2 diabetic cardiomyopathy has been linked to Ca2+ signaling alterations, notably a decreased mitochondrial Ca2+ uptake. Recent discovery of Ca2+ microdomains between cardiac mitochondria and reticulum launched a new investigation avenue for cardiometabolic diseases. Objective Hereby, we aimed to investigate if the mitochondrial Ca2+ mishandling is due to a dysregulation of the reticulum-mitochondria interactions or of the mitochondrial Ca2+ uniporter in the diabetic mouse heart. Methods and results Mice fed for 16 weeks with a high fat high sucrose diet (HFHSD) displayed a cardiac insulin resistance combined to cardiac fibrosis, hypertrophy and contractile dysfunction (decreased strain rate) versus standard diet-fed mice (SD). Fractionation of HFHSD mice hearts revealed a decreased protein content of their mitochondria associated-reticulum membranes, together with a lower expression of tethering proteins quantified by mass spectrometry. On functional terms, reduced IP3R-VDAC proximity and co-immunoprecipitation were associated with a reduced IP3-stimulated Ca2+ transfer to mitochondria in isolated HFHSD cardiomyocytes, yet no changes in mitochondrial calcium uniporter protein expression and function were detected. Moreover, HFHSD cardiomyocytes displayed no significant changes in the reticular Ca2+ release level and the amplitude of cytosolic Ca2+ transients. Eight weeks of diet reversal restored the cardiac insulin signaling, MAM content, Ca2+ transfer and cardiac function in HFHSD mice. Conclusion Therefore, our data indicate that disruption of the cardiac reticulum-mitochondria interactions is an early and reversible event impairing mitochondrial Ca2+ handling and contributing to the cardiac contractile dysfunction in the diabetic mouse heart, which could be counteracted by diet reversal.

Cellular senescence is induced by stresses and results in a stable proliferation arrest accompanied by a pro-inflammatory secretome. Senescent cells accumulate during aging, promoting various age-related pathologies and limiting lifespan. The endoplasmic reticulum (ER) inositol 1,4,5-trisphosphate receptor, type 2 (ITPR2) calcium-release channel and calcium fluxes from the ER to the mitochondria are drivers of senescence in human cells. Here we show that Itpr2 knockout (KO) mice display improved aging such as increased lifespan, a better response to metabolic stress, less immunosenescence, as well as less liver steatosis and fibrosis. Cellular senescence, which is known to promote these alterations, is decreased in Itpr2 KO mice and Itpr2 KO embryo-derived cells. Interestingly, ablation of ITPR2 in vivo and in vitro decreases the number of contacts between the mitochondria and the ER and their forced contacts induce premature senescence. These findings shed light on the role of contacts and facilitated exchanges between the ER and the mitochondria through ITPR2 in regulating senescence and aging.

International audience; Inflammation and oxidative stress are thought to be involved in, or associated with, the development of obesity, dyslipidemia, hepatic steatosis, and insulin resistance. This work was designed to determine the evolution of inflammation and oxidative stress during onset and progression of hepatic steatosis and glucose intolerance. Seventy-five male Wistar rats were divided to control and high-fat high-fructose (HFHFr) groups. A subgroup of each group was sacrificed at 4, 8, 12, 16, and 20 weeks. HFHFr-fed rats exhibited overweight, glucose intolerance, and hepatic steatosis with increased contents of hepatic diacylglycerols and ceramides. The HFHFr diet increased hepatic interleukin 6 (IL-6) protein and adipose tissue CCL5 gene expression and hepatic nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity but not mitochondrial reactive oxygen species (ROS) production. The HFHFr diet decreased plasma and liver levels of isoprostanoid metabolites as well as plasma thiobarbituric acid-reactive substance (TBARS) levels. Hepatic glutathione content was decreased with a moderate decrease in superoxide dismutase (SOD) and glutathione peroxidase (GPx) with the HFHFr diet. Overall, HFHFr diet led to hepatic lipid accumulation and glucose intolerance, which were accompanied by only moderate inflammation and oxidative stress. Most of these changes occurred at the same time and as early as 8 or 12 weeks of diet treatment. This implies that oxidative stress may be the result, not the cause, of these metabolic alterations, and suggests that marked hepatic oxidative stress should probably occur at the end of the steatotic stage to result in frank insulin resistance and steatohepatitis. These findings need to be further evaluated in other animal species as well as in human studies.

