Search

The short-term adverse effects of anticancer drugs (AD) in patients with type 2 diabetes (T2D) are poorly studied and their management still represents an important challenge for clinicians. We carried out a retrospective single-center study in 168 patients with T2D and cancer, evaluating both the short-term effects of first-line AD on glycemic control and chronic diabetes complications. Average glycated hemoglobin significantly increased after AD compared to values before treatment (7.5 vs. 7.1%, p < 0.005). In 46.4% of patients, diabetes therapy had to be potentiated, in most cases (82.1%) by shifting to insulin. The use of alkylating agents and high-dose glucocorticoids predicted the need to potentiate diabetes therapy. After AD transaminase values significantly increased, whereas the estimated glomerular filtration rate decreased (in 12.5%

Obesity represents a major risk factor for metabolic disorders, but some individuals, &ldquo, metabolically healthy&rdquo, (MHO), show less clinical evidence of these complications, in contrast to &ldquo, metabolically unhealthy&rdquo, (MUO) individuals. The aim of this cross-sectional study is to assess the prevalence of the MHO phenotype in a cohort of 246 overweight/obese Italian children and adolescents, and to evaluate their characteristics and the role of insulin resistance. Homeostasis model assessment&ndash, insulin resistance (HOMA-IR), insulin sensitivity index (ISI), insulinogenic index (IGI) and disposition index (DI) were all calculated from the Oral Glucose Tolerance Test (OGTT). MHO was defined by either: (1) HOMA-IR &lt, 2.5 (MHO-IRes), or (2) absence of the criteria for metabolic syndrome (MHO-MetS). The MHO prevalence, according to MHO-MetS or MHO-IRes criteria, was 37.4% and 15.8%, respectively. ISI was the strongest predictor of the MHO phenotype, independently associated with both MHO-IRes and MHO-MetS. The MHO-MetS group was further subdivided into insulin sensitive or insulin resistant on the basis of HOMA-IR (either &lt, or &ge, 2.5). Insulin sensitive MHO-MetS patients had a better metabolic profile compared to both insulin resistant MHO-MetS and MUO-MetS individuals. These data underscore the relevance of insulin sensitivity to identifying, among young individuals with overweight/obesity, the ones who have a more favorable metabolic phenotype.

Objective: To analyze the clinical outcomes of patients with type 2 diabetes (T2D) before and after implementation of a personalized care plan in the primary health care setting. Design: Observational, retrospective, real-world study. Setting: All T2D patients with a care plan in Rovaniemi Health Center, Rovaniemi, Finland, for whom data were available from a baseline visit (in 2013-2015 during which the care plan was written) and from a follow-up visit, including an updated care plan by the year 2017. Subjects: In total, 447 patients were included. Mean age was 66.9 (SD 10.1) years, 58.8% were male, 15.4% were smokers, 33.1% had vascular disease, and 17.0% were receiving insulin treatment. The mean follow-up time was 14.4 months. Main Outcome Measures: Glycosylated hemoglobin A1 (HbA1c), low-density lipoprotein (LDL), blood pressure (BP), and body mass index (BMI). Clinical values were taken at both baseline and follow-up. Results: LDL decreased by 0.2 mmol/L, systolic blood pressure by 2.2 mm Hg, diastolic blood pressure by 1.5 mm Hg, and BMI by 0.5 kg/m2 ( P < .05 for each). The decrease in HbA1c was 0.8 mmol/mol ( P = .07). Conclusion: We observed statistically significant decreases in LDL, BP, and BMI. Our results indicate that, over 14 months of follow-up, implementation of a written care plan was associated with small improvements in the clinical outcomes of T2D patients in a primary health care study population in a real-world setting.

Alzheimer’s disease (AD) is the most common form of senile dementia, accounting for up to 70% of dementia cases. AD is a slowly progressive disease, which causes global mental deterioration by affecting various cognitive areas. A growing body of evidence has demonstrated that lifestyle habits and nutritional patterns could delay the natural course of the neurodegeneration process. There is no single dietary pattern unequivocally proven to prevent AD. Nevertheless, epidemiological data suggest that by adopting several dietary habits, especially if accompanied with a healthy lifestyle, the negative consequences of AD could potentially be delayed. Alongside with others, two specific eating patterns have been well investigated concerning their potential beneficial effect on cognitive status: the Mediterranean diet (MedDi) and the Ketogenic Diet (KD). Despite the different underlying mechanisms, both of them have demonstrated a fairly profitable role in reducing or delaying cognitive impairment. The aim of the present narrative review is to overview the existing research on the efficacy of MedDi and KD against AD-related cognitive decline, focusing on the proposed protective mechanisms of action. Although the current knowledge on this complex topic does not allow us, at this point, to make exhaustive conclusions, this information could be of help in order to better characterize the possible role of MedDi and KD as nonpharmacological therapies in the treatment of AD and, more generically, of neurodegenerative disorders.

Insulin degludec is an extra‐long‐acting insulin analog that allows for enhanced efficacy and flexibility in the injection time. However, it is not approved for use during pregnancy. We report the pregnancy outcome and newborn conditions in two women with type 1 diabetes who continued preconception degludec treatment during embryogenesis. No pregnancy complication or congenital neonatal malformation was observed. Both babies presented with hypoglycemia and required neonatal intensive care unit admission. Degludec treatment did not cause adverse effects in the mothers or malformations in the newborns. The observed neonatal complications were probably independent of early pregnancy degludec treatment.

Aims Hyperinsulinemia is a recognized risk factor for cancer and plays a major role for the increased cancer incidence in diabetic patients. Whether insulin analogs, and particularly long-acting analogs, worsen the pro-cancer effect of excess insulin is still controversial. Data synthesis In this paper we summarize the biological bases for the potential detrimental effect of long-acting analogs on cancer cells and review the in vitro and in vivo evidence on this issue. Because of their different molecular structure relative to native insulin, insulin analogs may activate the insulin receptor (IR) and the post receptor pathways differently. Most, but not all, in vitro evidence indicate that long-acting analogs may have a stronger mitogenic potency than insulin on cancer cells. Notably insulin glargine, the most studied long-acting analog, also has a higher affinity for the insulin-like growth factor (IGF)-1 receptor, a potent growth mediator. In vitro observations, however, may not reflect what occurs in vivo when analogs are metabolized to derivatives with a different mitogenic activity. Clinical studies, mostly retrospective and predominantly concerning glargine, provide contrasting results. The only perspective trial found no cancer increase in patients treated with glargine. All these studies, however, have severe weaknesses because of the insufficient evaluation of important factors such as dose administered, length of exposure, patient follow-up duration and site-specific cancer investigation. Moreover, whether cancer promotion is a long-acting analog class characteristic or a specific effect of a single agent is not clear. Conclusions In conclusion the carcinogenic risk of long-acting analogs, and specifically glargine, can be neither confirmed nor excluded. A personalized and shared decision, considering all the individual risk factors (metabolic and non-metabolic), is the suggestion for the clinician.

