Audrey Ayer

Resveratrol induces a mitochondrial complex I-dependent increase in NADH oxidation responsible for sirtuin activation in liver cells.

Background: The mechanism of action of resveratrol on sirtuin and mitochondrial metabolism remains elusive. Results: Resveratrol increases the mitochondrial NAD+ level by direct stimulation of complex I, leading to a SIRT3-dependent increase in substrate supplies. Conclusion: Our results link the direct stimulation by resveratrol of NADH oxidation to the SIRT3 activation. Significance: Mitochondrial NAD+/NADH ratio is a critical parameter mediating resveratrol effect on mitochondrial function. Resveratrol (RSV) has been shown to be involved in the regulation of energetic metabolism, generating increasing interest in therapeutic use. SIRT1 has been described as the main target of RSV. However, recent reports have challenged the hypothesis of its direct activation by RSV, and the signaling pathways remain elusive. Here, the effects of RSV on mitochondrial metabolism are detailed both in vivo and in vitro using murine and cellular models and isolated enzymes. We demonstrate that low RSV doses (1–5 μm) directly stimulate NADH dehydrogenases and, more specifically, mitochondrial complex I activity (EC50 ∼1 μm). In HepG2 cells, this complex I activation increases the mitochondrial NAD+/NADH ratio. This higher NAD+ level initiates a SIRT3-dependent increase in the mitochondrial substrate supply pathways (i.e. the tricarboxylic acid cycle and fatty acid oxidation). This effect is also seen in liver mitochondria of RSV-fed animals (50 mg/kg/day). We conclude that the increase in NADH oxidation by complex I is a crucial event for SIRT3 activation by RSV. Our results open up new perspectives in the understanding of the RSV signaling pathway and highlight the critical importance of RSV doses used for future clinical trials.

The SGLT2 Inhibitor Dapagliflozin Prevents Cardiomyopathy in a Diabetic Lipodystrophic Mouse Model

Type 2 diabetes mellitus (T2DM) is a well-recognized independent risk factor for heart failure. T2DM is associated with altered cardiac energy metabolism, leading to ectopic lipid accumulation and glucose overload, the exact contribution of these two parameters remaining unclear. To provide new insight into the mechanism driving the development of diabetic cardiomyopathy, we studied a unique model of T2DM: lipodystrophic Bscl2−/− (seipin knockout [SKO]) mice. Echocardiography and cardiac magnetic resonance imaging revealed hypertrophic cardiomyopathy with left ventricular dysfunction in SKO mice, and these two abnormalities were strongly correlated with hyperglycemia. Surprisingly, neither intramyocardial lipid accumulation nor lipotoxic hallmarks were detected in SKO mice. [18F]Fludeoxyglucose positron emission tomography showed increased myocardial glucose uptake. Consistently, the O-GlcNAcylated protein levels were markedly increased in an SKO heart, suggesting a glucose overload. To test this hypothesis, we treated SKO mice with the hypoglycemic sodium–glucose cotransporter 2 (SGLT2) inhibitor dapagliflozin and the insulin sensitizer pioglitazone. Both treatments reduced the O-GlcNAcylated protein levels in SKO mice, and dapagliflozin successfully prevented the development of hypertrophic cardiomyopathy. Our data demonstrate that glucotoxicity by itself can trigger cardiac dysfunction and that a glucose-lowering agent can correct it. This result will contribute to better understanding of the potential cardiovascular benefits of SGLT2 inhibitors.

Resveratrol Directly Binds to Mitochondrial Complex I and Increases Oxidative Stress in Brain Mitochondria of Aged Mice.

Resveratrol is often described as a promising therapeutic molecule for numerous diseases, especially in metabolic and neurodegenerative disorders. While the mechanism of action is still debated, an increasing literature reports that resveratrol regulates the mitochondrial respiratory chain function. In a recent study we have identified mitochondrial complex I as a direct target of this molecule. Nevertheless, the mechanisms and consequences of such an interaction still require further investigation. In this study, we identified in silico by docking study a binding site for resveratrol at the nucleotide pocket of complex I. In vitro, using solubilized complex I, we demonstrated a competition between NAD+ and resveratrol. At low doses (<5μM), resveratrol stimulated complex I activity, whereas at high dose (50 μM) it rather decreased it. In vivo, in brain mitochondria from resveratrol treated young mice, we showed that complex I activity was increased, whereas the respiration rate was not improved. Moreover, in old mice with low antioxidant defenses, we demonstrated that complex I activation by resveratrol led to oxidative stress. These results bring new insights into the mechanism of action of resveratrol on mitochondria and highlight the importance of the balance between pro- and antioxidant effects of resveratrol depending on its dose and age. These parameters should be taken into account when clinical trials using resveratrol or analogues have to be designed.