Glucotoxicity-induced beta cell dysfunction in type 2 diabetes is associated with alterations of mitochondria and endoplasmic reticulum (ER). Both organelles interact at contact sites, defined as mitochondria-associated membranes (MAMs), which were recently implicated in the regulation of glucose homeostasis. The role of MAMs in beta cells is still largely unknown and their implication in glucotoxicity-associated beta cell dysfunction remains to be defined. Here, we report that acute glucose stimulation stimulated ER-mitochondria interactions and calcium (Ca(2+)) exchange in INS-1E cells, whereas disruption of MAMs altered glucose-stimulated insulin secretion (GSIS). Conversely, chronic incubations with high glucose of either INS-1E cells or human pancreatic islets altered GSIS, and concomitantly reduced ER Ca(2+) store, increased basal mitochondrial Ca(2+) and reduced ATP-stimulated ER-mitochondria Ca(2+) exchanges, despite an increase of organelle interactions. Furthermore, glucotoxicity-induced perturbations of Ca(2+) signaling are associated with ER stress, altered mitochondrial respiration and mitochondria fragmentation, and these organelle stresses may participate to increased organelle tethering, as a protective mechanism. Lastly, sustained induction of ER-mitochondria interactions using a linker reduced organelle Ca(2+) exchange, induced mitochondrial fission and altered GSIS. Therefore, dynamic organelle coupling participates to GSIS in beta cells and over-time disruption of organelle Ca(2+) exchange might be a novel mechanism contributing to glucotoxicity-induced beta cell dysfunction.

Exercise is important to maintain skeletal muscle mass through stimulation of protein synthesis, which is a major ATP-consuming process for cells. However, muscle cells have to face high energy demand during contraction. The present study aimed to investigate protein synthesis regulation during aerobic exercise in mouse hindlimb muscles. Male C57Bl/6J mice ran at 12 m/min for 45 min or at 12 m/min for the first 25 min followed by a progressive increase in velocity up to 20 m/min for the last 20 min. Animals were injected intraperitoneally with 40 nmol/g of body weight of puromycin and euthanized by cervical dislocation immediately after exercise cessation. Analysis of gastrocnemius, plantaris, quadriceps, soleus, and tibialis anterior muscles revealed a decrease in protein translation assessed by puromycin incorporation, without significant differences among muscles or running intensities. The reduction of protein synthesis was associated with a marked inhibition of mammalian target of rapamycin complex 1 (mTORC1)-dependent phosphorylation of eukaryotic translation initiation factor 4E-binding protein 1, a mechanism consistent with reduced translation initiation. A slight activation of AMP-activated protein kinase consecutive to the running session was measured but did not correlate with mTORC1 inhibition. More importantly, exercise resulted in a strong upregulation of regulated in development and DNA damage 1 (REDD1) protein and gene expressions, whereas transcriptional regulation of other recognized exercise-induced genes ( IL-6, kruppel-like factor 15, and regulator of calcineurin 1) did not change. Consistently with the recently discovered role of REDD1 on mitochondria-associated membranes, we observed a decrease in mitochondria-endoplasmic reticulum interaction following exercise. Collectively, these data raise questions concerning the role of mitochondria-associated endoplasmic reticulum membrane disruption in the regulation of muscle proteostasis during exercise and, more generally, in cell adaptation to metabolic stress. NEW & NOTEWORTHY How muscles regulate protein synthesis to cope with the energy demand during contraction is poorly documented. Moreover, it is unknown whether protein translation is differentially affected among mouse hindlimb muscles under different physiological exercise modalities. We showed here that 45 min of running decreases puromycin incorporation similarly in 5 different mouse muscles. This decrease was associated with a strong increase in regulated in development and DNA damage 1 protein expression and a significant disruption of the mitochondria and sarcoplasmic reticulum interaction.

