Pituitary autoimmunity in patients with diabetes mellitus and other endocrine disorders.

OBJECTIVE: Pituitary autoimmunity is often found in association with other endocrine autoimmune or non-autoimmune diseases. Aim of the study was to assess the prevalence of serum pituitary antibodies (PitAb) in patients with Type 1 diabetes mellitus (T1DM) or Type 2 diabetes mellitus (T2DM). RESEARCH DESIGN AND METHODS: In this casecontrol study 111 patients with T1DM, 110 patients with T2DM, and 214 healthy controls were enrolled in a tertiary referral center. Pituitary, thyroperoxidase, thyroglobulin, 21-hydroxylase, and parietal cell antibodies were assessed in all cases. Endocrine function was further assessed by basal hormone measurement and by dynamic tests, as well as a pituitary magnetic resonance imaging (MRI) was performed in those patients found positive for PitAb. RESULTS: PitAb prevalence was higher in T1DM (4 out of 111, 3.6%) than in T2DM (0 out of 110, p=0.045) and in healthy subjects (1 out of 214, 0.5% p=0.029). Prevalence of other autoimmune diseases was significantly higher in patients with T1DM (45 out of 111, 40.5%) when compared with patients with T2DM (18 out of 110 T2DM, 16.3%, p<0.001). Patients with T1DM and PitAb positivity were found with a pituitary lesion at MRI in 2 cases and pituitary dysfunction in one case. CONCLUSIONS: A significant association between pituitary autoimmunity and T1DM was found, in particular in subjects with one or more other endocrine autoimmune diseases.

Aminotransferase and gamma-glutamyltranspeptidase levels in obesity are associated with insulin resistance and the metabolic syndrome.

Fatty liver at ultrasounds, with/ without raised plasma levels of hepatic enzymes, is common in obesity. In most cases, it is the hallmark of non-alcoholic fatty liver disease (NAFLD), a potentially progressive disease associated with insulin resistance and the metabolic syndrome (MS). We tested the hypothesis that insulin resistance per se might be associated with hepatocellular necrosis. Alanine and aspartate aminotransferases (ALT and AST; no.=799) and gamma-glutamyltranspeptidase (GGT; no.=459) were analyzed in a group of treatment-seeking obese patients recruited in 12 Italian medical centers. Insulin resistance was calculated by the homeostasis model assessment method (HOMA-IR; no.=522). Median ALT and AST increased with increasing obesity class (p=0.001 and p=0.005) and exceeded normal limits in 21.0% of cases. Also HOMA-IR increased with the obesity class (p<0.0001), and was higher in subjects with elevated ALT (median, 4.93 vs 2.89; p<0.0001). A significant correlation was observed between HOMA-IR and ALT (R2=0.208; p<0.0001), as well as between HOMA-IR and AST or GGT (R2=0.112 and R2=0.080; p<0.0001). The correlation was maintained when cases with elevated enzyme levels were omitted from analysis. Diabetes and hypertriglyceridemia were the features of the MS most commonly associated with raised liver enzymes. In logistic regression, after correction for age, gender, BMI and features of the MS, HOMA-IR maintained a highly predictive value for raised ALT, AST and GGT. We conclude that in obesity insulin resistance is a risk factor for raised liver enzyme levels, possibly related to NAFLD.

Serum haptoglobin: a novel marker of adiposity in humans.

Haptoglobin (Hp) is a glycoprotein involved in the acute phase response to inflammation. Our previous findings indicate that Hp mRNA and protein are present in the adipose tissue of rodents and that Hp gene expression is up-regulated in obese models. The aim of the present study was to establish whether Hp could be considered a marker of obesity in humans. In 312 subjects, serum Hp was correlated directly with body mass index (BMI), leptin, C-reactive protein (CRP), and age. In a multivariate stepwise regression analysis, BMI and CRP were independent determinants of serum Hp in females, with BMI having the strongest effect. CRP and age were independent determinants of serum Hp in males, although explaining only a modest percentage of the total variability. Serum Hp was positively associated with body fat, as assessed by dual-energy x-ray absorptiometry, both in female and in male groups. The level of significance improved when serum Hp was analyzed against fat mass adjusted for lean mass. Finally, Northern and Western blot analyses performed in biopsies of sc abdominal fat from 20 obese individuals showed the presence of Hp mRNA and protein in the human adipose tissue. In conclusion, serum Hp constitutes a novel marker of adiposity in humans, and the adipose tissue likely contributes to determine its levels.