OBJECTIVE Muscle cramps occur in >50% of diabetic patients and reduce the quality of life. No effective treatment is available. We evaluated the clinical effectiveness of botulinum toxin A (BTX-A) injections for treating cramps in diabetic patients with neuropathy. METHODS This single-center, double-blind, placebo-controlled perspective study investigated the efficacy and safety of BTX-A intramuscular injection for treating calf or foot cramps refractory to common pharmacological drugs. Fifty diabetic patients with peripheral neuropathy and cramps were randomly assigned to 2 matched groups. BTX-A (100 or 30 units) or saline was injected on each side into the gastrocnemius or the small flexor foot muscles. Changes in pain intensity (primary outcome) and cramp frequency were evaluated over the course of 20 weeks after BTX-A administration. Cramp interference in daily life and the electrophysiological cramp threshold frequency were also measured. The treatment was repeated 5 months after first injection in 19 responders. RESULTS All outcome measures improved significantly after BTX-A compared with placebo. The changes with respect to baseline were already significant after 1 week and persisted up to week 14. Only 5 of 25 (20%) patients were nonresponders (

Cytosolic PLA2 (cPLA2) and Ca(2+)-independent PLA2 (iPLA2) play a significant role in insulin β-cells secretion. Bacterial infections may be responsible of the onset of diabetes. The mechanism by which Staphylococcus aureus infection of INS-1 cells alters glucose-induced insulin secretion has been examined. After acute infection, insulin secretion and PLA2 activities significantly increased. Moreover, increased expressions of phospho-cPLA2, phospho-PKCα and phospho-ERK 1/2 were observed. Chronic infection causes a decrease in insulin release and a significant increase of iPLA2 and COX-2 protein expression. Moreover, insulin secretion in infected cells could be restored using specific siRNAs against iPLA2 isoform and specific COX-2 inhibitor.

Insulin resistance (IR) and cardiovascular disease (CVD) share a common soil. We investigated the combined role of single nucleotide polymorphisms (SNPs) affecting insulin signaling (ENPP1 K121Q, rs1044498; IRS1 G972R, rs1801278; TRIB3 Q84R, rs2295490) on CVD, age at myocardial infarction (MI), in vivo insulin sensitivity and in vitro insulin-stimulated nitric oxide synthase (NOS) activity.1. We first studied, incident cardiovascular events (a composite endpoint comprising myocardial infarction-MI, stroke and cardiovascular death) in 733 patients (2186 person-years, 175 events). 2. In a replication attempt, age at MI was tested in 331 individuals. 3. OGTT-derived insulin sensitivity index (ISI) was assessed in 829 individuals with fasting glucose126 mg/dl. 4. NOS activity was measured in 40 strains of human vein endothelial cells (HUVECs).1. Risk variants jointly predicted cardiovascular events (HR = 1.181; p = 0.0009) and, when added to clinical risk factors, significantly improved survival C-statistics; they also allowed a significantly correct reclassification (by net reclassification index) in the whole sample (135/733 individuals) and, even more, in obese patients (116/204 individuals). 2. Risk variants were jointly associated with age at MI (p = 0.006). 3. A significant association was also observed with ISI (p = 0.02). 4. Finally, risk variants were jointly associated with insulin-stimulated NOS activity in HUVECs (p = 0.009).Insulin signaling genes variants jointly affect cardiovascular disease, very likely by promoting whole body and endothelium-specific insulin resistance. Further studies are needed to address whether their genotyping help identify very high-risk patients who need specific and/or more aggressive preventive strategies.

It is suspected that microbial infections take part in the pathogenesis of diabetes mellitus type 1 (T1DM). Glucose-induced insulin secretion is accompanied by the release of free arachidonic acid (AA) mainly by cytosolic- and calcium independent phospholipases A2 (cPLA2 and iPLA2). Insulinoma cell line (INS-1E) was infected with E. coli isolated from the blood culture of a patient with sepsis. Invasion assay, Scanning Electron Microscopy and Transmission Electron Microscopy demonstrated the capacity of E. coli to enter cells, which was reduced by PLA2 inhibitors. Glucose-induced insulin secretion was significantly increased after acute infection (8h) but significantly decreased after chronic infection (72h). PLA2 activities, cPLA2, iPLA2, phospho-cPLA2, and COX-2 expressions were increased after acute and, even more, after chronic E. coli infection. The silencing of the two isoforms of PLA2s, with specific cPLA2- or iPLA2-siRNAs, reduced insulin secretion after acute infection and determined a rise in insulin release after chronic infection. Prostaglandins E2 (PGE2) production was significantly elevated in INS-1E after long-term E. coli infection and the restored insulin secretion in presence of L798106, a specific EP3 antagonist, and NS-398, a COX-2 inhibitor, and the reduction of insulin secretion in presence of sulprostone, a specific EP3 agonist, revealed their involvement in the effects triggered by bacterial infection. The results obtained demonstrated that cPLA2 and iPLA2 play a key role in insulin secretion process after E. coli infection. The high concentration of AA released is transformed into PGE2, which could be responsible for the reduced insulin secretion.

OBJECTIVE— To study the role of the ENPP1 Q121 variant on glucose homeostasis in whites from Italy.RESEARCH DESIGN AND METHODS— We conducted case-control studies in 764 adults (from two independent samples of 289 nonobese and 485 obese individuals) and 240 overweight/obese children undergoing oral glucose tolerance testing (OGTT). Early-phase insulin secretion and insulin sensitivity (the insulinogenic index and the insulin sensitivity index) and their interplay (the disposition index) were calculated.RESULTS— In adult subjects, glucose profiles during OGTT were significantly (P = 2 × 10−2) different across K121Q genotype groups and higher in QQ than KK individuals (P = 5 × 10−2). The insulinogenic index was significantly reduced in QQ (18.5 ± 3.4) compared with both KK (31.6 ± 1.0; P = 2.2 × 10−7) and KQ (30.5 ± 1.5; P = 3.2 × 10−6) individuals. KQ individuals also showed a reduced insulin sensitivity index compared with KK subjects (P = 3.6 × 10−2). The disposition index was lower in QQ carriers than in KQ and KK individuals (P = 8 × 10−3 and 4 × 10−4, respectively) and lower in KQ than in KK individuals (P = 3 × 10−2). Data obtained in overweight/obese children were very similar to those observed in adults, with QQ individuals showing (compared with KQ and KK subjects) a reduced insulinogenic index (P = 7 × 10−3 and 2 × 10−2, respectively) and disposition index (P = 2 × 10−2 and 7 × 10−3, respectively).CONCLUSIONS— Homozygous carriers of the ENPP1 Q121 variant are characterized by an altered glucose homeostasis. Reduced early-phase insulin secretion and inefficient interplay between insulin secretion and sensitivity, which occur at early ages, are major determinants of this defect.

Free fatty acids (FFAs) acutely stimulate but chronically impair glucose-stimulated insulin secretion from beta-cells. The G protein-coupled transmembrane receptor 40 (GPR40) mediates both acute and chronic effects of FFAs on insulin secretion and plays a role in glucose homeostasis. Limited information is available on the effect of GPR40 genetic abnormalities on insulin secretion and metabolic regulation in human subjects.For in vivo studies, we screened 734 subjects for the coding region of GPR40 and identified a new single-nucleotide mutation (Gly180Ser). The mean allele frequency was 0.75%, which progressively increased (P0.05) from nonobese subjects (0.42%) to moderately obese (body mass index = 30-39.9 kg/m2, 1.07%) and severely obese patients (body mass indexor = 40 kg/m2, 2.60%). The relationship between the GPR40 mutation, insulin secretion, and metabolic alterations was studied in 11 Gly/Ser mutation carriers. In these subjects, insulin secretion (insulinogenic index derived from oral glucose tolerance test) was significantly lower than in 692 Gly/Gly carriers (86.0 +/- 48.2 vs. 183.7 +/- 134.4, P0.005). Moreover, a case-control study indicated that plasma insulin and C-peptide responses to a lipid load were significantly (P0.05) lower in six Gly/Ser than in 12 Gly/Gly carriers. In vitro experiments in HeLa cells cotransfected with aequorin and the mutated Gly/Ser GPR40 indicated that intracellular Ca2+ concentration increase after oleic acid was significantly lower than in Gly/Gly GPR40-transfected cells. This fact was confirmed using fura-2 acetoxymethyl ester.This newly identified GPR40 variant results in a loss of function that prevents the beta-cell ability to adequately sense lipids as an insulin secretory stimulus because of impaired intracellular Ca2+ concentration increase.

