Serum levels of osteopontin predict diabetes remission after bariatric surgery.

AIM: Bariatric surgery has been shown to effectively improve glycaemic control in morbidly obese subjects. However, the molecular bases of this association are still elusive and may act independently of weight loss. Here, our retrospective study has investigated the inflammatory molecule osteopontin (OPN) as a potential predictor of type 2 diabetes mellitus (T2DM) remission. METHODS: Baseline serum levels of OPN were analyzed in 41 T2DM patients who underwent bariatric surgery. Anthropometric measures and biochemical variables, including insulin sensitivity indices (HOMA2), were assessed at baseline and at 1 and 3 years after surgery. RESULTS: At baseline, patients who experienced T2DM remission had increased waist circumference, body weight and BMI, and higher serum OPN, compared with non-remitters. Patients with and without T2DM remission improved their lipid and glucose profiles, although insulin resistance indices were only improved in the T2DM remission group. In the overall cohort of both T2DM remission and non-remission patients, baseline circulating levels of OPN significantly correlated with reductions of body weight and BMI over time, and insulin sensitivity improved as well. However, only the HOMA2-%S remained independently associated with serum OPN on multivariate linear regression analysis (B: 0.227, 95% CI: 0.067-0.387, ß = 0.831; P = 0.010). Baseline values of OPN predicted 3-year T2DM remission independently of body weight loss, lower BMI and duration of diabetes (OR: 1.046, 95% CI: 1.004-1.090; P = 0.033). CONCLUSION: Although larger studies are still needed to confirm our preliminary results, pre-operative OPN serum levels might be useful for predicting 3-year T2DM remission independently of weight loss in patients undergoing bariatric surgery.

Changes in adiponectin and leptin concentrations during glucocorticoid treatment: a pilot study in patients with polymyalgia rheumatica.

This study is concerned with an evaluation of the effects of glucocorticoids (GC) on adiponectin and leptin concentrations in patients with polymyalgia rheumatica (PMR). Seven patients diagnosed with PMR were studied at baseline and after one and three months of prednisone treatment. Serum leptin and adiponectin, serum glucose and insulin, erythrocyte sedimentation rate, C-reactive protein, and IL-6 were all measured by commercial assays. The treatment with GC normalized inflammation markers and significantly increased adiponectin and leptin concentrations without any impairment of insulin sensitivity measured by HOMA-IR. Adiponectin significantly increased only between baseline and 1 month (P= 0.013). A significant correlation was found between adiponectin and leptin concentrations both at baseline and after 3 months of treatment (both rho = 0.89, P= 0.03). In addition, adiponectin correlated also with serum glucose at baseline (rho = 0.81, P= 0.047). According to our results, adiponectin concentrations seem to be driven by inflammation, whereas leptin seems to be increased directly by the use of steroids.

Absence of thyrotropin-induced increase in leptin levels in patients with history of differentiated thyroid carcinoma undergoing recombinant human thyrotropin testing.

BACKGROUND: Some extra-thyroid effects of TSH have been described in vitro and in vivo. TSH has recently been suggested to induce interleukin-6 secretion by adipocytes. Leptin is the main protein secreted by adipose tissue. OBJECTIVE: The aim of our study was to evaluate the acute effect of the recombinant human TSH (rhTSH)-induced TSH surge on serum leptin levels in thyroidectomized patients undergoing levothyroxine (L-T4) suppressive therapy for differentiated thyroid carcinoma (DTC). DESIGN: A cohort of 15 female DTC patients was evaluated. Standard rhTSH testing was performed. Leptin, TSH, thyroid hormones, and thyroglobulin were measured before and 3, 6, and 9 days after rhTSH testing. Some metabolic parameters were also evaluated at the baseline. RESULTS: Baseline leptin levels were 12.2+/-3.2 microg/l. Only body mass index (BMI) correlated significantly (p<0.05) with leptin levels. After rhTSH administration, TSH levels increased significantly (p<0.001), while thyroid hormones remained unchanged. Twenty hours after the last rhTSH administration, leptin (11.8+/-3.0 microg/l) levels were unchanged. The maximal TSH level was negatively related with BMI (p<0.05), but no correlation between maximal TSH and leptin levels after rhTSH was noted. CONCLUSIONS: Our in vivo experimental model suggests that an acute TSH increase after rhTSH testing is ineffective in changing circulating leptin levels.

