Frédéric Lamarche

Interactions between mitochondria and the endoplasmic reticulum, known as MAMs, are altered in the liver in obesity, which contributes to disruption of the insulin signaling pathway. In addition, the plasma level of glycine is decreased in obesity, and the decrease is strongly correlated with the severity of insulin resistance. Certain nutrients have been shown to regulate MAMs; therefore, we tested whether glycine supplementation could reduce insulin resistance in the liver by promoting MAM integrity. Glycine (5 mM) supported MAM integrity and insulin response in primary rat hepatocytes cultured under control and lipotoxic (palmitate 500 µM) conditions for 18 h. In contrast, in C57 BL/6 JOlaHsd mice (male, 6 weeks old) fed a high-fat, high-sucrose diet (HFHS) for 16 weeks, glycine supplementation (300 mg/kg) in drinking water during the last 6 weeks (HFHS-Gly) did not reverse the deleterious impact of HFHS-feeding on liver MAM integrity. In addition, glycine supplementation worsened fasting glycemia and glycemic response to intraperitoneal pyruvate injection compared to HFHS. The adverse impact of glycine supplementation on hepatic gluconeogenesis was further supported by the higher oxaloacetate/acetyl-CoA ratio in the liver in HFHS-Gly compared to HFHS. Although glycine improves MAM integrity and insulin signaling in the hepatocyte in vitro, no beneficial effect was found on the overall metabolic profile of HFHS-Gly-fed mice.

Rationale: Intermittent hypoxia (IH) is one of the main features of sleep-disordered breathing (SDB). Recent findings indicate that hypoxia inducible factor-1 (HIF-1) promotes cardiomyocytes apoptosis during chronic IH, but the mechanisms involved remain to be elucidated. Here, we hypothesize that IH-induced ER stress is associated with mitochondria-associated ER membrane (MAM) alteration and mitochondrial dysfunction, through HIF-1 activation. Methods: Right atrial appendage biopsies from patients with and without SDB were used to determine HIF-1α, Grp78 and CHOP expressions. Wild-type and HIF-1α^+/− mice were exposed to normoxia (N) or IH (21–5% O₂, 60 cycles/h, 8 h/day) for 21 days. Expressions of HIF-1α, Grp78 and CHOP, and apoptosis, were measured by Western blot and immunochemistry. In isolated cardiomyocytes, we examined structural integrity of MAM by proximity ligation assay and their function by measuring ER-to-mitochondria Ca²⁺ transfer by confocal microscopy. Finally, we measured mitochondrial respiration using oxygraphy and calcium retention capacity (CRC) by spectrofluorometry. MAM structure was also investigated in H9C2 cells incubated with 1 mM CoCl₂, a potent HIF-1α inducer. Results: In human atrial biopsies and mice, IH induced HIF-1 activation, ER stress and apoptosis. IH disrupted MAM, altered Ca²⁺ homeostasis, mitochondrial respiration and CRC. Importantly, IH had no effect in HIF-1α^+/− mice. Similar to what observed under IH, HIF-1α overexpression was associated with MAM alteration in H9C2. Conclusion: IH-induced ER stress, MAM alterations and mitochondrial dysfunction were mediated by HIF-1; all these intermediate mechanisms ultimately inducing cardiomyocyte apoptosis. This suggests that HIF-1 modulation might limit the deleterious cardiac effects of SDB.

Background: Animal studies and clinical data support the interest of citrulline as a promising therapeutic for sarcopenia. Citrulline is known to stimulate muscle protein synthesis, but how it affects energy metabolism to support the highly energy‐dependent protein synthesis machinery is poorly understood.Methods: Here, we used myotubes derived from primary culture of mouse myoblasts to study the effect of citrulline on both energy metabolism and protein synthesis under different limiting conditions.Results: When serum/amino acid deficiency or energy stress (mild uncoupling) were applied, citrulline stimulated muscle protein synthesis by +22% and +11%, respectively. Importantly, this increase was not associated with enhanced energy status (ATP/ADP ratio) or mitochondrial respiration. We further analysed the share of mitochondrial respiration and thus of generated ATP allocated to different metabolic pathways by using specific inhibitors. Our results indicate that addition of citrulline allocated an increased share of mitochondrially generated ATP to the protein synthesis machinery under conditions of both serum/amino acid deficiency (+28%) and energy stress (+21%). This reallocation was not because of reduced ATP supply to DNA synthesis or activities of sodium and calcium cycling ion pumps.Conclusions: Under certain stress conditions, citrulline increases muscle protein synthesis by specifically reallocating mitochondrial fuel to the protein synthesis machinery. Because ATP/ADP ratios and thus Gibbs free energy of ATP hydrolysis remained globally constant, this reallocation may be linked to decreased activation energies of one or several ATP (and GTP)‐consuming reactions involved in muscle protein synthesis.

Islets of Langerhans transplantation is a promising therapy for type 1 diabetes mellitus, but this technique is compromised by transplantation stresses including inflammation. In other tissues, co-transplantation with mesenchymal stem cells has been shown to reduce damage by improving anti-inflammatory and anti-oxidant defences. Therefore, we probed the protection afforded by bone marrow mesenchymal stem cells to islets under pro-inflammatory cytokine stress. In order to evaluate the cytoprotective potential of mesenchymal stem cells on rat islets, co-cultures were exposed to the interleukin-1, tumour necrosis factor α and interferon γ cocktail for 24 h. Islet viability and functionality tests were performed. Reactive oxygen species and malondialdehyde were measured. Expression of stress-inducible genes acting as anti-oxidants and detoxifiers, such as superoxide dismutases 1 and 2, NAD(P)H quinone oxidoreductase 1, heme oxygenase-1 and ferritin H, was compared to non-stressed cells, and the corresponding proteins were measured. Data were analysed by a two-way ANOVA followed by a Holm-Sidak post hoc analysis. Exposure of rat islets to cytokines induces a reduction in islet viability and functionality concomitant with an oxidative status shift with an increase of cytosolic ROS production. Mesenchymal stem cells did not significantly increase rat islet viability under exposure to cytokines but protected islets from the loss of insulin secretion. A drastic reduction of the antioxidant factors heme oxygenase-1 and ferritin H protein levels was observed in islets exposed to the cytokine cocktail with a prevention of this effect by the presence of mesenchymal stem cells. Our data evidenced that MSCs are able to preserve islet insulin secretion through a modulation of the oxidative imbalance mediated by heme and iron via heme oxygenase-1 and ferritin in a context of cytokine exposure.

Background: Exercise and citrulline (CIT) are both regulators of muscle protein metabolism. However, the combination of both has been under-studied yet may have synergistic effects on muscle metabolism and performance. Methods: Three-month-old healthy male rats were randomly assigned to be fed ad libitum for 4 weeks with either a citrulline-enriched diet (1 g·kg⁻¹·day⁻¹) (CIT) or an isonitrogenous standard diet (by addition of nonessential amino acid) (Ctrl) and trained (running on treadmill 5 days·week⁻¹) (ex) or not. Maximal endurance activity and body composition were assessed, and muscle protein metabolism (protein synthesis, proteomic approach) and energy metabolism [energy expenditure, mitochondrial metabolism] were explored. Results: Body composition was affected by exercise but not by CIT supplementation. Endurance training was associated with a higher maximal endurance capacity than sedentary groups (PCITex group than the Ctrlex group (139±4 min versus 122±6 min, PCtrl, PCtrl, PConclusion: CIT supplementation and endurance training in healthy male rats modulates both muscle protein and energy metabolisms, with synergic effects on an array of parameters, including performance and protein synthesis.