Search

Expression of the cDNA encoding a human insulin receptor with replacement of alanine for lysine at residue 1018 in the ATP binding domain of the beta subunit results in a receptor that is not only kinase-defective, but also biologically inactive. Interestingly, this mutated receptor shows a decreased insulin binding affinity when expressed at high level. We, therefore, studied the binding property of this mutant receptor expressed in Rat 1 fibroblasts. The association rate (Ka) of insulin to the mutant receptor was comparable to normal, but the dissociation rate (Kd) was twice as fast. Furthermore, the Kd of the mutant receptor was also more sensitive to changes in pH, accelerating more rapidly with pH changes than did the Kd of normal receptors. Despite this difference, the mutant receptor still exhibited negative cooperativity. These results indicate that the loss of tyrosine kinase activity of the beta subunit of the insulin receptor leads to alteration of the ligand binding affinity of the alpha subunit.

Receptor-binding kinetics and degradation of tyrosine A-14 and A-19 125I-labelled insulin was studied using cultured human lymphocytes. Receptor-binding ability of A-14 insulin was 1.5-times as high as that of A-19 insulin. Dissociation from receptors on lymphocytes showed no difference between these two labelled insulins. In association studies percent bound of A-14 insulin was 1.5-times as high as that of A-19 insulin at any time after incubation. These results suggested that lower binding affinity of A-19 insulin was due to decreased association rate, but not due to increased dissociation rate. Degradation of A-14 insulin by incubation media of lymphocytes was also 1.5-times as high as that of A-19 insulin.

We studied the in vivo and in vitro effect of p-chlorophenoxyisobutyrate (CPIB) on insulin binding and glucose transport in isolated rat adipocytes. In the in vitro study, adipocytes were incubated with 1mM of CPIB for 2 h at 37 degrees C, pH 7.4, and then insulin binding (37 degrees C, 60 min) and 3-0-methylglucose transport (37 degrees C, 2s) were measured. Incubation with CPIB did not affect either insulin binding or glucose transport in the cells. The addition of insulin (10 ng/ml) with CPIB to the incubation media also did not affect the following insulin binding and glucose transport. In the in vivo study, rats were fed a high sucrose-diet containing 0.25% CPIB for 7 days. Serum cholesterol, plasma free fatty acid, and insulin levels were significantly decreased in the CPIB-treated rats. The treated rats demonstrated an almost 2 fold increased maximal binding capacity for insulin (189,000 sites/cell for treated vs 123,000 sites/cell for control cells). Basal glucose transport (glucose transport in the absence of insulin) significantly decreased in the CPIB-treated rats, although insulin-stimulated glucose transport was comparable in treated and control cells. Thus, CPIB might have no direct effect on glucose transport and insulin binding, as determined by the in vitro studies. Furthermore, a relatively short-term in vivo treatment with CPIB, such as 7 days, did not stimulate glucose transport.

Rat 1 fibroblasts have been transfected with the cDNA encoding a kinase-defective mutant human insulin receptor (A/K1018). Expression of this cDNA results in a receptor that is not only biologically inactive but also inhibits normal insulin action through the normal endogenous rat receptors in this fibroblast line (McClain, D. A., Maegawa, H., Lee, J., Dull, T. J., Ullrich, A., and Olefsky, J. M. (1987) J. Biol. Chem. 262, 14663-14671). We have investigated the mechanism of this inhibition and show that: 1) rat receptors are expressed at normal to increased levels in two cell lines which also express A/K1018 receptors at low (A/K1018-A, 5700 total receptors) or high (A/K1018-B, 2.2 x 10(5) total receptors) levels. 2) The rat receptors in the A/K1018 lines can be normally autophosphorylated under the control of insulin in vitro. 3) A/K1018 receptors do not inhibit the kinase activity of normal receptors when mixed together in vitro. 4) In intact A/K1018-B cells, the ability of insulin to stimulate autophosphorylation of the rat receptor is unimpaired; furthermore, the autophosphorylated rat receptor becomes normally activated as a tyrosine kinase. 5) The expression of receptors for insulin-like growth factor I and stimulation of hexose uptake mediated by this receptor are unaffected in cells expressing inhibitory A/K1018 receptors. 6) Expression of the A/K1018 receptor inhibits insulin-stimulated phosphorylation of two endogenous protein substrates (pp220 and pp170) by the native rat receptors. We conclude that the inhibition of insulin action seen in the A/K1018 cells is not mediated at the levels of native receptor expression or activation, nor is the effector (hexose uptake) mechanism affected by the A/K1018 receptors. The expression of this kinase-defective receptor does, however, inhibit the phosphorylation of substrate molecules by the normally activated endogenous rat receptors.

