Insulin Target Cells Research Articles

11 Treatment—metformin

The hyperglycaemia of NIDDM is associated with insulin resistance due, in part, to reduced insulin receptor binding and more especially postreceptor defects. Metformin is an antihyperglycaemic agent which can be used to ameliorate insulin resistance. It appears to act directly on insulin target cells to enhance insulin action. Although metformin may increase insulin-receptor binding, its main effect appears to be directed at the postreceptor level of insulin action. Accordingly the drug potentiates insulin-suppression of hepatic gluconeogenesis and increases insulin-mediated peripheral glucose uptake and metabolism. It does not stimulate insulin release, does not cause weight gain and does not cause clinical hypoglycaemia. The risk of lactate accumulation should be appreciated in patients with renal insufficiency, liver dysfunction and following acute illness with hypoxia, when therapy should be stopped. Although metformin is often bracketed with phenformin in the context of lactic acidosis, different pharmacodynamics and adherence to prescribing guidelines render such a comparison unwarranted.

Characterization of the insulin receptor kinase from human erythrocytes.

The insulin receptor from human erythrocytes was studied for receptor-associated tyrosine kinase activity. Receptor phosphorylation was performed by incubation of the receptor with [gamma-32P]ATP and Mn2+ in the presence or absence of insulin, and the phosphorylated receptor was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis. In Triton X-100-solubilized, and partially purified, receptor preparations, insulin stimulated the phosphorylation of a 95,000 dalton protein in a dose-dependent fashion. Immunoprecipitation with antiinsulin receptor antibodies indicates that this 95,000 dalton protein corresponds to the beta-subunit of the insulin receptor. Phosphoaminoacid analysis revealed that 32P incorporation occurred predominantly on tyrosine residues of the beta-subunit. In addition, the insulin receptor kinase catalyzed the phosphorylation of Histone H2b. Dose response curves for insulin-stimulated phosphorylation of the beta-subunit and Histone H2b were sigmoidal. Both half-maximal effects were observed at 3 X 10(-9) M insulin with maximal effects at 10(-6) M. When insulin binding was examined under the same conditions as phosphorylation, the dose-response curves for receptor occupancy and the kinase activation were nearly superimposable, indicating few or no spare receptors for this response to insulin. Insulin-stimulated receptor phosphorylation was diminished by treatment with N-ethylmaleimide, indicating that the receptor possesses sulfhydryl groups, which are important for its enzyme activity. This study demonstrates that insulin receptor kinase from human erythrocytes shares characteristics which are similar to those found in other classical insulin target cells and tissues.

Insulin binding and biological activities in the FRTL-5 rat thyroid cell line

A cloned rat thyroid cell line (FRTL-5) was examined for both insulin binding and responsiveness. The characteristics of insulin binding to thyroid cells were similar to those observed in typical insulin target cells. The 125I-insulin binding was time and temperature dependent and Scatchard analysis suggested the presence of two major binding sites with high and low affinity constant (Kd = 1.4 × 10 −10 mol/L and 1.5 × 10 −9 mol/L, respectively). 125I-insulin was also internalized and degraded in a temperature-dependent manner. IGF 1 was weakly effective in completing 125I-insulin binding to FRTL-5 cells (57% inhibition at 333 nmol/L), whereas noninsulin-related peptide hormones were ineffective. Exposure of FRTL-5 cells to insulin stimulated both methyl-aminoisobutyric acid (M-AIB) and 2-deoxy-D-glucose (2DG) transport. These effects were evident at 10 −9 mol/L and maximal at 10 −7 mol/L insulin. Maximal stimulation was three- to four-fold over basal value for both M-AIB and 2DG transport. These data suggest that insulin specifically binds to FRTL-5 cells and regulates different biological functions of this thyroid cell line.

Pancreatic hormone receptors on islet cells.