Snoring, hypertension and Type 2 diabetes in obesity. Protection by physical activity.

Sleep-related breathing disorders are recognized as major health problems in obesity. They are involved in both hypertension and Type 2 diabetes, through mechanisms possibly related to increased sympathetic tone. We studied the association of habitual snoring with diabetes, hypertension, weight cycling and physical activity in a large Italian database of treatment-seeking obese subjects. Clinical and behavioral data were assessed by standardized questionnaires. Consecutive data of 1890 obese patients were analyzed [average body mass index (BMI), 38.2 kg/m2, median age: 46 yr, 78% females], from 25 obesity Italian centers, with low prevalence of clinical manifestations of cardiovascular disease. Habitual snoring was reported in 56% of the cases, and was associated with day-time sleepiness. The prevalence increased with obesity class and waist circumference, and was positively associated with weight cycling and weight gain since the age of 20, and smoking. Regular physical activity had a protective effect. Snoring was associated with diabetes and hypertension at univariate analysis, but in multivariate analysis an independent effect was only observed for hypertension. After adjustment for age, gender and BMI, physical activity maintained an independent, protective effect on both snoring (odds ratio 0.65, 95% confidence interval 0.49-0.84; p=0.001), diabetes (0.50, 0.30-0.86; p=0.011) and hypertension (0.71, 0.53-0.95; p=0.023). We conclude that in treatment-seeking, obese subjects with low prevalence of cardiovascular disease, snoring independently increases the risk of hypertension, whereas physical activity exerts a protection on both snoring and complications. These data underline the importance of lifestyle interventions to limit the burden of obesity and associated diseases.

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.

Intracellular hyperinsulinism: a metabolic characteristic of obesity with and without Type 2 diabetes: intracellular insulin in obesity and Type 2 diabetes.

There is evidence that intracellular insulin may carry out some insulin mediated actions, including glucose transport. As intracellular insulin has never been quantitatively assessed in human cells, we evaluated its concentrations in monocytes from normal subjects (n = 7) and obese patients without (n = 9) and with Type 2 diabetes mellitus (n = 10). After the incubation of cells with labeled insulin for 60 min at 37 degrees C, intracellular intact insulin concentrations were measured by HPLC and expressed as pmol x 10(-6). Insulin concentrations were higher (ANOVA P < 0.01) within cells from obese (115.4 +/- 26.4 pmol x 10(-6)/2 x 10(5) cells) and obese diabetic patients (93.2 +/- 36.3 pmol x 10(-6)/2 x 10(5) cells) compared with normal cells (28.5 +/- 13.1 pmol x 10(-6)/2 x 10(5) cells). Moreover, after insulin was removed from the incubation medium the decrease of intracellular insulin was significantly lower (P < 0.01) in cells from both obese and obese diabetic patients than in normal subjects. Intracellular undissociated insulin-insulin receptor complexes on average, increased 2-fold (P < 0.01) in cells from insulin resistant patients compared with normal cells. Finally, in downregulated cells from obese and obese diabetic patients, the recycling of the internalized insulin receptor was completely disrupted. In conclusion, monocytes from obese patients with and without Type 2 diabetes mellitus, present increased intracellular insulin concentrations and these conditions are associated with a significant impairment of insulin receptor processing. Increased intracellular insulin concentration in cells from these patients may be necessary in order to overcome insulin resistance.

Inhibition of endosomal acidification in normal cells mimics the derangements of cellular insulin and insulin-receptor metabolism observed in non-insulin-dependent diabetes mellitus.