Introduction The prevalence of type 2 diabetes (T2D) is increasing dramatically in our modern world. Cardiovascular complications are the leading cause of T2D-related morbi-mortality, notably diabetic cardiomyopathy (DCM). This cardiac dysfunction is characterized by hypertrophy and diastolic dysfunction. DCM animal models are required to better define the underlying mechanisms. Objective We aim to characterize the cardiac and contractile function of the leptin-deficient ob/ob mouse as a potential DCM model. Methods Twelve weeks-old ob/ob mice were characterized at the metabolic level. Contractile function was evaluated by echocardiography while Ca2+ transients were measured by imaging. Results Ob/ob mice displayed increased body weight, hyperglycemia and cardiac insulin resistance. Despite a slight thickening of the posterior left ventricular wall, the heart of ob/ob mice does not exhibit any cardiac hypertrophy, further validated by no change in the cardiomyocyte membrane capacitance measured by electrophysiology. Echocardiography revealed no diastolic dysfunction in the ob/ob mice but a mild strain rate reduction with preserved ejection fraction. Peak amplitudes of Ca2+ transients were unchanged but time to peak was decreased in the ob/ob cardiomyocyte. Histological analysis showed triglyceride accumulation in the ob/ob heart. Conclusion Our data demonstrate that the 12 weeks-old ob/ob mice, well characterized as a T2D and obese mouse model, do not recapitulate the main hallmarks of DCM, i.e. diastolic dysfunction and cardiac hypertrophy. The high accumulation of triglycerides and the subsequent lipotoxicity in the ob/ob cardiomyocytes may rather rely on their obese phenotype than their diabetic one, probably contributing to the early systolic dysfunction.

KEY POINTS Vascular brain lesions and atherosclerosis are two similar conditions that are characterized by increased inflammation and oxidative stress. Non-invasive imaging in a murine model of atherosclerosis showed vascular brain damage and peripheral inflammation. In this study, exercise training reduced magnetic resonance imaging-detected abnormalities, insulin resistance and markers of oxidative stress and inflammation in old ApoE-/- mice. Our results demonstrate the protective effect of exercise on neurovascular damage in the ageing brain of ApoE-/- mice. ABSTRACT Vascular brain lesions, present in advanced atherosclerosis, share pathological hallmarks with peripheral vascular lesions, such as increased inflammation and oxidative stress. Physical activity reduces these peripheral risk factors, but its cerebrovascular effect is less documented, especially by non-invasive imaging. Through a combination of in vivo and post-mortem techniques, we aimed to characterize vascular brain damage in old ApoE-/- mice fed a high-cholesterol (HC) diet with dietary controlled intake. We then sought to determine the beneficial effects of exercise training on oxidative stress and inflammation in the brain as a treatment option in an ageing atherosclerosis mouse model. Using in vivo magnetic resonance imaging (MRI) and biological markers of oxidative stress and inflammation, we evaluated the occurrence of vascular abnormalities in the brain of HC-diet fed ApoE-/- mice >70 weeks old, its association with local and systemic oxidative stress and inflammation, and whether both can be modulated by exercise. Exercise training significantly reduced both MRI-detected abnormalities (present in 71% of untrained vs. 14% of trained mice) and oxidative stress (lipid peroxidation, 9.1 ± 1.4 vs. 5.2 ± 0.9 μmol mg-1 ; P

Communication between the endoplasmic reticulum (ER) and mitochondria plays a pivotal role in Ca