Protein tyrosine phosphatase 1B (PTP1B) inhibits insulin signaling and, when overexpressed, plays a role in insulin resistance (Ahmad et al. 1997). We identified, in the 3' untranslated region of the PTP1B gene, a 1484insG variation that, in two different populations, is associated with several features of insulin resistance: among male individuals, higher values of the insulin resistance HOMA(IR) index (P=.006), serum triglycerides (P=.0002), and total/HDL cholesterol ratio (P=.025) and, among female individuals, higher blood pressure (P=.01). Similar data were also obtained in a family-based association study by use of sib pairs discordant for genotype (Gu et al. 2000). Subjects carrying the 1484insG variant showed also PTP1B mRNA overexpression in skeletal muscle (6,166 plus minus 1,879 copies/40 ng RNA vs. 2,983 plus minus 1,620; P.01). Finally, PTP1B mRNA stability was significantly higher (P.01) in human embryo kidney 293 cells transfected with 1484insG PTP1B, as compared with those transfected with wild-type PTP1B. Our data indicate that the 1484insG allele causes PTP1B overexpression and plays a role in insulin resistance. Therefore, individuals carrying the 1484insG variant might particularly benefit from PTP1B inhibitors, a promising new tool for treatment of insulin resistance (Kennedy and Ramachandran 2000).

Glucagon-like peptide-1 (GLP-1) is a potent gluco-incretin hormone, which plays a central role on pancreatic beta cell proliferation, survival and insulin secreting activity and whose analogs are used for treating hyperglycemia in type 2 diabetes mellitus. Notably, abnormal insulin signaling affects all the above-mentioned aspects on pancreatic beta cells. The aim of our study was to investigate whether the protective effects of GLP1-1 on beta cells are affected by altered insulin receptor signaling. To this end, several effects of GLP-1 were studied in INS-1E rat beta cells transfected either with an inhibitor of insulin receptor function (i.e., the Ectonucleotide Pyrophosphatase Phosphodiesterase 1, ENPP1), or with insulin receptor small interfering RNA, as well as in control cells. Crucial experiments were carried out also in a second cell line, namely the βTC-1 mouse beta cells. Our data indicate that in insulin secreting beta cells in which either ENPP1 was up-regulated or insulin receptor was down-regulated, GLP-1 positive effects on several pancreatic beta cell activities, including glucose-induced insulin secretion, cell proliferation and cell survival, were strongly reduced. Further studies are needed to understand whether such a scenario occurs also in humans and, if so, if it plays a role of clinical relevance in diabetic patients with poor responsiveness to GLP-1 related treatments.

Background The aim of this study was to determine the efficacy of real-time continuous glucose monitoring in T1D patients treated with insulin pump therapy or multiple daily insulin therapy. Methods Twenty adult patients (ten insulin pump therapy and ten multiple daily insulin) with poor glycaemic control (HbA1c > 8.0%) were randomized into two groups for 6 months: the continuous glucose monitoring arm (using real-time continuous glucose monitoring) and the SMBG arm. After 2 months of wash-out, the participants crossed over. The primary outcome was HbA1c reduction. The secondary outcomes were hypoglycaemia and hyperglycaemia risk assessment (area under the curve 200 mg/dL/day, respectively) and glucose variability. Results Fourteen patients (eight multiple daily insulin, six insulin pump therapy) used continuous glucose monitoring appropriately (at least 40% of the time). In these patients, the improvement in glycaemic control was more evident during the real-time continuous glucose monitoring period (7.76% ± 0.4 vs 8.54% ± 0.4, p

The human plasma-cell membrane differentiation antigen-1 (PC-1) has been shown to inhibit insulin receptor tyrosine kinase activity. Recently, a K121Q polymorphism in the human PC-1 gene was found in a Sicilian population and was shown to be strongly associated with insulin resistance. The objectives of the present investigation were to examine in the Danish Caucasian population whether the K121Q variant was associated with type 2 diabetes or, in glucose-tolerant subjects, with impaired whole-body insulin sensitivity. We genotyped 404 Danish type 2 diabetic patients and found that the allele frequency of the variant was 0.14 (95% CI 0.12-0.16), whereas the allele frequency was 0.16 (95% CI 0.13-0.19) among 237 matched glucose-tolerant control subjects (P = 0.6). In the control subjects, there were no significant differences among wild-type, heterozygous, or homozygous subjects in regard to 1) serum insulin and plasma glucose levels at fasting, 60 min, or 120 min during an oral glucose tolerance test (OGTT) or 2) the estimates of insulin resistance obtained from the homeostasis model assessment (HOMA). Furthermore, we investigated the impact of the variant in 2 other Danish population samples that comprised 356 young healthy subjects and 226 glucose-tolerant offspring of type 2 diabetic probands, respectively. In all of the study populations, the polymorphism was not associated with an altered insulin sensitivity index as estimated from an intravenous glucose tolerance test in combination with an intravenous injection of tolbutamide. In addition, among the 226 offspring, the variations in serum insulin and serum C-peptide responses measured during an OGTT were not related to the PC-1 genotype. In conclusion, the K121Q polymorphism of the human PC-1 gene is not associated with type 2 diabetes or insulin resistance among Danish Caucasians.

Background To verify whether insulin levels influence PC-1 tissue content, we studied PC-1 gene expression and protein content in skeletal muscle of patients with insulinoma, a model of primary hyperinsulinemia. Data were compared with those obtained in matched insulin sensitive or resistant healthy subjects. In addition, the effect of high insulin concentration on PC-1 protein content was studied in HepG2 cells. Methods The following measurements were performed: insulin sensitivity by euglycemic clamp; PC-1 protein content and insulin receptor autophosphorylation by specific ELISAs; PC-1 gene expression by competitive polymerase chain reaction (PCR); phosphatidyl-inositol-3 kinase by immunoprecipitation and thin layer chromatography; glycogen synthesis by 14C-glucose incorporation. Results Muscle PC-1 content was similar in the insulinoma patients and in insulin sensitive controls but higher (p

Defects in insulin receptor tyrosine kinase activity have been demonstrated in tissues from insulin resistant subjects, but mutations in the insulin receptor gene are rare. Therefore, other molecules that are capable of modulating the insulin receptor most likely play a major role in insulin resistance. In cultured fibroblasts from an insulin resistant patient with Type 2 diabetes, we first identified membrane glycoprotein PC-1 as an inhibitor of the insulin receptor tyrosine kinase activity. PC-1 is overexpressed in fibroblasts from other insulin resistant subjects, both with and without Type 2 diabetes. PC-1 is a large class II exoprotein whose function is unknown. Studies in muscle and fat of insulin resistant subjects, two primary tissues for insulin activation, reveal that elevated levels of PC-1 are inversely correlated with decreased insulin action both in vivo and in vitro. Transfection and expression of PC-1 in cultured cells demonstrate that overexpression of PC-1 produces impairments in insulin receptor tyrosine kinase activity, and the subsequent cellular responses to insulin. These studies indicate, therefore, that PC-1 is a major factor in the etiology of insulin resistance, and is a potential new therapeutic target for antidiabetic therapy.

Peripheral resistance to insulin is a major component of non-insulin dependent diabetes mellitus. Defects in insulin receptor tyrosine kinase activity have been demonstrated in several tissues from insulin resistant subjects, but mutations in the insulin receptor gene occur in only a small fraction of cases. Therefore, other molecules that are capable of modulating the function of the insulin receptor are likely candidates in the search for the cellular mechanisms of insulin resistance. We have isolated an inhibitor of insulin receptor tyrosine kinase activity from cultured fibroblasts of an insulin resistant NIDDM patient and identified it as membrane glycoprotein PC-1. Subsequently we have demonstrated that expression of PC-1 is elevated in fibroblasts from other insulin resistant subjects, both with and without NIDDM. Studies in muscle, the primary site for insulin-mediated glucose disposal, have shown that the levels of PC-1 in this tissue are inversely correlated to insulin action both in vivo and in vitro. Transfection of PC-1 into cultured cells has confirmed that overexpression of PC-1 can produce impairments in insulin receptor tyrosine kinase activity and the subsequent cellular responses to insulin. Preliminary data suggests a direct interaction between PC-1 and the insulin receptor. However, the mechanisms whereby PC-1 inhibits insulin receptor signaling remain to be determined.