Dissociation of the insulin-insulin receptor complex plays a crucial role in the processing of both insulin and the insulin receptor, and the acidification of endocytic vesicles may be the mechanism by which internalized insulin is dissociated from its receptor and properly sorted and processed. Internalized insulin-insulin receptor complexes are abnormally processed in cells from patients with non-insulin-dependent diabetes mellitus (NIDDM). Accordingly, to further investigate the mechanisms of the derangements observed in NIDDM cells, we examined the effects of the ionophore monensin, which inhibits endosomal acidification, on the cellular processing of insulin and insulin receptor in monocytes from control subjects (n = 12) and NIDDM patients (n = 14). This study confirms that monocytes from NIDDM patients, compared with cells from normal controls, had reduced binding (P < .01), internalization (P < .01), and degradation (P < .01) of insulin. In addition, the release of intracellular radioactivity was slower (P < .01), and recycling of the insulin receptor was inhibited (P < .01). Moreover, these defects were associated with a significant (P < .01) decrease of dissociation of the internalized insulin-insulin receptor complex. In cells from normal controls, incubation with monensin decreased insulin binding (P < .01), but not insulin internalization. High-performance liquid chromatography (HPLC) analysis of intracellular radioactivity showed that after monensin intracellular intact insulin significantly increased (P < .01), thus suggesting a decrease of intracellular insulin degradation. Moreover, insulin receptor recycling was completely disrupted. All of these derangements were associated with a significant decrease (P < .01) of dissociation of insulin-insulin receptor complexes. On the contrary, in diabetic monocytes, monensin had no significant additional effect on NIDDM-linked alterations. Comparison of the results obtained in cells from NIDDM patients to those found in monensin-treated normal cells demonstrates that NIDDM and monensin gave rise to a superimposable impairment of dissociation of the intracellular insulin-insulin receptor complex, associated with similar abnormal sorting and processing of insulin and its receptor. The only defect present in NIDDM cells but not in monensin-treated cells is the decrease of insulin internalization, which thus seems independent of the action of monensin on the processing of internalized insulin-insulin receptor complex. These results suggest that the impairment of dissociation of the insulin-insulin receptor complex may play a crucial role in the subsequent altered processing of insulin and insulin receptor. Moreover, they raise the question as to a possible similar alteration of the same intracellular mechanism by NIDDM and monensin, and point out that the derangements found in cells from NIDDM patients could be localized within the endosomal apparatus and consist mainly of a defective acidification of its interior.

Effects of tryptophan load on amino acid metabolism in type 1 diabetic patients.

Tolerance to an oral tryptophan load (50 mg/kg body weight) was evaluated in a group of 15 insulin-dependent diabetic patients of both sexes in poor metabolic control. Tryptophan was measured fluorometrically, and the plasma levels of the other physiological amino acids were determined by HPLC. The ratio of the plasma concentration of each large neutral amino acid (LNAA) to the sum of the others was calculated to serve as an index for the competitive transport of these amino acids into the brain. The results show that post-loading plasma tryptophan levels in diabetic patients increased less than in healthy controls, suggesting enhanced liver catabolism of this amino acid (as reported for diabetic animals). Small changes were observed in the post-loading plasma concentrations of other amino acids. Therefore, the increment in the tryptophan/LNAA ratio in controls (basal, 0.12 +/- 0.01; 120 min after the load, 0.89 +/- 0.04; 240 min, 0.51 +/- 0.03) was greatly attenuated in diabetic patients (basal, 0.11 +/- 0.01, NS; 120 min, 0.46 +/- 0.04, p < 0.01; 240 min, 0.31 +/- 0.04, p < 0.01). Post-loading excursions in some other ratios were slightly larger in control than diabetic subjects. These differences, which may occur to a lesser extent after a protein-rich meal, could modify the availability of precursor amino acids to the brain for synthesis of neurotransmitters. Thus, as happens in certain animal species, an impairment of the post-absorptive accumulation of tryptophan and serotonin in the brain may occur in diabetic patients as a result of altered metabolic disposal of tryptophan.

A prevalence study of known diabetes mellitus in Tuscany assessed from pharmaceutical prescriptions and other independent sources.

This study evaluates the prevalence of diabetes mellitus (DM) in Pisa (Tuscany, Italy) using four independent data sources. The main source, represented by computerized prescriptions for anti-diabetic agents collected over a 4-month period, was validated using three secondary sources: (a) the list of diabetic patients who receive material of self-care from the National Health Service; (b) the clinical records of diabetic patients obtained from a random sample of family doctors; (c) the clinical records of diabetic patients attending our outpatient clinic. The main source provided 3806 patients, and 697 patients were added from the secondary sources, thus identifying a total number of 4503. The prevalence of known DM in the "Pisa area" exclusively reckoned by the main source, was 2.01%, and the prevalence corrected by the addition of the various sources resulted in 2.4%. The capture-recapture method showed a completeness of ascertainment of the survey of 90.1%, and thus an estimated prevalence of known diabetes of 2.64%. Of these, 141 patients had insulin-dependent diabetes mellitus (IDDM) corresponding to 3.2% of identified diabetic subjects (prevalence 0.07% inhabitants); 4362 patients had non-insulin-dependent diabetes mellitus (NIDDM), 96.8% of identified diabetic subjects (prevalence 2.36%). Of patients with NIDDM 10.5% was treated by diet, 65% with oral hypoglycaemic agents (OHA), 23% with insulin and 1.5% with insulin plus OHA. This study shows that the method used in this survey is suitable for epidemiological studies because it does not demand the cooperation of the diabetic patients, is addressed to the entire diabetic population without age discrimination and singles out the diabetic population in a very reliable way.