International audience; Communication between the endoplasmic reticulum (ER) and mitochondria plays a pivotal role in Ca2+ signaling, energy metabolism, and cell survival. Dysfunction in this cross-talk leads to metabolic and neurodegenerative diseases. Wolfram syndrome is a fatal neurodegenerative disease caused by mutations in the ER-resident protein WFS1. Here, we showed that WFS1 formed a complex with neuronal calcium sensor 1 (NCS1) and inositol 1,4,5-trisphosphate receptor (IP3R) to promote Ca2+ transfer between the ER and mitochondria. In addition, we found that NCS1 abundance was reduced in WFS1-null patient fibroblasts, which showed reduced ER-mitochondria interactions and Ca2+ exchange. Moreover, in WFS1-deficient cells, NCS1 overexpression not only restored ER-mitochondria interactions and Ca2+ transfer but also rescued mitochondrial dysfunction. Our results describe a key role of NCS1 in ER-mitochondria cross-talk, uncover a pathogenic mechanism for Wolfram syndrome, and potentially reveal insights into the pathogenesis of other neurodegenerative diseases.

Glucotoxicity-induced β-cell dysfunction in type 2 diabetes is associated with alterations of mitochondria and the endoplasmic reticulum (ER). Both organelles interact at contact sites, defined as mitochondria-associated membranes (MAMs), which were recently implicated in the regulation of glucose homeostasis. The role of MAMs in β-cells is still largely unknown, and their implication in glucotoxicity-associated β-cell dysfunction remains to be defined. Here, we report that acute glucose treatment stimulated ER-mitochondria interactions and calcium (Ca

International audience; Living organisms have the capacity to sense both nutrients and immune signals in order to adapt their metabolism to the needs, and both metabolic inflexibility and exacerbated immune responses are associated with metabolic diseases. Over the past decade, mitochondria emerged as key nutrient and immune sensors regulating numerous signalling pathways, and mitochondria dysfunction has been extensively implicated in metabolic diseases. Interestingly, mitochondria interact physically and functionally with the endoplasmic reticulum (ER, in contact sites named mitochondria-associated membranes (MAMs), in order to exchange metabolites and calcium and regulate cellular homeostasis. Emerging evidences suggest that MAMs provide a platform for hormone and nutrient signalling pathways and for innate immune responses, then regulating mitochondrial bioenergetics and apoptosis. Here, I thus propose the concept that MAMs could be attractive nutrient and immune sensors that regulate mitochondria physiology in order to adapt metabolism and cell fate, and that organelle miscommunication could be involved in the metabolic inflexibility and the pro-inflammatory status associated with metabolic diseases. (C) 2017 Elsevier Inc. All rights reserved.

International audience; Modifications of the interactions between endoplasmic reticulum (ER) and mitochondria, defined as mitochondria-associated membranes (MAMs), were recently shown to be involved in the control of hepatic insulin action and glucose homeostasis, but with conflicting results. Whereas skeletal muscle is the primary site of insulin-mediated glucose uptake and the main target for alterations in insulin-resistant states, the relevance of MAM integrity in muscle insulin resistance is unknown. Deciphering the importance of MAMs on muscle insulin signaling could help to clarify this controversy. Here, we show in skeletal muscle of different mice models of obesity and type 2 diabetes (T2D) a marked disruption of ER-mitochondria interactions as an early event preceding mitochondrial dysfunction and insulin resistance. Furthermore, in human myotubes, palmitate-induced insulin resistance is associated with a reduction of structural and functional ER-mitochondria interactions. Importantly, experimental increase of ER-mitochondria contacts in human myotubes prevents palmitate-induced alterations of insulin signaling and action, whereas disruption of MAM integrity alters the action of the hormone. Lastly, we found an association between altered insulin signaling and ER-mitochondria interactions in human myotubes from obese subjects with or without T2D compared with healthy lean subjects. Collectively, our data reveal a new role of MAM integrity in insulin action of skeletal muscle and highlight MAM disruption as an essential subcellular alteration associated with muscle insulin resistance in mice and humans. Therefore, reduced ER-mitochondria coupling could be a common alteration of several insulin-sensitive tissues playing a key role in altered glucose homeostasis in the context of obesity and T2D.