Effects of phloretin and dextran-linked phloretin on pancreatic islet metabolism and insulin release

The effects of phloretin on islet metabolism and insulin release have been studied in isolated pancreatic islets of the rat. At a concentration of 0.18 mM phloretin inhibited insulin release stimulated by glucose or leucine but did not affect the oxidation rates of glucose or leucine, the rate of glucose utilization and the islet content of ATP. Higher concentrations of phloretin caused inhibition of the rate of glucose metabolism, but stimulation of insulin release. Insulin release stimulated by phloretin was inhibited by mannoheptulose but was independent of extracellular Ca²⁺ and was not potentiated by caffeine. Both inhibitory and stimulatory effects of dextran-linked phloretin on insulin release were also seen; a concentration of dextran-linked phloretin that did not inhibit islet metabolism inhibited glucose-stimulated insulin release, but not release stimulated by leucine or glyceraldehyde. Higher concentrations of dextran-linked phloretin inhibited glucose oxidation but stimulated insulin release. These data are discussed in terms of current models of the β-cell glucose-sensor mechanism.     

Effect of a brewer's yeast extract on growth and glucose metabolism of cells in culture.

Brewer's yeast preparations influence glucose metabolism in vivo and in isolated tissues. We have studied the effect of a brewer's yeast extract on glucose metabolism and grwoth of rat hepatoma and human embryonic cells. Growth of the rat hepatoma cells was very much stimulated by the extract in a concentration-dependent manner. Glucose uptake was, on the other hand, appreciably inhibited, and lactate uptake completely abolished by the extract. Insulin stimulated cell growth and inhibited lactate uptake but did not affect the glucose level. Insulin and the extract had additive effects on growth and lactate uptake of the hepatoma cells. The inhibition by the brewer's yeast extract of glucose uptake was, however, antagonized by insulin. Niacin or Cr³⁺, which are suggested to be components of a “glucose tolerance factor” of brewer's yeast, did not affect growth or glucose and lactate uptake. The glucose uptake of the human embryonic cells was strongly inhibited by the brewer's yeast extract. Cell growth and lactate production were not influenced by the extract or by insulin; however, when both insulin and extract were present simultaneously, a slight stimulation of growth and inhibition of lactate production was observed. The results indicate that brewer's yeast can have appreciable direct effects on cells and that not all of these effects are “insulin-like”.     

Evaluation of the isolated perfused rat hindquarter for the study of muscle metabolism

1. The metabolic integrity of a new isolated rat hindquarter preparation was studied. The hindquarter was perfused with a semi-synthetic medium containing aged human erythrocytes. More than 95% of the oxidative metabolism of the preparation was due to muscle, the remainder being due to bone, adipose tissue and, where present, skin. 2. Consumption of O(2), glucose utilization, glycerol release and lactate production were similar in the presence and in the absence of the skin, indicating that the latter contributed little to the overall metabolism of the preparation. 3. After 40min of perfusion, tissue concentrations of creatine phosphate, ATP and ADP were similar to those found in muscle taken directly from intact animals. The muscle also appeared normal under the electron microscope. 4. The hindquarter did not lose K(+) to the medium during a 30min perfusion. In the presence of insulin it had a net K(+) uptake. 5. Insulin caused a sixfold increase in glucose uptake, stimulated O(2) consumption by nearly 40% and depressed glycerol release to less than half the control value. 6. Bilateral sciatic-nerve stimulation caused severalfold increases in O(2) consumption and lactate production. In the absence of insulin nerve stimulation also enhanced glucose uptake; in the presence of insulin it did not further increase the already high rate of glucose uptake. 7. Rates of lactate production and O(2) consumption of the rat hindquarter in vivo and the isolated perfused hindquarter were very similar. 8. Ketone bodies were a major oxidative fuel in vivo of the hindquarter of a rat starved for 2 days. If the acetoacetate and 3-hydroxybutyrate removed by the tissue were completely oxidized, they would have accounted for 77% of the O(2) consumption. 9. Acetoacetate accounted for 84% of the ketone bodies removed by the hindquarter in vivo even though its arterial concentration was half that of 3-hydroxybutyrate. 10. Similar rates of acetoacetate and 3-hydroxybutyrate utilization were observed in the perfused hindquarter. 11. Acetoacetate utilization by the perfused hindquarter was not diminished by the addition of either oleate or insulin to the perfusate. 12. Oxidation of glucose to CO(2) accounted for less than 4% of the O(2) consumed by the perfused hindquarter in both the presence and the absence of insulin. 13. The results indicate that the isolated perfused hindquarter is a useful tool for studying muscle metabolism. They also suggest that ketone bodies, if present in sufficient concentration, are the preferred oxidative fuel of resting muscle.     

