Role of Sphingosine Kinase 1 in Glucolipotoxicity-Induced Early Activation of Autophagy in INS-1 Pancreatic ß Cells.

Insulin-producing pancreatic ß cells play a crucial role in the regulation of glucose homeostasis, and their failure is a key event for diabetes development. Prolonged exposure to palmitate in the presence of elevated glucose levels, termed gluco-lipotoxicity, is known to induce ß cell apoptosis. Autophagy has been proposed to be regulated by gluco-lipotoxicity in order to favor ß cell survival. However, the role of palmitate metabolism in gluco-lipotoxcity-induced autophagy is presently unknown. We therefore treated INS-1 cells for 6 and 24 h with palmitate in the presence of low and high glucose concentrations and then monitored autophagy. Gluco-lipotoxicity induces accumulation of LC3-II levels in INS-1 at 6 h which returns to basal levels at 24 h. Using the RFP-GFP-LC3 probe, gluco-lipotoxicity increased both autophagosomes and autolysosmes structures, reflecting early stimulation of an autophagy flux. Triacsin C, a potent inhibitor of the long fatty acid acetyl-coA synthase, completely prevents LC3-II formation and recruitment to autophagosomes, suggesting that autophagic response requires palmitate metabolism. In contrast, etomoxir and bromo-palmitate, inhibitors of fatty acid mitochondrial ß-oxidation, are unable to prevent gluco-lipotoxicity-induced LC3-II accumulation and recruitment to autophagosomes. Moreover, bromo-palmitate and etomoxir potentiate palmitate autophagic response. Even if gluco-lipotoxicity raised ceramide levels in INS-1 cells, ceramide synthase 4 overexpression does not potentiate LC3-II accumulation. Gluco-lipotoxicity also still stimulates an autophagic flux in the presence of an ER stress repressor. Finally, selective inhibition of sphingosine kinase 1 (SphK1) activity precludes gluco-lipotoxicity to induce LC3-II accumulation. Moreover, SphK1 overexpression potentiates autophagic flux induced by gluco-lipotxicity. Altogether, our results indicate that early activation of autophagy by gluco-lipotoxicity is mediated by SphK1, which plays a protective role in ß cells.

[Endoplasmic reticulum stress: from physiology to pathogenesis of type 2 diabetes]. / Le stress du réticulum endoplasmique : de la physiologie à la pathogenèse du diabète de type 2.

Type 2 diabetes is characterized by a dysfunction of pancreatic ß cells producing insulin and by impaired insulin responses in liver and skeletal muscle. This dysregulation of insulin secretion and action leads to chronic hyperglycaemia. The main causes that have been proposed to explain the pathogenesis of type 2 diabetes are lipotoxicity, glucotoxicity, oxidative stress and inflammation. Interestingly, these alterations converge towards the activation of a cellular pathway called "Unfolded Protein Response" which is set up in ß cells and insulin-sensitive tissues. This cellular pathway is central to the pathogenesis of type 2 diabetes and emerges as an important therapeutic target in the treatment of this disease.

Hypothalamic AgRP-neurons control peripheral substrate utilization and nutrient partitioning.

Obesity-related diseases such as diabetes and dyslipidemia result from metabolic alterations including the defective conversion, storage and utilization of nutrients, but the central mechanisms that regulate this process of nutrient partitioning remain elusive. As positive regulators of feeding behaviour, agouti-related protein (AgRP) producing neurons are indispensible for the hypothalamic integration of energy balance. Here, we demonstrate a role for AgRP-neurons in the control of nutrient partitioning. We report that ablation of AgRP-neurons leads to a change in autonomic output onto liver, muscle and pancreas affecting the relative balance between lipids and carbohydrates metabolism. As a consequence, mice lacking AgRP-neurons become obese and hyperinsulinemic on regular chow but display reduced body weight gain and paradoxical improvement in glucose tolerance on high-fat diet. These results provide a direct demonstration of a role for AgRP-neurons in the coordination of efferent organ activity and nutrient partitioning, providing a mechanistic link between obesity and obesity-related disorders.

Regulation of hepatic mitochondrial metabolism in response to a high fat diet: a longitudinal study in rats

Mitochondrial dysfunctions have been detected in non-alcoholic steatohepatitis, but less information exists regarding adaptation of mitochondrial function during the initiation of hepatic steatosis. This study aimed to determine in rat liver the sequence of mitochondrial and metabolic adaptations occurring during the first 8 weeks of a moderate high fat diet (HFD). Sprague-Dawley rats were fed a HFD during 2, 4, and 8 weeks. Mitochondrial oxygen consumption, respiratory chain complexes activity, and oxidative phosphorylation efficiency were assessed in isolated liver mitochondria. Gene expression related to fat metabolism and mitochondrial biogenesis were determined. Results were compared to data collected in a group of rats sacrificed before starting the HFD feeding. After 2 and 4 weeks of HFD, there was a development of fatty liver and a concomitant increase the expression of mitochondrial glycerol-3-phosphate acyltransferase (mtGPAT) and peroxisome proliferator-activated receptor Gammma. Higher serum Beta-hydroxybutyrate levels and enhanced hepatic pyruvate dehydrogenase kinase 4 expression suggested increased fatty acid oxidation. However, mitochondrial respiration and respiratory chain activity were normal. After 8 weeks of HFD, lower accumulation of liver triglycerides was associated with reduced expression of mtGPAT. At this time, oxygen consumption with palmitoyl-L-carnitine was decreased whereas oxidative phosphorylation efficiency (ATP/O) with succinate was enhanced. Hepatic levels of mtDNA were unchanged whatever the time points. This longitudinal study in rats fed a HFD showed that hepatic lipid homeostasis and mitochondrial function can adapt to face the increase in fatty acid availability (AU)

