Studies on the role of insulin in the regulation of glyceride synthesis in rat epididymal adipose tissue.
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ABSTRACT:
SUBMITTER: Sooranna SR
PROVIDER: S-EPMC1165759 | biostudies-other | 1975 Sep
REPOSITORIES: biostudies-other
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Project description:1. Evidence is presented that exposure of epididymal fat-pads from fed rats to insulin leads to a marked diminution in the Km for phosphoenolpyruvate of pyruvate kinase. Effects of insulin may be readily demonstrated in experiments both in vivo and in vitro and are not secondary to the activation by the hormone of glucose transport. No effect of insulin is apparent in tissues from 48 h-starved animals. 2. The mechanism of the effect of insulin on pyruvate kinase was not established. The observed changes in Km do not appear to be the result of alterations in the amounts of bound effectors such as fructose 1,6-bisphosphate and alanine. Rather, as the effect persists in incubated extracts, it appears that a change in the degree of phosphorylation or some other covalent modification of the enzyme may be involved.
| S-EPMC1161090 | biostudies-other
Project description: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.
| S-EPMC1179343 | biostudies-other
Project description:1. Epididymal adipose tissues obtained from rats that had been previously starved, starved and refed a high fat diet for 72h, starved and refed bread for 144h or fed a normal diet were incubated in the presence of insulin+glucose or insulin+glucose+acetate. 2. Measurements were made of the whole-tissue concentrations of hexose phosphates, triose phosphates, glycerol 1-phosphate, 3-phosphoglycerate, 6-phosphogluconate, adenine nucleotides, acid-soluble CoA, long-chain fatty acyl-CoA, malate and citrate after 1h of incubation. The release of lactate, pyruvate and glycerol into the incubation medium during this period was also determined. 3. The rates of metabolism of glucose in the hexose monophosphate pathway, the glycolytic pathway, the citric acid cycle and into glyceride glycerol, fatty acids and lactate+pyruvate were also determined over a 2h period in similarly treated tissues. The metabolism of acetate to CO(2) and fatty acids in the presence of glucose was also measured. 4. The activities of acetyl-CoA carboxylase, fatty acid synthetase and isocitrate dehydrogenase were determined in adipose tissues from starved, starved and fat-refed, and alloxan-diabetic animals and also in tissues from animals that had been starved and refed bread for up to 96h. Changes in these activities were compared with the ability of similar tissues to incorporate [(14)C]glucose into fatty acids in vitro. 5. The activities of acetyl-CoA carboxylase and fatty acid synthetase roughly paralleled the ability of tissues to incorporate glucose into fatty acids. 6. Rates of triglyceride synthesis and fatty acid synthesis could not be correlated with tissue concentrations of long-chain fatty acyl-CoA, citrate or glycerol 1-phosphate. In some cases changes in phosphofructokinase flux rates could be correlated with changes in citrate concentration. 7. The main lesion in fatty acid synthesis in tissues from starved, starved and fat-refed, and alloxan-diabetic rats appeared to reside at the level of pyruvate utilization and to be related to the rate of endogenous lipolysis. 8. It is suggested that pyruvate utilization by the tissue may be regulated by the metabolism of fatty acids within the tissue. The significance of this in directing glucose utilization away from fatty acid synthesis and into glyceride-glycerol synthesis is discussed.