We have previously shown that a mutant human insulin receptor with a COOH-terminal 43-amino acid deletion (HIR delta CT), when expressed in Rat 1 fibroblasts, binds insulin normally, autophosphorylates, and undergoes endocytosis after insulin binding in a manner comparable to the normal human insulin receptor (HIRc). In this paper we have examined the biologic activity of the truncated and normal insulin receptors. In vitro, the HIR delta CT receptors caused a 1.8-fold greater phosphorylation of a Glu4/Tyr1 polypeptide than did the HIRc receptors, but the two receptor types were nearly equivalent in their ability to phosphorylate a src-derived peptide. Furthermore, insulin preactivation of HIRc and HIR delta CT receptors in intact cells led to equivalent stimulation of tyrosine kinase activity as subsequently determined for histone in vitro. Expression of HIRc receptors in cells led to enhanced sensitivity to insulin of 2-deoxy-D-glucose uptake and glycogen synthase activation. This increased sensitivity was proportional to receptor number at low (Ro = 6400) but not at high (Ro = 1.25 X 10(6] levels of receptor expression. However, expression of HIR delta CT receptors (Ro = 2.5 X 10(5] led to little, if any, increase in insulin sensitivity of either 2-deoxy-D-glucose uptake or glycogen synthase activation. Furthermore, compared with HIRc cells, HIR delta CT cells respond poorly to an agonistic monoclonal antibody specific for the human insulin receptor. In conclusion, the HIR delta CT receptor retains intact protein kinase activity in vitro. Despite this, however, the receptor displays low activity in mediating the metabolic effects of insulin.

We have studied the restriction fragment-length polymorphism in the 5'-flanking region of the human insulin gene in 47 nondiabetic Japanese subjects and in 52 subjects with non-insulin-dependent diabetes (NIDDM) to elucidate the ethnic variation of the genetic polymorphism and its relationship with NIDDM. Allelic frequencies in the nondiabetic subjects were 0.957 in class 1 (Bgl I fragments of 2800 ± 300 bp), 0 in class 2 (fragments of 3500 ± 300 bp), and 0.043 in class 3 (fragments of >3900 bp with a mean of 4500 bp). Corresponding frequencies in the NIDDM subjects were 0.962, 0, and 0.038, respectively. Four subjects with NIDDM who had the class 3 allele did not exhibit any particular clinical characteristics compared with the rest of the patients. Thus, the class 3 allele or the large insertion of the human insulin gene is much less frequent in Japanese than reported in other races, including Caucasians, and this class of allele is not associated with NIDDM in Japanese. Ethnic homogeneity is, thus, important in the analysis and interpretation of the genetic polymorphism.

We studied the effect of the duration of diabetic state on insulin action in skeletal muscle by measuring insulin binding, 2-deoxyglucose uptake, and intracellular glucose metabolism in isolated soleus muscles from streptozotocin-induced diabetic rats. Insulin binding to soleus muscles from diabetic rats was increased over that from controls. Glucose transport activity was determined by measuring the 2-deoxyglucose uptake at the concentration of 1 mmol/L at 25 degrees C. In the rats with diabetes of one week duration, insulin-stimulated 2-deoxyglucose uptake was not impaired, whereas basal 2-deoxyglucose uptake was decreased. However, the diabetic rats with two weeks duration revealed a 35.6% decrease in the insulin-stimulated 2-deoxyglucose uptake. Furthermore, four week duration of diabetic state led to a 60% decrease both in basal and insulin-stimulated 2-deoxyglucose uptake. Total glucose utilization was estimated as the total amount of glucose incorporated into muscle and lactate released into the medium following incubation at 37 degrees C, with 5 mmol/L glucose. The diabetic rats with one week duration did not demonstrate any changes in total glucose utilization both in basal and insulin-stimulated state. However more than two weeks duration of diabetes led to a 30% to 35% decrease both in basal and insulin-stimulated total glucose utilization, similar to the findings in the 2-deoxyglucose uptake study. We concluded that prolonged insulinopenia led to decreased glucose transport and intracellular glucose metabolism and resulted in insulin resistance in skeletal muscles.

