Central Acting Hsp10 Regulates Mitochondrial Function, Fatty Acid Metabolism, and Insulin Sensitivity in the Hypothalamus

Mitochondria are critical for hypothalamic function and regulators of metabolism. Hypothalamic mitochondrial dysfunction with decreased mitochondrial chaperone expression is present in type 2 diabetes (T2D). Recently, we demonstrated that a dysregulated mitochondrial stress response (MSR) with reduced chaperone expression in the hypothalamus is an early event in obesity development due to insufficient insulin signaling. Although insulin activates this response and improves metabolism, the metabolic impact of one of its members, the mitochondrial chaperone heat shock protein 10 (Hsp10), is unknown. Thus, we hypothesized that a reduction of Hsp10 in hypothalamic neurons will impair mitochondrial function and impact brain insulin action. Therefore, we investigated the role of chaperone Hsp10 by introducing a lentiviral-mediated Hsp10 knockdown (KD) in the hypothalamic cell line CLU-183 and in the arcuate nucleus (ARC) of C57BL/6N male mice. We analyzed mitochondrial function and insulin signaling utilizing qPCR, Western blot, XF96 Analyzer, immunohistochemistry, and microscopy techniques. We show that Hsp10 expression is reduced in T2D mice brains and regulated by leptin in vitro. Hsp10 KD in hypothalamic cells induced mitochondrial dysfunction with altered fatty acid metabolism and increased mitochondria-specific oxidative stress resulting in neuronal insulin resistance. Consequently, the reduction of Hsp10 in the ARC of C57BL/6N mice caused hypothalamic insulin resistance with acute liver insulin resistance.

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Abstract 330: Glycogen Synthase Kinase-3a Negatively Regulates Fatty Acid Metabolism During Lipid-induced Insulin Resistance

Circulation Research ◽

10.1161/res.113.suppl_1.a330 ◽

2013 ◽

Vol 113 (suppl_1) ◽

Author(s):

Michinari Nakamura ◽

Junichi Sadoshima

Keyword(s):

Insulin Resistance ◽

Fatty Acid ◽

Palmitic Acid ◽

Fatty Acid Metabolism ◽

Lipid Accumulation ◽

Acid Metabolism ◽

Glycogen Synthase ◽

Acid Oxidation ◽

Peroxisome Proliferator

Obesity and insulin resistance lead to ectopic lipid accumulation and impaired cardiac metabolism, resulting in cardiovascular diseases. Peroxisome proliferator-activated receptor α (PPARα) is highly expressed in the heart and serves as a key regulator of fatty acid metabolism. However, the underlying mechanisms responsible for the development of cardiac dysfunction in these pathologies are still poorly understood. GSK-3α was activated, as evidenced by a decrease in S21 phosphorylation, during insulin resistance with normoglycemia in the hearts of obese mice fed a high-fat diet and ob/ob mice. To evaluate the functional significance of GSK-3α upregulation with regard to metabolism, we applied 50 μM of BSA-conjugated palmitic acid to cardiomyocytes in vitro for three days. This intervention elicited ectopic lipid accumulation, as evaluated with Oil Red O staining, and a 2.0-fold activation of GSK-3α, similar to lipid-induced insulin resistance and dyslipidemia in the heart in vivo . In this condition, downregulation of GSK-3α with shRNA-GSK-3α in cardiomyocytes increased cell viability, ATP synthesis, and fatty acid oxidation, but not glycolysis. Downregulation of GSK-3α also increased the activity of PPRE-luciferase (1.5 fold, p<0.05) and mRNA expression of genes involved in fatty acid metabolism in response to palmitic acid, including Acox1 and Cpt1b . Overexpression of GSK-3α induced a rightward shift of the dose response curve where the activity of the PPARα reporter was plotted against the dose of WY14643, a PPARα agonist. GSK-3α, but not GSK-3β, directly interacted with and phosphorylated PPARα in vitro . Collectively, these results suggest that GSK-3α negatively regulates ligand-dependent activity of PPARα through phosphorylation of PPARα, thereby inhibiting fatty acid metabolism during lipid-induced insulin resistance. GSK-3α may be a novel therapeutic target for metabolic disorders.

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Lipid and glucose metabolism in hepatocyte cell lines and primary mouse hepatocytes: a comprehensive resource for in vitro studies of hepatic metabolism

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00365.2018 ◽

2019 ◽

Vol 316 (4) ◽

pp. E578-E589 ◽

Cited By ~ 15

Author(s):

Shilpa R. Nagarajan ◽

Moumita Paul-Heng ◽

James R. Krycer ◽

Daniel J. Fazakerley ◽

Alexandra F. Sharland ◽

...

Keyword(s):

Fatty Acid ◽

Glucose Metabolism ◽

Cell Lines ◽

Fatty Acid Metabolism ◽

Acid Metabolism ◽

Primary Hepatocytes ◽

Primary Mouse Hepatocytes ◽

Primary Mouse ◽

Mouse Hepatocytes

The liver is a critical tissue for maintaining glucose, fatty acid, and cholesterol homeostasis. Primary hepatocytes represent the gold standard for studying the mechanisms controlling hepatic glucose, lipid, and cholesterol metabolism in vitro. However, access to primary hepatocytes can be limiting, and therefore, other immortalized hepatocyte models are commonly used. Here, we describe substrate metabolism of cultured AML12, IHH, and PH5CH8 cells, hepatocellular carcinoma-derived HepG2s, and primary mouse hepatocytes (PMH) to identify which of these cell lines most accurately phenocopy PMH basal and insulin-stimulated metabolism. Insulin-stimulated glucose metabolism in PH5CH8 cells, and to a lesser extent AML12 cells, responded most similarly to PMH. Notably, glucose incorporation in HepG2 cells were 14-fold greater than PMH. The differences in glucose metabolic activity were not explained by differential protein expression of key regulators of these pathways, for example glycogen synthase and glycogen content. In contrast, fatty acid metabolism in IHH cells was the closest to PMHs, yet insulin-responsive fatty acid metabolism in AML12 and HepG2 cells was most similar to PMH. Finally, incorporation of acetate into intracellular-free cholesterol was comparable for all cells to PMH; however, insulin-stimulated glucose conversion into lipids and the incorporation of acetate into intracellular cholesterol esters were strikingly different between PMHs and all tested cell lines. In general, AML12 cells most closely phenocopied PMH in vitro energy metabolism. However, the cell line most representative of PMHs differed depending on the mode of metabolism being investigated, and so careful consideration is needed in model selection.

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