Pertussis toxin reversal of the antilipolytic action of insulin in rat adipocytes in the presence of forskolin does not involve cyclic AMP

Insulin inhibition of lipolysis in the presence of forskolin was reversed by a four hour exposure of adipocytes to pertussis toxin. In contrast, the antilipolytic action of insulin against lipolysis due to theophylline was unaffected by pertussis toxin as was the ability of insulin to lower cyclic AMP in the presence of either forskolin or theophylline. The stimulation of adenylate cyclase by norepinephrine in crude plasma membranes obtained from rat adipocytes was inhibited by N6-(Phenylisopropyl)adenosine (PIA) and abolished by pretreating rat adipocytes with pertussis toxin. The stimulation of glucose metabolism by insulin was not altered by pertussis toxin pretreatment of rat adipocytes. These findings suggest that pertussis toxin selectively abolishes the antilipolytic effect of insulin in the presence of forskolin through a cyclic AMP independent mechanism.     

Leptin secretion and protein kinase A activity

Leptin is an adipocyte-derived hormone participating in the regulation of food intake and energy balance. Its secretion from fat cells is potentiated by insulin and by substrates providing ATP, whereas factors increasing cAMP level attenuate hormone release stimulated by insulin and glucose. The present experiments were aimed to determine the effect of cAMP on leptin secretion stimulated by glucose, alanine or leucine in the presence of insulin. Moreover, the effect of protein kinase A inhibition on leptin secretion was tested. To stimulate leptin secretion, isolated rat adipocytes were incubated for 2 h in the buffer containing 5 mmol/l glucose, 10 mmol/l alanine or 10 mmol/l leucine, all in the presence of 10 nmol/l insulin. Inhibition of protein kinase A (PKA) by H-89 (50 micromol/l) slightly enhanced leptin release stimulated by glucose and leucine but not by alanine. Activation of this enzyme by dibutyryl-cAMP (1 mmol/l) substantially restricted leptin secretion stimulated by glucose, alanine and leucine. The inhibitory influence of dibutyryl-cAMP on leptin secretion was totally (in the case of stimulation induced by glucose) or partially (in the case of stimulation by alanine and leucine) suppressed by H-89. These results demonstrate that leptin secretion induced by glucose, alanine and leucine is profoundly attenuated by cAMP in PKA-dependent manner. Therefore, the action of different stimulators of leptin secretion may be restricted by agents increasing the cAMP content in adipocytes. Moreover, it has also been shown that inhibition of PKA evokes the opposite effect and enhances leptin release.     

Inhibition of tyrosine autophosphorylation of the solubilized insulin receptor by an insulin-stimulating peptide derived from bovine serum albumin

A polypeptide from a tryptic digest of bovine serum albumin potentiates glucose oxidation stimulated by insulin in isolated rat adipocytes. We studied whether this effect is related to a modification of the insulin receptor kinase. In a solubilized rat adipocytes receptor system, the peptide caused dose-dependent inhibition of the stimulation by insulin of phosphorylation of the 95,000 dalton subunit of insulin receptor. The peptide also inhibited stimulation by vanadate of tyrosine autophosphorylation of the beta subunit of the receptor, though it enhanced vanadate-stimulated glucose oxidation. During the phosphorylation reaction, no phosphorylated forms of the peptide could be detected. The peptide had no effect on dephosphorylation of the phosphorylated beta subunit of the insulin receptor. These results strongly suggest that the inhibition of phosphorylation by the peptide is due not to either simple substrate competition or activation of phosphoprotein phosphatase, but to specific inhibition of tyrosine-specific protein kinase.     

The potentiations by insulin-stimulating peptide from bovine serum albumin of the effects of insulin mimickers and insulin in stimulating glucose utilization by rat adipocytes

An insulin-stimulating peptide derived from bovine serum albumin by digestion with trypsin was shown to inhibit insulin degradation. Addition of this peptide (1.2 microM) to the medium of isolated rat adipocytes markedly inhibited the degradation of insulin in the medium, but had a little effect on degradation of cell-associated insulin. Moreover, this peptide did not prevent dissociation of cell-associated insulin, suggesting that it is a bacitracin-type, not a chloroquine-type inhibitor of insulin degradation. The peptide also potentiated the stimulation by insulin mimickers of glucose oxidation by rat adipocytes, strongly indicating that it has some other effects besides inhibition of insulin degradation. Therefore, the effect of the peptide on activation of pyruvate dehydrogenase (PDH), one of the postbinding actions of insulin, was studied. Addition of the peptide (4 microM) to adipocytes was found to activate PDH in the absence or presence of insulin. This stimulatory effect of the peptide on PDH was dose-dependent and was observed in both whole cells and subcellular fractions of rat adipocytes. The peptide also stimulated PDH in a subcellular system of either plasma membranes and mitochondria or mitochondria only. Sodium fluoride, an inhibitor of phosphatase, blocked the action of the peptide almost completely, suggesting that the stimulatory effect of the peptide on PDH activity is at least partly due to its activation of PDH phosphatase. The mechanisms of action of the peptide are discussed. The peptide should be useful in studies on modulation of the action of insulin.     

