Endoplasmic reticulum-mitochondria miscommunication is an early and causal trigger of hepatic insulin resistance and steatosis.

BACKGROUND & AIMS: Hepatic insulin resistance in obesity and type 2 diabetes was recently associated with endoplasmic reticulum (ER)-mitochondria miscommunication. These contact sites (mitochondria-associated membranes: MAMs) are highly dynamic and involved in many functions; however, whether MAM dysfunction plays a causal role in hepatic insulin resistance and steatosis is not clear. Thus, we aimed to determine whether and how organelle miscommunication plays a role in the onset and progression of hepatic metabolic impairment. METHODS: We analyzed hepatic ER-mitochondria interactions and calcium exchange in a time-dependent and reversible manner in mice with diet-induced obesity. Additionally, we used recombinant adenovirus to express a specific organelle spacer or linker in mouse livers, to determine the causal impact of MAM dysfunction on hepatic metabolic alterations. RESULTS: Disruption of ER-mitochondria interactions and calcium exchange is an early event preceding hepatic insulin resistance and steatosis in mice with diet-induced obesity. Interestingly, an 8-week reversal diet concomitantly reversed hepatic organelle miscommunication and insulin resistance in obese mice. Mechanistically, disrupting structural and functional ER-mitochondria interactions through the hepatic overexpression of the organelle spacer FATE1 was sufficient to impair hepatic insulin action and glucose homeostasis. In addition, FATE1-mediated organelle miscommunication disrupted lipid-related mitochondrial oxidative metabolism and induced hepatic steatosis. Conversely, reinforcement of ER-mitochondria interactions through hepatic expression of a synthetic linker prevented diet-induced glucose intolerance after 4 weeks' overnutrition. Importantly, ER-mitochondria miscommunication was confirmed in the liver of obese patients with type 2 diabetes, and correlated with glycemia, HbA1c and HOMA-IR index. CONCLUSIONS: ER-mitochondria miscommunication is an early causal trigger of hepatic insulin resistance and steatosis, and can be reversed by switching to a healthy diet. Thus, targeting MAMs could help to restore metabolic homeostasis. LAY SUMMARY: The literature suggests that interactions between the endoplasmic reticulum and mitochondria could play a role in hepatic insulin resistance and steatosis during chronic obesity. In the present study, we reappraised the time-dependent regulation of hepatic endoplasmic reticulum-mitochondria interactions and calcium exchange, investigating reversibility and causality, in mice with diet-induced obesity. We also assessed the relevance of our findings to humans. We show that organelle miscommunication is an early causal trigger of hepatic insulin resistance and steatosis that can be improved by nutritional strategies.

Loss and gain of function of Grp75 or mitofusin 2 distinctly alter cholesterol metabolism, but all promote triglyceride accumulation in hepatocytes.

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. 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.

Regulation of Mitochondria-Associated Membranes (MAMs) by NO/sGC/PKG Participates in the Control of Hepatic Insulin Response.

Under physiological conditions, nitric oxide (NO) produced by the endothelial NO synthase (eNOS) upregulates hepatic insulin sensitivity. Recently, contact sites between the endoplasmic reticulum and mitochondria named mitochondria-associated membranes (MAMs) emerged as a crucial hub for insulin signaling in the liver. As mitochondria are targets of NO, we explored whether NO regulates hepatic insulin sensitivity by targeting MAMs. In Huh7 cells, primary rat hepatocytes and mouse livers, enhancing NO concentration increased MAMs, whereas inhibiting eNOS decreased them. In vitro, those effects were prevented by inhibiting protein kinase G (PKG) and mimicked by activating soluble guanylate cyclase (sGC) and PKG. In agreement with the regulation of MAMs, increasing NO concentration improved insulin signaling, both in vitro and in vivo, while eNOS inhibition disrupted this response. Finally, inhibition of insulin signaling by wortmannin did not affect the impact of NO on MAMs, while experimental MAM disruption, using either targeted silencing of cyclophilin D or the overexpression of the organelle spacer fetal and adult testis-expressed 1 (FATE-1), significantly blunted the effects of NO on both MAMs and insulin response. Therefore, under physiological conditions, NO participates to the regulation of MAM integrity through the sGC/PKG pathway and concomitantly improves hepatic insulin sensitivity. Altogether, our data suggest that the induction of MAMs participate in the impact of NO on hepatocyte insulin response.

Disruption of Mitochondria-Associated Endoplasmic Reticulum Membrane (MAM) Integrity Contributes to Muscle Insulin Resistance in Mice and Humans.

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.

Mitochondria-associated endoplasmic reticulum membrane (MAM) integrity is required for insulin signaling and is implicated in hepatic insulin resistance.

Mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) are functional domains between both organelles involved in Ca(2+) exchange, through the voltage-dependent anion channel (VDAC)-1/glucose-regulated protein 75 (Grp75)/inositol 1,4,5-triphosphate receptor (IP3R)-1 complex, and regulating energy metabolism. Whereas mitochondrial dysfunction, ER stress, and altered Ca(2+) homeostasis are associated with altered insulin signaling, the implication of MAM dysfunctions in insulin resistance is unknown. Here we validated an approach based on in situ proximity ligation assay to detect and quantify VDAC1/IP3R1 and Grp75/IP3R1 interactions at the MAM interface. We demonstrated that MAM integrity is required for insulin signaling and that induction of MAM prevented palmitate-induced alterations of insulin signaling in HuH7 cells. Disruption of MAM integrity by genetic or pharmacological inhibition of the mitochondrial MAM protein, cyclophilin D (CypD), altered insulin signaling in mouse and human primary hepatocytes and treatment of CypD knockout mice with metformin improved both insulin sensitivity and MAM integrity. Furthermore, ER-mitochondria interactions are altered in liver of both ob/ob and diet-induced insulin-resistant mice and improved by rosiglitazone treatment in the latter. Finally, increasing organelle contacts by overexpressing CypD enhanced insulin action in primary hepatocytes of diabetic mice. Collectively, our data reveal a new role of MAM integrity in hepatic insulin action and resistance, providing a novel target for the modulation of insulin action.