| S-EPMC1179344 | biostudies-other
Project description:1. Attempts were made to define the role of phosphofructokinase in glycolytic control and the factors regulating the concentration of l-glycerol 3-phosphate in rat epididymal fat pads incubated in vitro. 2. Glycolysis rates were altered by anoxia or by additions of insulin, adrenaline or both to the incubation medium, and the changes in rate were related to changes in the steady-state concentrations of hexose phosphates, adenine nucleotides, l-glycerol 3-phosphate and citrate in the whole tissue. Measurements were also made of the lactate/pyruvate concentration ratio in the medium after incubation. 3. The mass-action ratios of phosphofructokinase, calculated from the whole-tissue concentrations of products and substrates, were less than 0.1% of the value of the ratio at pH7.4 at equilibrium. 4. Only in the presence of adrenaline could the observed stimulation of glycolytic flux be related to a possible activation of phosphofructokinase since, in this situation, the concentration of one substrate, fructose 6-phosphate, was not altered and the concentration of the other, ATP, was decreased. Increased glycolytic flux in the presence of insulin may be explained by an observed increase in the concentration of the substrate, fructose 6-phosphate. Under anaerobic conditions, glycolytic flux was decreased but this did not appear to be the result of inhibition of phosphofructokinase, since the concentrations of both substrates, fructose 6-phosphate and ATP, were decreased. The changes in glycolytic flux with insulin and anoxia may be secondary to changes in the rate of glucose uptake. 5. Changes in l-glycerol 3-phosphate concentration appear to be related both to changes in the concentration of dihydroxyacetone phosphate and to changes in the NADH/NAD(+) concentration ratio in the cytoplasm. They do not seem to be related directly to alterations in glycolytic rate.
| S-EPMC1184621 | biostudies-other
Project description:1. Most of the cyclic-nucleotide-independent acetyl-CoA carboxylase kinase activity in an extract of rat epididymal adipose tissue was evaluated from a Mono Q column by 0.175 M-NaCl at pH 7.4. The activity of the kinase in this fraction (fraction 1) was increased after exposure of intact tissue to insulin. 2. Incubation of purified adipose-tissue acetyl-CoA carboxylase with [gamma-32P]ATP and samples of fraction 1 led to the incorporation of up to 0.4 mol of 32P/mol of enzyme subunit. Most of the phosphorylation was on serine residues within a single tryptic peptide. This peptide, on the basis of two-dimensional t.l.c. analysis, h.p.l.c. and Superose 12 chromatography, appeared to be the same as the acetyl-CoA carboxylase peptide ('I'-peptide) which exhibits increased phosphorylation in insulin-treated tissue. 3. Phosphorylation of purified acetyl-CoA carboxylase by the kinase in fraction 1 was found to be associated with a parallel 4-fold increase in activity. However, increases in both phosphorylation and activity were much diminished if fraction 1 was treated by Centricon centrifugation to remove low-Mr components. Among these components was a potent inhibitor of acetyl-CoA carboxylase activity which appeared to be necessary for the kinase in fraction 1 to be fully active. 4. The inhibitor remains to be identified, but inhibition requires MgATP, although the inhibitor itself does not cause any phosphorylation of the carboxylase. No effects of insulin were observed on the activity of the inhibitor. 5. It is concluded that the kinase probably plays an important role in the mechanism whereby insulin brings about the well-established increases in phosphorylation and activation of acetyl-CoA carboxylase in adipose tissue.
| S-EPMC1131803 | biostudies-other
Project description:The sensitivity of rat epididymal-adipose-tissue pyruvate dehydrogenase phosphate phosphatase, NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase to Ca2+ ions was studied both in mitochondrial extracts and within intact coupled mitochondria. It is concluded that all three enzymes may be activated by increases in the intramitochondrial concentration of Ca2+ and that the distribution of Ca2+ across the mitochondrial inner membrane is determined, as in rat heart mitochondria, by the relative activities of a uniporter (which transports Ca2+ into mitochondria and is inhibited by Mg2+ and Ruthenium Red) and an antiporter (which allows Ca2+ to leave mitochondria in exchange for Na+ and is inhibited by diltiazem). Previous studies with incubated fat-cell mitochondria have indicated that the increases in the amount of active non-phosphorylated pyruvate dehydrogenase in rat epididymal tissue exposed to insulin are the result of activation of pyruvate dehydrogenase phosphate phosphatase. In the present studies, no changes in the activity of the phosphatase were found in extracts of mitochondria, and thus it seemed likely that insulin altered the intramitochondrial concentration of some effector of the phosphatase. Incubation of rat epididymal adipose tissue with medium containing a high concentration of CaCl2 (5mM) was found to increase the active form of pyruvate dehydrogenase to much the same extent as insulin. However, the increases caused by high [Ca2+] in the medium were blocked by Ruthenium Red, whereas those caused by insulin were not. Moreover, whereas the increases resulting from both treatments persisted during the preparation of mitochondria and their subsequent incubation in the absence of Na+, only the increases caused by treatment of the tissue with insulin persisted when the mitochondria were incubated in the presence of Na+ under conditions where the mitochondria are largely depleted of Ca2+. It is concluded that insulin does not act by increasing the intramitochondrial concentration of Ca2+. This conclusion was supported by finding no increases in the activities of the other two Ca2+-responsive intramitochondrial enzymes (NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase) in mitochondria prepared from insulin-treated tissue compared with controls.