[SerB24]-insulin, the second structurally abnormal mutant insulin, and [SerB25]-insulin were semisynthesized and were studied for receptor binding and biological activity. Receptor binding and biological activity determined by its ability to increase 2-deoxy-glucose uptake in rat adipocytes were 0.7-3% of native insulin for [SerB24]-insulin and 3-8% for [SerB25]-insulin. Negative cooperative effect of these analogues was also markedly decreased. Immunoreactivity of [SerB24]-insulin was decreased whereas that of [SerB25]-insulin was normal. Markedly decreased receptor binding of [SerB24]-insulin appeared to be due to substitution of hydrophobic amino acid, Phe, with a polar amino acid, Ser, at B24.

We studied the effect of chronic exposure to insulin on insulin receptors of cultured lymphocytes (RPMI-1788 line). The cells treated with insulin (2 micrograms/ml) at 37 degrees C for 12 h, showed a 36.5% decrease in the number of binding sites. Solubilized extract from the cells treated with insulin showed a 35.9% decrease of binding capacity, suggesting that insulin exposure induced the loss of total (cell surface and intracellular) receptors. Insulin-induced loss of receptors was blocked by chloroquine, suggesting that receptor loss is mediated by a chloroquine sensitive pathway. Bacitracin, which inhibited the insulin degradation, had no effect on insulin-induced receptor loss in this cell line. We also found that vitamin K5, one of the insulin mimickers, induced a 31.5% loss of insulin receptors. Therefore, the post-receptor process appeared to mediate down regulation. In cultured lymphocytes, insulin exposure caused a significant loss of total receptors, suggesting that insulin-induced receptor loss may be due to receptor degradation. Insulin-induced receptor loss is mediated by a chloroquine sensitive pathway, and is related to the post-binding process stimulated by vitamin K5.

A modified erythrocyte insulin receptor assay was developed. The major advantage of our method is the smaller amount of blood necessary for the assay and consequent simple handling in the binding assay. Three ml of blood is enough for this assay, making it possible to assess insulin receptors in a group of pediatric patients. There was a close relationship between mononuclear cells and erythrocyte insulin binding assays (r = 0.900, p0.05). With this method insulin binding studies were done on normal subjects and adult onset non-obese diabetics. Decreased insulin binding was seen in the diabetic group, and this was due to a decreased number of insulin receptors. This erythrocyte insulin receptor assay can be useful tool for the study of the mechanism of insulin resistance.

The in vitro effect of glucocorticoid on insulin binding and glucose transport was studied with rat adipocytes. Isolated rat adipocytes were incubated with or without 0.70 microgram/ml (1.9 mumol) of hydrocortisone in TCM 199 medium at 37 degrees C, 5% CO2/95% air (v/v), pH 7.4, for 2, 4, and 8 h, and then fat cell insulin binding and insulin-stimulated 3-O-methylglucose transport were measured. Hydrocortisone did not affect insulin binding in terms of affinity or receptor number. Glucose transport in the absence of insulin was significantly decreased at the incubation time of 2 h and continued to decrease up to 8 h of incubation with hydrocortisone. Decreased insulin sensitivity of glucose transport (i.e., a right-ward shift of the dose response curve) was also demonstrated after 2 h incubation with hydrocortisone, and the ED50 of insulin was maximally increased at 4 h of incubation (0.53 ng/ml for treated vs. 0.22 ng/ml for control cells). Maximal insulin responsiveness was also significantly decreased in treated cells after 8 h incubation with hydrocortisone. When percent maximum glucose transport was expressed relative to receptor-bound insulin, the ED50 values of treated and control cells were 10.5 and 7.2 pg of bound insulin, per 2 X 10(5) cells, respectively. Thus, it was evident that glucocorticoid induced a post-receptor coupling defect in the signal transmission of insulin-receptor complex.

