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

Research output: Contribution to journalArticle

57 Citations (Scopus)

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

Fingerprint

Diglycerides
Muscle Cells
Insulin
Phosphatidic Acids
Phosphatidylcholines
Glycerol
Labeling
Hydrolysis
Chemical activation
Radioactivity
Type C Phospholipases
Phosphates
Glucose
Clostridium
Clostridium perfringens
Ionophores
Calcimycin
Myristic Acid
Phorbol Esters
Phenylephrine

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

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

Mechanisms whereby insulin increases diacylglycerol in BC3H-1 myocytes. / Farese, R. V.; Cooper, D. R.; Konda, T. S.; Nair, G.; Standaert, M. L.; Davis, J. S.; Pollet, R. J.

In: Biochemical Journal, Vol. 256, No. 1, 01.01.1988, p. 175-184.

Research output: Contribution to journalArticle

Farese, RV, Cooper, DR, Konda, TS, Nair, G, Standaert, ML, Davis, JS & Pollet, RJ 1988, 'Mechanisms whereby insulin increases diacylglycerol in BC3H-1 myocytes', Biochemical Journal, vol. 256, no. 1, pp. 175-184. https://doi.org/10.1042/bj2560175
Farese RV, Cooper DR, Konda TS, Nair G, Standaert ML, Davis JS et al. Mechanisms whereby insulin increases diacylglycerol in BC3H-1 myocytes. Biochemical Journal. 1988 Jan 1;256(1):175-184. https://doi.org/10.1042/bj2560175
Farese, R. V. ; Cooper, D. R. ; Konda, T. S. ; Nair, G. ; Standaert, M. L. ; Davis, J. S. ; Pollet, R. J. / Mechanisms whereby insulin increases diacylglycerol in BC3H-1 myocytes. In: Biochemical Journal. 1988 ; Vol. 256, No. 1. pp. 175-184.
@article{626770d63df0433195f81b846472e433,
title = "Mechanisms whereby insulin increases diacylglycerol in BC3H-1 myocytes",
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.",
author = "Farese, {R. V.} and Cooper, {D. R.} and Konda, {T. S.} and G. Nair and Standaert, {M. L.} and Davis, {J. S.} and Pollet, {R. J.}",
year = "1988",
month = "1",
day = "1",
doi = "10.1042/bj2560175",
language = "English (US)",
volume = "256",
pages = "175--184",
journal = "Biochemical Journal",
issn = "0264-6021",
publisher = "Portland Press Ltd.",
number = "1",

}

TY - JOUR

T1 - Mechanisms whereby insulin increases diacylglycerol in BC3H-1 myocytes

AU - Farese, R. V.

AU - Cooper, D. R.

AU - Konda, T. S.

AU - Nair, G.

AU - Standaert, M. L.

AU - Davis, J. S.

AU - Pollet, R. J.

PY - 1988/1/1

Y1 - 1988/1/1

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0023723253&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0023723253&partnerID=8YFLogxK

U2 - 10.1042/bj2560175

DO - 10.1042/bj2560175

M3 - Article

C2 - 3146971

AN - SCOPUS:0023723253

VL - 256

SP - 175

EP - 184

JO - Biochemical Journal

JF - Biochemical Journal

SN - 0264-6021

IS - 1

ER -