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Abstract
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.
Highlights
Within the hepatocyte, mitochondria and the endoplasmic reticulum (ER) communicate through contact points, called mitochondria-associated ER membranes (MAMs), in order to exchange phospholipids and calcium and regulate cellular functions [1]
We confirmed by Western blotting that endothelial NO synthase (eNOS) was the main isoform expressed in the liver of male C57BL/6JOlaHsd mice, and that isoform of NOS (iNOS) was not significantly expressed (Figure S1A)
nitric oxide (NO) derived from iNOS was shown to induce combined alterations in mitochondria and ER, resulting in modified calcium fluxes between the two organelles [36]
Summary
Mitochondria and the endoplasmic reticulum (ER) communicate through contact points, called mitochondria-associated ER membranes (MAMs), in order to exchange phospholipids and calcium and regulate cellular functions [1]. The exact composition of MAMs is still unclear, key proteins participating in the MAM structure and function have been identified, such as inositol. We recently demonstrated that MAMs are major nutrient sensors in the liver, as they adapt to glucose availability in order to regulate mitochondrial dynamics and bioenergetics [3]. MAMs are presented as subcellular signaling platforms important for hepatic metabolism. Understanding key mechanisms regulating MAM integrity in the liver could help identify new strategies to modulate hepatic insulin sensitivity, which is a key lever to prevent metabolic disorders associated with obesity [6,7]
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