To assess whether islet cells are equipped with recognition units which allow an intra-islet regulation via released hormones, the presence of insulin and glucagon receptors is investigated on purified pancreatic A and B cells. Mono-[125I]glucagon is shown to bind specifically to islet B cells, with similar binding characteristics as in isolated hepatocytes but involving less receptors per cell (2.10(4) per B cell vs. 8.10(5) per liver cell). Binding is half-maximally displaced by 5.10(-9) M glucagon, a concentration known to induce half-maximal biological effects in isolated B cells. These results are compatible with a regulatory role of glucagon in the insulin release process. No specific binding of [125I]tyr-A14-insulin is detected on pancreatic A cells. In order to increase receptor assay sensitivity, [123I]tyr-A14-insulin is prepared with at least 5-fold higher specific activity. Its validity for in vitro receptor analysis is demonstrated in IM-9 lymphocytes, where insulin binding is detectable down to 10(4) cells/ml. However, no insulin-binding sites are identified on pancreatic A cells, even at 10(6) cells/ml. If isolated A cells contain high affinity insulin receptors, their number should be inferior to 400 per cell, which is 50- to 500-fold lower than in classical insulin target cells. These findings explain the insensitivity of the glucagon release process to acute exposure to insulin.

Identification of functional insulin receptors on membranes from an insulin-producing cell line (RINm5F).

Insulin receptors on RINm5F cell membranes (an insulin-producing rat pancreatic cell line) were studied. To study the insulin receptor alpha-subunit, 125I-labelled photoreactive insulin was covalently bound to the membranes in the absence or presence of unlabelled insulin. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis under reducing conditions showed specific labelling of an Mr 130 000 protein. The receptor beta-subunit was studied by using a cell-free phosphorylation assay. Analysis under reducing conditions showed a phosphoprotein of Mr 95 000 whose level of phosphorylation was selectively increased by insulin, and which was specifically immunoprecipitated by antibodies to the insulin receptor. Further, covalent hormone-receptor complexes purified with anti-insulin antibodies were able to undergo autophosphorylation, indicating the existence of operational receptor subunit arrangements. RINm5F cell insulin receptors (and, by analogy, possibly those of native B-cells) thus display structural and functional integrity comparable with those of conventional insulin target cells.

Identification and characterization of insulin receptors on foetal-mouse brain-cortical cells.

The occurrence of insulin receptors was investigated in freshly dissociated brain-cortical cells from mouse embryos. By analogy with classical insulin-binding cell types, binding of 125I-insulin to foetal brain-cortical cells was time- and pH-dependent, only partially reversible, and competed for by unlabelled insulin and closely related peptides. Desalanine-desasparagine-insulin, pig proinsulin, hagfish insulin and turkey insulin were respectively 2%, 4%, 2% and 200% as potent as bovine insulin in inhibiting 125I-insulin binding to brain-cortical cells, which corresponds to their relative biological potencies in classical insulin-target cells; no competition was observed with glucagon and nerve growth factor, even at high concentrations. Scatchard analysis of competitive-binding data resulted in curvilinear plots with a high-affinity binding of Ka = 3.6 X 10(8) M-1. Insulin binding to foetal brain-cortical cells differed, however, in two distinct aspects from that to classical insulin-binding cell types. Firstly, dilution of 125I-insulin-bound cells in the presence of unlabelled insulin did not accelerate dissociation of the labelled hormone. Secondly, exposure of brain-cortical cells to insulin before the binding assay enhanced insulin binding, suggesting up-regulation of insulin receptors in response to insulin. In conclusion, foetal-mouse brain-cortical cells bear specific binding sites for insulin. Their insulin receptor shows a marked specificity and affinity for insulin, but differs in at least two properties from most classical insulin receptors. These differences in hormone-receptor interaction could reflect structural differences between insulin receptors on embryonic and differentiated cells.

Downregulation occurs normally in cultured Epstein-Barr virus-transformed lymphocytes from patients with extreme insulin resistance. Discrepancy between downregulation in vivo and in vitro.