Biological effects of sulphated insulin in adipocytes and hepatocytes

Summary The binding affinity of sulphated insulin compared with unmodified, neutral insulin has been reported to be approximately four times lower in human and rat adipocytes but over twenty times lower in rat hepatocytes. In the present study the biological action of sulphated insulin was assesed in rat hepatocytes and human and rat adipocytes. To achieve half-maximal stimulation of fatty acid synthesis in rat hepatocytes about twenty one times higher concentrations of sulphated than neutral insulin were required (15.07±5.50 vs 0.71±0.34 nmol/l), this ratio being similar to the ratio of binding affinity in rat hepatocytes. In human adipocytes, half-maximal stimulation of initial rates of glucose uptake was observed at 11.6±5.1 vs 2.9±1.3 pmol/l for sulphated and neutral insulin respectively, and half-maximal inhibition of lipolysis at 31.0±13.5 vs 7.3+2.5 pmol/I respectively. These data are consistent with the four-fold lower binding affinity of sulphated insulin to human adipocytes. However, in rat adipocytes the biological potency of sulphated insulin was found to be much lower than anticipated from the binding data, half-maximal stimulation of initial rates of glucose uptake being observed at 757±299 vs 35±13 pmol/l respectively and half-maximal inhibition of lipolysis at 35.9±12.1 vs 1.5±0.5 pmol/l respectively. Thus, in rat adipocytes, approximately 22 times the concentration of sulphated insulin was required to achieve equivalent biological effect. A discrepancy between binding affinity and biological action with respect to sulphated insulin was identified in rat adipocytes but not human adipocytes nor rat hepatocytes suggesting differences in the binding-action linkage in these cells.     

Adenosine modulation of fat cell responsiveness to insulin and oxytocin

We have investigated the effects of adenosine on the stimulation of glucose oxidation and lipogenesis by oxytocin and insulin in rat epididymal adipocytes. The addition of adenosine deaminase (1 U/ml) to the assay medium reduced the maximal oxytocin response (glucose oxidation and lipogenesis) to between 25 and 50% of the maximum response in control cells. The maximal response to insulin was not appreciably affected under these conditions. The addition of adenosine (10 or 30 microM) increased the cell sensitivity to oxytocin by elevating the maximum rate of oxytocin-stimulated glucose metabolism. Adenosine also increased the cell sensitivity to insulin by decreasing its ED50. A change in ED50, however, was observed only when control or adenosine-treated cells were compared to adenosine deaminase-treated cells; but not when control and adenosine-treated cells were compared. On its own, adenosine also caused an appreciable increase in both glucose oxidation and lipogenesis (ED50 approximately equal to 3 microM adenosine). The difference in the effect of adenosine on oxytocin action, compared with the effect on insulin action, points to differences in the mechanisms by which insulin and oxytocin stimulate glucose metabolism in adipocytes.     

Effects of pertussis toxin treatment on the metabolism of rat adipocytes

The protein toxin present in Bordetella pertussis vaccine blocks the inhibition of adenylate cyclase by prostaglandins and adenosine which may be secondary to ADP-ribosylation of an inhibitory guanine nucleotide-binding protein. The stimulatory effects of alpha 1-catecholamine agonists on 32P uptake into phosphatidic acid and phosphatidylinositol in isolated rat adipocytes were virtually abolished by pertussis toxin treatment. In contrast, the stimulatory effects of insulin were increased in adipocytes after pertussis toxin treatment. Pertussis toxin treatment did not alter insulin stimulation of glucose oxidation and actually increased glucose conversion to lipid. Basal lipolysis was elevated in adipocytes by pertussis toxin treatment but not basal cyclic AMP. However, the increases in cyclic AMP and lipolysis due to low concentrations of catecholamines and forskolin were markedly potentiated by pertussis toxin treatment. The inhibitory effects of adenosine on cyclic AMP stimulation due to catecholamines were abolished by pertussis toxin. These data indicate that pertussis toxin selectively interferes with inhibition of cyclic AMP accumulation in rat adipocytes by adenosine, potentiates the increases in cyclic AMP due to catecholamines, increases the stimulatory effects of insulin on adipocyte metabolism, and interferes with alpha 1-catecholamine stimulation of phosphatidylinositol turnover.     

