Luca Benzi

Identification of cathepsin K as a novel marker of adiposity in white adipose tissue.

In obesity, adipocytes undergo dramatic morphological and molecular changes associated with alterations in their gene expression profile. To identify genes differentially modulated in white adipose tissue (WAT) of obese db/db mice compared to wild type (wt) mice, we utilized RNA fingerprinting. Among the 52 candidates that we identified, we focused here on cathepsin K (ctsk), a cysteine protease, prevalently localized in lysosomes and involved in bone extracellular matrix degradation. In db/db mice, WAT ctsk mRNA was elevated 5.9‐fold, as were Mitf and TFE3 (2‐ and 3.3‐fold respectively), two transcription factors involved in ctsk induction in osteoclasts. Moreover, the level of WAT ctsk mRNA was increased in other obese models including Ay, fat, and tubby (2.8‐, 3.2‐, and 4.9‐fold respectively) and decreased in mice undergoing weight loss. Despite the ubiquitous distribution of the ctsk transcript, we demonstrated that the obesity related increase is specific to the adipocytes. Further, in vitro experiments proved that the abundance of ctsk transcript increases upon adipose conversion of the established cell line of preadipocytes 3T3‐F442A. In addition, ctsk gene expression was examined in adipose tissue of 21 lean and obese male subjects and significant correlations with BMI (r = 0.54, P = 0.012) and plasma leptin levels (r = 0.54, P = 0.015) were found. In conclusion, the WAT of obese db/db mice exhibits a different expression profile from that of the wt mice, and cathepsin K can be considered a novel marker of obesity and a target for the inhibition of adipose mass growth.

C-reactive protein and metabolic syndrome in women with previous gestational diabetes.

This study evaluates the presence of metabolic syndrome (MS) and its association with C‐reactive protein (CRP) and other cardiovascular (CV) risk factors, in a sample of women with and without previous Gestational Diabetes (pGDM).

Obesity modulates the expression of haptoglobin in the white adipose tissue via TNFα

Increase in adipose mass results in obesity and modulation of several factors in white adipose tissue (WAT). Two important examples are tumor necrosis factor α (TNFα) and leptin, both of which are upregulated in adipose tissue in obesity. In order to isolate genes differentially expressed in the WAT of genetically obese db/db mice compared to their lean littermates, we performed RNA fingerprinting and identified haptoglobin (Hp), which is significantly upregulated in the obese animals. Hp is a glycoprotein induced by a number of cytokines, LPS (Lipopolysaccharide), and more generally by inflammation. A significant upregulation of WAT Hp expression was also evident in several experimental obese models including the yellow agouti (/) Ay, ob/ob and goldthioglucose‐treated mice (10‐, 8‐, and 7‐fold, respectively). To identify the potential signals for an increase in Hp expression in obesity, we examined leptin and TNFα in vivo. Wild type animals treated with recombinant leptin did not show any alteration in WAT Hp expression compared to controls that were food restricted to the level of intake of the treated animals. On the other hand, Hp expression was induced in mice transgenically expressing TNFα in adipose tissue. Finally, a significant downregulation of WAT Hp mRNA was observed in ob/ob mice deficient in TNFα function, when compared to the ob/ob controls. These results demonstrate that haptoglobin expression in WAT is increased in obesity in rodents and TNFα is an important signal for this regulation. J. Cell. Physiol. 190: 251–258, 2002.

Agonist‐Induced Internalization and Recycling of the Human A3 Adenosine Receptors