New insights into ER stress-induced insulin resistance.

Insulin resistance is a major characteristic of obesity and type 2 diabetes (T2DM). During the last decade, endoplasmic reticulum (ER) stress has emerged as a new player in this field and a considerable number of recent studies have pointed out its role in the onset of insulin resistance (IR). ER stress appears to act directly as a negative modulator of the insulin signaling pathway but also indirectly by promoting lipid accumulation. This review aims to summarize and decipher the abundant new literature concerning the emerging and multifaceted involvement of ER stress in the development of metabolic dysfunctions in insulin target tissues.

Regulation of hepatic mitochondrial metabolism in response to a high fat diet: a longitudinal study in rats.

Mitochondrial dysfunctions have been detected in non-alcoholic steatohepatitis, but less information exists regarding adaptation of mitochondrial function during the initiation of hepatic steatosis. This study aimed to determine in rat liver the sequence of mitochondrial and metabolic adaptations occurring during the first 8 weeks of a moderate high fat diet (HFD). Sprague-Dawley rats were fed a HFD during 2, 4, and 8 weeks. Mitochondrial oxygen consumption, respiratory chain complexes activity, and oxidative phosphorylation efficiency were assessed in isolated liver mitochondria. Gene expression related to fat metabolism and mitochondrial biogenesis were determined. Results were compared to data collected in a group of rats sacrificed before starting the HFD feeding. After 2 and 4 weeks of HFD, there was a development of fatty liver and a concomitant increase the expression of mitochondrial glycerol-3-phosphate acyltransferase (mtGPAT) and peroxisome proliferator-activated receptor γ. Higher serum ß-hydroxybutyrate levels and enhanced hepatic pyruvate dehydrogenase kinase 4 expression suggested increased fatty acid oxidation. However, mitochondrial respiration and respiratory chain activity were normal. After 8 weeks of HFD, lower accumulation of liver triglycerides was associated with reduced expression of mtGPAT. At this time, oxygen consumption with palmitoyl-L: -carnitine was decreased whereas oxidative phosphorylation efficiency (ATP/O) with succinate was enhanced. Hepatic levels of mtDNA were unchanged whatever the time points. This longitudinal study in rats fed a HFD showed that hepatic lipid homeostasis and mitochondrial function can adapt to face the increase in fatty acid availability.

Dynamic regulation of mitochondrial network and oxidative functions during 3T3-L1 fat cell differentiation

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Mitochondria have been shown to be impaired in insulin resistance-related diseases but have not been extensively studied during the first steps of adipose cell development. This study was designed to determine the sequence of changes of the mitochondrial network and function during the first days of adipogenesis. 3T3-L1 preadipocytes were differentiated into adipocytes without using glitazone compounds. At days 0, 3, 6, 9, and 12, mitochondrial network imaging, mitochondrial oxygen consumption, membrane potential, and oxidative phosphorylation efficiency were assessed in permeabilized cells. Gene and protein expressions related to fatty acid metabolism and mitochondrial network were also determined. Compared to preadipocytes (day 0), new adipocytes (days 6 and 9) displayed profound (..) (AU)

Dynamic regulation of mitochondrial network and oxidative functions during 3T3-L1 fat cell differentiation.

Mitochondria have been shown to be impaired in insulin resistance-related diseases but have not been extensively studied during the first steps of adipose cell development. This study was designed to determine the sequence of changes of the mitochondrial network and function during the first days of adipogenesis. 3T3-L1 preadipocytes were differentiated into adipocytes without using glitazone compounds. At days 0, 3, 6, 9, and 12, mitochondrial network imaging, mitochondrial oxygen consumption, membrane potential, and oxidative phosphorylation efficiency were assessed in permeabilized cells. Gene and protein expressions related to fatty acid metabolism and mitochondrial network were also determined. Compared to preadipocytes (day 0), new adipocytes (days 6 and 9) displayed profound changes of their mitochondrial network that underwent fragmentation and redistribution around lipid droplets. Drp1 and mitofusin 2 displayed a progressive increase in their gene expression and protein content during the first 9 days of differentiation. In parallel with the mitochondrial network redistribution, mitochondria switched to uncoupled respiration with a tendency towards decreased membrane potential, with no variation of mtTFA and NRF1 gene expression. The expression of PGC1α and NRF2 genes and genes involved in lipid oxidation (UCP2, CD36, and CPT1) was increased. Reactive oxygen species (ROS) production displayed a nadir at day 6 with a concomitant increase in antioxidant enzyme gene expression. This 3T3-L1-based in vitro model of adipogenesis showed that mitochondria adapted to the increased number of lipid droplets by network redistribution and uncoupling respiration. The timing and regulation of lipid oxidation-associated ROS production appeared to play an important role in these changes.