Roles of alpha and beta adrenergic receptors in control of glucose oxidation in hamster epididymal adipocytes.

Oxidation of [14C] glucose in isolated epididymal adipocytes from Golden hamsters was stimulated by isoproterenol, epinephrine and norepinephrine, which all interact with beta-adrenergic receptors and by adrenocorticotrophic hormone. In contrast alpha-receptor agonists, such as phenylephrine, methoxamine or clonidine did not increase basal glucose oxidation. The beta-adrenergic blocking drug propranolol inhibited both lipolysis and glucose oxidation when these had been stimulated by isoproterenol, epinephrine or norepinephrine. Conversely, the alpha-adrenergic blocking drugs phentolamine and phenoxybenzamine did not influence lipolysis or glucose oxidation when isoproterenol provided the stimulus and increased both lipolysis and glucose metabolism in the present of either epinephrine or norepinephrine. All alpha-adrenergic agonists tested (phenylephrine, methoxamine and clonidine) lowered lipolysis and glucose oxidation isolated adipocytes exposed to isoproterenol. However, when adrenocorticotropin provided the stimulus for glucose oxidation and lipolysis, only clonidine produced a significant reduction in lipolysis and glucose oxidation. None of the alpha-agonists influenced glucose metabolism which had been increased by insulin. These data confirm the presence of both alpha and beta adrenergic receptors on hamster epididymal adipocytes and suggest that they exert antagonistic influences on lipolysis and glucose oxidation. These data are also consistent with the view that adrenergic stimulation of glucose oxidation and lipolysis in adipocytes are both mediated through beta receptors.     

Action of insulin modulated by pertussis toxin in rat adipocytes

We studied the effect of pertussis toxin (PT) treatment on the ability of insulin to inhibit lipolysis and to stimulate glucose oxidation in isolated rat adipocytes. In cells maximally modified by PT (100% ADP ribosylation of a 41-kdalton protein in membranes), the ability of insulin to inhibit lipolysis stimulated either by PT alone or in combination with a catecholamine was abolished. In cells wherein ADP ribosylation was submaximal (about 67% modification), a small but variable antilipolytic action of insulin could still be detected. In cells maximally modified by PT, both basal and insulin-stimulated glucose oxidation were markedly reduced (to 10-15% of control levels). However, relative to the basal oxidation level, the fold stimulation by insulin in PT-treated cells was equivalent to the fold stimulation in control cells. Nonetheless, PT treatment caused a rightward shift in the dose-response curve for insulin-stimulated glucose oxidation as well as a small reduction in insulin binding. Our results point strongly not only to a link between the inhibitory guanine nucleotide regulatory complex (Gi) and the antilipolytic action of insulin but also to a link between the Gi complex and the overall regulation of glucose metabolism in adipocytes.     

Effects of insulin and norepinephrine on glucose transport and metabolism in rat brown adipocytes. Potentiation by insulin of norepinephrine-induced glucose oxidation

Glucose is an important fuel for rat brown adipose tissue in vivo and its utilization is highly sensitive to insulin. In this study, the different glucose metabolic pathways and their regulation by insulin and norepinephrine were examined in isolated rat brown adipocytes, using [6-14C]glucose as a tracer. Glucose utilization was stimulated for insulin concentrations in the range of 40-1000 microU/ml. Furthermore, the addition of adenosine deaminase (200 mU/ml) or adenosine (10 microM) did not alter insulin sensitivity of glucose metabolism. The major effect of insulin (1 mU/ml) was a respective 7-fold and 5-fold stimulation of lipogenesis and lactate synthesis, whereas glucose oxidation remained very low. The 5-fold stimulation of total glucose metabolism by 1 mU/ml of insulin was accompanied by an 8-fold increase in glucose transport. In the presence of norepinephrine (8 microM), total glucose metabolism was increased 2-fold. This was linked to a 7-fold increase of glucose oxidation, whereas lipogenesis was greatly inhibited (by 72%). In addition, norepinephrine alone did not modify glucose transport. The addition of insulin to adipocytes incubated with norepinephrine, induced a potentiation of glucose oxidation, while lipogenesis remained very low. In conclusion, in the presence of insulin and norepinephrine glucose is a oxidative substrate for brown adipose tissue. However the quantitative importance of glucose as oxidative fuel remains to be determined.     