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.     

A comparison of the cerebral uptake and metabolism of labeled glucose and deoxyglucose in vivo in rats

Experimental evidence indicated that: 1) [¹⁴C]deoxyglucose-6-phosphate (¹⁴C-DG-6-P) in brain (and other rat tissues) did not increase with time after injection of¹⁴C-DG, 2)¹⁴C-DG-6-P in rat brain (and other tissues) did not correlate with glucose metabolism 3)¹⁴C-DG-6-P in rat brain (and other tissues) had a significant negative correlation with glucose-6-phosphatase activity. Further, arterio-venous studies in rats, in which the cerebral uptake and metabolism of labeled glucose were compared directly with those of labeled DG (and labeled fluorodeoxyglucose, FDG), employing double labeled techniques, showed that DG (and FDG) cannot be used to measure glucose uptake and/or metabolism.     

Tissue kallikrein and bradykinin do not have direct insulin-like actions on skeletal muscle glucose utilization

Studies suggest that the actions of insulin on glucose metabolism may be mediated through activation of a membrane-bound serine protease with properties similar to a kallikrein-like enzyme. Also, bradykinin, a vasoactive product of kallikrein's action upon kininogen substrates, increases glucose uptake when infused into the human forearm. To determine whether a kallikrein or a kinin directly affects cellular glucose metabolism or participates in mediating insulin's actions, we studied their effects on isolated rat soleus muscle. Although trypsin (1.34 microM) increased incorporation of glucose into muscle glycogen to the same extent as insulin (200 mu units/ml), a purified rat tissue (urinary) kallikrein (0.4-1.34 microM) produced no such effect. Furthermore, the tissue kallikrein inhibitor, aprotinin, or a polyclonal kallikrein antiserum did not inhibit the action of insulin on incorporation of glucose into muscle glycogen. Treatment of the muscle preparation with bradykinin (1nM - 10 microM) did not result in any change in basal or insulin-stimulated (20 - 2000 mu units/ml) entry of glucose into glycogen or the glycolytic pathway. Bradykinin (1nM - 10 microM) also did not influence basal or insulin-stimulated (1000 mu units/ml) initial rates of glucose transport. These studies suggest that the previously observed in vivo effects of bradykinin on peripheral glucose uptake are probably mediated by changes in tissue perfusion rather than direct kinin effects on skeletal muscle, and that the putative membrane serine protease involved in the insulin-effector system is not tissue kallikrein.     

Studies on the biological activity of an insulin-stimulating peptide from a tryptic digest of bovine serum albumin

Summary A two-chain polypeptide, which corresponds to amino acid residues 115–143 and 144–184(185) of bovine serum albumin, connected to each other by a disulfide bridge, potentiated the effects of insulin on glucose transport and glucose metabolism in isolated rat adipocytes. Although the peptide alone had little activity, it shifted the concentration-response curves of insulin-stimulated D-[I-¹⁴C]glucose oxidation, 2-deoxyglucose transport, and lipid synthesis from D-[U-¹⁴C]glucose to lower insulin concentrations. It also increased the maximal responses of these parameters to insulin. However, it did not affect insulin binding to adipocytes. The peptide protected insulin considerably from degradation, but this effect alone cannot account for its effect in increasing the maximal responses to the hormone, and even when degradation of a submaximal concentration of insulin was suppressed by bacitracin, the peptide still had an enhancing effect. These results suggest not only that the peptide influences a step distal to receptor-mediated insulin binding but also that inhibition of insulin degradation alone cannot explain its total effect.The peptide lost its insulin-stimulating activity completely when it was further digested with V8 or lysinespecific endopeptidase, or when it was reduced and then carboxamidomethylated or oxidized with performic acid. Similar active tryptic fragments were obtained from human and rat albumins.Insulin-stimulating peptides should be useful in studies on the mechanisms of insulin action including both the sensitivities and responsiveness of target cells to the hormone.Abbreviations ISP insulin-stimulating peptide - HEPES N-(2-hydroxyethyl)piperazine-N-2-ethanesulfonic acid - HPLC high-performance liquid chromatography - SDS sodium dodecyl sulfate     

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.     