Central Role of P2Y6 UDP Receptor in Arteriolar Myogenic Tone

Objective—Myogenic tone (MT) of resistance arteries ensures autoregulation of blood flow in organs and relies on the intrinsic property of smooth muscle to contract in response to stretch. Nucleotides released by mechanical strain on cells are responsible for pleiotropic vascular effects, including vasoconstriction. Here, we evaluated the contribution of extracellular nucleotides to MT. Approach and Results—We measured MT and the associated pathway in mouse mesenteric resistance arteries using arteriography for small arteries and molecular biology. Of the P2 receptors in mouse mesenteric resistance arteries, mRNA expression of P2X1 and P2Y6 was dominant. P2Y6 fully sustained UDP/UTP-induced contraction (abrogated in P2ry6−/− arteries). Preventing nucleotide hydrolysis with the ectonucleotidase inhibitor ARL67156 enhanced pressure-induced MT by 20%, whereas P2Y6 receptor blockade blunted MT in mouse mesenteric resistance arteries and human subcutaneous arteries. Despite normal hemodynamic parameters, P2ry6−/− mice were protected against MT elevation in myocardial infarction–induced heart failure. Although both P2Y6 and P2Y2 receptors contributed to calcium mobilization, P2Y6 activation was mandatory for RhoA–GTP binding, myosin light chain, P42–P44, and c-Jun N-terminal kinase phosphorylation in arterial smooth muscle cells. In accordance with the opening of a nucleotide conduit in pressurized arteries, MT was altered by hemichannel pharmacological inhibitors and impaired in Cx43+/− and P2rx7−/− mesenteric resistance arteries. Conclusions—Signaling through P2 nucleotide receptors contributes to MT. This mechanism encompasses the release of nucleotides coupled to specific autocrine/paracrine activation of the uracil nucleotide P2Y6 receptor and may contribute to impaired tissue perfusion in cardiovascular diseases.

Dual Effects of Resveratrol on Arterial Damage Induced By Insulin Resistance in Aged Mice

Aging leads to increased insulin resistance and arterial dysfunction, with oxidative stress playing an important role. This study explored the metabolic and arterial effects of a chronic treatment with resveratrol, an antioxidant polyphenol compound that has been shown to restore insulin sensitivity and decrease oxidative stress, in old mice with or without a high-protein diet renutrition care. High-protein diet tended to increase insulin resistance and atheromatous risk. Resveratrol improved insulin sensitivity in old mice fed standard diet by decreasing homeostasis model of assessment-insulin resistance and resistin levels. However, resveratrol did not improve insulin resistance status in old mice receiving the high-protein diet. In contrast, resveratrol exhibited deleterious effects by increasing inflammation state and superoxide production and diminishing aortic distensibility. In conclusion, we demonstrate that resveratrol has beneficial or deleterious effects on insulin sensitivity and arterial function, depending on nutritional status in our models.

High-protein-low-carbohydrate diet: deleterious metabolic and cardiovascular effects depend on age

High-protein-low-carbohydrate (HP-LC) diets have become widespread. Yet their deleterious consequences, especially on glucose metabolism and arteries, have already been underlined. Our previous study (2) has already shown glucose intolerance with major arterial dysfunction in very old mice subjected to an HP-LC diet. The hypothesis of this work was that this diet had an age-dependent deleterious metabolic and cardiovascular outcome. Two groups of mice, young and adult (3 and 6 mo old), were subjected for 12 wk to a standard or to an HP-LC diet. Glucose and lipid metabolism was studied. The cardiovascular system was explored from the functional stage with Doppler-echography to the molecular stage (arterial reactivity, mRNA, immunohistochemistry). Young mice did not exhibit any significant metabolic modification, whereas adult mice presented marked glucose intolerance associated with an increase in resistin and triglyceride levels. These metabolic disturbances were responsible for cardiovascular damages only in adult mice, with decreased aortic distensibility and left ventricle dysfunction. These seemed to be the consequence of arterial dysfunctions. Mesenteric arteries were the worst affected with a major oxidative stress, whereas aorta function seemed to be maintained with an appreciable role of cyclooxygenase-2 to preserve endothelial function. This study highlights for the first time the age-dependent deleterious effects of an HP-LC diet on metabolism, with glucose intolerance and lipid disorders and vascular (especially microvessels) and cardiac functions. This work shows that HP-LC lead to equivalent cardiovascular alterations, as observed in very old age, and underlines the danger of such diet.

Seipin deficiency alters brown adipose tissue thermogenesis and insulin sensitivity in a non-cell autonomous mode

Loss-of-function mutations in BSCL2 are responsible for Berardinelli-Seip congenital lipodystrophy, a rare disorder characterized by near absence of adipose tissue associated with insulin resistance. Seipin-deficient (Bscl2−/−) mice display an almost total loss of white adipose tissue (WAT) with residual brown adipose tissue (BAT). Previous cellular studies have shown that seipin deficiency alters white adipocyte differentiation. In this study, we aimed to decipher the consequences of seipin deficiency in BAT. Using a brown adipocyte cell-line, we show that seipin knockdown had very little effect on adipocyte differentiation without affecting insulin sensitivity and oxygen consumption. However, when submitted to cold acclimation or chronic β3 agonist treatment, Bscl2−/− mice displayed altered thermogenic capacity, despite several signs of BAT remodeling. Under cold activation, Bscl2−/− mice were able to maintain their body temperature when fed ad libitum, but not under short fasting. At control temperature (i.e. 21 °C), fasting worsened Bscl2−/− BAT properties. Finally, Bscl2−/− BAT displayed obvious signs of insulin resistance. Our results in these lipodystrophic mice strongly suggest that BAT activity relies on WAT as an energetic substrate provider and adipokine-producing organ. Therefore, the WAT/BAT dialogue is a key component of BAT integrity in guaranteeing its response to insulin and cold-activated adrenergic signals.