We studied insulin degradation in human cultured lymphocytes (RPMI-1788 line) with a small but significant number of lysosomes under the electron microscope. Insulin degradation determined by the TCA solubility method was 64.6 +/- 1.2% (mean +/- SEM) at a trace concentration after the incubation with 2.0 x 10(7) cells (4.0 x 10(7) cells/ml) for 60 min at 37 degrees C. Because insulin degradation was 54.6 +/- 7.0% in the cell-free buffer in which 2.0 x 10(7) cells were previously incubated, most of the insulin was degraded outside of the cells. Gel filtration of the radioactive materials also revealed that most of the labeled insulin in the medium was degraded, and the main peak of the cell-associated radioactivities was intact labeled insulin. Chloroquine, a lysosomotropic agent, failed not only to increase insulin binding but also to decrease the insulin degradation. Other lysosomal protease inhibitors, antipain and leupeptin had also no effect on insulin degradation. In contrast, bacitracin (500 micrograms/ml) significantly decreased the insulin degradation analyzed by TCA solubility, receptor-rebinding, and the gel filtration method. These results suggest that insulin molecules are degraded by the enzymes leaked from the cells. The non-receptor mediated process, which is the bacitracin sensitive pathway, might be a general mechanism of insulin degradation in human cultured lymphocytes in vitro.

We compared A-14 and A-19 125I-labelled insulin in receptor-binding and degradation. Percent receptor-binding of A-14 and A-19 125I-labelled insulin to 2.4 X 10(9)/ml erythrocytes after 210 min incubation at 15 degrees C was 7.8 and 4.9%, respectively. Percent insulin-receptor binding of A-14 insulin was 1.6 times greater than that of A-19 insulin. A similar result was obtained in an adipocytes insulin binding study. Percent receptor-binding of A-14 and A-19 insulin to 2 X 10(5)/ml fat cells after 30 min incubation in the above buffer was 3.9 and 2.4%, respectively. Degradation of A-14 and A-19 insulin in rat adipocytes was also studied by molecular sieve column chromatography. Isolated rat adipocytes were allowed to associate with A-14 and A-19 125I-insulin for 60 min at 37 degrees C, pH 8.0 in a HEPES-phosphate buffer, and then cells were separated from the buffer by centrifugation. After solubilization with triton X-100, both the solubilized cells and the incubation medium were applied to the Bio-Gel P-30 column to assess the insulin degradation. Degradation of A-14 125I-insulin by the isolated rat adipocytes was 1.6 times greater than that of A-19 125I-insulin. Furthermore, the peak which was thought to be intermediate degradation products of insulin was obtained between the peak of intact insulin and that of 125I-tyrosine. Such a peak of intermediates was much smaller in the incubation media than in the cell-associated materials.(ABSTRACT TRUNCATED AT 250 WORDS)

[D-Phe B24 ]- and [D-Phe B25 ]-human insulin were semisynthesized from porcine insulin by enzyme assisted coupling method. Receptor binding ability of [D-Phe B24 ]- and [D-Phe B25 ]-insulin was 180% and 4%, respectively, of that of human insulin. Increased affinity of [D-Phe B24 ]-insulin was ascribed to markedly decreased dissociation rate in binding to human cultured lymphocytes. Negative cooperative effect of [D-Phe B24 ]insulin was also increased to twice of that of human insulin. Biological activity of these analogues was assessed by 2-deoxy-glucose uptake studies in isolated adipocytes and the ability of [D-Phe B24 ]- and [D-Phe B25 ]-insulin was 140% and 4%, respectively, of that of human insulin. These findings suggest that B25 L-Phe is more crucial for receptor binding and that [D-Phe B24 ]-insulin is the first semisynthetic insulin to show increased affinity for insulin receptors.