Mechanism of insulin resistance in the post receptor events in sheep: 3-O-methylglucose transport in ovine adipocytes

A severe resistance to the stimulatory action of insulin on glucose metabolism has been shown in ruminant adipose tissue or isolated adipocytes as compared to that of rats. To elucidate the mechanism of insulin resistance in ruminants, we measured the stimulatory effect of insulin on 3-O-methylgulose transport and on intracellular glucose metabolism in isolated adipocytes from sheep and rats. At a glucose concentration (0.1 mM) where transport is thought to be rate-limiting for metabolism, lipogenesis from [U-14C]glucose by ovine adipocytes was markedly less than by rat adipocytes in both the basal state and at all insulin concentrations. The responsiveness to insulin assessed by percent increase above basal was reduced to about 15% of that in rat adipocytes, but the insulin sensitivity was similar, because the insulin concentration giving half-maximal stimulation, ED50, did not differ significantly between ovine and rat adipocytes. The maximal insulin-stimulated 3-O-methylglucose transport in ovine adipocytes per cell was less than 20% of that in rat adipocytes, with a significant lowering in basal rates of transport. However, when data was expressed per 3-O-methylglucose equilibrium space no significant differences were found between ovine and rat in the basal transport rates, but a lowered ability of insulin to stimulate glucose transport was still seen in ovine adipocytes. The dose-response curve for glucose transport was slightly shifted to the right in ovine adipocytes compared to rat adipocytes, indicating a small decrease in insulin sensitivity. The decrease in glucose transport was due to 60% reduction in the maximum velocity in the insulin--stimulated state, with no change in the Km.     

Effects of melittin on adipocyte metabolism unrelated to lysophospholipid accumulation

Melittin addition to rat or hamster adipocytes resulted in inhibition of lipolysis, cyclic AMP accumulation and glucose oxidation. Low concentrations of melittin were not insulin-like with respect to either stimulation of glucose metabolism or inhibition of lipolysis. Higher concentrations of melittin lysed adipocytes. In the presence of melittin, cellular phospholipids were released to the medium and hydrolyzed with little accumulation of lysophospholipids. Only in adipocytes incubated with melittin contaminated with phospholipase A2 was any appreciable accumulation of lysophospholipids seen and this was in the medium. These data suggest that the toxic effects of melittin on adipocytes are not due to the accumulation of lysophospholipids but rather to the loss of membrane phospholipids or alterations in membrane proteins.     

Roles of alpha and beta adrenergic receptors in control of glucose oxidation in hamster epididymal adipocytes

Oxidation of [¹⁴C]glucose in isolated epididymal adipocytes from Golden hamsters was stimulated by isoproterenol and norepinephrine, which all interact with β-adrenergic receptors and by adrenorticotrophic hormone. In contrast α-receptor agonists, such as phenylephrine, methoxamine or clonidine did not increase basal glucose oxidation. The β-adrenergic blocking drug propranolol inhibited both lipolysis and glucose oxidation when these had been stimulated by isoproterenol, ephinephrine and phenoxybenzamine did not the α-adrenergic blocking drugs phentolamine and phenoxybenzamine did not influence lipolysis or glucose oxidation when isoproterenol provided the stimulus and increased both liposlysis and glucose metabolism in the presence of either epinephrine or norepinephrine. All α-adrenergic agonists tested (phenylephrine, methoxamine and clonidine) lowered liposlysis and glucose oxidation in isolated adipocytes exposed to isoproterenol. However, when adrenorcortropin provided the stimulus for glucose oxidation and lipolysis, only clonidine produced a significant reduction in lipolysis and glucose oxidation. None of the α-agonists influenced glucose metabolism which had been increased by insulin. These data confirm the presence of both α and β adrenergic receptors on hamster epididymal adipocytes and suggests that they exert antagonistic influences on lipolysis and glucose oxidation. These data are also consistent with the view that adrenergic stimulation of glucose oxidation and lipolysis in adipocytes are both mediated through β receptors.     

Effect of lactation on insulin sensitivity of glucose metabolism in rat adipocytes

During lactation glucose metabolism in paraovarian adipocytes is characterized by a 40 and 80% decrease of glucose incorporation into CO2 and fatty acids in the presence of insulin. In contrast with the stimulation by insulin of glucose incorporation into lactate, glycerol remains unchanged. As a result, insulin sensitivity of total glucose metabolism (oxidation and lipid synthesis) is not altered in adipocytes from lactating rats.     