In the present study we measured PC-1 content, tumour necrosis factor (TNF)-α gene expression, and insulin stimulation of insulin receptor tyrosine-kinase activity in adipose tissue from non-obese, non-diabetic subjects. These parameters were correlated with in vivo insulin action as measured by the intravenous insulin tolerance test (Kitt values). PC-1 content was negatively correlated with Kitt values (r = –0.5, p = 0.04) and positively with plasma insulin levels both fasting (r = 0.58, p = 0.009) and after 120 min during oral glucose tolerance test (OGTT) (r = 0.67, p = 0.002). Moreover, adipose tissue PC-1 content was higher in relatively insulin-resistant subjects (Kitt values lower than 6) than in relatively insulin-sensitive subjects (Kitt values higher than 6) (525 ± 49 ng/mg protein vs 336 ± 45, respectively, p = 0.012). Adipose tissue insulin receptor tyrosine-kinase activity in response to insulin was significantly lower at all insulin concentrations tested (p = 0.017, by two-way analysis of variance test) in insulin-resistant than in insulin-sensitive subjects (Kitt values lower or higher than 6, respectively). In contrast to PC-1, no significant correlation was observed between adipose tissue TNF-α mRNA content and Kitt values, and plasma insulin levels, both fasting and at after 120 min during OGTT. Also, no difference was observed in TNF-α mRNA content between subjects with Kitt values higher or lower than 6. These studies in adipose tissue, together with our previous studies in skeletal muscle raise the possibility that PC-1, by regulating insulin receptor function, may play a role in the degree of insulin sensitivity in non-obese, non-diabetic subjects. [Diabetologia (1997) 40: 282–289]

Context: Reduced insulin signaling in insulin secreting β-cells causes defective insulin secretion and hyperglycemia in mice. Objective: We investigated whether functional polymorphisms affecting insulin signaling (ie, ENPP1 K121Q, rs1044498; IRS1 G972R, rs1801278; and TRIB3 Q84R, rs2295490) exert a joint effect on insulin secretion and abnormal glucose homeostasis (AGH). Design: Insulin secretion was evaluated by 1) the disposition index (DI) from an oral glucose tolerance test (OGTT) in 829 individuals; 2) insulin secretion stimulation index (SI) in islets from nondiabetic donors after glucose (n = 92) or glibenclamide (n = 89) stimulation. AGH (including impaired fasting glucose and/or impaired glucose tolerance or type 2 diabetes; T2D) was evaluated in case-control studies from the GENetics of Type 2 Diabetes in Italy and the United States (GENIUS T2D) Consortium (n = 6607). Results: Genotype risk score, obtained by totaling individual weighted risk allele effects, was associated with the following: 1) DI (P = .005); 2) glucose and glibenclamide SI (P = .046 and P = .009); or 3) AGH (odds ratio 1.08, 95% confidence interval 1.03–1.13; P = .001). We observed an inverse relationship between genetic effect and age at AGH onset, as indicated by a linear correlation between AGH-genotype risk score odds ratios and age-at-diagnosis cutoffs (R2 = 0.80, P < .001). Conclusions: Functional polymorphisms affecting insulin signaling exert a joint effect on both in vivo and in vitro insulin secretion as well as on early-onset AGH. Our data provide further evidence that abnormal insulin signaling reduces β-cell function and impairs glucose homeostasis.

The expression of adiponectin receptors has been demonstrated in human and rat pancreatic beta cells, where globular (g) adiponectin rescues rat beta cells from cytokine and fatty acid-induced apoptosis. The aim of our study was to evaluate whether adiponectin has a direct effect on insulin secretion and the metabolic pathways involved. Purified human pancreatic islets and rat beta cells (INS-1E) were exposed (1 h) to g-adiponectin, and glucose-induced insulin secretion was measured. A significant increase in glucose-induced insulin secretion was observed in the presence of g-adiponectin (1 nmol/l) with respect to control cells in both human pancreatic islets (n = 5, p < 0.05) and INS-1E cells (n = 5, p < 0.001). The effect of globular adiponectin on insulin secretion was independent of AMP-dependent protein kinase (AMPK) activation or glucose oxidation. In contrast, g-adiponectin significantly increased oleate oxidation (n = 5, p < 0.05), and the effect of g-adiponectin (p < 0.001) on insulin secretion by INS-1E was significantly reduced in the presence of etomoxir (1 μmol/l), an inhibitor of fatty acid beta oxidation. g-Adiponectin potentiates glucose-induced insulin secretion in both human pancreatic islets and rat beta cells via an AMPK independent pathway. Increased fatty acid oxidation rather than augmented glucose oxidation is the mechanism responsible. Overall, our data indicate that, in addition to its anti-apoptotic action, g-adiponectin has another direct effect on beta cells by potentiating insulin secretion. Adiponectin, therefore, in addition to its well-known effect on insulin sensitivity, has important effects at the pancreatic level.

The aim of this study was to deeper investigate the mechanisms through which ENPP1, a negative modulator of insulin receptor (IR) activation, plays a role on insulin signaling, insulin secretion and eventually glucose metabolism. ENPP1 cDNA (carrying either K121 or Q121 variant) was transfected in HepG2 liver-, L6 skeletal muscle- and INS1E beta-cells. Insulin-induced IR-autophosphorylation (HepG2, L6, INS1E), Akt-Ser(473), ERK1/2-Thr(202)/Tyr(204) and GSK3-beta Ser(9) phosphorylation (HepG2, L6), PEPCK mRNA levels (HepG2) and 2-deoxy-D-glucose uptake (L6) was studied. GLUT 4 mRNA (L6), insulin secretion and caspase-3 activation (INS1E) were also investigated. Insulin-induced IR-autophosphorylation was decreased in HepG2-K, L6-K, INS1E-K (20%, 52% and 11% reduction vs. untransfected cells) and twice as much in HepG2-Q, L6-Q, INS1E-Q (44%, 92% and 30%). Similar data were obtained with Akt-Ser(473), ERK1/2-Thr(202)/Tyr(204) and GSK3-beta Ser(9) in HepG2 and L6. Insulin-induced reduction of PEPCK mRNA was progressively lower in untransfected, HepG2-K and HepG2-Q cells (65%, 54%, 23%). Insulin-induced glucose uptake in untransfected L6 (60% increase over basal), was totally abolished in L6-K and L6-Q cells. GLUT 4 mRNA was slightly reduced in L6-K and twice as much in L6-Q (13% and 25% reduction vs. untransfected cells). Glucose-induced insulin secretion was 60% reduced in INS1E-K and almost abolished in INS1E-Q. Serum deficiency activated caspase-3 by two, three and four folds in untransfected INS1E, INS1E-K and INS1E-Q. Glyburide-induced insulin secretion was reduced by 50% in isolated human islets from homozygous QQ donors as compared to those from KK and KQ individuals. Our data clearly indicate that ENPP1, especially when the Q121 variant is operating, affects insulin signaling and glucose metabolism in skeletal muscle- and liver-cells and both function and survival of insulin secreting beta-cells, thus representing a strong pathogenic factor predisposing to insulin resistance, defective insulin secretion and glucose metabolism abnormalities.

