Infusion of insulin impairs human adipocyte glucose metabolism in vitro without decreasing adipocyte insulin receptor binding

Summary To determine whether hyperinsulinaemia can cause insulin resistance in man and, if so, whether this occurs at a receptor or post-receptor site, nine normal volunteers were infused with insulin for 6 h at a rate (2 mU·kg^-1·min^-1) which resulted in steady-state plasma insulin concentrations of 140±13mU/l and four subjects were infused with saline (0.45%). Isolated adipocytes and monocytes were used as models for studying insulin binding, while adipocytes were also used to study insulin action in vitro. Adipocyte insulin binding did not decrease following infusion of insulin (4.6±0.5 versus 4.4±0.4% per 2×10⁵ cells, before and after, respectively), whereas monocyte insulin binding did (7.2±0.6 versus 6.2±0.6% per 10⁷ cells, p<0.05). Initial rates of adipocyte 3-0-methyl glucose transport were decreased in the absence of insulin (basal) and at submaximally effective (33.3pmol/l) but not at maximally effective insulin concentrations. At all insulin concentrations and in the absence of insulin, rates of glucose conversion to lipids were decreased more than 50% (p<0.05), whereas rates of glucose oxidation were unaffected. This decrease in the rates of conversion of glucose to lipids could not be accounted for by the decrease in rates of glucose transport. These results suggest that hyperinsulinaemia can cause insulin resistance in man and that, at least initially, this occurs at a post-receptor site. Furthermore, the discordant effect of hyperinsulinaemia on monocyte and adipocyte insulin binding indicates that monocyte insulin binding may not always reflect insulin binding in insulin-sensitive tissues.     

Stimulatory and cytotoxic effects of an antiserum to adipocyte plasma membranes on adipose tissue metabolism in vitro and in vivo

Antibodies to rat adipocyte plasma membranes raised in sheep had a dual effect in vitro; at low concentrations they mimicked the actions of insulin whilst higher concentrations inhibited glucose incorporation into lipid. The insulin-like effects of the antibody appeared to be due to direct activation of the glucose transport system since the antibodies did not bind to the insulin receptor, as judged by their inability to immunoprecipitate the receptor or to inhibit insulin binding, and antibodies were able to stimulate glucose transport in cells which had had their insulin receptors removed by trypsinization. The inhibitory effects of the antiserum were due to cytotoxicity since, in the presence of antiserum, adipocytes began to release large quantities of the intracellular enzyme, lactate dehydrogenase, and were ultimately lysed. This cytotoxic effect of the antiserum was complement-dependent since heat-inactivated antiserum or a crude immunoglobulin fraction of the serum possessed only stimulatory effects on lipid metabolism. When injected into rats for 4 days the antiserum produced gross abnormality of adipose tissue depots. Dissolution of adipocytes and massive infiltration by lymphocytes and polymorphs were evident. Preliminary observations suggest that such treatment results in long-term reduction of the number of adipocytes in internal fat depots.     

Studies of acute effects of insulin-like growth factors I and II in human fat cells

The acute metabolic effects and receptor binding of insulin-like growth factors (IGFs) I and II were studied in human adipose tissue. The IGFs inhibited fat cell glycerol release and stimulated adipocyte 3-O-methylglucose transport and adipose tissue glucose oxidation as effectively as did insulin, but the biological potencies of the IGFs, on a molar basis, were 600-1000 times less than that of insulin. The insulin dose-response curve for antilipolysis gradually shifted to the left in the presence of submaximally and maximally effective IGF-I concentrations, whereas no additive response was found when fat cells were incubated with maximally effective concentrations of insulin and the IGFs. Adipocyte [125I]IGF-I and -II binding was low and was not inhibited by excess unlabeled IGF. In contrast, IGF-I inhibited [125I]insulin binding with a molar potency 1600 times lower than that of native insulin. In adipose tissue segments obtained from patients with untreated noninsulin-dependent diabetes mellitus, IGF-I and insulin inhibited glycerol release in a normal way. Conversely, neither insulin nor IGF-I increased the rate of glucose oxidation significantly above the nonhormone-stimulated level. We conclude that human fat cells lack specific cell surface IGF-binding sites. However, the IGFs definitely produce acute insulin-like effects in the human adipocyte, which seems to be mediated via the insulin receptor.     

