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SummaryAutophagy is a homeostatic degradative process for cell components that enables stress resilience and can determine cellular fate and function. However, lipid sources for the rapid membrane expansions of autophagosomes, the workhorses of autophagy, are poorly understood. Here, we identify de novo lipogenesis (DNL) as a critical source of fatty acids (FA) to fuel autophagosome dynamics in adipocytes. Adipocyte fatty acid synthase (Fasn) deficiency markedly impairs autophagy, evident by autophagosome accumulation, and severely compromises degradation of the autophagic substrate p62. Autophagy dependence on FA produced by Fasn is not fully alleviated by exogenous FA in cultured adipocytes even though lipid droplet size is restored. Imaging studies reveal that Fasn colocalizes with nascent autophagosomes, while loss of Fasn decreases certain membrane phosphoinositides known to be required for autophagosome assembly. Together, our studies highlight a newly appreciated function for adipocyte DNL in autophagosome membrane formation and provide evidence that localized FA synthesis contributes to autophagosome dynamics.

Hepatic steatosis associated with high fat diets, obesity and type 2 diabetes is thought to be the major driver of severe liver inflammation, fibrosis, and cirrhosis. Cytosolic acetyl-coenzyme A (AcCoA), a central metabolite and substrate for de novo lipogenesis (DNL), is produced from citrate by ATP-citrate lyase (ACLY) and from acetate through AcCoA synthase short chain family member 2 (ACSS2). However, the relative contributions of these two enzymes to hepatic AcCoA pools and DNL rates in response to high fat feeding is unknown. We report here that hepatocyte-selective depletion of either ACSS2 or ACLY caused similar 50% decreases in liver AcCoA levels in obese mice, showing that both pathways contribute to generation of this DNL substrate. Unexpectedly however, the hepatocyte ACLY depletion in obese mice paradoxically increased total DNL flux measured by D2O incorporation into palmitate, while in contrast ACSS2 depletion had no effect. The increase in liver DNL upon ACLY depletion was associated with increased expression of nuclear sterol regulatory element-binding protein 1c (SREBP1c) and of its target DNL enzymes. This upregulated DNL enzyme expression explains the increased rate of palmitate synthesis in ACLY depleted livers. Furthermore, this increased flux through DNL may also contribute to the observed depletion of AcCoA levels due to its increased conversion to Malonyl CoA (MalCoA) and palmitate. Together, these data indicate that in HFD fed obese mice, hepatic DNL is not limited by its immediate substrates AcCoA or MalCoA, but rather by activities of DNL enzymes.

Leptin is a potent endocrine hormone produced by adipose tissue and regulates a broad range of metabolism including glucose and lipid metabolism, with and without insulin. It is evident that central leptin signaling can lower hyperglycemia in insulin-deficient rodents via multiple mechanisms including restoration of dyslipidemia. However, the specific neurons that regulate these glucose-lowering and anti-dyslipidemia effects of leptin remain unidentified. Here we report that leptin receptors (LEPRs) in neurons expressing Cre recombinase driven by a short fragment of a promoter region ofIns2gene (RIP-Cre25Mgnneurons) are required for central leptin signaling to reverse hyperglycemia and dyslipidemia in insulin-deficient mice. Ablation of LEPRs in RIP-Cre25Mgnneurons completely blocks glucose-lowering effects of leptin in insulin-deficient mice. Further investigations reveal that insulin-deficient mice lacking LEPRs in RIP-Cre25Mgnneurons (RIP-CreΔLEPRmice) exhibit greater lipid levels in blood and liver compared to wild-type controls, and that leptin injection into the brain does not suppress dyslipidemia in insulin-deficient RIP-CreΔLEPRmice. Leptin administration into the brain combined with acipimox, which lowers blood lipids by suppressing triglyceride lipase activity, can restore normal glycemia in insulin-deficient RIP-CreΔLEPRmice, suggesting that excess circulating lipids are a driving-force of hyperglycemia in insulin-deficient RIP-CreΔLEPRmice. Collectively, our data demonstrate that LEPRs in RIP-Cre25Mgnneurons significantly contribute to glucose-lowering effects of leptin in an insulin-independent manner by suppression of dyslipidemia.

Mitochondrial dysfunction includes heritable diseases, acquired pathologies, and age-related declines in health. Szeto-Schiller (SS) peptides comprise a class of amphipathic tetrapeptides that have demonstrated efficacy in treating a wide array of mitochondrial disorders, and are believed to target mitochondrial membranes due to their enrichment in the anionic phospholipid cardiolipin (CL). However, little is known regarding how SS peptides interact with or alter the physical properties of lipid bilayers. In this study, we have analyzed the interactions of the lead compound SS-31 (Elamipretide) with model and mitochondrial membranes using biophysical and computational approaches. Our results show that this polybasic peptide partitions into the membrane interfacial region with affinity and binding density that are directly related to surface charge. SS-31 binding does not destabilize lamellar bilayers even at the highest binding concentrations; however, it does cause saturable alterations in lipid packing. Most notably, SS-31 modulates the surface electrostatic properties of model and mitochondrial membranes, which could play a significant role in the mitoprotective properties of this compound. As a proof of concept, we show that SS-31 alters ion distribution at the membrane interface with implications for maintaining mitochondrial membranes subject to divalent cation (calcium) stress. Taken together, these results support a mechanism of action in which SS peptides interact with lipid bilayers and alter the biophysical (primarily electrostatic) properties of mitochondrial membranes as their primary mechanism of action. Understanding this molecular mechanism is key to the development of future compound variants with enhanced efficacy.SignificanceSzeto-Schiller (SS) peptides are among the most promising therapeutic compounds for mitochondrial dysfunction. However, the molecular target(s) and the mechanism of action of SS peptides are poorly understood. In this study, we evaluate the interaction of the lead compound SS-31 (Elamipretide) with mitochondrial and synthetic model membranes using a host of biophysical techniques. Our results show that SS-31 membrane interaction is driven largely by the negative surface charge of mitochondrial membranes and that SS-31 alters lipid bilayer properties, most notably electrostatics at the membrane interface. This work supports a mechanism in which SS peptides act on a key physical property of mitochondrial membranes rather than with a specific protein complex, consistent with the exceptionally broad therapeutic efficacy of these compounds.