Levels of fasting plasma insulin are generally inversely correlated with 125I-insulin binding to circulating blood cells. In disease states associated with hyperinsulinemia (e.g., obesity and non-insulin-dependent diabetes mellitus), 125I-insulin binding is usually low. In contrast, 125I-insulin binding to circulating cells may be normal in patients with certain forms of extreme insulin resistance despite marked hyperinsulinemia. To explain this paradox, it has been proposed that postbinding defects in insulin action may give rise to defects in downregulation. We have employed cultured Epstein-Barr virus (EBV)-transformed lymphocytes from eight patients with extreme insulin resistance to address the question of whether there is a defect in the downregulation process in vitro. In this cell type, insulin leads to a decrease in the number of insulin receptors on the cell surface by accelerating the rate of degradation of insulin receptors. We could not detect any abnormality in in vitro down-regulation with cultured EBV-transformed lymphocytes from insulin-resistant patients. The apparent discrepancy between the in vivo and in vitro studies raises the possibility that some factor in the patient's internal milieu may prevent insulin-induced downregulation. An alternative possible explanation might be that the mechanism of downregulation in vitro differs from the mechanism whereby receptor number is regulated in vivo in insulin's target cells.

INSULIN IS A GROWTH-PROMOTING PEPTIDE IN THE RAT BRAIN

Previous work in this laboratory documented specific insulin binding (IB) in various areas of the developing rat brain (J Neurochem, Jan. 1984, in press). IB was correlated with brain growth and increments in protein content in these areas during the first 21 days of postnatal life, supporting our hypothesis that insulin stimulates brain growth directly.We recently studied the effect of insulin on protein and nucleic acid synthesis in newborn rat brain glial cells in culture to further substantiate the role of insulin as a growth factor for the brain.IB to glial cells is rapid, reversible and specific (particularly with respect to insulin-like growth factors which competed only 3% as well as insulin for binding). Insulin stimulated the incorporation of valine into glial cell protein beginning at a concentration of 0.8nM. The stimulation was dose-dependent and became significant at 3.5nM, which was well below the empty site dissociation constant for IB by glial cells. A dose-dependent stimulation of thymidine incorporation into DNA nucleo-protein was also seen, but at higher concentrations of insulin.The studies document that glial cells have specific receptors for IB. In addition, and of particular importance, glial cells in culture are target cells for insulin with respect to those metabolic processes (i.e. protein and nucleic acid synthesis) which mediate brain growth.

Increased insulin binding to adipocytes and monocytes and increased insulin sensitivity of glucose transport and metabolism in adipocytes from non-insulin-dependent diabetics after a low-fat/high-starch/high-fiber diet

Nine non-insulin-dependent diabetics were studied before and after 3 weeks on an isoenergetic high-fiber/high-starch/low-fat diet (alternative diet), and nine non-insulin-dependent diabetics were studied on their usual diet. In the group that ate the alternative diet, the intake of fiber and starch increased 120% and 53%, whereas fat intake decreased 31%. Diabetes control improved as demonstrated by decreased fasting plasma glucose ( P < 0.05) and 24-hour urinary glucose excretion ( P < 0.05). The in vivo insulin action increased (K IVITT increased, P < 0.05) with no change in fasting serum insulin levels. In fat cells obtained from patients in the alternative-diet group, insulin receptor binding increased ( P < 0.05) after the change of diet. Insulin binding to purified monocytes (more than 95% monocytes) also increased ( P < 0.05), whereas no change was found in insulin binding to erythrocytes. When lipogenesis was studied at a tracer glucose concentration at which glucose transport seems to be rate limiting, insulin sensitivity increased ( P < 0.02). This is the predicted consequence of increased receptor binding. Moreover, when CO 2 production and lipogenesis were studied at a higher glucose concentration, where steps beyond transport seem to be rate limiting for glucose metabolism, increased insulin sensitivity was also observed. In contrast, no change was found in maximal insulin responsiveness. Fat and blood cells from the patients who continued on their usual diet showed no changes of the mentioned quantities. In the total group of non-insulin-dependent diabetics, fat cell insulin binding as well as rates (both basal and maximal insulin-stimulated rates) of glucose transport, CO 2 production, and lipogenesis were very heterogeneous, but when results from the first and second fat biopsy from the same patient were compared, these values varied only slightly. We conclude that the beneficial effect of a high-fiber/high-starch/low-fat diet on metabolic control in diabetics may in part be mediated through an increased insulin-binding ability of target cells for insulin, which causes an increased insulin sensitivity in these cells.