Acute growth hormone deficiency rapidly alters glucose metabolism in rat adipocytes. Relation to insulin responses and binding

To investigate cellular aspects of the antagonism between endogenous GH and insulin in adipose tissue, we examined glucose metabolism, insulin responses, and insulin binding in adipocytes isolated from rats made GH deficient by treatment with antibodies to rat GH (ArGH), which neutralize the biological actions of GH. Adipocytes were incubated in the presence or absence of insulin, and their ability to convert [14C] glucose to CO2 and lipid was measured. Basal glucose utilization was significantly elevated in adipocytes from ArGH-treated rats after 6, 3, or even 1 h of serum treatment compared to that in controls treated with nonimmune serum. This suggests that endogenous GH suppresses glucose metabolism in adipocytes from normal rats. In the presence of insulin, the absolute values of glucose utilization were also higher in cells from ArGH-treated rats than in controls. However, because basal glucose oxidation was higher with GH deficiency, the percent stimulation by insulin was not different in adipocytes from ArGH-treated rats and controls. In keeping with this, the binding of [125I]iodoinsulin was not altered by GH deficiency. These findings indicate that endogenous GH suppresses basal glucose metabolism in adipocytes, and that this effect is rapidly reversed by treatment of normal rats with ArGH. Furthermore, this effect of GH on adipocyte metabolism is independent of changes in insulin binding and appears to account for the enhanced responses to insulin observed with GH deficiency.     

Rottlerin inhibits insulin-stimulated glucose transport in 3T3-L1 adipocytes by uncoupling mitochondrial oxidative phosphorylation

There is increasing evidence that protein kinase C (PKC) isoforms modulate insulin-signaling pathways in both positive and negative ways. Recent reports have indicated that the novel PKCdelta mediates some of insulin's actions in muscle and liver cells. Many studies use the specific inhibitor rottlerin to demonstrate the involvement of PKCdelta. In this study, we investigated whether PKCdelta might play a role in 3T3-L1 adipocytes. We found that PKCdelta is highly expressed in mouse adipose tissue and increased on 3T3-L1 adipocyte differentiation, and insulin-stimulated glucose transport is blocked by rottlerin. The phosphorylation state and activity of PKCdelta are not altered by insulin, but the protein translocates to membranes following insulin treatment. In contrast to the results with rottlerin, inhibition of PKCdelta activity or expression has no effect on glucose transport in adipocytes, unlike muscle cells. Lastly, we found that rottlerin lowers adenosine triphosphate levels in 3T3-L1 cells by acting as a mitochondrial uncoupler, and this is responsible for the observed inhibition of glucose transport.     

Insulin receptor binding and insulin action in human fat cells: effects of obesity and fasting

We have studied (125I)-insulin binding and insulin dose response relationships of (14C)-methylglucose transport conversion of (14C)-glucose to CO2 and total lipids, and lipolysis at 37 degrees C and pH 7.4 in adipocytes from obese patients before (n = 15) and after fasting for 10 days (n = 6). Studies of adipocytes from obese before fasting showed a significant reduction of insulin binding when expressed to cell surface area and rightward shifts of the insulin dose response curves (decreased insulin sensitivity) for glucose transport, glucose oxidation, lipogenesis and antilipolysis. The decreased insulin sensitivity of adipocytes from obese was most likely the functional consequence of the impaired insulin binding. Moreover, decreased maximal glucose transport capacities were present in rat cells from obese both in the basal and maximally insulin stimulated states. Similarly, the percentage response above basal level to maximal insulin stimulation of glucose oxidation and lipogenesis was impaired to these cells. The latter findings suggest post receptor defects localized both to the transport system per se and to intracellular mechanisms involved in the metabolism of glucose. Conversely, the post receptor pathways for the insulin induced antilipolysis was intact in fat cells from obese man. Studies after fasting showed an increase of adipocyte insulin binding accompanied by an increased sensitivity to the antilipolytic effect of insulin with unchanged maximal responsiveness. However, due to marked post receptor alterations, the insulin stimulated glucose utilization was severely blunted. Thus, the glucose transport system of adipocytes from all fasted subjects was totally unresponsive to insulin, while some of the fasted patients had a slight response of glucose oxidation and lipogenesis in the presence of insulin in maximally effective concentrations.     

