The regulation of triglyceride synthesis and fatty acid synthesis in rat epididymal adipose tissue. Effects of insulin, adrenaline and some metabolites in vitro

Abstract

1. Adipose tissues from rats fed a balanced diet were incubated in the presence of glucose (20mm) with the following additions: insulin, anti-insulin serum, insulin+acetate, insulin+pyruvate, insulin+lactate, insulin+phenazine methosulphate, insulin+oleate+albumin, insulin+adrenaline+albumin, insulin+6-N-2'-O-dibutyryl 3':5'-cyclic AMP+albumin. 2. Measurements were made of the whole tissue concentrations of adenine nucleotides, hexose phosphates, triose phosphates, glycerol 1-phosphate, 3 phosphoglycerate, 6-phosphogluconate, long-chain fatty acyl-CoA, acid-soluble CoA, citrate, isocitrate, malate and 2-oxoglutarate, and of the release into the incubation medium of lactate, pyruvate and glycerol after 1h of incubation. 3. Fluxes of [(14)C]glucose carbon through the major pathways of glucose metabolism were calculated from the yields of (14)C in various products after 2h of incubation. Fluxes of [(14)C]acetate, [(14)C]pyruvate or [(14)C]lactate carbon in the presence of glucose were also determined. 4. Measurements were also made of the whole-tissue concentrations of metabolites in tissues taken directly from Nembutal-anaesthetized rats. 5. Whole tissue mass-action ratios for phosphofructokinase, phosphoglucose isomerase and the combined (aldolasextriose phosphate isomerase) reaction were similar in vivo and in vitro. The reactants of phosphofructokinase appeared to be far from mass-action equilibrium. In vitro, the reactants of hexokinase also appeared to be far from mass-action equilibrium. 6. Correlation of observed changes in glycolytic flux with changes in fructose 6-phosphate concentration suggested that phosphofructokinase may show regulatory behaviour. The enzyme appeared to be activated in the presence of oleate or adrenaline and to be inhibited in the presence of lactate or pyruvate. 7. Evidence is presented that the reactants of lactate dehydrogenase and glycerol 1-phosphate dehydrogenase may be near to mass-action equilibrium in the cytoplasm. 8. No satisfactory correlations could be drawn between the whole-tissue concentrations of long-chain fatty acyl-CoA, citrate and glycerol 1-phosphate and the observed rates of triglyceride and fatty acid synthesis. Under the conditions employed, the concentration of glycerol 1-phosphate appeared to depend mainly on the cytoplasmic [NAD(+)]/[NADH] ratios. 9. Calculated hexose monophosphate pathway flux rates roughly correlated with fatty acid synthesis rates and with whole tissue [6-phosphogluconate]/[glucose 6-phosphate] ratios. The relative rates of production of NADPH for fatty acid synthesis by the hexose monophosphate pathway and by the ;malic enzyme' are discussed. It is suggested that all NADH produced in the cytoplasm may be used in that compartment for reductive synthesis of fatty acids, lactate or glycerol 1-phosphate.

Regulation of adipose tissue pyruvate dehydrogenase by insulin and other hormones

“…Further, the enzyme activity after exposure to insulin is in 'the range 10-20,umol/h per g of wet tissue at 370C (assuming enzyme activities double for each 100C rise in temperature). This would be sufficient to account for published rates of carbon atom flow through pyruvate dehydrogenase when fatty acids are synthesized from glucose with insulin (Saggerson & Greenbaum, 1970a;Flatt & Ball, 1964;Katz & Rognstad, 1966). It is also noteworthy that the observed increase in pyruvate dehydrogenase activity due to insulin is comparable with the magnitude of the hormonal effect on fatty acid synthesis from fructose (Table 1).…”

“…The increase in fatty acid synthesis from glucose induced by insulin in rat epididymal adipose tissue is often in excess of 500%, whereas the associated increase in pyrtvate output is rarely greater than 200% (Denton, Halperin & Randle, 1968;Saggerson & Greenbaum, 1970a;Halperin & Robinson, 1970). This preferential incorporation of pyruvate carbon atoms into fatty acids compared with its release from fat-pads in the presence of insulin led to the suggestion that insulin might specifically activate the conversion of pyruvate carbon atoms into fatty acid in addition to its well-established effect on glucose uptake .…”

“…This preferential incorporation of pyruvate carbon atoms into fatty acids compared with its release from fat-pads in the presence of insulin led to the suggestion that insulin might specifically activate the conversion of pyruvate carbon atoms into fatty acid in addition to its well-established effect on glucose uptake . Adrenaline, on the other hand, may antagonize an action of insulin on the conversion of pyruvate carbon atoms into fatty acid because it increases the output of lactate and pyruvate from glucose and either inhibits or leaves unchanged the rate of incorporation of glucose carbon atoms into fatty acid Saggerson & Greenbaum, 1970a).…”

Evidence that noradrenaline increases pyruvate dehydrogenase activity and decreases acetyl-CoA carboxylase activity in rat interscapular brown adipose tissue in vivo

“…For measurement of pyruvate dehydrogenase and acetyl-CoA carboxylase activities, anaesthetized rats were injected with glucose, noradrenaline and/or propranolol as given in the legend to Fig. 1 (Denton & Halperin, 1968;Saggerson & Greenbaum, 1970). There is much evidence that the mechanism involves activation of cyclic AMP-dependent protein kinase, and hence increased phosphorylation of acetyl-CoA carboxylase, which results in the inactivation of this enzyme (Lee & Kim, 1978;Brownsey et al, 1979;Brownsey & Denton, 1982;Witters et al, 1983;Holland et al, 1985).…”

Inactivation of rat adipocyte pyruvate dehydrogenase by palmitate. Protection against this effect by insulin in the presence of glucose

“…Samples of media were deproteinized, and lactate and pyruvate assayed enzymically as described by Saggerson & Greenbaum (1970). Non-esterified fatty acids were assayed as described by Sooranna & Saggerson (1975).…”