Techniques of Sleeve Gastrectomy and Modified Roux-en-Y Gastric Bypass in Mice

Obesity is a major public health issue, with a prevalence of 4 to 28% for men and 6.2 to 36.5% for women in Europe (from 2003 to 2008). Morbid obesity is frequently associated with metabolic complications, such as type 2 diabetes, hypertension, and dyslipidemia, reducing life expectancy and quality. In the absence of any effective noninvasive treatments, bariatric surgery is a valuable therapeutic option for patients with morbid obesity (body mass index (BMI) >40 kg/m2), leading to long-term, sustained weight loss and improvements in metabolic complications. However, the underlying cellular and molecular mechanisms sustaining the beneficial effects of bariatric surgery are not yet fully understood. Due to the numerous genetically-modified strains available, the mouse model is the most convenient animal model to explore the molecular mechanisms behind the pleiotropic beneficial effects of bariatric surgeries. Here, we detailed the optimized healthcare methods and surgical protocols in mice for the two most widely-used bariatric surgeries: the sleeve gastrectomy and the modified Roux-en-Y gastric bypass. Deciphering the molecular mechanisms underlying the therapeutic effects of bariatric surgeries offers the promise of identifying new therapeutics targets.

FGF21 Improves the Adipocyte Dysfunction Related to Seipin Deficiency.

Fibroblast growth factor 21 (FGF21) was shown to improve metabolic homeostasis, at least partly by controlling white adipocyte profile and adiponectin secretion. Here, we studied its effect on adipocyte dysfunction in the context of Berardinelli-Seip congenital lipodystrophy (BSCL) linked to seipin deficiency. Bscl2−/− mice displayed a progressive adipose tissue loss with aging as evidenced by the altered profile of residual fat pads and the decrease in adiponectin plasma levels in 12- vs. 4-week-old animals. Aiming to prevent this impairment, we treated 6-week-old Bscl2−/− mice with an FGF21 analog (LY2405319) for a period of 28 days. FGF21 treatment increased adiponectin plasma levels and normalized insulin sensitivity in Bscl2−/− mice by improving the white adipose tissue gene expression pattern. To further decipher the molecular pathways altered by seipin deficiency in mature adipocytes, we developed a unique inducible seipin knockdown cell line (SKD). SKD showed chronic activation of the p38 MAPK pathway associated with apoptotic cell death. Interestingly, FGF21 treatment exerted an antistress effect on SKD cells, reducing p38 MAPK phosphorylation and limiting mature adipocyte loss. Our data demonstrate that FGF21 treatment improves the metabolic profile of Bscl2−/− lipodystrophic mice, partly by improving mature adipocyte maintenance through suppression of cellular stress via inhibition of p38 MAPK activity.

Roux-en-Y gastric bypass reduces plasma cholesterol in diet-induced obese mice by affecting trans-intestinal cholesterol excretion and intestinal cholesterol absorption

Objective:Bariatric surgery appears as the most efficient therapeutic alternative in morbidly obese patients. In addition to its efficiency to decrease body weight, it also improves metabolic complications associated to morbid obesity, including dyslipidemia. Although the cholesterol-lowering effect varies with the bariatric procedures, the underlying molecular mechanisms remain poorly defined. This study aims to assess the consequence of both restrictive (sleeve gastrectomy; SG) and malabsorptive (Roux-en-Y gastric bypass; RYGB) procedures on cholesterol metabolism in mice.Subjects:Ten-week-old C57BL6/J males were fed with a high-fat diet for 8–14 weeks before sleeve or RYGB surgery.Results:SG has a modest and transient effect on plasma cholesterol levels, linked to a reduction in food intake. In contrast, modified RYGB led to a sustained ≈35% reduction in plasma cholesterol concentrations with a drastic increase in fecal cholesterol output. Mechanistically, RYGB exerts a synergystic effect on cholesterol metabolism by inducing the trans-intestinal cholesterol efflux and reducing the intestinal cholesterol absorption.Conclusions:In mice, RYGB, but not sleeve, strongly favors plasma cholesterol elimination by concomitantly increasing trans-intestinal cholesterol excretion and by decreasing intestinal cholesterol absorption. Our models open new perspective for deciphering the hypocholesterolemic effects of bariatric procedures.