We characterized the structure and the function of insulin receptors isolated from 10 human breast cancer specimens. We observed that the insulin receptor content, as determined by a specific radioimmunoassay, was four fold increased in human breast cancer tissue when compared to normal breast tissues. In both cancer and normal breast tissues, insulin receptor mRNA consisted of two major species of approximately 11.0 and 8.5 kilobases. The size of the insulin receptor alpha subunit was determined by 125I-insulin cross-linking followed by immunoprecipitation and polyacrylamide gel electrophoresis; a value of 135kDa was observed for receptors from both breast cancer and normal breast tissues. The functional binding ability of insulin receptors from cancer tissues was slightly lower as compared to normal tissue derived insulin receptor (% B/T = 2.22 +/- 0.50 per ng of insulin receptor as determined by radioimmunoassay vs. 2.96 +/- 0.49, mean +/- S.E.M.). The concentration of insulin that caused half maximal inhibition of 125I-insulin binding was very similar for both cancer and normal breast receptors (80pM). The size of the insulin receptor beta subunit as determined by receptor autophosphorylation was 95kDa. Basal and maximal insulin (100nM) stimulated receptor tyrosine kinase activity, in terms of both receptor autophosphorylation and phosphorylation of an exogenous substrate, was similar in malignant and normal breast tissue derived insulin receptor. Also, a very similar insulin stimulated Km value for ATP was showed by the tyrosine kinase of insulin receptors from breast cancer and normal breast tissue (11.1 and 10.8 microM ATP, respectively). However, in insulin receptors from breast cancer tissue the average tyrosine kinase sensitivity to insulin, as calculated on the exogenous substrate, was higher, although not significantly, with respect to normal breast tissue (ED50 at 0.28 +/- 0.09 and 1.08 +/- 0.33 nM insulin, respectively). A similarly different sensitivity to insulin was observed also for receptor autophosphorylation. In conclusion, this study demonstrates that breast cancer tissues have an increased number of structurally and functionally normal insulin receptors. In some breast cancer tissues, however, the sensitivity of the receptor tyrosine kinase activity to insulin is greatly increased. These data suggest that, in vivo, the mitogenic effect of insulin may play a role in the biology of certain breast cancers.

With a two-step purification procedure employing sequential affinity chromatography with insulin receptor monoclonal antibody followed by wheat germ agglutinin, we isolated from the plasma of two healthy individuals a material that reacted in a specific RIA for insulin receptors. This material produced dilution curves that were parallel to a human placental insulin receptor standard. This material also bound [125I]insulin; competition-inhibition curves revealed an ED50 of 0.3 nM, a value similar to that obtained with placental insulin receptors. The material purified from plasma was then labeled with [125I] Bolton-Hunter reagent, followed by polyacrylamide gel electrophoresis under reducing conditions and autoradiography. A band at 135 kilodaltons (kDa) was observed, corresponding to the alpha-subunit of the insulin receptor. Several bands ranging from 82-46 kDa were also detected. One or more of these fragments had intrinsic autophosphorylation activity, but only the 82-kDa band activity was responsive to insulin. In addition, employing the synthetic substrate poly(Glu4:Tyr1), no insulin-sensitive tyrosine kinase activity was present. These studies demonstrate, therefore, that insulin receptor-derived material is present in human plasma. This material retains high affinity insulin binding, but has an altered beta-subunit that is devoid of insulin-responsive tyrosine kinase activity.

Objective— Insulin resistance induces increased pulse pressure (PP), endothelial dysfunction (ED), and reduced bioavailability of endothelium-derived nitric oxide (NO). The genetic background of these 3 cardiovascular risk factors might be partly common. The ENPP1 K121Q polymorphism is associated with insulin resistance and cardiovascular risk. Methods and Results— We investigated whether the K121Q polymorphism is associated with increased PP in white Caucasians and with ED in vitro. In 985 individuals, (390 unrelated and 595 from 248 families), the K121Q polymorphism was associated with PP ( P =8.0×10 −4 ). In the families, the Q121 variant accounted for 0.08 of PP heritability ( P =9.4×10 −4 ). This association was formally replicated in a second sample of 475 individuals ( P =2.6×10 −2 ) but not in 2 smaller samples of 289 and 236 individuals ( P =0.49 and 0.21, respectively). In the individual patients’ data meta-analysis, comprising 1985 individuals, PP was associated with the Q121 variant ( P =1.2×10 −3 ). Human endothelial cells carrying the KQ genotype showed, as compared to KK cells, reduced insulin-mediated insulin receptor autophosphorylation ( P =0.03), Ser 473 -Akt phosphorylation ( P =0.03), and NO synthase activity ( P =0.003). Conclusions— Our data suggest that the ENPP1 Q121 variant is associated with increased PP in vivo and reduced insulin signaling and ED in vitro, thus indicating a possible pathogenic mechanism for the increased cardiovascular risk observed in ENPP1 Q121 carriers.

A radioimmunoassay for the human insulin-like growth factor-I (IGF-I) receptor was developed using a rabbit polyclonal antibody to the human IGF-I receptor and a highly purified IGF-I receptor. The purified receptor was radiolabeled with 125I-Bolton-Hunter reagent. Over 18% of the radiolabeled receptor was immunoprecipitated with the polyclonal antireceptor antibody. Purified IGF-I receptor concentrations as low as 5 ng/0.5 mL inhibited the radiolabeled IGF-I receptor binding. Purified insulin receptor weakly inhibited this binding, while the ligand IGF-I did not show inhibition. The radioimmunoassay was applicable to the measurements of IGF-I receptors in the Triton X-100 extracts of various tissues and cells. Breast cancer tissues and cells showed detectable IGF-I receptors, which correlated with IGF-I ligand binding. Receptor content was measurable in placenta and IM-9 cells, but receptor content was not measurable in liver and muscle extracts.

Insulin resistance is a major feature of most patients with type 2 diabetes mellitus (T2D). A number of laboratories have observed that PC-1 (membrane [corrected] glycoprotein plasma cell antigen 1; also termed [corrected] ectonucleotide pyrophosphatase phosphodiesterase 1 or ENPP1) [corrected] is either overexpressed or overactive in muscle, adipose tissue, fibroblasts, and other tissues of insulin-resistant individuals, both nondiabetic and diabetic. Moreover, PC-1 (ENPP1) overexpression [corrected] in cultured cells in vitro and in transgenic mice in vivo, [corrected] impairs insulin stimulation of insulin receptor (IR) activation and downstream signaling. PC-1 binds to the connecting domain of the IR alpha-subunit that is located in residues 485-599. The connecting domain transmits insulin binding in the alpha-subunit to activation of tyrosine kinase activation in the beta-subunit. When PC-1 is overexpressed, it inhibits insulin [corrected]induced IR beta-subunit tyrosine kinase activity. In addition, a polymorphism of PC-1 (K121Q) in various ethnic populations is closely associated with insulin resistance, T2D, and cardio [corrected] and nephrovascular diseases. The product of this polymorphism has a 2- to 3-fold increased binding affinity for the IR and is more potent than the wild-type PC-1 protein (K121K) in inhibiting the IR. These data suggest therefore that PC-1 is a candidate protein that may play a role in human insulin resistance and T2D by its overexpression, its overactivity, or both.