Phosphorylation of the hepatic insulin receptor: Stimulating effect of insulin on intact cells and in a cell-free system

The current understanding of the molecular basis of insulin action is rather limited despite the impressive effort made by several laboratories to elucidate the mechanisms of action of this hormone (l-31. However, the first step in insulin action is accepted to be binding of the hormone to cellsurface membrane components termed insulin receptors [4]. Several new techniques have allowed us to define the structural features of the insulin receptor [5-121. In brief, the insulin receptor is an integral membrane glycoprotein, and has been found, in all tissues studied thus far, to be composed of 2 major subunits (a respectively /3) of 135 000 and 95 000 M, respectively. All subunits are linked together by disulfide bridges into a large receptor complex of app. M, 350 000. Both the (Y(M, 135 000) and the P-subunit (M, 95 000) are glycoproteins. Further, we have shown that both subunits of the insulin receptor are specifically precipitated with autoantibodies against insulin receptor [9121. The observation that the entire spectrum of metabolic effects (both acute and late) of insulin can be initiated by the interaction of ligands other than insulin (i.e., autoantibodies to insulin receptor) with the receptor has been taken as evidence that the receptor contains all the necessary information, and/or might be the ‘second messenger’ for insulin action (13,141. Further, information has been gathered suggesting that insulin action results in phosphorylation-dephosphorylation reactions in some cellular proteins 131. Thus an important question arises as to whether the receptor itself possesses an enzymatic activity leading to phosphorylation-dephosphorylation, or is a substrate for such enzymatic activity. In intact rat hepatoma cells and cultured human IM-9 lymphocytes insulin increases the phosphorylation of its own M, = 95 000 receptor subunit [ 151. Here, we have investigated the phosphorylation of the insulin receptor in normal rat hepatocytes, which are major target cells for insulin’s actions. We demonstrate in intact hepatocytes that insulin specifically stimulates the phosphorylation of its M, 95 000 receptor subunit. More importantly, we present the first demonstration of this insulininduced covalent modification of its receptor subunit in a cell-free system.

Insulin binding, internalization, and receptor regulation in cultured human fibroblasts.

Insulin binding to receptors was studied using monolayers of cultured normal human fibroblasts. Binding was rapid and inversely related to the incubation temperature; prolonged periods of steady-state binding were achieved at all temperatures studied and the amount of degradation of extracellular insulin was minimal. Competition curves demonstrated half-maximal inhibition of 125I-insulin binding at an unlabeled insulin concentration of 125I-insulin binding at an unlabeled insulin concentration of 7 ng/ml. Scatchard plots of the binding data were curvilinear and revealed that fibroblasts contained about 7,000 receptor sites per cell. Bound 125I-insulin dissociated from fibroblasts with a t 1/2 of 10 min at 30 degrees C and 35 min at 16 degrees C. After 60 min dissociation at 30 degrees C, 45% of the dissociated radioactivity consisted of 125I-insulin degradation products, whereas only 8% of the dissociated material was in the form of degraded products after 60 min of dissociation at 16 degrees C. This indicates that fibroblasts possess a temperature-sensitive receptor-mediated process for insulin degradation. Preincubation of the monolayers with insulin led to a hormone-induced loss of insulin receptors. Thus, incubating cells with 25 ng/ml insulin for 6 h at 37 degrees C caused a 50% reduction in subsequently measured 125I-insulin binding. This hormone-induced receptor loss was sensitive to physiologic insulin levels, with approximately 5 ng/ml causing a half-maximal receptor loss. When monolayers were treated with the lysosomotropic agent chloroquine and subsequently incubated with 5 X 10(-11) M 125I-insulin, a 130% increase in cell-associated radioactivity was observed after 120 min at 30 degrees C. In summary, 1) cultured normal human fibroblasts possess insulin receptors that exhibit kinetic properties and specificity identical to that of other insulin target cells; 2) incubation of fibroblasts with physiologic concentrations of insulin causes a marked loss of cell-surface insulin receptors; and 3) receptor-bound 125I-insulin is internalized through an energy-dependent endocytotic pathway and subsequently degraded by a chloroquine-sensitive reaction.