| S-EPMC1153330 | biostudies-other
Project description:1. Flow of carbon atoms from glucose and glycogen glucose to glyceride glycerol, glyceride fatty acids and glycerol was calculated in the perfused rat heart and incubated epididymal adipose tissue from the incorporation of (14)C from [U-(14)C]-glucose (into glyceride glycerol, glyceride fatty acids and glycerol in the medium), and from measurements of the specific activity of l-glycerol 3-phosphate, and the effects of insulin, adrenaline and alloxan-diabetes were studied. Measurements were also made of the uptake of glucose and the outputs of lactate, pyruvate and glycerol. 2. New methods are described for the measurement of radioactivity in small amounts of metabolites (glycerol, glucose 6-phosphate and fructose 6-phosphate and l-glycerol 3-phosphate) in which use has been made of alterations in charge induced by enzymic conversions to effect resolution by ion-exchange chromatography. 3. In hearts the specific activity of l-glycerol 3-phosphate was less than that of glucose in the medium but similar to that of lactate released during perfusion. Because repeated measurements of the specific activity of l-glycerol 3-phosphate was impracticable, the specific activity of lactate has been used as an indirect measurement of glycerol phosphate specific activity. 4. In fat pads, specific activity of lactate was the same as that of glucose in the medium and thus the specific activity of l-glycerol 3-phosphate was taken to be the same as that of medium glucose. 5. In hearts from alloxan-diabetic rats, despite decreased glucose uptake and l-glycerol 3-phosphate concentration, flow of carbon atoms through l-glycerol 3-phosphate to glyceride glycerol was increased about threefold. 6. In fat pads, flow of carbon atoms through l-glycerol 3-phosphate to glyceride glycerol was increased by insulin (twofold), by adrenaline in the presence of insulin (fivefold) and by diabetes in pads incubated with insulin (1.5-fold). These increases could not be correlated either with increases in glucose uptake, which was unchanged by adrenaline and decreased in diabetes, or with the concentration of l-glycerol 3-phosphate, which was decreased by adrenaline and unchanged in diabetes. 7. These results are discussed in relation to the control of glyceride synthesis in heart and adipose tissue and to the regulation of glyceride fatty acid oxidation in the perfused rat heart.