Alterations in the autonomic control of heart rate variability in patients with anorexia or bulimia nervosa: correlations between sympathovagal activity, clinical features, and leptin levels.

Changes in body composition, hormone secretions, and heart function with increased risk of sudden death occur in eating disorders. In this observational clinical study, we evaluated sympathovagal modulation of heart rate variability (HRV) and cardiovascular changes in response to lying-to-standing in patients with anorexia (AN) or bulimia nervosa (BN) to analyze: a) differences in autonomic activity between AN, BN, and healthy subjects; b) relationships between autonomic and cardiovascular parameters, clinical data and leptin levels in patients with eating disorders. HRV, assessed by power spectral analysis of R-R intervals, blood pressure (BP) and heart rate (HR) were studied by tilt-table test in 34 patients with AN, 16 with BN and 30 healthy controls. Autonomic and cardiovascular findings were correlated with clinical data, and serum leptin levels. Leptin levels were lowered in AN vs BN and healthy subjects (p<0.0001), but both AN and BN patients showed unbalanced sympathovagal control of HRV due to relative sympathetic failure, prevalent vagal activity, impaired sympathetic activation after tilting, independently from their actual body weight and leptin levels. No significant correlations were obtained between HRV data vs clinical data, BP and HR findings, and leptin levels in eating disorders. Body mass indices (BMI) (p<0.02), and leptin levels (p<0.04) correlated directly with BP values. Our data showed alterations of sympathovagal control of HRV in eating disorders. These changes were unrelated to body weight and BMI, diagnosis of AN or BN, and leptin levels despite the reported effects of leptin on the sympathetic activity.

The extracellular portion of the insulin receptor beta-subunit regulates the cellular trafficking of the insulin-insulin receptor complex. Studies on Chinese hamster ovary cells carrying the Cys 860-->Ser insulin receptor mutation.

OBJECTIVE: Chinese hamster ovary (CHO) cells transfected with human engineered insulin receptor (IR) cDNA to mutate Cys 860 to Ser (CHO-IR(C860S)) showed a defective insulin internalization without affecting insulin binding and IR autophosphorylation. Moreover, this mutation reduces insulin receptor substrate (IRS)-1 tyrosine phosphorylation and insulin-induced metabolic and mitogenic effects. Altogether, these observations support a role of the extracellular domain of IR beta-subunit in insulin and receptor intracellular targeting as well as in insulin signaling. DESIGN AND METHODS: This study assesses in more details the effect of IR(C860S) mutation on the trafficking of the insulin-IR complex. In particular, IR internalization, phosphorylation, dissociation and recycling, as well as insulin degradation and retroendocytosis have been investigated in CHO cells overexpressing either wild type (CHO-IR(WT)) or mutated IRs. RESULTS: the C860S mutation significantly decreases IR internalization both insulin stimulated and constitutive. In spite of a similar dissociation of internalized insulin-IR complex, recycling of internalized IR was significantly faster (half life (t(1/2)): 21 min vs 40 min, P<0.001) and more extensive (P<0.01) for IR(C860S) than for IR(WT). On the other hand, insulin degradation and retroendocytosis were superimposable in both cell lines. As expected, insulin-induced phosphorylation was similar in both IRs, however dephosphorylation was much more rapid and was greater (P<0.01) in CHO-IR(WT) as compared with CHO-IR(C860S) cells. CONCLUSIONS: Transmembrane and intracellular domain of IR seem to be determinants for IR internalization. Now we report that Cys 860 in the IR beta-subunit ectodomain may be of relevance in ensuring a proper internalization and intracellular trafficking of the insulin-IR complex.

Cys860 in the extracellular domain of insulin receptor beta-subunit is critical for internalization and signal transduction.