Endoplasmic reticulum stress: a new actor in the development of hepatic steatosis.

PURPOSE OF REVIEW: To examine the role of endoplasmic reticulum stress in the regulation of hepatic lipid metabolism and its contribution to the development of hepatic steatosis. RECENT FINDINGS: Endoplasmic reticulum stress activation has been reported in most models of hepatic steatosis in rodents and humans and its contribution to hepatic fat deposition has been recently documented. The main metabolic pathway affected by endoplasmic reticulum stress is lipogenesis. Endoplasmic reticulum stress activates the proteolytic cleavage of the lipogenic transcription factor sterol regulatory element binding protein-1c leading to the induction of lipogenic enzyme expression. A role for X box-binding protein 1, an endoplasmic reticulum stress-activated transcription factor, has also recently emerged. Endoplasmic reticulum stress, by inhibiting apoB100 secretion, has associated with impaired VLDL secretion. In rodents, treatments with molecular or chemical chaperones that reduce endoplasmic reticulum stress markers have fully demonstrated their efficiency in the treatment of hepatic steatosis. SUMMARY: Manipulating endoplasmic reticulum stress pathway yields encouraging results for the treatment of hepatic steatosis in rodents. However, activation of unfolded protein response is a physiological mechanism, which is particularly important for secretory cells such as hepatocytes and the long-term consequences of such treatments should be cautiously evaluated.

Effects of the cannabinoid CB1 antagonist rimonabant on hepatic mitochondrial function in rats fed a high-fat diet.

The aim of this study was to investigate the effect of rimonabant treatment on hepatic mitochondrial function in rats fed a high-fat diet. Sprague-Dawley rats fed a high-fat diet (35% lard) for 13 wk were treated with rimonabant (10 mg·kg(-1)·day(-1)) during the last 3 wk and matched with pair-fed controls. Oxygen consumption with various substrates, mitochondrial enzyme activities on isolated liver mitochondria, and mitochondrial DNA quantity were determined. Body weight and fat mass were decreased in rats treated with rimonabant compared with pair-fed controls. Moreover, the serum adiponectin level was increased with rimonabant. Hepatic triglyceride content was increased, while serum triglycerides were decreased. An increase of mitochondrial respiration was observed in rats treated with rimonabant. The increase of mitochondrial respiration with palmitoyl-CoA compared with respiration with palmitoyl-l-carnitine stating that the entry of fatty acids into mitochondria via carnitine palmitoyltransferase I was increased in rats treated with rimonabant. Moreover, rimonabant treatment led to a reduction in the enzymatic activity of ATP synthase, whereas the quantity of mitochondrial DNA and the activity of citrate synthase remained unchanged. To summarize, rimonabant treatment leads to an improvement of hepatic mitochondrial function by increasing substrate oxidation and fatty acid entry into mitochondria for the ß-oxidation pathway and by increasing proton leak. However, this increase of mitochondrial oxidation is regulated by a decrease of ATP synthase activity in order to have only ATP required for the cell function.

Fatty liver and insulin resistance in obese Zucker rats: no role for mitochondrial dysfunction.

The relationship between insulin resistance and mitochondrial function is of increasing interest. Studies looking for such interactions are usually made in muscle and only a few studies have been done in liver, which is known to be a crucial partner in whole body insulin action. Recent studies have revealed a similar mechanism to that of muscle for fat-induced insulin resistance in liver. However, the exact mechanism of lipid metabolites accumulation in liver leading to insulin resistance is far from being elucidated. One of the hypothetical mechanisms for liver steatosis development is an impairment of mitochondrial function. We examined mitochondrial function in fatty liver and insulin resistance state using isolated mitochondria from obese Zucker rats. We determined the relationship between ATP synthesis and oxygen consumption as well as the relationship between mitochondrial membrane potential and oxygen consumption. In order to evaluate the quantity of mitochondria and the oxidative capacity we measured citrate synthase and cytochrome c oxidase activities. Results showed that despite significant fatty liver and hyperinsulinemia, isolated liver mitochondria from obese Zucker rats display no difference in oxygen consumption, ATP synthesis, and membrane potential compared with lean Zucker rats. There was no difference in citrate synthase and cytochrome c oxidase activities between obese and lean Zucker rats in isolated mitochondria as well as in liver homogenate, indicating a similar relative amount of hepatic mitochondria and a similar oxidative capacity. Adiponectin, which is involved in bioenergetic homeostasis, was increased two-fold in obese Zucker rats despite insulin resistance. In conclusion, isolated liver mitochondria from lean and obese insulin-resistant Zucker rats showed strictly the same mitochondrial function. It remains to be elucidated whether adiponectin increase is involved in these results.