GOLM1 depletion modifies cellular sphingolipid metabolism and adversely affects cell growth.

Golgi membrane protein 1 (GOLM1) is a Golgi-resident type 2 transmembrane protein known to be overexpressed in several cancers, including hepatocellular carcinoma (HCC), as well as in viral infections. However, the role of GOLM1 in lipid metabolism remains enigmatic. In this study, we employed siRNA-mediated GOLM1 depletion in Huh-7 HCC cells to study the role of GOLM1 in lipid metabolism. Mass spectrometric lipidomic analysis in GOLM1 knockdown cells showed an aberrant accumulation of sphingolipids, such as ceramides, hexosylceramides, dihexosylceramides, sphinganine, sphingosine, and ceramide phosphate, along with cholesteryl esters. Furthermore, we observed a reduction in phosphatidylethanolamines and lysophosphatidylethanolamines. In addition, Seahorse extracellular flux analysis indicated a reduction in mitochondrial oxygen consumption rate upon GOLM1 depletion. Finally, alterations in Golgi structure and distribution were observed both by electron microscopy imaging and immunofluorescence microscopy analysis. Importantly, we found that GOLM1 depletion also affected cell proliferation and cell cycle progression in Huh-7 HCC cells. The Golgi structural defects induced by GOLM1 reduction might potentially affect the trafficking of proteins and lipids leading to distorted intracellular lipid homeostasis, which may result in organelle dysfunction and altered cell growth. In conclusion, we demonstrate that GOLM1 depletion affects sphingolipid metabolism, mitochondrial function, Golgi structure, and proliferation of HCC cells.

Compounds that modulate AMPK activity and hepatic steatosis impact the biosynthesis of microRNAs required to maintain lipid homeostasis in hepatocytes.

BACKGROUND: While the impact of metformin in hepatocytes leads to fatty acid (FA) oxidation and decreased lipogenesis, hepatic microRNAs (miRNAs) have been associated with fat overload and impaired metabolism, contributing to the pathogenesis of non-alcoholic fatty liver disease (NAFLD). METHODS: We investigated the expression of hundreds of miRNAs in primary hepatocytes challenged by compounds modulating steatosis, palmitic acid and compound C (as inducers), and metformin (as an inhibitor). Then, additional hepatocyte and rodent models were evaluated, together with transient mimic miRNAs transfection, lipid droplet staining, thin-layer chromatography, quantitative lipidomes, and mitochondrial activity, while human samples outlined the translational significance of this work. FINDINGS: Our results show that treatments triggering fat accumulation and AMPK disruption may compromise the biosynthesis of hepatic miRNAs, while the knockdown of the miRNA-processing enzyme DICER in human hepatocytes exhibited increased lipid deposition. In this context, the ectopic recovery of miR-30b and miR-30c led to significant changes in genes related to FA metabolism, consistent reduction of ceramides, higher mitochondrial activity, and enabled ß-oxidation, redirecting FA metabolism from energy storage to expenditure. INTERPRETATION: Current findings unravel the biosynthesis of hepatic miR-30b and miR-30c in tackling inadequate FA accumulation, offering a potential avenue for the treatment of NAFLD. FUNDING: Instituto de Salud Carlos III (ISCIII), Govern de la Generalitat (PERIS2016), Associació Catalana de Diabetis (ACD), Sociedad Española de Diabetes (SED), Fondo Europeo de Desarrollo Regional (FEDER), Xunta de Galicia, Ministerio de Economía y Competitividad (MINECO), "La Caixa" Foundation, and CIBER de la Fisiopatología de la Obesidad y Nutrición (CIBEROBN).

Depletion of TM6SF2 disturbs membrane lipid composition and dynamics in HuH7 hepatoma cells.

A polymorphism of TM6SF2 associates with hepatic lipid accumulation and reduction of triacylglycerol (TAG) secretion, but the function of the encoded protein has remained enigmatic. We studied the effect of stable TM6SF2 knock-down on the lipid content and composition, mitochondrial fatty acid oxidation and organelle structure of HuH7 hepatoma cells. Knock-down of TM6SF2 resulted in intracellular accumulation of TAGs, cholesterol esters, phosphatidylcholine (PC) and phosphatidylethanolamine. In all of these lipid classes, polyunsaturated lipid species were significantly reduced while saturated and monounsaturated species increased their proportions. The PCs encountered relative and absolute arachidonic acid (AA, 20:4n-6) depletion, and AA was also reduced in the total cellular fatty acid pool. Synthesis and turnover of the hepatocellular glycerolipids was enhanced. The TM6SF2 knock-down cells secreted lipoprotein-like particles with a smaller diameter than in the controls, and more lysosome/endosome structures appeared in the knock-down cells. The mitochondrial capacity for palmitate oxidation was significantly reduced. These observations provide novel clues to TM6SF2 function and raise altered mebrane lipid composition and dynamics among the mechanism(s) by which the protein deficiency disturbs hepatic TAG secretion.