Somatostatin-induced inhibition of insulin secretion from isolated islets of rat pancreas in presence of glucagon.

In order to study the oeffect of somatostatin on the endocrine pancreas directly, islets isolated from rat pancreas by collagenase were incubated for 2 hrs 1) at 50 and 200 mg/100 ml glucose in the absence and presence of somatostatin (1, 10 and 100 mg/ml) and2) at 200 mg/100 ml glucose together with glucagon (5 mug/ml), with or without somatostatin (100 ng/ml). Immunologically measurable insulin was determined in the incubation media at 0, 1 and 2 hrs. Insulin release was not statistically affected by any concentration stomatostatin. On the other hand, somatostatin exerted a significant inhibitory action on glucagon-potentiated insulin secretion (mean +/- SEM, mu1/2 hrs/10 islets: glucose and glucagon: 1253 +/- 92; glucose, glucagon and somatostatin: 786 +/- 76). The insulin output in th epresence of glucose, glucagon and somatostatin was also significantly smaller than in thepresence of glucose alone (1104 +/- 126) or of glucose and somatostatin (1061 +/- 122). The failure of somatostatin to affect glucose-stimulated release of insulin from isolated islets contrasts its inhibitory action on insulin secretion as observed in the isolated perfused pancreas and in vivo. This discrepancy might be ascribed to the isolation procedure using collagenase. However, somatostatin inhibited glucagon-potentiated insulin secretion in isolated islets which resulted in even lower insulin levels than obtained in the parallel experiments without glucagon. It is concluded that the hormone of the alpha cells, or the cyclic AMP system, might play a part in the machanism of somatostatin-induced inhibition of insulin release from the beta-cell.     

Effect of growth hormone on glycogenesis in cerebral cortical slices

The uptake of glucose by cerebral cortical slices of rats was found to be enhanced by insulin by Rafaelsen (1961) and Genes and Charnaya (1966). This was confirmed by Prasannan and Subrah-manyam (1965) and more recently by Nelson , Schultz , Pasoneau and Wry (1968). Eisenberg and Seltzer (1962) and Gotistein , Held , Sebenng and Walpurger (1965) obtained evidence for a direct effect of insulin on the entry of glucose into brain and on its metabolism in this tissue. A marked resynthesis of glycogen was demonstrated with glucose as substrate by Lebaron (1955) and Mcilwain and Tresize (1956) in cerebral cortical slices of the guinea pig. Prasannan and Subrahmanyam (1965) obtained evidence for a similar resynthesis of glycogen in cerebral cortical slices of the rat. Addition of 0.2 unit of insulin per 3.5 ml of incubating medium gave rise to an increase of 60 per cent in the resynthesis of glycogen in these slices. The incorporation of ¹⁴C from labelled glucose into glycogen and CO₂ by cerebral cortical slices of normal and alloxan diabetic rats and the stimulation of the incorporation into glycogen by insulin in vitro was reported by Visweswaran , Prasannan and Subrahmanyam (1969). An insulin-like action of growth hormone on the carbohydrate metabolism was reported by Ketterer , Randle and Young (1967) and Manchester and Young (1961). It was believed to be due to the formation of a polypeptide breakdown product of growth hormone which has biological insulin-like properties. Park , Brown , Cornbluth , Daughaday and Krahl . (1952) reported an increased uptake of glucose by isolated rat diaphragm due to the action of growth hormone which is similar to that of insulin. Hence, it was considered appropriate to study the incorporation of ¹⁴C from labelled glucose into glycogen and CO₂ by cerebral slices of growth hormone treated rats and the effect of growth hormone treatment on the activities of the enzymes concerned with glycogenesis in rat cerebral cortex.     

Effects of phloretin and dextran-linked phloretin on pancreatic islet metabolism and insulin release.

The effects of phloretin on islet metabolism and insulin release have been studied in isolated pancreatic islets of the rat. At a concentration of 0.18 mM phloretin inhibited insulin release stimulated by glucose or leucine but did not affect the oxidation rates of glucose or leucine, the rate of glucose utilization and the islet content of ATP. Higher concentrations of phloretin caused inhibition of the rate of glucose metabolism, but stimulation of insulin release. Insulin release stimulated by phloretin was inhibited by mannoheptulose but was independent on extracellular Ca2+ and was not potentiated by caffeine. Both inhibitory and stimulatory effects of dextran-linked phloretin on insulin release were also seen; a concentration of dextran-linked phloretin that did not inhibit islet metabolism inhibited glucose-stimulated insulin release, but not release stimulated by leucine or glyceraldehyde. Higher concentrations of dextran-linked phloretin inhibited glucose oxidation but stimulated insulin release. These data are discussed in terms of current models of the beta-cell glucose-sensor mechanism.     