The C860S mutation (IRC860S) in the extracellular domain of the insulin receptor beta-subunit has previously been shown to result in an inhibition of insulin receptor internalization. The present work aims at further dissecting the consequences of this mutation not only on insulin receptor internalization, but also on the signaling of the receptor. Following transfection of Chinese hamster ovary (CHO) cells with insulin receptors with the C860S mutation (CHO-IRC860S) and quantitative electron microscopic analysis of [125I]insulin localization in these cells, the inhibition of receptor internalization appears to be due to an inhibition of the lateral translocation of the receptor from microvilli to nonvillous domains of the cell surface. At 37 C, insulin-stimulated insulin receptor substrate-1 (IRS-1) phosphorylation is inhibited by 50% in CHO-IRC860S, whereas Shc phosphorylation remains unaffected. The inhibition of IRS-1 phosphorylation is still present when experiments are conducted at 4 C, a temperature at which insulin receptor internalization is prevented, suggesting that the defect in IRS-1 phosphorylation is not due to the reduced internalization of the receptor. In terms of biological effects, the mutation has negative consequences on insulin-stimulated c-fos expression and DNA synthesis as well as on glycogen synthase activity. Eventually, the events observed are specific for Cys860, as individual substitution of the two more proximal Cys residues (795 and 872) to Ser is not accompanied by any change in either insulin-induced insulin receptor internalization or IRS-1 phosphorylation. Thus, the present analysis of CHO-IRC860S 1) reveals that insulin receptor surface redistribution is not solely dependent on receptor autophosphorylation, 2) emphasizes that IRS-1 phosphorylation is not dependent on receptor internalization and can be triggered from microvilli, and 3) stresses divergent aspects between two of the major signaling pathways of the insulin receptor.

A ser for Cys mutation in the extracellular portion of insulin receptor beta subunit impairs the insulin-insulin receptor complex internalization in CHO cells.

We investigated the effects of a ser for cys 860 mutation, located in the extracellular portion of insulin receptor beta subunit, on several receptor functions. CHO cells, stably transfected with the mutated cDNA, were used for this study. In the present paper, we show that the ser 860 mutation does not affect the 125I-insulin binding, but severely impairs the insulin-insulin receptor complex internalization. The kinetic analysis of internalization indicates that this process is inhibited at steps preceding the coated pit endocytosis. The beta subunit autophosphorylation of the mutated receptor is higher both in the basal and insulin stimulated states, compared with autophosphorylation measured in wild type insulin receptors. The ser 860 mutation impairs also the insulin receptor down regulation, thus suggesting an effect on the intracellular sorting of insulin-insulin receptor complex. On the basis of these results we suggest that the cys 860 plays an important role in insulin receptor lateral moving on cell surface, after insulin binding, and on the intracellular sorting to degradation pathways.

The overexpression of insulin receptor makes CHO cells resistant to the action of IGF-1: role of IRS-1.

We investigated the influence of the relative abundance of insulin and IGF-1 receptors on cellular growth, by measuring the stimulation of c-fos expression and of H3-thymidine incorporation into DNA by insulin and IGF-1 in CHO cells overexpressing insulin Receptor (CHO-IR). We found that CHO-IR cells were resistant to the action of IGF-1, but were more responsive to insulin, compared to parental clone. This result suggest the presence of a limiting step, distal to the IGF-1 receptor, in the transduction pathway. To address this question we measured the IGF-1 effect on c-fos expression in CHO-IR cells, transiently transfected with the cDNA for IRS-1, the common intracellular substrate for both insulin and IGF-1 receptors (CHO-IR/IRS-1 cells). In these cells IGF-1 stimulated a 10 fold higher c-fos expression compared to CHO-IR cells. These results suggest that the abundance of IRS-1, relative to the number of insulin and IGF-1 receptors, represents a limiting step for the intracellular transduction of insulin and IGF-1 biological messages.

Substitution of Leu for Pro-193 in the insulin receptor in a patient with a genetic form of severe insulin resistance.