MicroRNA-192* impairs adipocyte triglyceride storage.

We investigated the expression of miR-192* (miR-192-3p) in the visceral adipose tissue (VAT) of obese subjects and its function in cultured human adipocytes. This miRNA is a 3' arm derived from the same pre-miRNA as miR-192 (miR-192-5p) implicated in type 2 diabetes, liver disease and cancers, and is predicted to target key genes in lipid metabolism. In morbidly obese subjects undergoing bariatric surgery preceded by a very low calorie diet, miR-192* in VAT correlated negatively (r=-0.387; p=0.046) with serum triglyceride (TG) and positively with high-density lipoprotein (HDL) concentration (r=0.396; p=0.041). In a less obese patient cohort, the miRNA correlated negatively with the body mass index (r=-0.537; p=0.026). To characterize the function of miR-192*, we overexpressed it in cultured adipocytes and analyzed the expression of adipogenic differentiation markers as well as cellular TG content. Reduced TG and expression of the adipocyte marker proteins aP2 (adipocyte protein 2) and perilipin 1 were observed. The function of miR-192* was further investigated by transcriptomic profiling of adipocytes expressing this miRNA, revealing impacts on key lipogenic genes. A number of the mRNA alterations were validated by qPCR. Western analysis confirmed a marked reduction of the lipogenic enzyme SCD (stearoyl coenzyme A desaturase-1), the fatty aldehyde dehydrogenase ALDH3A2 (aldehyde dehydrogenase 3 family member A2) and the high-density lipoprotein receptor SCARB1 (scavenger receptor B, type I). SCD and ALDH3A2 were demonstrated to be direct targets of miR-192*. To conclude, the present data identify miR-192* as a novel controller of adipocyte differentiation and lipid homeostasis.

Regulation of Angiopoietin-Like Proteins (ANGPTLs) 3 and 8 by Insulin.

OBJECTIVE: Circulating ANGPTL8 has recently been used as a marker of insulin action. We studied expression and insulin regulation of ANGPTL8 and ANGPTL3 in vivo and in vitro. DESIGN AND METHODS: Expression of ANGPTL8 and ANGPTL3 was studied in 34 paired samples of human liver and adipose tissue. Effects of insulin on 1) plasma concentrations and adipose tissue expression of ANGPTL8 and ANGPTL3 (in vivo 6-h euglycemic hyperinsulinemia; n = 18), and 2) ANGPTL8 and ANGPTL3 gene and protein expression in immortalized human hepatocytes (IHH) and adipocytes were measured. Effect of ANGPTL3 on secretion of ANGPTL8 in cells stably overexpressing ANGPTL3, -8, or both was determined. RESULTS: ANGPTL3 was only expressed in the liver, whereas ANGPTL8 was expressed in both tissues. In vivo hyperinsulinemia significantly decreased both plasma ANGPTL8 and ANGPTL3 at 3 and 6 hours. Insulin increased ANGPTL8 expression in human adipose tissue 14- and 18-fold at 3 and 6 hours and ANGPTL8 was the most insulin-responsive transcript on microarray. Insulin also increased ANPGTL8 in cultured adipocytes and IHH but the protein mainly remained intracellular. In vitro in IHH, insulin decreased ANGPTL3 gene expression and secretion of ANGPTL3 into growth medium. Overexpression of ANGPTL8 in CHO cells did not result in its release into culture medium while abundant secretion occurred in cells co-expressing ANGPTL3 and -8. CONCLUSIONS: Insulin decreases plasma ANGPTL3 by decreasing ANGPTL3 expression in the liver. Insulin markedly increases ANGPTL8 in adipose tissue and the liver but not in plasma. These data show that measurement of plasma ANGPTL3 but not -8 reflects insulin action in target tissues.