Insulin resistance, which is pathogenic for type 2 diabetes (T2D), is under the control of largely unknown genetic determinants. LAR, a protein-tyrosine phosphatase which inhibits insulin signalling, is overexpressed in animal and human models of insulin resistance. We studied the entire sequence of the LAR gene by SSCP analysis and automatic DNA sequencing, with the aim of verifying whether its sequence variants might be associated with insulin resistance. In the 276 bp sequence upstream of the transcriptional start site (i.e. a region we have identified as having basal promoter activity) a -127 bp T--A SNP (5% frequency) was associated with lower body mass index (BMI) ( P=0.03), waist circumference ( P=0.01), blood pressure ( P=0.01) and urinary albumin/creatinine ratio ( P=0.04) in 589 non-diabetic unrelated individuals from the Gargano region (central east coast of Italy). To quantify the risk for a high body weight conferred by the -127 T--A SNP, the whole cohort was divided into tertiles according to the individual BMI. The risk of belonging to the heavier tertile, as compared to the leaner one, was reduced by approximately 60%. In a population from East Sicily ( n=307), T/A genotype carriers ( n=13) showed lower triglyceride levels ( P=0.04) and higher insulin sensitivity as indicated by lower plasma glucose ( P=0.03) and serum insulin ( P=0.006) during oral glucose tolerance testing (OGTT). Promoter activity, studied by cDNA transfection experiments, was similar for the A and T alleles. In conclusion, a genetic variant of the LAR gene promoter is consistently associated with features of insulin resistance in two different Caucasian populations. Although the biological relevance of this variant has yet to be determined, this finding underlines the potential importance of the LAR gene in dysregulation of insulin sensitivity and related disorders.

Insulin resistance is believed to be under the control of several genes often interacting each other. However, whether genetic epistasis does in fact modulate human insulin sensitivity is unknown. In 338 healthy unrelated subjects from Sicily, all nondiabetic and not morbidly obese, we investigated whether two gene polymorphisms previously associated with insulin resistance (namely PC-1 K121Q and PPARgamma2 P12A) affect insulin sensitivity by interacting. PC-1 X121Q subjects showed higher level of fasting glucose, lower insulin sensitivity (by both the Matsuda insulin sensitivity index and M values at clamp, the latter performed in a subgroup of 113 subjects representative of the overall cohort) and higher insulin levels during the oral glucose tolerance test (OGTT) than PC-1 K121K subjects. In contrast, no difference in any of the measured variables was observed between PPARgamma2 P12P and X12A individuals. The deleterious effect of the PC-1 X121Q genotype on each of these three variables was significant and entirely dependent upon the coexistence of the PPARgamma2 P12P genotype. Among PPARgamma2 P12P carriers also fasting insulin and glucose levels during OGTT were higher in PC-1 X121Q than in K121K individuals. In contrast, no deleterious effect of the PC-1 X121Q genotype was observed among PPARgamma2 X12A carriers; rather, in these subjects a lower body mass index and consequently lower fasting insulin level was observed in PC-1 X121Q than in K121K carriers. Overall, a significant interaction between the two genes was observed on body mass index, insulin levels (both fasting and after OGTT) and both insulin sensitivity (i.e., insulin sensitivity index and M value) and insulin secretion (i.e., HOMA-B%) indexes.

We investigated the significance of Gly1057Asp and Leu647Val insulin receptor substrate (IRS)-2 polymorphisms in two Italian cohorts comprising 186 glucose-tolerant subjects and 240 subjects with type 2 diabetes from the Lazio region (i.e. representative of central Italy), and 123 glucose-tolerant subjects from the Sicily region (i.e. representative of south Italy). The allelic frequency of Gly1057Asp variant did not differ between diabetics (32.9%) and nondiabetic subjects, whatever their ethnicity was (35.8% and 33.7% from Lazio and Sicily, respectively). As compared with Gly/Gly subjects within each group, Asp/Asp individuals showed no differences in quantitative traits, including fasting insulin and C-peptide, and several indices of insulin sensitivity and secretion. Only one of the diabetic patients was heterozygous for the Leu647Val variant, and none of the control subjects carried this variant. This patient had three children who were also heterozygous for this variant. They were glucose tolerant, and their insulin sensitivity and insulin secretion indices were within the range of age-matched controls. We also analyzed IRS-2 function in fibroblasts from carriers of Gly1057Asp or Leu647Val variant. No defects in IRS-2 expression, insulin-stimulated phosphorylation, or binding to the p85 subunit of phosphatidylinositol 3-kinase were observed. These results strongly argue against a major role of IRS-2 polymorphisms in the pathogenesis of type 2 diabetes.

The current study sought to verify whether glucosamine (GlcN)-induced insulin resistance is associated with impaired insulin receptor (IR) autophosphorylation. Rats were given either saline or primed continuous GlcN infusion (5 micromol x kg(-1) x min(-1)) 10 minutes prior to and during euglycemic hyperinsulinemic clamp (primed continuous infusion of 20 mU x kg(-1) x min(-1) insulin for 2 hours). IR autophosphorylation was measured in skeletal muscle after in vivo insulin stimulation (ie, during clamp) by Western blot and then retested after subsequent in vitro 0.1 to 100 nmol/L insulin stimulation (by enzyme-linked immunosorbent assay [ELISA]). Tissue PC-1 enzymatic activity was also measured. In vivo, insulin/GlcN rats had decreased (P

The insulin resistance syndrome (IRS) is a common clinical condition whose aetiology is poorly understood and which is known to recognize a genetic background. Impairment in insulin receptor (IR) tyrosine-kinase activity has been demonstrated in tissues from insulin-resistant subjects. Several inhibitors of the insulin receptor tyrosine-kinase activity have been recently described and associated with human insulin resistance. Genes encoding for two of these inhibitors, namely PC-1 and PTP1-B, have been deeply studied. The obtained results are here reported. PC-1, a class II transmembrane ectoenzyme, modulates insulin sensitivity by different mechanisms, unravelled by the discovery of two genetic variants. A polymorphism in exon 4, namely K121Q, has been described to be strongly associated with insulin resistance. As compared to the more common K variant, the Q variant has a greater inhibitory activity on insulin receptor function and action, by interacting more strongly with the insulin receptor and significantly reducing receptor autophosphorylation, PC-1 Q variant also identifies type 1 diabetic patients with a faster progression of diabetic nephropathy (DN), a condition that may share some common genetic determinants with insulin resistance. Furthermore, a haplotype in the 3′-untranslated region modulates PC-1 expression in skeletal muscle and confers, therefore, an increased risk for insulin resistance. The mechanism of PC-1 overexpression was explained by an observed significant increase in haplotype-specific mRNA half life. PTP1-B, a nonreceptor-type protein-tyrosine phosphatase (PTPase) that physically interacts with and dephosphorylates the activated insulin receptor, is a major regulator of insulin sensitivity and body fat, as shown from experimental evidences in animal models. A number of polymorphic variants, identified in different populations, have been associated to insulin resistance and its associated clinical conditions. A 1484insG variation in the 3′-UTR region was associated with several features of insulin resistance. Subjects carrying the 1484insG variant showed PTP1-B mRNA overexpression in skeletal muscle, supposedly due to an mRNA stabilization, as demonstrated by transfection experiments. A rare missense variation, namely P387L, was found significantly associated with the risk of type 2 diabetes (T2D). Also, a silent third base polymorphism, namely 981CT, was found to be associated with higher risk of either impaired glucose tolerance (IGT) or type 2 diabetes. Altogether, these data clearly indicate that subtle mutations affecting genes encoding for proteins able to inhibit insulin receptor tyrosine-kinase activity may play a role in modulating susceptibility for insulin resistance and/or type 2 diabetes mellitus.

