Carlo Maria Rotella

Regulation of extracellular matrix synthesis by transforming growth factor β1 in human fat-storing cells

BACKGROUND Fat storing cells (FSC) are nonparenchymal liver cells generally considered the major source of the hepatic extracellular matrix (ECM). Transforming growth factor beta 1 (TGF-beta 1) is a potent regulator of ECM synthesis in various cell types. In this study, the effect of TGF-beta 1 on procollagen types I, III, IV, laminin (Lam), and fibronectin (FN) synthesis in cultured human FSCs was analyzed. METHODS FSCs were isolated from wedge sections of normal human livers. Morphological studies were performed by immunofluorescence and electron microscopy. ECM components in human FSC cultures were measured by an enzyme-linked immunosorbent assay. The expression of messenger RNA (mRNA) was evaluated by Northern blot and in situ hybridization. RESULTS Cultured human FSCs displayed numerous fat droplets in the perinuclear zone, and immunoreactivity for vimentin and alpha-smooth muscle actin. A weak nonfibrillar staining was observed by using a polyclonal antidesmin antibody. TGF-beta 1 induced a dose-dependent increase of procollagen I, III, and FN accumulation in human FSC cultures, whereas procollagen IV and Lam production was not affected. Furthermore, TGF-beta 1 increased the expression of alpha 1 (I), alpha 1 (III) procollagen, FN and TGF-beta 1 mRNA in human FSC cultures. CONCLUSIONS These data indicate that TGF-beta 1 is able to increase the synthesis of procollagen I, III, and FN in cultured human FSCs. Moreover, TGF-beta 1 can induce its own mRNA in the same cells.

Quality of life and overweight: the obesity related well-being (Orwell 97) questionnaire.

The development and validation of a self-reported measure of obesity-related quality of life, the Obesity Related Well-Being (ORWELL 97), were undertaken to examine the intensity and the subjective relevance of physical and psychosocial distress. The questionnaire was validated in a sample of 147 obese patients (99 females, 48 males). The Eating Disorder Examination 12.0D interview, a structured diagnostic interview for DSM-III-R (DSM-IV criteria for binge eating disorder), Beck Depression Inventory, Binge Eating Scale, and the State-Trait Anxiety Inventory 1 and 2 scales were also applied. Internal consistency and test-retest reliability were satisfactory. Factor analysis allowed the identification of two subscales: ORWELL 97-1 related to psychological status and social adjustment, and ORWELL 97-2 related to physical symptoms impairment. Obese female patients showed a lower quality of life, and the severity of obesity appeared to interfere with physical functioning rather than psychological status and social adjustment. The ORWELL 97 questionnaire appears to be a simple and reliable measure of obesity-related quality of life, which can be used in current clinical practice.

Upregulation of Mesangial Growth Factor and Extracellular Matrix Synthesis by Advanced Glycation End Products Via a Receptor-Mediated Mechanism

Enhanced advanced glycosylation end product (AGE) formation has been shown to participate in the patho-genesis of diabetes-induced glomerular injury by mediating the increased extracellular matrix (ECM) deposition and altered cell growth and turnover leading to mesangial expansion. These effects could be exerted via an AGE-receptor-mediated upregulation of growth factors, such as the IGFs and transforming growth factor-β (TGF-β). We tested this hypothesis in human and rat mesangial cells grown on nonglycated or native bovine serum albumin (BSA), glycated BSA with AGE formation (BSA-AGE), or glycated BSA in which AGE formation was prevented by the use of aminoguanidine (BSA-AM), in the presence or absence of an antibody, α-p60, directed against the p60/OST protein named AGE-receptor 1 (AGE-R1), or normal control (pre-immune) serum. The mRNA and/or protein levels of IGF-I, IGF-II, IGF receptors, IGF binding proteins (IGFBPs), TGF-β1 and the ECM components fibronectin, laminin, and collagen IV were measured, together with cell proliferation. Both human and rat mesangial cells grown on BSA-AGE showed increased IGF-I and total and bioac-tive TGF-β medium levels and enhanced IGF-I, IGF-II, and TGF-β1 gene expression, compared with cells grown on BSA, whereas total IGFBP and IGFBP-3 medium content, IGF receptor density and affinity, and IGF-I receptor transcripts were unchanged. Moreover, cells grown on BSA-AGE showed increased ECM protein and mRNA levels versus cells cultured on BSA, whereas cell proliferation was unchanged in human mesangial cells and slightly reduced in rat mesangial cells. Growing cells on BSA-AM did not affect any of the measured parameters. Co-incubation of BSA-AGE with anti-AGE-R1, but not with pre-immune serum, prevented AGE-induced increases in IGF-I, TGF-β1, and ECM production or gene expression; anti-AGE-R1 also reduced growth factor and matrix synthesis in cells grown on BSA. These results demonstrate that mesangial IGF and TGF-β1 synthesis is upregulated by AGE-modified proteins through an AGE-receptor-mediated mechanism. The parallelism with increased ECM production raises the speculation that the enhanced synthesis of these growth factors resulting from advanced nonen-zymatic glycation participates in the pathogenesis of hyperglycemia-induced mesangial expansion.