Mutations have been identified in the insulin receptor (IR) gene in patients who are insensitive to insulin action. We studied an extremely insulin resistant patient whose insulin binding to Epstein-Barr virus (EBV) transformed lymphocytes was severely reduced. Transmembrane signalling, evaluated as insulin receptor autophosphorylation, was normal. The patient's IR was immunoprecipitated normally by AbP6, a polyclonal antibody directed to the beta subunit. However, there was an approximately 50% decrease in the affinity of IR immunoprecipitation by a monoclonal antibody (MA-10) directed against the alpha subunit. These observations suggested that there were likely to be a mutation in the patient's insulin receptor that caused misfolding of the IR alpha subunit. Analysis of gene structure by Southern blotting experiments did not reveal any major deletion in the IR gene of the proband. Northern blot analysis showed a normal level of expression of IR gene. We applied denaturing gradient gel electrophoresis (DGGE) as well as direct sequence analysis to study the 22 exons of IR gene amplified by polymerase chain reaction (PCR) using the proband's genomic DNA as a template. We identified a new missense mutation substituting leucine (CTG) for proline (CCG) in homozygous state at codon 193 in exon 3. Both parents are heterozygous for the Leu193 mutation. The Leu193 mutation was not detected in any of 75 normal subjects (150 chromosomes), indicating that it is not a common sequence variant of the insulin receptor. In addition, during the course of screening the patient's DNA with perpendicular DGGE, we identified two previously unreported silent substitutions in exon 9.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of in vivo vanadate treatment on insulin receptor tyrosine kinase activity in partially pancreatectomized diabetic rats.

Liver-purified insulin receptor tyrosine kinase (IRTK) activity was examined in partially pancreatectomized rats following normalization of blood glucose concentration by either phlorizin or vanadate treatment. Chronic moderate hyperglycemia did not modify the IRTK activity, despite the presence of in vivo and in vitro insulin resistance. Oral vanadate administration for 3 weeks normalized glucose tolerance and caused a 2.5-fold increase in basal IRTK activity. In contrast, correction of hyperglycemia with phlorizin, an inhibitor of renal glucose reabsorption, did not change the IRTK activity, although glucose tolerance was returned to normal. The vanadate-induced effect on basal IRTK was due to an increase in Vmax of the enzyme; the Km remained unchanged. The insulin-stimulated IRTK activity was not affected by either vanadate or phlorizin treatment. These results suggest that: 1) partial (90%) pancreatectomy in rats causes insulin resistance in the absence of in vitro alterations in IRTK and 2) correction of chronic hyperglycemia with vanadate, but not with phlorizin, is associated with an increased basal activation of the protein tyrosine kinase in liver insulin receptors.

Effect of metformin treatment on insulin action in diabetic rats: in vivo and in vitro correlations.

The mechanism (both at the whole body and cellular level) by which metformin improves insulin sensitivity has yet to be defined. In the present study, we examined in vivo insulin-mediated whole-body glucose disposal, glycogen synthesis, hepatic glucose production, and insulin secretion, as well as in vitro muscle insulin receptor tyrosine kinase activity in eight control, eight neonatal streptozotocin diabetic rats, and eight diabetic rats before and after treatment with metformin. Ten weeks after birth diabetic rats had higher fasting (132 + 5 v 101 + 2 mg/dL) and postmeal (231 + 10 v 133 + 3) plasma glucose levels compared with controls (P less than .001). Metformin treatment was followed by a significant decrease in the growth rate and normalized glucose tolerance without enhancing the deficient insulin response. Insulin-mediated glucose uptake in diabetic versus control rats was reduced (P less than .01) during the high-dose (15.4 + 0.6 v 18.3 + 1.0 mg/kg.min) insulin clamp study and was increased to values greater (P less than .05) than controls following metformin treatment. Muscle glycogen synthetic rate in vivo, measured by incorporation of 3H-3-glucose radioactivity, was diminished by 25% (P less than .01) in diabetic rats, restored to normal values with metformin, and correlated closely (r = .82, P less than .002) with total-body glucose uptake during the insulin clamp in all three groups. Insulin receptor tyrosine kinase activity, measured in partially purified insulin receptors, was reduced in diabetic rats and increased to supernormal levels after metformin. The decrease in muscle tyrosine kinase activity in diabetic versus control animals was entirely accounted for by a reduction in maximal velocity (Vmax) (32 v 45 pmol/mg.min, P less than .01) and increased to supernormal levels following metformin (91 pmol/mg.min, P less than .001) without any change in affinity (Km). Muscle tyrosine kinase activity was closely correlated with both the muscle glycogen synthetic rate (r = .82, P less than .002) and total-body insulin-mediated glucose disposal (r = .64, P less than .01) in vivo. The close correlation between in vivo insulin action, muscle glycogen synthesis, and muscle insulin receptor tyrosine kinase activity is consistent with an important role of the enzyme in the insulin resistance of diabetes and its improvement following metformin treatment.