Abstract: A3 adenosine receptors have been proposed to play an important role in the pathophysiology of cerebral ischemia with a regimen‐dependent nature of the therapeutic effects probably related to receptor desensitization and down‐regulation. Here we studied the agonist‐induced internalization of human A3 adenosine receptors in transfected Chinese hamster ovary cells, and then we evaluated the relationship between internalization and signal desensitization and resensitization. Binding of N6‐(4‐amino‐3‐[125I]iodobenzyl)adenosine‐5′‐N‐methyluronamide to membranes from Chinese hamster ovary cells stably transfected with the human A3 adenosine receptor showed a profile typical of these receptors in other cell lines (KD = 1.3 ± 0.08 nM; Bmax = 400 ± 28 fmol/mg of proteins). The iodinated agonist, bound at 4°C to whole transfected cells, was internalized by increasing the temperature to 37°C with a rate constant of 0.04 ± 0.034 min‐1. Agonist‐induced internalization of A3 adenosine receptors was directly demonstrated by immunogold electron microscopy, which revealed the localization of these receptors in plasma membranes and intracellular vesicles. Moreover, short‐term exposure of these cells to the agonist caused rapid desensitization as tested in adenylyl cyclase assays. Subsequent removal of the agonist led to restoration of the receptor function and recycling of the receptors to the cell surface. The rate constant of receptor recycling was 0.02 ± 0.0017 min‐1. Blockade of internalization and recycling demonstrated that internalization did not affect signal desensitization, whereas recycling of internalized receptors was implicated in the signal resensitization.

Defects in Insulin-Receptor Internalization and Processing in Monocytes of Obese Subjects and Obese NIDDM Patients

We investigated intracellular processing of the insulin-receptor complex in monocytes from 12 healthy control subjects, 11 obese nondiabetic subjects, and 13 obese patients with non-insulin-dependent diabetes mellitus (NIDDM) by measuring receptor internalization, recovery of cell-surface insulin binding after receptor internalization, and the release of intracellular intact insulin (insulin retroendocytosis). When monocytes from the three groups of subjects were exposed to 100 nM unlabeled insulin for 30 min at 37°C, the subsequent cell-surface 125I-labeled insulin binding was reduced, but the total number of insulin receptors, measured by radioimmunoassay, was not changed. These findings indicate a redistribution of insulin receptors from the surface to the cell interior. Insulin-receptor internalization was significantly lower in monocytes of obese NIDDM patients (mean ± SE 17.8 ± 4.7%) than in obese subjects and healthy control subjects (33.5 ± 4.5%, P < .05, and 34.4 ± 3.7%, P < .02, respectively). Moreover, in downregulated cells, a complete recovery of the initial insulin binding was observed in control subjects but not in obese NIDDM patients or obese nondiabetic subjects. The release of internalized insulin was also reduced in obese NIDDM patients and obese subjects (f1/2 = 49.0 ± 2.4 min, P < .02; 47.4 ± 5.7 min, P < .05; and 32.9 ± 3.8 in NIDDM patients, obese subjects, and control subjects, respectively). In the radioactivity released from monocytes of obese subjects and obese NIDDM patients, the percentage of intact insulin was higher (P < .05) than in control subjects, suggesting reduced intracellular insulin degradation in obese subjects and obese NIDDM patients. This study indicates that insulin-resistant obese subjects and obese NIDDM patients have multiple postbinding defects of the insulin-receptor intracellular processing. Among these defects, decreased insulin-receptor internalization is specifically associated with diabetic patients.

Maternal Metabolic Control and Perinatal Outcome in Women With Gestational Diabetes Mellitus Treated With Lispro or Aspart Insulin Comparison with regular insulin

Gestational diabetes mellitus (GDM) is associated with increased risk of maternal and neonatal morbidity with macrosomia being the most common neonatal complication (1). The risk of macrosomia and/or disproportionate fetal growth is closely related to 1-h postprandial glucose concentration (2). Therefore, the treatment of GDM should be aimed at normalizing maternal glycemia including the early postprandial response. Insulin therapy is needed whenever strict normoglycemia cannot be achieved by medical nutritional therapy alone (3). Because of their pharmacokinetic properties, short-acting insulin analogs (Insulin Aspart [ASP] and Insulin Lispro [LIS]) could be more effective in pregnancy than human regular insulin (HI) (4). Nevertheless, data …

Improvement with metformin in insulin internalization and processing in monocytes from NIDDM patients.