Insulin degradation in vivo: a high-performance liquid chromatographic analysis.

The metabolism of insulin in vivo was investigated using an isocratic reversed-phase high-performance liquid chromatographic (RP-HPLC) method. After intravenous injection of A14-[125I]insulin into normals, eight labelled insulin derivatives were found in plasma (peaks 1-8). Two of them (peaks 1 and 7) showed an elution pattern identical with those of reference [125I]monoiodotyrosine and intact A14-[125I]insulin, respectively. Of the other six peaks, five (2-6) eluted before and one (peak 8) after insulin. This pattern was highly reproducible in terms of capacity factors and peak heights. Radioactivity separated by RP-HPLC was further characterized for its trichloroacetic acid precipitability and immunoprecipitability. Fractions corresponding to peaks 4-6 and 8, which showed an immunoprecipitability higher than 50%, were pooled in order to obtain sufficient radioactivity and were found to be insulin separated by Sephadex G-50 chromatography, containing in its structure, after sulphitolysis, intact A-chain and to be partially rebindable to monocyte insulin receptors. These data demonstrate that in blood, products of insulin metabolism circulate which retain a part of the immunological and biological properties of the hormone. These products are clearly separated from one another and from intact insulin by RP-HPLC, suggesting that the appropriate use of this technique may allow a further and more accurate qualitative and quantitative characterization of in vivo insulin metabolism in physiological and pathological conditions.

[Efficacy of sulfonylurea and sulfonylurea-benfluorex therapy in patients with type 2 diabetes treated with commercial sulfonylurea-biguanide combinations]. / Efficacia della terapia con sulfaniluree e sulfaniluree-benfluorex in pazienti con diabete di tipo II trattati con associazioni sulfaniluree-biguanidi del commercio.

This study tests the possibility to avoid the use of phenformin in 40 type 2 (non insulin dependent) diabetic patients treated with the commercial sulphonylurea-phenformin combinations. In diabetic patients treated with sulphonylureas and phenformin at low dosage (glibenclamide 5 mg and phenformin 50 mg) it was possible to maintain good glycometabolic control using only the sulphonylurea gliclazide (160 mg/die). The diabetic patients on treatment with sulphonylureas and phenformin at higher dosage (glibenclamide 7.5 mg and phenformin 75 mg) may further improve their metabolic control when transferred to a gliclazide-benfluorex combination 160 mg and 300 mg/die, respectively. These results suggest the possibility of withdrawing or replacing phenformin in the therapy of type 2 diabetic patients without modifying their glycometabolic control.

Ibopamine does not affect insulin resistance in patients with type I diabetes.

Diabetes mellitus was assessed by investigating 8 type I diabetic patients, in good metabolic control and with congestive heart failure, who were studied after 3 weeks of placebo and ibopamine (100 mg t.i.d.) treatment. Metabolic control and daily insulin dose did not change in any patient during the study. The insulin-mediated glucose uptake during the clamp studies showed no variation after placebo or ibopamine therapy. Total cholesterol, HDL-cholesterol and triglycerides concentrations remained unchanged. This study clearly suggests that ibopamine administered at a daily dose of 300 mg for 3 weeks presents a metabolic safety in type I diabetic patients.

Metabolic control affects plasma lipid and apolipoprotein levels in women, but not in men, with IDDM.

