Abstract
Glycerol is taken up by human muscle in vivo and incorporated into lipids, but little is known about regulation of glycerol metabolism in this tissue. In this study, we have analyzed the role of glycerol kinase (GlK) in the regulation of glycerol metabolism in primary cultured human muscle cells. Isolated human muscle cells exhibited lower GlK activity than fresh muscle explants, but the activity in cultured cells was increased by exposure to insulin. [U-(14)C]Glycerol was incorporated into cellular phospholipids and triacylglycerides (TAGs), but little or no increase in TAG content or lactate release was observed in response to changes in the medium glycerol concentration. Adenovirus-mediated delivery of the Escherichia coli GlK gene (AdCMV-GlK) into muscle cells caused a 30-fold increase in GlK activity, which was associated with a marked rise in the labeling of phospholipid or TAG from [U-(14)C]glycerol compared with controls. Moreover, GlK overexpression caused [U-(14)C]glycerol to be incorporated into glycogen, which was dependent on the activation of glycogen synthase. Co-incubation of AdCMV-GlK-treated muscle cells with glycerol and oleate resulted in a large accumulation of TAG and an increase in lactate production. We conclude that GlK is the limiting step in muscle cell glycerol metabolism. Glycerol 3-phosphate is readily used for TAG synthesis but can also be diverted to form glycolytic intermediates that are in turn converted to glycogen or lactate. Given the high levels of glycerol in muscle interstitial fluid, these finding suggest that changes in GlK activity in muscle can exert important influences on fuel deposition in this tissue.
Highlights
Glycerol levels in human muscle interstitial fluid are much higher than plasma levels and approach the concentrations found in adipose tissue [1, 2]
Isolated human muscle cells exhibited lower glycerol kinase (GlK) activity than fresh muscle explants, but the activity in cultured cells was increased by exposure to insulin. [U-14C]Glycerol was incorporated into cellular phospholipids and triacylglycerides (TAGs), but little or no increase in TAG content or lactate release was observed in response to changes in the medium glycerol concentration
Since insulin is known to increase GlK activity in hepatocytes [17], we examined its potential effect in cultured human muscle cells
Summary
Human Muscle Primary Cultures and Transduction with Adenovirus—Human muscle primary cultures were initiated from a bank of satellite cells of muscle biopsies obtained from patients considered free of muscle disease (biopsies were obtained with informed consent and approval of the Human Use Committee of Hospital Vall d’Hebron, Barcelona, Spain). The fatty acid-salt solution was immediately added to Dulbecco’s modified Eagle’s medium containing fatty acid-free bovine serum albumin (BSA) (Sigma) with continuous agitation to avoid precipitation. Acylglyceride Determination—To determine the TAG content, extracts were prepared by scraping cell monolayers into a buffer consisting of 50 mM Tris, 100 mM KCl, 20 mM KF, 0.5 mM EDTA, 0.05% Lubrol PX, pH 7.9 and three rounds of sonication for 5 s each [16]. Incorporation of [U-14C]Glycerol into Cellular Lipids: Thin-layer Chromatography Analysis—Cells were incubated with 5 mM [U-14C] glycerol (100 Ci/mmol) (Amersham Biosciences, Inc.), 5 mM glucose, and 1 mM sodium oleate (BSA:oleate molar ratio of 1:5) for 15 h. The cell monolayers were washed three times in Hanks’ balanced salt solution, and the lipids were extracted twice with hexane:isopropanol (3:2). Statistics—Differences between groups were assessed by Student’s t test
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