The long-term in vitro effects of insulin on insulin binding and glucose transport were studied using rat adipocytes in a time-dependent manner. Isolated fat cells were incubated with insulin (100 ng/ml) for 4, 8 and 24 h in a TCM 199 medium, at 37 degrees C, and then insulin binding (37 degrees C, 60 min) and 3-O-methylglucose transport (37 degrees C, 2 s) were determined. Decreased insulin binding was demonstrated in the cells incubated with insulin for 8 h, and Scatchard analysis revealed that receptor number was decreased to 61.7% of that of control cells. Thus, insulin-induced down regulation of receptors was evident after 8 h incubation with insulin. On the other hand, 8 h incubation with insulin resulted in markedly increased basal (i.e., in the absence of insulin) glucose transport up to 246% of control values. In the cells incubated with insulin for 24 h, maximally insulin-stimulated glucose transport was significantly increased up to 248% of control value. Thus, these results suggested that insulin-induced down regulation of receptors appeared to be coupled with increased cell-surface glucose transporters, and that there was a time-lag between down regulation of insulin receptors and increase of available glucose transporters in the plasma membrane.

A new potentiator of insulin action, 5-[4-(1-methylcyclohexylmethoxy)-benzyl]thiazolidine-2,4-dione, was tested for activation of insulin action in vitro. The agent (50 mg.kg−1.day−1) was orally administered to rats for 14 days and adipocytes from treated rats were used to assess insulin-binding, glucose uptake and glucose oxidation. In obese rats, the agent increased glucose uptake and oxidation without any change in insulin binding, whereas in lean or streptozotocin-treated rats it failed to increase glucose metabolism. Fat tissues were cultured with the agent for 24 h and were tested for insulin action. In the presence of insulin (10 ng/ml) in the culture media, the agent increased glucose oxidation in these cells without any change in insulin binding. However, without insulin in the culture media the agent did not increase glucose oxidation. Thus, the agent appeared to potentiate insulin action at the post receptor process.

In rodents a high-fructose diet induces metabolic derangements similar to those in metabolic syndrome. Previously we suggested that in mouse liver an unidentified nuclear protein binding to the sterol regulatory element (SRE)-binding protein-1c (SREBP-1c) promoter region plays a key role for the response to high-fructose diet. Here, using MALDI-TOF MASS technique, we identified an X-chromosome-linked RNA binding motif protein (RBMX) as a new candidate molecule. In electrophoretic mobility shift assay, anti-RBMX antibody displaced the bands induced by fructose-feeding. Overexpression or suppression of RBMX on rat hepatoma cells regulated the SREBP-1c promoter activity. RBMX may control SREBP-1c expression in mouse liver in response to high-fructose diet.

Renal sinus fat is partially characteristic of peri-vascular adipose tissue, however, RSF volume (RSFV) is associated with visceral adipose tissue (VATV). Therefore, the ratio of RSFV to VATV (RSFV/VATV ratio) can distinguish the importance of RSF as an extension of VAT versus its perivascular effects. We assessed the association of RSFV/VATV ratio with coronary artery calcification score (CACS) in 189 patients with suspected coronary artery disease. RSFV of the right kidney and VATV were quantified by using image data of unenhanced abdominal CT. CACS were measured on unenhanced ECG-gated CT images. This article contains data on explanatory scheme of how to measure RSFV on unenhanced abdominal CT, CT indication and exclusion criteria of study population, sex-adjusted association between RSFV with risk factors of coronary vascular diseases and metabolic indices, multivariate linear regression analysis with CACS as the dependent variable in the total study population. The data are supplemental to our original research article describing detailed association between RSFV/VATV ratio and CACS including sub-groups analyses classified by the age of 70 “Renal sinus fat volume on computed tomography in middle-aged patients at risk for cardiovascular disease and its association with coronary artery calcification” Murakami et al. [1]. Keywords: Renal sinus fat, Coronary calcification, Atherosclerosis, Visceral adipose tissue, Computed tomography