Long-term treatment with interleukin-1β induces insulin resistance in murine and human adipocytes

Aims/hypothesis Adipose tissue inflammation has recently been implicated in the pathogenesis of insulin resistance and is probably linked to high local levels of cytokines. IL1B, a proinflammatory cytokine, may participate in this alteration.Materials and methods We evaluated the chronic effect (1–10 days) of IL1B (0.1–20 ng/ml) on insulin signalling in differentiating 3T3-F442A and differentiated 3T3-L1 murine adipocytes and in human adipocytes. We also assessed expression of the gene encoding IL1B in adipose tissue of wild-type and insulin-resistant mice (diet-induced and genetically obese ob/ob mice).Results IL1B inhibited insulin-induced phosphorylation of the insulin receptor β subunit, insulin receptor substrate 1, Akt/protein kinase B and extracellular regulated kinase 1/2 in murine and human adipocytes. Accordingly, IL1B suppressed insulin-induced glucose transport and lipogenesis. Long-term treatment of adipose cells with IL1B decreased cellular lipid content. This could result from enhanced lipolysis and/or decreased expression of genes involved in lipid metabolism (acetyl-CoA carboxylase, fatty acid synthase). Down-regulation of peroxisome proliferating-activated receptor γ and CCAAT/enhancer-binding protein α in response to IL1B may have contributed to the altered phenotype of IL1B-treated adipocytes. Moreover, IL1B altered adipocyte differentiation status in long-term cultures. IL1B also decreased the production of adiponectin, an adipocyte-specific protein that plays a positive role in insulin sensitivity. Expression of the gene encoding IL1B was increased in epididymal adipose tissue of obese insulin-resistant mice.Conclusions/interpretation IL1B is upregulated in adipose tissue of obese and insulin-resistant mouse models and may play an important role in the development of insulin resistance in murine and human adipose cells.Electronic supplementary material Supplementary material is available in the online version for this article at and is accessible for authorized users.     

Potentiation of insulin stimulation of hexose transport by kallikrein and bradykinin in isolated rat adipocytes

Kallikrein and bradykinin additively increased adipocyte hexose transport under conditions of maximal intrinsic insulin stimulation, while no such effect occurred in the absence of insulin. This potentiation of insulin action follows a dose-response relationship with kallikrein and bradykinin concentrations consistent with a physiological role for the latter in the modulation of insulin action. Insulin degradation by isolated adipocytes and insulin binding to its receptors on adipocyte plasma membranes were not affected by either kallikrein or bradykinin. Thus, the kallikrein and bradykinin potentiation of insulin action occur at post-insulin binding sites. In conclusion, the kallikrein-bradykinin system increases the supply of substrates to target tissues through vasodilation and augmented blood perfusion, and it also stimulates glucose uptake and metabolism via its potentiation of insulin action. These actions suggest that the kallikrein-bradykinin system regulate both the availability and utilization of metabolic substrates, in target tissues.     

Vanadate increases cell surface insulin binding and improves insulin sensitivity in both normal and insulin-resistant rat adipocytes

Summary The aim of this study was to elucidate the acute effects of vanadate on cell surface insulin binding and insulin sensitivity in rat adipocytes. The cells were preincubated at 37° for 20 min followed by energy depletion with potassium cyanide, extensive washing and ¹²⁵I-insulin binding. The presence of vanadate or insulin during the preincubation period dose-dependently enhanced ¹²⁵I-insulin binding to normal adipocytes (maximally 4–5-fold) through an increased number of binding sites without any change in receptor affinity. Submaximal concentrations of vanadate added together with insulin enhanced the cellular sensitivity to the effect of insulin to stimulate 3-O-methylglucose transport. Vanadate, but not insulin, was also capable of increasing insulin binding as well as insulin sensitivity in insulin-resistant cells (treatment with N⁶-monobutyryl cAMP or amiloride and adipocytes from obese, aging rats). There was a correlation between the effect of vanadate to augment insulin binding and its ability to enhance cellular insulin sensitivity. Thus, the data suggest that short-term vanadate treatment improves insulin sensitivity through enhanced receptor binding and that this occurs in both normal and insulin-resistant cells.     