An extracellular domain of the insulin receptor beta-subunit with regulatory function on protein-tyrosine kinase.

Anti-insulin receptor monoclonal antibody MA-10 inhibits insulin receptor autophosphorylation of purified rat liver insulin receptors without affecting insulin binding (Cordera, R., Andraghetti, G., Gherzi, R., Adezati, L., Montemurro, A., Lauro, R., Goldfine, I. D., and De Pirro, R. (1987) Endocrinology 121, 2007-2010). The effect of MA-10 on insulin receptor autophosphorylation and on two insulin actions (thymidine incorporation into DNA and receptor down-regulation) was investigated in rat hepatoma Fao cells. MA-10 inhibits insulin-stimulated receptor autophosphorylation, thymidine incorporation into DNA, and insulin-induced receptor down-regulation without affecting insulin receptor binding. We show that MA-10 binds to a site of rat insulin receptors different from the insulin binding site in intact Fao cells. Insulin does not inhibit MA-10 binding, and MA-10 does not inhibit insulin binding to rat Fao cells. Moreover, MA-10 binding to down-regulated cells is reduced to the same extent as insulin binding. In rat insulin receptors the MA-10 binding site has been tentatively localized in the extracellular part of the insulin receptor beta-subunit based on the following evidence: (i) MA-10 binds to insulin receptor in intact rat cells; (ii) MA-10 immunoprecipitates isolated insulin receptor beta-subunits labeled with both [35S]methionine and 32P; (iii) MA-10 reacts with rat insulin receptor beta-subunits by the method of immunoblotting, similar to an antipeptide antibody directed against the carboxyl terminus of the insulin receptor beta-subunit. Moreover, MA-10 inhibits autophosphorylation and protein-tyrosine kinase activity of reduced and purified insulin receptor beta-subunits. The finding that MA-10 inhibits insulin-stimulated receptor autophosphorylation and reduces insulin-stimulated thymidine incorporation into DNA and receptor down-regulation suggests that the extracellular part of the insulin receptor beta-subunit plays a role in the regulation of insulin receptor protein-tyrosine kinase activity.

Direct modulation of insulin receptor protein tyrosine kinase by vanadate and anti-insulin receptor monoclonal antibodies.

Sodium vanadate activates "in vitro" insulin receptor autophosphorylation and protein tyrosine kinase in a dose-dependent manner. Insulin receptor protein tyrosine kinase is directly activated also by the anti-insulin receptor beta subunit monoclonal antibody 18-44. We previously demonstrated that the anti-insulin receptor monoclonal antibody MA-10 decreases insulin-stimulated receptor protein tyrosine kinase activity "in vitro", without inhibiting insulin receptor binding. In this report we show that insulin receptor protein tyrosine kinase, activated by sodium vanadate or by monoclonal antibody 18-44, is inhibited by MA-10 antibody. These data suggest that insulin receptor protein tyrosine kinase activity can be either activated and inhibited through mechanisms different from insulin binding.

[Preparation of 125I-labelled monoclonal antibodies of the insulin receptor]. / Préparation d'anticorps monoclonaux du récepteur de l'insuline marqués par 125I.