Pentitols and insulin release by isolated rat islets of Langerhans

1. Insulin secretion was studied in isolated islets of Langerhans obtained by collagenase digestion of rat pancreas. In addition to responding to glucose and mannose as do whole pancreas and pancreas slices in vitro, isolated rat islets also secrete insulin in response to xylitol, ribitol and ribose, but not to sorbitol, mannitol, arabitol, xylose or arabinose. 2. Xylitol and ribitol readily reduce NAD(+) when added to a preparation of ultrasonically treated islets. 3. Adrenaline (1mum) inhibits the effects of glucose and xylitol on insulin release. Mannoheptulose and 2-deoxy-glucose, however, inhibit the response to glucose but not that to xylitol. 4. The intracellular concentration of glucose 6-phosphate is increased when islets are incubated with glucose but not with xylitol, suggesting that xylitol does not promote insulin release by conversion into glucose 6-phosphate. 5. Theophylline (5mm) potentiates the effect of 20mm-glucose on insulin release from isolated rat islets of Langerhans, but has no effect on xylitol-mediated release. These results indicate that xylitol does not stimulate insulin release by alterations in the intracellular concentrations of cyclic AMP. 6. A possible role for the metabolism of hexoses via the pentose phosphate pathway in the stimulation of insulin release is discussed.     

Development of insulin sensitivity in white adipose tissue during the suckling-weaning transition in the rat. Involvement of glucose transport and lipogenesis.

The changes of insulin responsiveness of white adipose tissue during the suckling-weaning transition in the rat were investigated in vitro on isolated adipocytes. Insulin binding, glucose transport and glucose metabolism in adipocytes from suckling rats and from rats weaned on to a high-carbohydrate (HC) or a high-fat (HF) diet were compared. Despite similar insulin binding, insulin-stimulated glucose transport rate is lower in adipocytes from suckling rats and HF-weaned rats than in adipocytes from HC-weaned rats. Moreover, whereas insulin markedly stimulates glucose metabolism in adipocytes from HC-weaned rats, glucose metabolism is totally unresponsive to insulin in adipocytes from suckling and HF-weaned rats. This insulin resistance is associated with a very low rate of lipogenesis and low activities of acetyl-CoA carboxylase, fatty acid synthase and pyruvate dehydrogenase.     

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.     

cAMP-enhancing agents “permit” stimulus-secretion coupling in canine pancreatic islet β-cells

Isolated canine islets of Langerhans differ from isolated islets of other species (including rodents and man) in that elevated glucose concentrations are unable to stimulate insulin secretion. Here we demonstrate that addition to the perifusate of isobutylmethylxanthine (IBMX), forskolin or 8-CPT-cAMP, all of which enhance cytosolic cAMP, permits insulin secretion in response to glucose, leucine or tolbutamide. These cAMP enhancers increase secretogogue-induced electrical activity in β-cells and restore depolarization-induced, Ca²⁺-dependent granule exocytosis measured as stepwise increases in membrane capacitance. We propose that the primary permissive action of cAMP is to tightly link Ca²⁺ entry to insulin granule release, while a secondary action is to tighten the link between glucose metabolism and cell depolarization.     

Stimulation of Immunoreactive Insulin Release by Glucose in Rat Brain Synaptosomes

The effect of glucose on the release of immunoreactive insulin (IRI) in synaptosomes isolated from rat brain was studied. In the absence of glucose synaptosomes release about 4% (0.77 IU/mg protein) of total content. Glucose increases significantly the IRI released by synaptosomes. Addition of the glycolytic inhibitor iodoacetic acid (IAA), decreased the glucose-induced release of IRI by about 50%, suggesting that glucose metabolism is involved. The observation that glucose provides a concentration related signal for IRI release indicates that this synaptosomal preparation may be useful as a model for research on the mechanism of insulin release in brain.     

Kinetics of early insulin response by the isolated and perfused rat pancreas. Suggestion for two different actions of glucose on the insulin releasing process.