Influence of ambient glucose and insulin concentrations on adipocyte insulin binding

To elucidate factors of importance for insulin binding, fat cells from humans and rats were incubated under various experimental conditions for different periods of time. Human adipocytes incubated for 24 hours in the absence of insulin showed no significant difference in insulin binding compared with cells from freshly excised tissue. After 48 hours, however, an increased rate of binding (average 54%; P less than 0.05) was obtained. The addition of insulin (2000 microU/ml) to the culture medium resulted in a decrease in insulin binding (average 33%; P less than 0.05) compared with cells maintained in the absence of insulin. There was no apparent difference in receptor affinity, indicating that the altered binding was due to a change in receptor number. In the absence of insulin, elevating the glucose concentration of the medium from 0.8 mM to 22.4 mM did not significantly influence insulin binding. Rat adipocytes showed similar but more rapid changes. Thus, incubation for 24 hours without insulin caused an increase in insulin binding (average 37%; P less than 0.05). This up-regulation was seen even in a high glucose concentration (28 mM) but was completely prevented by the presence of insulin in the medium. Furthermore, when rat adipocytes were incubated with insulin in the presence of a high glucose concentration (28 mM) there was a significant further decrease in insulin binding compared with that of parallel incubations performed in 5.6 mM glucose. Thus, even in the absence of TRIS buffer, insulin-dependent regulation of the number of binding sites is shown for both human and rat adipocyte tissue in vitro. Although this perturbation could be directly due to hormone-receptor interaction at the membrane level, the finding of rat adipocytes that the ambient glucose concentration can modulate this effect suggests the importance of post-receptor events.     

In vivo specific uptake of labeled insulin by liver,adipose tissue,pituitary, and adrenals in the turtle Chrysemys dorbigni

Insulin labeled with ¹²⁵I was injected into turtles (Chrysemys dorbigni) to study its specific uptake by tissues. The maximum specific uptake of radioactivity by turtle tissues was obtained 1 hr after administration of [¹²⁵I]iodoinsulin. Besides liver and adipose tissue, specific uptake of labeled insulin was detected in some endocrine glands, such as pituitary and adrenals. Both glands were as active in concentrating labeled insulin as liver and adipose tissue. A significant reduction of the uptake was observed when unlabeled insulin was injected together with the labeled hormone. This reduction was dose dependent, and the concentration of unlabeled insulin that prevented 50% of the tissue uptake of [¹²⁵I]iodoinsulin was of 1 to 10 μg/kg body weight. These doses were able to induce blood glucose decrease in the turtle. Prolactin, growth hormone, or glucagon were unable to displace labeled insulin uptake. The major proportion of the radioactive material extracted from liver and pituitary 1 hr after [¹²⁵I]iodoinsulin injection into turtle coeluted with [¹²⁵I]iodoinsulin in Sephadex G-50 column. The presence of radioactive degradation products are consistent with the intracellular receptor mediated degradation hypothesis. These findings suggest the presence of specific insulin binding sites in liver, adipose tissue, pituitary, and adrenal glands from turtles.     

Binding and molecular weight properties of the insulin receptor from omental and subcutaneous adipocytes in human obesity

Summary The insulin binding properties and the molecular weights of the insulin receptor and its insulin binding subunit were studied in omental and subcutaneous adipocytes prepared from obese- and normal-weight subjects. Insulin binding by such adipocytes was decreased in obesity when the binding activity was expressed per unit of cell surface area. No significant difference from the lean controls was evident, however, when binding was calculated on a per cell basis, indicating that the total receptor content of the cells from the obese subjects was not altered. In addition, the normal difference in the receptor binding affinities previously reported between omental and subcutaneous cells from lean individuals was unaffected by the obese condition. Studies of the molecular weight of the non-reduced insulin receptor in fat cell membranes prepared from pieces of omental and subcutaneous fat demonstrated a major receptor species of 390–425K M_r. In contrast, adipocytes isolated by collagenase treatment of the fat had heterogenous non-reduced receptor species of M_r 355K, 285K and small amounts of 427K and 182K. Although different non-reduced receptor species were evident depending on the adipocyte receptor preparation (e.g. isolated adipocytes or fat cell membranes), no differences were found between obese and lean controls or between subcutaneous and omental receptors when the appropriate comparisons were made. Upon sulphydryl reduction, all receptor preparations had a major binding subunit of 125K M_r. In conclusion, obesity is characterized by a dilution of the insulin receptor over the adipocyte cell surface in the absence of a change in total cellular content of receptors. The difference in insulin binding affinities between omental and subcutaneous adipocytes could not be explained by an alteration in receptor molecular weight.     