Introduction Dapagliflozin is a selective sodium glucose co-transporter 2 inhibitor that improves glycemic control and reduces body weight and systolic blood pressure in patients with type 2 diabetes mellitus (T2DM). Dapagliflozin is effective and well tolerated over 12–24 weeks in Japanese patients with T2DM. In this study, the safety and efficacy of dapagliflozin administered as monotherapy and combination therapy were assessed over 52 weeks in Japanese patients with T2DM. Methods This was a 52-week open-label Phase 3 study consisting of a single treatment arm with no comparator. Dapagliflozin was administered as monotherapy (n = 249) or combination therapy (n = 479) with existing antihyperglycemic agents (sulfonylurea, glinides, metformin, alpha-glucosidase inhibitors, thiazolidinediones, dipeptidyl peptidase-4 inhibitors, or glucagon-like peptide-1 receptor agonists) to Japanese patients with T2DM and inadequate glycemic control for 52 weeks. Treatment with dapagliflozin was initiated at 5 mg/day and titrated to 10 mg/day as required. Results Dapagliflozin administered as monotherapy or combination therapy was well tolerated. The frequency of adverse events (AEs) over 52 weeks was similar between monotherapy (79.1%) and combination therapy (72.4%) groups, and AEs were mostly mild or moderate. The incidence of hypoglycemia at 52 weeks was 2.4% in the monotherapy group and 4.0% in the combination therapy group. In patients receiving dapagliflozin as monotherapy or combination therapy, reductions from baseline to week 52 were observed in glycosylated hemoglobin (HbA1c) (−0.7% in both groups), weight (−2.6 and −2.1 kg, respectively), and systolic blood pressure (−5.2 mmHg and −3.9 mmHg). In patients with insufficient response to 5 mg/day, dapagliflozin was increased to 10 mg/day, and a further decrease in HbA1c from the pre-titration value was observed in both groups. Conclusion Dapagliflozin was well tolerated and effective as monotherapy or combination therapy in Japanese patients with T2DM over 52 weeks. Electronic supplementary material The online version of this article (doi:10.1007/s13300-014-0086-7) contains supplementary material, which is available to authorized users.

We studied the effect of acute short-term exercise on insulin binding to erythrocytes in patients with non-insulin dependent diabetes mellitus and normal controls. In the normal controls, insulin binding was increased immediately after the exercise and returned to the basal level at 30 min after the exercise. This increase was due to an affinity change without any change in the insulin receptor number. In contrast, insulin binding tended to decrease in the diabetics after the exercise, and this change was due to a decreased affinity without any change in the number of insulin receptors. These results indicate that the control mechanism of insulin receptor affinity in response to exercise is different in non-insulin dependent diabetics and normals.

Insulin binding to erythrocytes from normal subjects over a wide range of ages (group I, cord blood; group II, Age 6M to 6 yrs; group III, 7 to 19 yrs; group IV, 20 to 59 yrs, group V, over 60 yrs of age) was determined using modified Gambhir's method and the effect of age and sex on the binding was evaluated. The highest insulin binding to cord blood erythrocytes was accounted for by the increased number of reticulocytes since there was a highly significant correlation between insulin binding and the reticulocyte count (r = 0.928, p less than 0.001). Group IV showed significantly higher binding than younger are groups II and III. The mean binding in group V was the highest among all age groups except for group I, although the differences failed to reach statistical significance. The receptor concentrations (R0) were comparable among groups except group I which had a slightly increased R0. The affinity of insulin receptors was the lowest in group I and II, and gradually increased with the age and a positive correlation (p less than 0.01) was obtained between age and Ke. This increase in affinity with age is considered an adaptive response to the intracellular metabolic derangement often seen in the aged. No effect of sex on insulin binding was seen before puberty. However, in normal adults insulin binding of the female tended to be lower than that of the male with slightly increased R0 and decreased affinity in the female. Thus, the slightly decreased binding of the female group was mainly accounted for by the decreased affinity although the difference in the affinity was not significant.

To investigate the difference of insulin action between skeletal muscle and adipose tissue in response to dietary manipulation, we studied the effect of high-sucrose (HS) and high-fat (HF) diet on insulin action by measuring insulin binding and insulin action both in soleus muscles and adipocytes. HS feeding led to a 14 and 28% decrease, and HF feeding led to a 25 and 36% decrease in insulin binding both to soleus muscles and adipocytes, respectively (P less than 0.01). In HF-fed rats, both rates of glucose uptake and intracellular glucose metabolism were impaired by 30-40% in soleus muscles (P less than 0.01), and adipocyte glucose uptake was also decreased by 30% in the submaximally insulin-stimulated state (P less than 0.05). On the other hand, in HS-fed rats with prominent hyperinsulinemia, glucose uptake was 2.3- to 2.7-fold increased in adipocytes (P less than 0.01). However, both rates of glucose uptake and intracellular glucose metabolism were not increased in soleus muscles from HS-fed rats. Steady-state plasma glucose (SSPG) level was unchanged in HS-fed rats during somatostatin, glucose, and insulin infusion, whereas the SSPG level of HF-fed rats was twice as much as that in controls (P less than 0.01). These results indicate that long-term regulation of glucose metabolism by ambient insulin in skeletal muscle may be different from that in adipocytes and that insulin action in muscle, rather than in adipocyte, may reflect insulin action of whole body.