Three monoclonal anti-insulin receptor antibodies have been labelled with 125I according to various methods (Cloramine T, Lactoperoxidase and IODO-GEN). The effect of labelling on antibody structure and function has been characterized using the following parameters: a) specific activity obtained in four different labelling procedures, at least; b) TCA labelled antibody precipitable 90 days after labelling; c) interaction between labelled antibodies and the insulin receptor; d) ability of antibodies to inhibit insulin-stimulated receptor auto-phosphorylation. Cloramine T method produced labelled antibody with constant specific activity; however, some preparations were unstable and showed reduced capacity to recognize the insulin receptor. Lactoperoxidase method produced stable antibodies; however, specific activity was highly variable and antibodies had low capacity to interact with the insulin receptor. The IODO-GEN method produced antibodies with constant specific activity, stable, high capacity to interact with the insulin receptor, and, moreover, maintaining in full the capacity to inhibit the insulin-stimulated auto-phosphorylation of the insulin receptor, since it does not induce antibody alterations which in turn affect antibody-receptor interaction biological action.

Species specificity of insulin binding and insulin receptor protein tyrosine kinase activity.

The effect of monoclonal anti-insulin receptor antibody MA 10 on [125I]insulin binding and on insulin receptor protein tyrosine kinase activity was investigated in human and rat tissues. It was observed that MA 10 inhibits insulin binding to human, but not rat, tissues while inhibiting insulin-stimulated receptor autophosphorylation and protein tyrosine kinase activity in both human and rat tissues. These data suggest that MA 10 is directed against a region of the insulin receptor that is in between the insulin-binding domain and the beta-subunit and that in human, but not rat, tissues, this region is involved in insulin binding.

Regulation of insulin receptor-associated tyrosine kinase by a polyclonal IgG.

The effect of a polyclonal anti-insulin receptor antibody (pIgG) on the insulin receptor tyrosine kinase (IRTK) activity toward poly-(Glu-Tyr) was examined using wheat germ agglutinin agarose-purified insulin receptors from rat liver membranes. The main effect of pIgG was a reduction of Vmax (from 60.8 to 31.8 pmol/min/mg), without changes of Km, when IRTK was activated by insulin. In contrast, when IRTK was activated by ATP preincubation, pIgG was unable to affect the reaction, suggesting that IRTK possesses at least two regulatory mechanisms, one of which can be affected by pIgG.

Insulin receptor autophosphorylation and kinase activity in streptozotocin diabetic rats. Effect of a short fast.

Insulin receptor associated kinase activity and its relationships with the insulin resistance of streptozotocin-induced diabetes were investigated in rats, using solubilized, partially purified insulin receptors from liver membranes. Insulin receptor kinase activity was measured by means of both autophosphorylation and phosphorylation of the exogenous substrate Glu4:Tyr1. Diabetes was associated with a 45% reduction in kinase activity, in the same number of insulin receptors, with no change in insulin binding affinity. To investigate the independent roles of hyperglycemia and hypoinsulinemia on the observed impairment of receptor kinase activity, diabetic rats were fasted for 24 h in order to normalize blood glucose levels only. After this short fast, no change in kinase activity, from the values measured in fed diabetic animals, was observed. Our findings suggest that streptozotocin diabetes is associated with a reduction of insulin receptor kinase activity, which a short fast is not able to reverse.

Insulin-like growth factor I (IGF I) receptor autophosphorylation and kinase activity. Effect of a human polyclonal antibody (pIgG)

IGF I receptor is a tyrosine kinase capable of phosphorylating the receptor itself and other substrates. A high degree of homology does exist in tyrosine kinase domain among receptors for several polypeptide growth factor receptors and this enzymic activity has been indicated as a possible mediator of biological action. Nevertheless growth factor receptors possess peculiar specificities both in their functions and tissue distribution. A human polyclonal IgG (pIgG), previously characterized as anti insulin receptor antibody, able to inhibit insulin receptor kinase activity, was used to further investigate subunit homologies and differences in antigenicity and functional regulation between IGF I and insulin receptors, IGF I receptor tyrosine kinase was stimulated by a IGF I analog (aIGF I), produced by DNA recombinant technology, pIgG was able to inhibit IGF I receptor kinase activity, thus revealing antigenic homologies between the kinase domains of insulin and IGF I receptors. However the more pronounced inhibition of IGF I receptor-compared with insulin receptor kinase activity by pIgG suggests the existence of different regulatory mechanisms.