Mechanisms whereby insulin increases diacylglycerol in BC3H-1 myocytes

R. V. Farese, D. R. Cooper, T. S. Konda, G. Nair, M. L. Standaert, J. S. Davis, R. J. Pollet

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Abstract

We previously suggested that insulin increases diacylglycerol (DAG) in BC3H-1 myocytes, both by increases in synthesis de novo of phosphatidic acid (PA) and by hydrolysis of non-inositol-containing phospholipids, such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE). We have now evaluated these insulin effects more thoroughly, and several potential mechanisms for their induction. In studies of the effect on PA synthesis de novo, insulin stimulated [2-3H]glycerol incorporation into PA, DAG, PC/PE and total glycerolipids of BC3H-1 myocytes, regardless of whether insulin was added simultaneously with, or after 2 h or 3 or 10 days of prelabelling with, [2-3H]glycerol. In prelabelled cells, time-related changes in [2-3H]glycerol labelling of DAG correlated well with increases in DAG content: both were maximal in 30-60 s and persisted for 20-30min. [2-3H]Glycerol labelling of glycerol 3-phosphate, on the other hand, was decreased by insulin, presumably reflecting increased utilization for PA synthesis. Glycerol 3-phosphate concentrations were 0.36 and 0.38 mM before and 1 min after insulin treatment, and insulin effects could not be explained by increases in glycerol 3-phosphate specific radioactivity. In addition to that of [2-3H]glycerol, insulin increased [U-14C]glucose and [1,2,3-3H]glycerol incorporation into DAG and other glycerolipids. Effects of insulin on [2-3H]glycerol incorporation into DAG and other glycerolipids were half-maximal and maximal at 2 nM- and 20 nM-insulin respectively, and were not dependent on glucose concentration in the medium, extracellular Ca2+ or protein synthesis. Despite good correlation between [3H]DAG and DAG content, calculated increases in DAG content from glycerol 3-phosphate specific radioactivity (i.e. via the pathway of PA synthesis de novo) could account for only 15-30% of the observed increases in DAG content. In addition to increases in [3H]glycerol labelling of PC/PE, insulin rapidly (within 30 s) increased PC/PE labelling by [3H]arachidonic acid, [3H]myristic acid, and [14C]choline. Phenylephrine, ionophore A23187 and phorbol esters did not increase [2-3H]glycerol incorporation into DAG or other glycerolipids in 2 h-prelabelling experiments; thus activation of the phospholipase C which hydrolyses phosphatidylinositol, its mono- and bis-phosphate, Ca2+ mobilization, and protein kinase C activation, appear to be ruled out as mechanisms to explain the insulin affect on synthesis de novo of PA, DAG and PC. Exogenously added non-specific phospholipase C (from Clostridium perfringens) increased [3H]DAG in [3H]glycerol-prelabelled cells, but apparently at the exposure of pre-existing [3H]PC/PE, rather than via synthesis of PA de novo. Thus, PC/PE hydrolysis may contribute to increases in DAG, but does not initiate the effect of insulin on synthesis of PA, DAG and PC de novo. The present findings provide further evidence that insulin increases synthesis de novo of PA, DAG and PC, but activation of this pathway can only partly account for observed increases in DAG content; and other mechanisms, such as hydrolysis of pre-existing lipids, also appear to be involved.

Original languageEnglish (US)
Pages (from-to)175-184
Number of pages10
JournalBiochemical Journal
Volume256
Issue number1
DOIs
StatePublished - Jan 1 1988

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Farese, R. V., Cooper, D. R., Konda, T. S., Nair, G., Standaert, M. L., Davis, J. S., & Pollet, R. J. (1988). Mechanisms whereby insulin increases diacylglycerol in BC3H-1 myocytes. Biochemical Journal, 256(1), 175-184. https://doi.org/10.1042/bj2560175