| S-EPMC1270603 | biostudies-other
Project description:Obesity is associated with insulin resistance, an important risk factor of type 2 diabetes, atherogenic dyslipidemia, and nonalcoholic fatty liver disease. The major purpose of this study was to test hypothesize that prophylactic removal of epididymal visceral adipose tissue (VAT) prevents obesity-induced multi-organ (liver, skeletal muscle, adipose tissue) insulin resistance. Accordingly, we surgically removed epididymal VAT pads from adult C57BL/6J mice and evaluated in vivo and cellular metabolic pathways involved in glucose and lipid metabolism following chronic high-fat diet (HFD) feeding. We found that VAT removal decreases HFD-induced body weight gain while increasing subcutaneous adipose tissue (SAT) mass. Strikingly, VAT removal prevents obesity-induced insulin resistance and hyperinsulinemia and markedly enhances insulin-stimulated AKT-phosphorylation at serine-473 (Ser473) and threonine-308 (Thr308) sites in SAT, liver, and skeletal muscle. VAT removal leads to decreases in plasma lipid concentrations and hepatic triglyceride (TG) content. In addition, VAT removal increases circulating adiponectin, a key insulin-sensitizing adipokine, whereas it decreases circulating interleukin 6, a pro-inflammatory adipokine. Consistent with these findings, VAT removal increases adenosine monophosphate-activated protein kinase C phosphorylation, a major downstream target of adiponectin signaling. Data obtained from RNA sequencing suggest that VAT removal prevents obesity-induced oxidative stress and inflammation in liver and SAT, respectively. Taken together, these findings highlight the metabolic benefits and possible action mechanisms of prophylactic VAT removal on obesity-induced insulin resistance and hepatosteatosis. Our results also provide important insight into understanding the extraordinary capability of adipose tissue to influence whole-body glucose and lipid metabolism as an active endocrine organ.
| S-EPMC8041347 | biostudies-literature
Project description:We analyzed the effects of castration on epididymal white adipose tissue (WAT) in C57BL/6J mice which were fed a regular or high-fat diet. Fourteen days following surgical castration profound effects on WAT tissue such as reductions in WAT wet weight and WAT/body weight ratio, induction of lipolysis and morphologic changes characterized by smaller adipocytes, and increased stromal cell compartment were documented in both dietary groups. Castrated animals had decreased serum leptin levels independent of diet but diet-dependent decreases in serum adiponectin and resistin. The castrated high-fat group had dramatically lower serum triglyceride levels. Immunohistochemical analysis revealed higher staining for smooth muscle actin, macrophage marker Mac-3, and Cxcl5 in the castrated than in the control mice in both dietary groups. We also detected increased fatty-acid synthase expression in the stromal compartment of WAT in the regular-diet group. Castration also reduces the expression of androgen receptor in WAT in the regular-diet group. We conclude that castration reduces tissue mass and affects biologic function of WAT in mice.
| S-EPMC3867123 | biostudies-literature
Project description:We recently showed that the peripheral cannabinoid receptor type 1 (CNR1) gene is upregulated by the synthetic glucocorticoid dexamethasone. CNR1 is highly expressed in the central nervous system and has been a drug target for the treatment of obesity. Here we explore the role of peripheral CNR1 in states of insulin resistance in human adipose tissue. Subcutaneous adipose tissue was obtained from well-controlled type 2 diabetes subjects and controls. Subcutaneous adipose tissue gene expression levels of CNR1 and endocannabinoid synthesizing and degrading enzymes were assessed. Furthermore, paired human subcutaneous adipose tissue and omental adipose tissue from non-diabetic volunteers undergoing kidney donation or bariatric surgery, was incubated with or without dexamethasone. Subcutaneous adipose tissue obtained from volunteers through needle biopsy was incubated with or without dexamethasone and in the presence or absence of the CNR1-specific antagonist AM281. CNR1 gene and protein expression, lipolysis and glucose uptake were evaluated. Subcutaneous adipose tissue CNR1 gene expression levels were 2-fold elevated in type 2 diabetes subjects compared with control subjects. Additionally, gene expression levels of CNR1 and endocannabinoid-regulating enzymes from both groups correlated with markers of insulin resistance. Dexamethasone increased CNR1 expression dose-dependently in subcutaneous adipose tissue and omental adipose tissue by up to 25-fold. Dexamethasone pre-treatment of subcutaneous adipose tissue increased lipolysis rate and reduced glucose uptake. Co-incubation with the CNR1 antagonist AM281 prevented the stimulatory effect on lipolysis, but had no effect on glucose uptake. CNR1 is upregulated in states of type 2 diabetes and insulin resistance. Furthermore, CNR1 is involved in glucocorticoid-regulated lipolysis. Peripheral CNR1 could be an interesting drug target in type 2 diabetes and dyslipidemia.
| S-EPMC5316391 | biostudies-literature