The permissive action of glucose (or glyceraldelhyde) is necessary to the glucagon induced insulin release. By collecting every 15 seconds the venous effluent of the perfused and isolated rat pancreas it was observed that the insulin response to glucagon (2 microgram/ml) was immediate if the pancreas was preperfused with low concentration of glucose (5 mM) or glyceraldelhyde (2,5 mM). On the other hand glucagon alone elicited no response, and the insulin discharge occurred 60 to 90 seconds after the addition of glucose (5 mM) or glyceraldelhyde (2,5 mM) this time being probably allowed to the metabolism of the sugar. The pancreatic response to 15 mM glucose occurred also 60 to 90 seconds after the stimulus. On the other hand when the medium contained a low concentration of glucose (or glyceraldelhyde) increasing the glucose concentration by 10 mM provoked an immediate insulin release. This suggests that glucose has two actions differing by their lag phase. One, "permissive", apparent after some delay, mimicked by glyceraldelhyde, necessary for glucagon induced insulin release, is mediated probably by the metabolic products of the sugar. The second, "triggering", initiates instantaneously the insulin release but appears dependent on the first action.     

IMMUNOASSAY OF ENDOGENOUS PLASMA INSULIN IN MAN

For years investigators have sought an assay for insulin which would combine virtually absolute specificity with a high degree of sensitivity, sufficiently exquisite for measurement of the minute insulin concentrations usually present in the circulation. Methods in use recently depend on the ability of insulin to exert an effect on the metabolism of glucose in vivo or in excised muscle or adipose tissue. Thus, the insulin concentration in plasma has been estimated: a) from the degree of hypoglycemia produced in hypophysectomized, adrenalectomized, alloxan-diabetic rats (1); b) from the augmentation of glucose uptake by isolated rat hemidiaphragm (2); or c) from the increased oxidation of glucose-1-C¹⁴ by the rat epididymal fat pad (3). Since there have been reports indicating the presence, in plasma, of inhibitors of insulin action (4) and of noninsulin substances capable of inducing an insulin-like effect (5,6), these procedures, while yielding interesting information regarding the effects of various plasmas on glucose metabolism in tissues, are of doubtful specificity for the measurement of insulin per se (5).     

Insulin-like effects of polyamines spermine binding to fat cells and fat cell membranes

Two naturally occurring polyamines, spermine and spermidine, mimic the action of insulin on lipid and glucose metabolism in adipocytes. To evaluate the role of cell membranes in the action of polyamines, studies of [¹⁴C] spermine binding using an oil separation method were conducted in isolated rat adipocytes and adipose cell membranes. Spermine binding and dissociation in fat cells and fat cell membranes were rapid and complete within 3–6 min. Following a 30-min incubation of [¹⁴C] spermine with fat cell membranes, over 90% of bound [¹⁴C] spermine was dissociable while under similar conditions only 25% of bound [¹⁴C] spermine was dissociable in cells. The non-dissociable fractions in cells likely represented intracellular accumulation. Binding and stimulation of glucose oxidation were demonstrated at similar concentrations. Bound spermine was displaced by spermine, spermidine and 1,8-diaminooctane with greater efficacy than putrescine (a polyamine devoid of insulin-like properties) or insulin. Similarly, polyamines did not complete with insulin for binding to isolated adipocytes. It appears, therefore, that polyamines initiate their insulin-like effects by interacting with the cell membrane at sites which are common to biologically active polyamines and which are distinct from the insulin receptor.     

Glucose as a lipolytic agent: studies on isolated rat adipocytes

In order to elucidate the direct effect of glucose on lipolysis in isolated rat adipocytes, cells were incubated in a buffer with different concentrations of this sugar: 2, 8 or 16 mmol/l. The increase in glucose concentration from 2 mmol/l to 8 or 16 mmol/l enhanced basal lipolysis by 30% and 47%, respectively. Epinephrine-induced lipolysis (1 micromol/l) was also increased by 31% and 32%, when glucose concentration was increased from 2 mmol/l to 8 or 16 mmol/l, respectively. The rise in lipolysis caused by glucose was restricted by H-89 (an inhibitor of protein kinase A, 30 micromol/l), but insulin (1 nmol/l) had no inhibitory action. The augmentation of lipolysis by glucose did not require its metabolism (as demonstrated using 2-deoxyglucose) and was due to the action of this sugar on the final steps of the lipolytic cascade, particularly on protein kinase A. However, short-term exposure of adipocytes to higher glucose concentrations did not restrict the inhibitory action of insulin on lipolysis induced by epinephrine.