Decrease in A1 adenosine receptors in adipocytes from spontaneously hypertensive rats

We have investigated whether the insulin resistance reported to occur in hypertension is due to decreased insulin receptors or to adenosine receptors in adipocyte membranes. Membranes were isolated from adipocytes from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKy) rats and assayed for insulin receptors and A1 adenosine receptors. The two groups of membranes bound 125I-insulin equally, but in contrast the SHR membranes bound approximately 25% less 125I-HPIA ([(-)-N6-p-hydroxyphenylisopropyl adenosine], an A1 adenosine receptor agonist) than the WKy (P less than .005). Scatchard analysis demonstrated that this was due to a lower number of receptors in the SHR. The affinity of the receptor for HPIA was approximately 0.7 nmol/L in both groups. 5'-Nucleotidase activity was approximately 40% higher in membranes from SHR than WKy (P less than .001), indicating that adipocytes from SHR have a higher capacity for adenosine production. This may cause increased adenosine concentrations in the SHR adipose tissue, leading to adenosine receptor down-regulation. Since we have previously demonstrated that adenosine receptor down-regulation can lead to insulin resistance, these findings may partly account for the insulin resistance of hypertension.     

Impaired insulin binding to isolated adipocytes in experimental diabetic ketoacidosis

Summary Insulin sensitivity in vivo and insulin binding in vitro to adipocytes have been studied in streptozotocin diabetic rats with ketoacidosis. Insulin sensitivity in vivo measured as the acute (20 min) fall in blood glucose in response to an insulin infusion of 1 U/kg body weight per hour correlated positively with arterial blood pH (r=0.92, p < 0.01: n=38). At pH < 6.9 there was no fall in blood glucose. For studies of insulin binding to adipocytes ketoacidotic animals were divided into a group with moderate ketoacidosis (pH > 7.0) and a second group with severe ketoacidosis (pH < 6.9). Insulin binding to adipocytes was maximal in cells from both ketoacidotic and from normal rats at pH 7.6–7.8. Total binding was decreased in the diabetic rats (p < 0.01) and this was more marked in the severely diabetic group (p < 0.001) at all pHs studied. At pH 7.4, ¹²⁵I-insulin binding was decreased in diabetics compared with normal rats (0.89±0.14 versus 2.0±0.24% with 2×10⁵ cells/ml: n=6; p < 0.01) and also in the severe compared with the moderate ketoacidotic rats (0.5± 0.08%/2×10⁵ cells; n=6, p < 0.05). Equilibrium binding studies showed that there was a small decrease in apparent affinity in adipocytes from both groups of diabetics (K_D = 2.8±0.2×10^-9 mol/l, n =6 in moderate ketoacidosis; 2.5±0.3×10^-9 mol/l, n=6 in severe ketoacidosis) compared with control animals (K_D = 1.8±0.15×10^-9 mol/l, n= 6). Scatchard analysis revealed that there was also a decrease in receptor concentration which was greater in the severely ketoacidotic group. These findings may explain in part the insulin resistance of severe ketoacidosis.     

Receptor binding and biological effect of insulin in human adipocytes

Summary The binding of¹²⁵I-labelled insulin to human adipocytes was studied at 37° C. The precipitability of the¹²⁵I-labelled insulin preparation (0.03 nmol/l) in trichloroacetic acid and the concentration of biologically active insulin (7.5 nmol/l) remained constant in buffer incubated with human adipocytes (100 l cells/ml suspension) for 30–60 minutes at 37° C, whereas more than half of the insulin was inactivated by rat fat cells under the same conditions. A constant level of binding of¹²⁵I-labelled insulin (0.03 nmol/l) to human adipocytes was obtained after 45 minutes. The apparent dissociation constant of receptor binding was about 0.2 nmol/l as compared to about 2 nmol/l for rat adipocytes. Conversion of [U-¹⁴C]glucose to lipids was stimulated half-maximally by about 0.05 nmol/l of insulin (similar to rat adipocytes). Thus, half-maximal stimulation of human adipocytes was obtained with a receptor occupancy of about 20–30 per cent.     