We examined antisera from patients treated with bovine-porcine mixture (hereafter referred to as bovine/porcine), porcine or human insulin, and compared their binding affinities to human insulin with those to porcine insulin. Patients treated with bovine/porcine insulin developed antisera with a higher affinity to porcine insulin compared with that to human insulin in five of nineteen cases. Furthermore, three of these five antisera had a comparable affinity to bovine and porcine insulin and appeared to recognize the amino acid residue at B-30. Treatment with porcine or human insulin, on the other hand, did not result in any significant difference in the affinity to porcine and human insulin in twenty-three patients. These results indicate the significant role of B-30 amino acid residue as an antigenic determinant, and suggest that the amino acid sequence of the A chain of bovine insulin may contribute to the development of antibody recognizing B-30 amino acid residue.

We studied insulin binding to cells from an insulin-resistant patient, a 5-year-old boy with clinical Rabson-Mendenhall syndrome. Decreased insulin binding was observed in three different cells: erythrocytes (37.4% of normal), cultured fibroblasts (53.3% of normal), and transformed lymphocytes (9.8% of normal). Decreased insulin binding in the cultured cells suggested that the patient had a primary defect in insulin receptors. In addition, insulin binding to the transformed lymphocytes from the patient was relatively high at lower pH compared with those in normal subjects. The cultured fibroblasts from the patient showed decreased glucose incorporation at the low insulin concentration with normal maximal stimulation, and the insulin dose response curve was shifted to the right. These results suggested that the defect resided in the receptor binding but not in the postreceptor steps. This was one of the rare cases showing decreased insulin binding clearly demonstrated in three different cells from a young male patient with extreme insulin resistance.

The first mutant insulin, [LeuB25]insulin, was semisynthesized, and its disappearance rate from the circulation was measured. Three micrograms of normal human or [LeuB25] insulin per kg were administered to three normal subjects. The half-lives of normal insulin and the mutant insulin were 4.5 and 24 min, respectively. The decrement in blood glucose levels after injection of mutant insulin was 22.6% that after injection of normal insulin. The blood glucose-lowering effect of the mutant insulin, evaluated by the time required to reach the nadir, was slightly prolonged (30 vs. 45 min). These results indicate that hyperinsulinemia in patients with abnormal insulin is due to prolonged disappearance because of decreased receptor binding.

A second mutant insulin, identified as [Ser B24 ]-insulin, has a highly hydrophilic character. To determine the biologic activity and the degradation of this mutant insulin, human [Ser B24 ]- and [Ser B25 ]-insulin analogues were semisynthesized from porcine insulin by an enzyme-assisted coupling method. All of the following studies on isolated rat adipocytes were performed at 37°C to directly correlate the binding potency and the biologic activity. The ability of these insulins to displace 125 I-porcine insulin bound to adipocytes was 0.5–2% and 1–4%, respectively, of porcine insulin. When the ability of these insulins to stimulate glucose transport and glucose oxidation was measured, both analogues had full activity at high concentrations (250 ng/ml). However, ED 50 of the porcine, [Ser B24 ]-, and [Ser B25 ]-insulins tostimulate glucose transport was 0.37 ± 0.05, 46.3 ± 5.4, and 23.3 ± 5.5 ng/ml, respectively. Similarly, for glucose oxidation, ED 50 was 0.38 ± 0.06, 33.8 ± 3.6, and 16.6 ± 3.4 ng/ml, respectively. Thus, the biologic activity of [Ser B24 ]- and [Ser B25 ]-insulins was reduced to 0.5–2% and 1–4% of that of porcine insulin, which was compatible with our previous studies under different conditions. No antagonistic effects were observed for either analogue. Degradation of 125 I-labeled [Ser B24 ]- and [Ser B25 ]-insulins was also decreased to 62.8% and 55.8%, respectively, of 125 I-porcine insulin. These results confirm the importance of the hydrophobic residues at B24 and B25 in the biologic activity of insulin; the patient having this hydrophilic insulin was considered to be in an insulinopenic state despite the hyperinsulinemia due to decreased degradation of the mutant insulin.