Insulin degradation by insulin target cells

Recent findings illustrate the complexities associated with the interaction between insulin and its target cells. These results suggest that the processes involved in insulin action and those involved in insulin degradation may have certain steps in common. Both apparently begin when insulin binds to the insulin receptor. The next step is unknown but it ultimately leads to the internalization of the hormone before insulin dissociates from the cell surface. Furthermore, internalization appears to be a requirement for efficient degradation of insulin since the vast majority (perhaps all in certain cells) of the degrading activity is intracellular. Internalization may not be required to produce certain actions of the hormone, however, and the two processes may diverge at this point. 162 It is not clear how insulin enters the target cell other than the process appears to be receptor-mediated. Also, further work is needed to more fully characterize the vesicles that contain internalized insulin. Finally, the actual location of insulin degradation and the enzyme(s) involved need further study, especially to clarify the relative contributions of lysosomes, cytosolic protease, and GIT to physiological insulin destruction. An understanding of the overall process of insulin degradation is required for a complete description of the physiologic disposition of the hormone at the target cell. Moreover, this system has subtle control mechanisms that may have important implications for the management of diabetes and other endocrine and metabolic disorders.

Insulin binding to erythrocytes in diabetic patients.

Insulin binding to circulating erythrocytes was studied in 32 maturity onset-type diabetic patients with varying degrees of insulin response to oral glucose. Specifically bound-insulin fraction to erythrocytes ranged from 4.9 to 12.7% at a tracer concentration of insulin. A negative correlation was found between the binding and fasting serum insulin concentration (r = -0.45). When the binding was compared with sum of serum insulin concentrations at 0, 30, 60, 90, and 120 min after the oral administration of 50 g glucose (sigma IRI), a better correlation was obtained (R = -0.63). Patients were classified into group A (sigma IRI less than or equal to 100 microunits/ml), group B (100 microunits/ml less than sigma IRI less than 200 muml) and group C (sigma IRI greater than or equal to 200 microunits/ml). Scatchard analysis of the competition data from each group indicated that erythrocytes from group a showed the highest concentration of receptors (60 sites/erythrocyte) while the lowest from group C (36 sites/erythrocyte). The receptor affinity was not significantly different among the groups. These results suggest that insulin receptors on human erythrocytes are regulated by the ambient insulin concentration as shown in receptors on insulin target cells. Therefore, erythrocytes seem useful for the study of insulin receptors in conditions associated with altered insulin sensitivity.

Insulin receptors in acute and chronic lymphoid leukaemias

Normal lymphocytes lack insulin receptors. The binding of this hormone was studied in twelve patients with acute lymphoblastic leukaemia (ALL) and fourteen patients with chronic lymphocytic leukaemia (CLL). Lymphoblasts from ALL have been found to possess hormone receptors with properties identical to those of the known target cells for insulin: specificity, pH and temperature dependence, and ligand-induced increase in dissociation rate. All patients with ALL displayed insulin receptors on their lymphoblasts. The null-type cells possessed higher numbers of binding sites than the T-type cells, without overlapping of the values. In the fourteen patients with CLL, eight had low levels of insulin receptors on their lymphocytes while six showed a complete lack of such binding sites. Our results suggest that measurement of insulin binding might be a useful non-immunological marker for the classification of human leukaemias.

Biomembrane cooperative enzymes. In vivo modulation of rat erythrocyte acetylcholinesterase by insulin in normal and diabetic conditions

The present study investigated the effect of insulin in vivo on the changes in the cooperativity of a membrane-bound enzyme. The allosteric inhibition by F − of the erythrocyte membrane acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) was studied during intravenous glucose tolerance tests in control and alloxan-induced diabetic rats. In the former group, the value of n decreased from 1.6 to 1.0 whereas it remained about 1.6 in the latter groups. Intravenous injection of insulin (30 U/kg) decreased the values of n in both groups. It is suggested that the in vivo insulin action on membrane cooperative enzymes could also take place in insulin target cells.