Relation of insulin receptors to insulin resistance.

The status of insulin-receptor interactions in a variety of insulin-resistant states is reviewed. Utilizing large adipocytes from adult rats and small fat cells from young rats, we have conducted a series of in vitro experiments in an attempt to determine the cellular alteration(s) responsible for the insulin resistance associated with obesity. Stimulation of glucose oxidation by insulin is reduced in large cells. Studies using a mimicker of insulin action, spermine, as well as measurements of 125I-insulin binding to large and small cells indicate that receptor number and affinity are not responsible for hormone resistance. Furthermore, when rapid and direct measurements of sugar uptake were made, insulin stimulation was virtually identical in both cell types. These findings indicate that large adipocytes have an efficient insulin-responsive D-glucose transport system and suggest that the apparent hormone resistance may be due to alterations in intracellular glucose metabolism. It has been proposed that altered insulin-receptor interaction underlies the insulin resistance of human obesity. We have investigated this particular aspect of insulin action by 125I-insulin binding studies. Similar numbers of insulin receptors per cell and affinity for insulin were observed in adipocytes obtained from normal weight subjects and morbidly obese patients. Thus, the initial step in insulin action is unaltered in human obesity.     

Changes in the properties of cytosolic acetyl-CoA carboxylase studied in cold-clamped liver samples from fed, starved and starved-refed rats.

The mechanism responsible for the insulin resistance described in vivo in brown adipose tissue (BAT) of lactating rats was investigated. The effect of insulin on glucose metabolism was studied on isolated brown adipocytes of non-lactating and lactating rats. Insulin stimulation of total glucose metabolism is 50% less in brown adipocytes from lactating than from non-lactating rats. This reflects a decreased effect of insulin on glucose oxidation and lipogenesis. However, the effect of noradrenaline (8 microM) on glucose metabolism was preserved in brown adipocytes from lactating rats as compared with non-lactating rats. The number of insulin receptors is similar in BAT of lactating and non-lactating rats. The insulin-receptor tyrosine kinase activity is not altered during lactation, for receptor autophosphorylation as well as tyrosine kinase activity towards the synthetic peptide poly(Glu4-Tyr1). The defect in the action of insulin is thus localized at a post-receptor level. The insulin stimulation of pyruvate dehydrogenase activity during euglycaemic/hyperinsulinaemic clamps is 2-fold lower in BAT from lactating than from non-lactating rats. However, the percentage of active form of pyruvate dehydrogenase is similar in non-lactating and lactating rats (8.6% versus 8.9% in the basal state, and 37.0% versus 32.3% during the clamp). A decrease in the amount of pyruvate dehydrogenase is likely to be involved in the insulin resistance described in BAT during lactation.     

Phenylarsine oxide inhibits insulin-dependent glucose transport activity in rat soleus muscles

Phenylarsine oxide (PAO) is known to block insulin-stimulated glucose transport activity in 3T3 L1 adipocytes at a post-receptor step. Herein, we demonstrate that, at right concentration, PAO also inhibits insulin activation of glucose uptake in rat soleus muscles but does not affect basal level of uptake. In control experiments, insulin stimulation of 2-deoxy-D-glucose uptake is about 400% of that of the control level. After PAO treatment, the stimulation reduces to 150% of the control. Since the intracellular level of ATP remains unchanged after PAO treatment, when measured by phosphorus-31 nmr spectroscopy, this inhibition is not due to depletion of ATP pool size. Moreover, PAO does not affect autophosphorylation of the insulin receptors purified from rat soleus muscles, implying that the PAO blockage of insulin-dependent glucose uptake in soleus muscles also may be post-receptor.     

Oxytocin action: lipid metabolism in adipocytes from homozygous diabetes insipidus rats (Brattleboro strain)

Oxytocin, like insulin, stimulates glucose oxidation in normal rat adipocytes. Fat cells from homozygous Brattleboro rats that exhibit diabetes insipidus (HoDI animals) and that have a normal number of oxytocin receptors, however, are unable to respond to oxytocin in terms of glucose oxidation. We now report that in adipocytes from HoDI animals that are responsive to insulin, oxytocin was also unable to stimulate lipogenesis. In contrast, oxytocin like insulin was able to inhibit epinephrine-stimulated lipolysis in adipocytes from HoDI animals. Thus, in HoDI adipocytes, the results indicate that the receptor-effector system is only partially defective.