BACKGROUND: Elevated levels of body mass index (BMI) and smoking are well established lifestyle risk factors for coronary heart disease (CHD) and stroke. If these two risk factors have a synergistic relationship, rigorous lifestyle modification may contribute to greater reduction in cardiovascular burden than previously expected. METHODS: A pooled analysis of individual participant data from 38 cohorts, involving 378,579 participants. Hazards ratios (HRs) and 95% confidence intervals (CIs) for BMI by cigarette smoking status were estimated using Cox proportional hazard models. RESULTS: During a mean follow-up of 3.8 years, 2706 CHD and 3264 strokes were recorded. There was a log-linear, positive relationship of BMI with CHD and stroke in both smokers and non-smokers with evidence of a synergistic effect of smoking on the association between BMI and CHD only: HRs (95% CIs) associated with a 2 kg/m2 higher BMI were 1.13 (1.10-1.17) in current smokers and 1.09 (1.06-1.11) in non-smokers (p-value for interaction=0.04). CONCLUSION: Smoking amplifies the positive association between BMI and CHD but not stroke. If confirmed, these results suggest that effective strategies that target smoking cessation and weight loss are likely to have a greater impact than anticipated on reducing the burden of CHD., published_or_final_version

Epidemiologic studies have investigated the relation between alcohol intake and coronary calcification, with controversial results. Furthermore, the influence of heavy drinking has not been well elucidated. In the present study, a random sample of community-based Japanese men aged 40 to 49 years without a history of cardiovascular disease (n=245) were examined for coronary artery calcium (CAC) determined by electron-beam computed tomography and drinking status. There was a J-shaped association between alcohol intake and CAC. There was an increase of CAC in heavy drinkers (or=46 g/day), and participants who were drinkingor=69 g/day showed a significant increase in CAC compared with never drinkers after adjusting for other cardiovascular risk factors.

In order to test the contribution of the insulin receptor COOH terminus to insulin action, a truncation of 43 COOH-terminal amino acids was engineered by cDNA-based deletion mutagenesis. This cDNA (HIR delta CT), as well as cDNA encoding the complete receptor (HIRc) was transfected into Rat 1 fibroblasts. Cells expressing 6.4 X 10(3) and 1.25 X 10(6) normal receptors and 2.5 X 10(5) HIR delta CT receptors, as well as control Rat 1 fibroblasts were selected for further analysis. All cell lines exhibited insulin binding of similar affinity. Partial tryptic digestion and immunoprecipitation by region-specific antibodies verified that the HIR delta CT receptors were truncated at the COOH terminus. Purified HIRc and HIR delta CT receptors underwent autophosphorylation with similar insulin and ATP sensitivity, although the HIR delta CT receptors were slightly more active in the absence of insulin. Transfected HIRc and HIR delta CT receptors undergo endocytosis in a normal fashion. Insulin internalization and degradation in both HIRc and HIR delta CT cells is increased in proportion to receptor number. Intracellular insulin processing, degradation, and release were qualitatively comparable among the transfected cell lines. Complete and truncated receptors internalize, recycle, and down-regulate normally. We conclude the following: 1) the COOH-terminal portion of the insulin receptor is not necessary for partial autophosphorylation or endocytosis; 2) following internalization the intracellular itinerary of the receptor and ligand appear normal with the truncated receptor; and 3) truncation of the COOH terminus does not impair recycling of the receptor or retroendocytosis of internalized ligand.

Levels of adiponectin are inversely associated with obesity levels. We examined the levels of adiponectin in American (n = 98) and Japanese (n = 92) men aged 40 to 49 years. Contrary to our expectations, the American men had higher levels of adiponectin than the Japanese men (13.3 ± 5.8 vs 7.3 ± 4.2 (μg/mL) despite higher levels of obesity. Smaller areas of visceral adipose tissue in American than in Japanese men may have resulted in the higher levels of adiponectin.