Diabetes is a complex disease that causes life-threatening complications and often requires life-long treatment. In the last few decades, drugs with different antidiabetes mechanisms have become available. These drugs include insulin-sensitizers (metformin and glitazones), secretagogues (sulphonylureas and incretins), and insulin-mimetics (both short- and long-acting analogs). These therapeutic tools have facilitated more tailored and efficacious therapy. When pancreatic insulin secretion is insufficient or absent, the injection of exogenous insulin or an insulin analog is the only available treatment to activate insulin receptors and restore the biological effects of insulin on target cells. This treatment, however, is associated with hypoglycemia, weight gain, and excess mitogenic activity that may promote the progression of pre-existing subclinical cancers. In these patients, an ideal treatment would activate the insulin receptor and replicate the beneficial metabolic actions of insulin, without causing adverse effects. In an innovative approach to treatment of insulin-dependent diabetes, a study by Bhaskar et al. (1) in this issue of Diabetes introduces this type of drug. Using phage display technology, the authors identified a human monoclonal antibody (XMetA) that binds with high-affinity (ED50 0.10 nmol/L) to the insulin receptor (IR) and has full glucoregulatory activity as well as a reduced risk of hypoglycemia and weight gain. XMetA is a partial IR …

Resistin is overexpressed in human adipose tissue of obese individuals and is likely to modulate insulin sensitivity. Resistin is, therefore, a candidate gene for insulin resistance. We searched for polymorphisms in the resistin gene by single strand conformation polymorphism and direct sequencing. An ATG triplet repeat in the 3-untranslated region was identified and considered for association with insulin resistance. Three alleles were identified (allele 1: 8 repeats, allele frequency, 0.3%; allele 2: 7 repeats; allele frequency, 94.5%; allele 3: 6 repeats; allele frequency, 5.2%). Two hundred and three unrelated white Caucasian nondiabetic subjects from Sicily and 456 from the Gargano area (center east coast of Italy) were analyzed. Among Sicilians, subjects carrying allele 3 had a lower fasting insulin and insulin resistance index (homeostasis model assessment of insulin resistance; P < 0.001 for both) and glucose (P 0.025) and insulin (P 0.002) levels during the oral glucose tolerance test. In subjects from Gargano, those carrying allele 3 had lower fasting plasma glucose levels and serum triglycerides (P 0.01 for both). When the 2 populations were analyzed together, subjects carrying allele 3 had lower fasting insulin levels (P < 0.005), homeostasis model assessment of insulin resistance (P < 0.005), and serum triglycerides (P 0.01). In conclusion, our data suggest that subjects carrying allele 3 of the resistin gene are characterized by relatively high insulin sensitivity. (J Clin Endocrinol Metab 87: 4403– 4406, 2002)

PC-1 is a membrane glycoprotein that impairs insulin receptor function. Its K121Q polymorphism is a genetic determinant of insulin resistance. We investigated whether the PC-1 gene modulates insulin sensitivity independently of weight status (i.e. both in nonobese and obese individuals). Nondiabetic subjects [164 males, 267 females; age, 37 +/- 0.6 yr, mean +/- SEM; body mass index (BMI), 32.7 +/- 0.5 kg/m(2)], who were subdivided into 220 nonobese (BMIor = 29.9) and 211 obese, were studied. Although subjects were nondiabetic by selection criteria, plasma insulin concentrations during oral glucose tolerance test were higher (P0.05) in Q allele-carrying subjects (K121Q or Q121Q genotypes), compared with K121K individuals, in both the nonobese and obese groups. Insulin sensitivity, measured by euglycemic clamp in a representative subgroup of 131 of 431 randomly selected subjects, progressively decreased (P0.001) from nonobese K121K [n = 61; glucose disposal (M) = 34.9 +/- 1.1 micromol/kg/min] to nonobese Q (n = 21; M = 29.9 +/- 2.0), obese K121K (n = 31, M = 18.5 +/- 1.2), and obese Q (n = 18, M = 15.5 +/- 1.2) carriers. The K121Q polymorphism was correlated with insulin sensitivity independently (P0.05) of BMI, gender, age, and waist circumference. In conclusion, the Q121 PC-1 variant and obesity have independent and additive effects in causing insulin resistance.

Hybrid receptors (HRs), insulin receptor (IR)/insulin-like growth factor I receptor (IGF-I-R) heterodimers have been reported increased in skeletal muscle of obese and type 2 diabetic patients and to contribute to the patient insulin resistance. To investigate whether or not the increased expression of hybrid receptors is an early defect (probably genetic) of insulin resistance, we measured by specific enzyme-linked immunosorbent assays both IR, IGF-I-R, and HR content in skeletal muscle of healthy nonobese, nondiabetic subjects either insulin sensitive or insulin resistant, and also in patients with moderate obesity. IR content was significantly reduced in insulin-resistant subjects both nonobese and obese, compared with insulin-sensitive subjects (2.32+/-0.26, 2.36+/-0.18, and 3.45+/-0.42 ng/mg protein, respectively, P = 0.002). In contrast, IGF-I-R content was similar in the three groups. Muscle HR content was not different in insulin-sensitive vs. insulin-resistant subjects (both nonobese and obese) (4.90+/-0.46, 4.69+/-0.29, and 4.91+/-0.25 ng/mg protein, respectively, P = not significant). These studies indicate that, in insulin-resistant subjects without diabetes or severe obesity, muscle IR content but not IGF-I-R or HR content is reduced. They do not suggest, therefore, a primary (genetic) role of increased HR as a cause of IR decrease and insulin resistance.

We studied whether there is an association between the single nucleotide polymorphism c.533A>C (K121Q) in the glycoprotein PC-1 gene and features of the metabolic syndrome in case-control and intrafamily association studies in 922 subjects from Finland and Sweden. No difference was observed in the Q allele frequency between control subjects and type 2 diabetic subjects (12.9 vs. 15.1%). The QK genotype was associated with higher fasting plasma glucose (FPG) concentrations than the KK genotype in type 2 diabetic patients (P

An increased tissue content of PC-1, an inhibitor of insulin receptor signaling, may play a role in insulin resistance. Large scale prospective studies to test this hypothesis are difficult to carry out because of the need for tissue biopsies. The aim of this study was to investigate whether PC-1 is measurable in human plasma and whether its concentration is related to insulin sensitivity. A soluble PC-1, with mol wt and enzymatic activity similar to those of tissue PC-1, was measurable in human plasma by a specific enzyme-linked immunosorbent assay and was inversely correlated to skeletal muscle PC-1 content (r = -0.5; P0.01). The plasma PC-1 concentration was decreased (P0.05) in insulin-resistant (22.7 +/- 3.0 ng/mL; n = 25) compared to insulin-sensitive (36.7 +/- 4.5; n = 25) nondiabetic subjects and was correlated negatively with the waist/hip ratio (r = -0.48; P0.001) and mean blood pressure (r = -0.3; P0.05) and positively with high density lipoprotein/total cholesterol (r = 0.38; P0.01) and both the M value and the plasma free fatty acid level decrement at clamp studies (r = 0.28; n = 50; P = 0.05 and r = 0.43; n = 22; P0.05, respectively). A plasma PC-1 concentration of 19 ng/mL or less identified a cluster of insulin resistance-related alterations with 75% accuracy. In conclusion, PC-1 circulates in human plasma, and its concentration is related to insulin sensitivity. This may help to plan studies aimed at understanding the role of PC-1 in insulin resistance.

The genes responsible for insulin resistance are poorly defined. Plasma cell differentiation antigen (PC-1) glycoprotein inhibits insulin receptor signaling and is associated with insulin resistance. We describe here a novel polymorphism in exon 4 of the PC-1 gene (K121Q) and demonstrate that it is strongly associated with insulin resistance in 121 healthy nonobese (BMI

To verify whether the short intravenous insulin tolerance test (ITT) (a safe, reproducible, inexpensive, rapid and easy to perform measurement of insulin sensitivity) is a suitable test for insulin resistance screening in a general population, we measured in 60 non diabetic subjects, either non-obese (no.=40) or obese (BMI28, no.=20) the K of glucose disappearance from plasma after ITT (K(ITT)), plasma glucose (PG) and insulin (IRI) both fasting (FPG, FIRI) and at 120 min of OGTT (PG- 120, IRI- 120), and also triglycerides (Tg), cholesterol (Chol) and blood pressure (BP). Subjects were subdivided into quartiles according to K(ITT) values. Average FPG, PG-120, FIRI, IRI-120, Tg and Chol values were progressively increased, and average HDL/Chol was progressively decreased from quartile 1 (the most insulin sensitive) to 4 (the most insulin resistant) (p0.05, by 1-way ANOVA test). Also BP was increased in the insulin resistant patients, but statistical significance was not reached. Three or more of the studied parameters (FPG and/or PG-120, FIRI and/or IRI-120, Tg, HDL/Chol, mean BP) were altered (below the worst 25 degree percentile) in 64% of subjects from quartile 4; none of the subjects in quartile 1 was affected by such a cluster of alterations. K(ITT) valuesor =4.8 identified the cluster of insulin resistance related alterations with an accuracy of 82% (sensitivity=83.3%, specificity=80.5%). In healthy subjects with a wide range of BMI the ITT is a reliable procedure for screening for the cluster of metabolic alterations related to insulin resistance.