Effect of 3'':5''-cyclic AMP on glucose transport in rat adipocytes.

An indirect method for obtaining a reliable measure of the rate of glucose transport into adipocytes is described. Evidence is presented that altered levels of 3':5'-cyclic AMP can influence the transport of glucose into adipocytes. When cyclic AMP levels were lowered with antilipolytic agents (insulin, nicotinic acid, or clofibrate), rates of glucose transport were increased. In contrast, when adipose tissue levels of cyclic AMP were elevated by lipolytic hormones or theophylline, glucose transport when cyclic AMP levels were elevated by lipolytic agents. Agents that can raise cyclic AMP but inhibit lipolytic (procaine, amitryptyline, and phenylethylbiguanide) reduced the rate of glucose transport. Other data are presented that are consistent with the conclusion that cyclic AMP inhibits glucose transport into adipocytes.     

Antagonistic effects of a covalently dimerized insulin derivative on insulin receptors in 3T3-L1 adipocytes.

In the present study we describe the antagonistic effects of the covalently dimerized insulin derivative B29,B29'-suberoyl-insulin on insulin receptors in 3T3-L1 mouse cells. In differentiated 3T3-L1 adipocytes, the derivative fully inhibits binding of 125I-labeled insulin to its receptor with about the same affinity as unlabeled insulin. In contrast, the dimerized derivative only partially (approximately 20%) mimics insulin's effects on glucose transport and DNA synthesis in the absence of insulin. In the presence of insulin, the agent competitively inhibits insulin-stimulated DNA synthesis ([3H]thymidine incorporation into total DNA), glucose transport activity (2-deoxyglucose uptake rate), and insulin receptor tyrosine kinase activity. In rat adipocytes, in contrast, the dimerized derivative stimulates glucose transport (initial 3-O-methylglucose as well as 2-deoxyglucose uptake rates) to the same extent as insulin does, and it fails to inhibit the effect of insulin. The data indicate that the dimerized insulin derivative B29,B29'-suberoyl-insulin is an insulin receptor antagonist (partial agonist) which retains a moderate intrinsic activity. The effects of this agent reveal a striking difference in insulin receptor-mediated stimulation of glucose transport between 3T3-L1 fatty fibroblasts and the mature rat adipocyte.     

Cellular mechanisms of insulin resistance in polycystic ovarian syndrome.

Insulin resistance is a predominant feature in women with polycystic ovarian syndrome (PCO). The cellular mechanisms for this insulin resistance have not been defined. In this study, major steps in the insulin action cascade, receptor binding, kinase activity, and glucose transport activity were evaluated in isolated adipocytes prepared from PCO subjects (n = 8) without acanthosis nigricans and in a group of age and weight-matched controls [normal cycling (NC) n = 8]. The PCO group was hyperinsulinemic and displayed elevated insulin responses to an iv glucose load. The binding of 125I-insulin to adipocytes was similar in cells from PCO and NC subjects. In PCO, autophosphorylation of the insulin receptor-subunit in the absence of insulin was normal but a significant decrease (30% below control) in maximal insulin stimulated autophosphorylation was observed. However, receptor kinase activity measured against the exogenous substrate poly glu:tyr (4:1) was normal. Cells from PCO subjects transported glucose at the same rate, in both the absence and presence of a maximal insulin concentration, as those from NC subjects. Strikingly, there was a large rightward shift in the insulin dose-response curve for transport stimulation in PCO cells (EC50 = 87 +/- 14 pmol in NC vs. 757 +/- 138 in PCO, P less than 0.0005); 8-fold greater insulin concentrations were required to attain comparable glucose transport rates in cells from PCO against NC. In conclusion, our results suggest that insulin resistance in PCO, as assessed in the adipocyte, is accompanied by normal function of insulin receptors, but involves a novel postreceptor defect in the insulin signal transduction chain between the receptor kinase and glucose transport.