In order to evaluate if in insulin-dependent diabetes lipid and apolipoprotein levels are differently affected by metabolic control in men and women, we measured the concentrations of fasting plasma glucose, mean plasma glucose, glycosylated hemoglobin, total cholesterol, HDL-cholesterol, LDL-cholesterol, triglycerides, and apolipoproteins A and B in 94 sex matched patients. Diabetic men and women were strictly comparable as far as age, relative body weight and metabolic control were concerned. In women, total and LDL cholesterol, triglycerides and apolipoprotein A correlated positively with HbA1 but not with fasting and mean plasma glucose. In men, no correlation between metabolic control and lipid and apolipoprotein levels was found. We conclude that, in diabetic women, the degree of metabolic control may affect the concentrations of plasma lipids, thus explaining, at least in part, the increased risk for coronary atherosclerosis in these patients.

Insulin degradation into monocytes from normal subjects: a high performance liquid chromatographic analysis.

In this work the fate of A14-125 I-insulin inside human cells has been investigated by the complementary use of gel permeation and reversed-phase high performance liquid chromatography to obtain a better resolution of the cell processed radioactive material resulting from the internalization of labeled insulin. Mononuclear leukocytes from 12 normals were incubated with pure A14-125 I insulin at 37 C and internalized radioactivity was characterized after 2, 15 and 60 min. Nearly 14% of intracellular radioactivity was associated to materials with a molecular weight of approximately 300,000. The remaining 86% had a molecular weight lower than 20,000. High molecular weight material showed an elution profile very similar to that obtained from purified human placental insulin receptor and was partially precipitable with antireceptor antibody. The reversed phase high performance liquid chromatography analysis of the low molecular weight material showed two main peaks corresponding to 125 I and A14-125 I-insulin and three intermediate peaks, a, b, c, accounting for about 8% of the recovered radioactivity. By increasing the incubation time of A14-125 I-insulin with monocytes a decrease of insulin peak (2 min: 38 +/- 18%; 15 min: 25 +/- 11%; 60 min: 6 +/- 4%) and a corresponding increase of iodide peak was observed. Immunoprecipitability with anti-insulin antibody was 0% for iodide and a peaks, 60% for peak b, 78% for peak c and 90% for A14-insulin peak. Our results show that intracellular insulin degradation procedes rapidly and in a time-dependent manner and that this process produces insulin derivatives which partially retain the immunological properties of intact A14-125 I insulin.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma biguanide levels are correlated with metabolic effects in diabetic patients.

Metabolic abnormalities occur in biguanide-treated diabetic patients. We investigated the relationship between plasma metformin and phenformin concentrations and metabolic effects. Drug levels were measured in 37 type II diabetic patients by HPLC. The method was sensitive, specific, and linear over a wide range of drug concentrations. Metformin and phenformin values ranged from 236 to 718 ng/ml and from 28 to 114 ng/ml, respectively. The plasma metformin level was correlated with triglycerides (r = -0.55; P less than 0.05) but not with drug dosage, plasma glucose, HbA1, creatinine, creatinine clearance, lactate, pyruvate, lipid, and clinical parameters. Plasma phenformin concentrations correlated with lactate (r = 0.49; P less than 0.05) and HbA1 (r = 0.50; P less than 0.05) but not with drug dosage, parameters of diabetes control, creatinine, creatinine clearance, pyruvate, and clinical parameters. The clinical usefulness of this HPLC method, the evidence that the increase of lactate is related to the circulating phenformin levels, and the demonstration that the metformin effect on triglyceride metabolism is correlated to plasma drug levels are the positive findings of this work.

Long-term therapy with biosynthetic human insulin: importance of short-acting/intermediate-acting insulin ratio on determining efficacy of treatment.

This report describes the efficacy of biosynthetic human insulin (BHI) in long-term (one year) therapy of type I diabetic patients previously treated with conventional insulins. The results were compared with those obtained in a group of diabetic patients kept on their usual treatment. In the latter, fasting plasma glucose, HbA1, insulin dose and relative proportions of insulin formulations remained constant throughout the study. In patients switched to BHI, hypoglycaemic episodes occurred during the first week of treatment and fasting plasma glucose was higher than basally at the first two visits (7th and 30th days). Both hypoglycaemia and high fasting plasma glucose were avoided by reducing the amount of short-acting insulin and increasing that of intermediate-acting insulin, so that the short-acting/intermediate-acting insulin ratio was significantly lower during BHI therapy, although the total daily insulin dose remained unchanged. HbA1 levels remained fairly constant throughout the study. It was concluded that in order to achieve full clinical efficacy of BHI, it is important to modify the proportions of short- and intermediate-acting insulin preparations accurately when switching patients from conventional insulin to biosynthetic human insulin.