Membrane glycoprotein PC-1 inhibits insulin receptor (IR) tyrosine kinase activity and subsequent cellular signaling. PC-1 content is elevated in muscle and adipose tissue from insulin-resistant subjects, and its elevation correlates with in vivo insulin resistance. To determine whether elevated PC-1 content is a primary cause of insulin resistance, we have now measured PC-1 content in cultured skin fibroblasts from nonobese nondiabetic insulin-resistant subjects and found that 1) PC-1 content was significantly higher in these cells when compared with cells from insulin-sensitive subjects (6.7 +/- 0.9 vs. 3.1 +/- 0.6 ng/0.1 mg protein, mean +/- SE, P < 0.01); 2) PC-1 content in fibroblasts was highly correlated with PC-1 content in muscle tissue (r = 0.95, P = 0.01); 3) PC-1 content in fibroblasts negatively correlated with both decreased in vivo insulin sensitivity and decreased in vitro IR autophosphorylation; and 4) in cells from insulin-resistant subjects, insulin stimulation of glycogen synthetase was decreased. These studies indicate, therefore, that the elevation of PC-1 content may be a primary factor in the cause of insulin resistance.

To determine the role of the insulin receptor overexpression in breast epithelial cell transformation, the 184B5 human breast epithelial cell line was transfected with human insulin receptor cDNA. In two cell lines transfected with and overexpressing human insulin receptors (IR) (223.8 and 184.5 ng IR/10(6) cells), but not in untransfected cells, insulin binding and tyrosine kinase activity were elevated, and insulin induced a dose-dependent increase in colony formation in soft agar.

The insulin receptor exists in two isoforms differing by the absence (HIR-A) or presence (HIR-B) of 12 amino acids in the COOH-terminus of the alpha-subunit as a consequence of alternative splicing of exon 11. In this study, we developed a radioimmunoassay for the two isoforms employing antibodies raised against two peptides, one (Pep-12) corresponding to residues encoded by exon 11, and the other (Pep-13) corresponding to a COOH-terminal domain of the alpha-subunit which is common to both HIR-A and HIR-B isoforms. These peptides were iodinated and used as both ligands and standards. The assay is specific, highly reproducible, and sensitive with a detection limit of 10 fmol of receptor. One mole of purified insulin receptor, measured by Scatchard analysis, is read as one mole of receptor in the radioimmunoassay with either Pep-12 or Pep-13 as standards. The radioimmunoassay is applicable to the measurement of total content and relative abundance of the two isoforms in extracts from various tissues. We applied the radioimmunoassay to measure the relative abundance of the two isoforms in fat and muscle from normal, obese non-diabetic and non-insulin-dependent diabetic (NIDDM) subjects. Results demonstrate that expression of the low-affinity HIR-B form is significantly increased in obese and NIDDM subjects compared with control subjects. In addition, the increased expression of the HIR-B isoform was significantly correlated with both body mass index (r = 0.52; p = 0.006) and fasting glucose levels (r = 0.59; p = 0.001).

We performed i. v. insulin tolerance test in 30 non-obese (BMI

Insulin sensitivity has been quantified by i. v. insulin tolerance test (0.1 U/kg of body weight) in 18 (11 male/7 female) non-obese (body mass index range 19–25 kg/m2) normoglycaemic subjects. We then compared the tyrosine kinase activity and internalization of insulin receptor in monocytes from the six most insulin-sensitive (group 1) and the six most insulin-resistant (group 3) subjects. Tyrosine kinase activity was measured on immunopurified receptors using 32P-ATP and poly-glutamic acid 4: tyrosine 1, sodium salt (poly-glu-tyr 4∶1). Insulin internalization was studied by incubating cells with 1 nmol/l 125I-insulin and measuring total cell-bound and intracellular 125I-insulin by an acid dissociation procedure. Basal (in the absence of insulin) receptor kinase activity was similar in both groups. Maximal (in the presence of 100 nmol/l insulin) kinase activity was 41% lower in group 3 (13.8±3.6 fmoles 32P-ATP incorporated vs 23.3±4.0, p=0.1). Delta increment of receptor kinase activity after insulin stimulation (calculated by subtracting basal from maximal activity) was significantly (p

Reduced insulin receptor tyrosine kinase activity and internalization have been reported in non-insulin-dependent diabetes mellitus (NIDDM) patients. To clarify whether in NIDDM the defective internalization is caused by the defective kinase activity, we studied receptor tyrosine kinase activity and internalization in monocytes from eight lean control and six obese subjects and 10 obese NIDDM patients. Receptor internalization was also stimulated by an anti-insulin receptor antibody (MA-10) that is unable to stimulate receptor kinase activity. Basal exogenous tyrosine kinase activity was not different in monocytes from the three groups of subjects. As compared with control subjects (2,690 +/- 637 fmol 32P incorporated), insulin (100 nmol/L)-stimulated tyrosine kinase activity was lower in NIDDM patients (1,262 +/- 318, P.05), but not in obese subjects (2,640 +/- 731). Basal receptor autophosphorylation did not differ between the three groups, whereas insulin-stimulated autophosphorylation in comparison to that in control subjects was reduced in NIDDM patients (P.05), but not in obese subjects. In NIDDM patients, receptor internalization induced by both insulin and MA-10, was lower (P.05) than that in control and obese subjects. No correlation was found between receptor internalization and exogenous tyrosine kinase activity (r = .30, NS) or autophosphorylation (r = .08, NS).(ABSTRACT TRUNCATED AT 250 WORDS)

Glycoprotein PC-1 inhibits insulin signaling and, when overexpressed, plays a role in human insulin resistance. Mechanisms of PC-1 overexpression are unknown. We have identified a haplotype in the 3′-untranslated region of the PC-1 gene that may modulate PC-1 expression and confer an increased risk for insulin resistance. Individuals from Sicily, Italy, carrying the “P” haplotype (i.e., a cluster of three single nucleotide polymorphisms: G2897A, G2906C, and C2948T) were at higher risk (P < 0.01) for insulin resistance and had higher (P < 0.05) levels of plasma glucose and insulin during an oral glucose tolerance test and higher levels of cholesterol, HDL cholesterol, and systolic blood pressure. They also had higher (P < 0.05–0.01) PC-1 protein content in both skeletal muscle and cultured skin fibroblasts. In CHO cells transfected with either P or wild-type cDNA, specific PC-1 mRNA half-life was increased for those transfected with P (t/2 = 3.73 ± 1.0 vs. 1.57 ± 0.2 h; P < 0.01). In a population of different ethnicity (Gargano, East Coast Italy), patients with type 2 diabetes (the most likely clinical outcome of insulin resistance) had a higher P haplotype frequency than healthy control subjects (7.8 vs. 1.5%, P < 0.01), thus replicating the association between the P allele and the insulin resistance–related abnormalities observed among Sicilians. In conclusion, we have identified a possible molecular mechanism for PC-1 overexpression that confers an increased risk for insulin resistance–related abnormalities.