This study investigated the relative effect of obesity alone and in combination with non-insulin-dependent diabetes mellitus (NIDDM) on the intracellular processing of insulin and evaluated the effect of metformin therapy on this process. Monocytes from 11 obese hyperinsulinemic subjects, 13 obese hyperinsulinemic NIDDM patients, and 7 nondiabetic control subjects were incubated with A14-125I-labeled insulin for 60 min at 37 degrees C, and intracellular insulin degradation was characterized by high-performance liquid chromatography. Total cell-associated insulin (insulin binding) and internalized and degraded insulin were decreased in obese subjects and significantly decreased in obese NIDDM patients compared with nondiabetic control subjects. In NIDDM patients, intracellular insulin degradation was inversely correlated with fasting plasma glucose (P less than 0.01). Eight obese subjects and 9 obese NIDDM patients were restudied after 4 wk of therapy with metformin (850 mg twice a day). Plasma levels of the drug were superimposable in the two groups. Metformin therapy did not change glucose and insulin levels in obese subjects but caused a decrease in blood glucose in obese NIDDM patients. Total cell-associated radioactivity (insulin binding) significantly increased in both groups (P less than 0.01). On the contrary, internalized radioactivity increased (0.83 +/- 0.3 vs. 1.31 +/- 0.35%, P less than 0.01), and similarly, insulin degradation was enhanced (54.6 +/- 8.9 vs. 74.22 +/- 9.15%, P less than 0.01) only in monocytes from obese NIDDM patients. However, the levels of these parameters were still lower than in control subjects (internalization, 2.94 +/- 0.68%; degradation, 93.03 +/- 3.7%).(ABSTRACT TRUNCATED AT 250 WORDS)

Early Subclinical Atherosclerosis in Women With Previous Gestational Diabetes Mellitus

To determine if women with previous gestational diabetes mellitus (pGDM), a population at high risk for type 2 diabetes and metabolic syndrome (1), have signs of subclinical atherosclerosis, we measured carotid intimal-medial thickness (IMT) and multiple cardiovascular risk factors in 28 women with and 24 without pGDM (control group) 2 years after delivery. A 75-g 2-h oral glucose tolerance test was performed for assessment of glucose tolerance, area under the glucose curve (AUCgluc), insulin sensitivity index, homeostasis model assessment of insulin resistance (HOMA-IR), lipid profile, oxidized LDL (oxLDL), C-reactive protein (CRP), adiponectin, and fibrinogen. Family history, anthropometric parameters, and blood pressure were recorded. IMT was measured at four segments of …

Salivary insulin concentrations in type 2 (non-insulin-dependent) diabetic patients and obese non-diabetic subjects: relationship to changes in plasma insulin levels after an oral glucose load

SummaryThe presence of immunoreactive insulin in saliva and its relationship to plasma immunoreactive insulin was investigated in healthy subjects, newly diagnosed non-obese Type 2 (non-insulin-dependent) diabetic patients and obese non-diabetic subjects, basally and after an oral glucose tolerance test. The mean ± SEM fasting values of plasma and salivary immunoreactive insulin were significantly higher in diabetic patients and obese non-diabetic subjects than in normal volunteers (p<0.05). During the glucose challenge, the increase of salivary insulin was related with that of plasma in the three groups of subjects, with a time lag in normal and obese subjects. In normal volunteers, plasma and salivary peak values were respectively 49.5 ± 13.4 μU/ml (p<0.05 vs obese subjects) at 60 min and 12.0±3.3μU/min (p<0.05 vs obese subjects) at 120 min; in diabetic patients, the values were 51.7 ± 5.6 μU/ml (p<0.05 vs obese subjects) and 14.6±4.1 μU/min at 120 min; in obese subjects, the peak value for plasma insulin was 111.5±40.1 μU/ml at 90 min and for salivary insulin 15.6 ± 5.1 μU/min at 120 min. A positive linear relationship was shown between plasma and salivary insulin during the oral glucose tolerance test. The identity of salivary insulin was assessed by reversed-phase HPLC. We conclude that salivary immunoreactive insulin can be found in Type 2 diabetic patients and in obese non-diabetic subjects, as well as normal volunteers, that plasma and salivary insulin are related after a glucose load, and that differences exist in salivary insulin secretion patterns among the three groups of subjects.

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 < 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 < 0.05) and HbA1 (r = 0.50; P < 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.