Psychopathological and clinical features of outpatients with an eating disorder not otherwise specified

In order to investigate similarities and differences between Eating Disorder Not Otherwise Specified (EDNOS) and Anorexia Nervosa (AN) and Bulimia Nervosa (BN), we studied a consecutive series of 189 female outpatients attending two Eating Disorder Units. The data were collected by means of interviews (Eating Disorder Examination, EDE 12.0D), the Structured Diagnostic Interview for DSM III-R, (SCID), and self-reported questionnaires (Beck Depression Inventory, BDI, and State and Trait Anxiety Inventory, STAI 1–2). The diagnosis of EDNOS was as frequent as that of AN and BN (43.8% versus 43.2%). There were no significant differences between EDNOS and AN/BN patients in terms of their general and specific psychopathological features, but significant differences were observed between bulimic-like and anorectic-like EDNOS patìents, as well as between those with AN and BN. In conclusion, in our clinical setting, the patients with EDNOS and those with typical eating disorders have similar psychopathological features, thus suggesting that EDNOS patients should be further divided into two groups, anorectic-like (similar to AN) and bulimic-like (similar to BN) patients.

Bone Fractures and Hypoglycemic Treatment in Type 2 Diabetic Patients A case-control study

OBJECTIVE—Hypoglycemic treatments could modulate the risk for fractures in many ways. Most studies have not explored the effect on the incidence of bone fractures of individual oral hypoglycemic agents, rather all oral treatments as a whole. The aim of this case-control study, nested within a retrospective cohort, is the assessment of the risk for bone fractures associated with exposure to insulin or different oral hypoglycemic agents. RESEARCH DESIGN AND METHODS—A case-control study nested within a cohort of 1,945 diabetic outpatients with a follow-up of 4.1 ± 2.3 years was performed, comparing 83 case subjects of bone fractures and 249 control subjects matched for age, sex, duration of diabetes, BMI, A1C, comorbidity, smoking, and alcohol abuse. Exposure to hypoglycemic drugs during the 10 years preceding the event (or matching index date) was assessed. RESULTS—In a model including treatment with insulin secretagogues metformin and insulin for at least 36 months during the previous 10 years, no significant association was observed between bone fractures and medications. In an alternative model considering treatments at the time of fracture, insulin treatment was significantly associated with bone fractures in men (OR 3.20 [95% CI 1.32–7.74]) but not in women (1.41 [0.73–2.73]). CONCLUSIONS—Insulin-sensitizing treatment with metformin is not associated with a higher incidence of bone fractures, suggesting that the negative effect of thiazolidinediones is due to a specific action on bone metabolism rather a reduction of insulinemia. Conversely, current treatment with insulin increases the risk of fractures; at the same time, exposure to this agent in the longer term does not appear to affect bone frailty.

Screening for binge eating disorder in obese outpatients

The prevalence of binge eating disorder (BED) in clinical samples of obese patients is controversial, and sensitive diagnostic protocols for use in routine clinical practice need to be further defined. Three hundred forty-four obese (body mass index [BMI] > or =30 kg/m2) patients were studied with the Structured Clinical Interview for DSM-III-R to investigate the lifetime prevalence of mental disorders. The current prevalence of BED was assessed using DSM-IV criteria. Eating attitudes and behavior were investigated with the Bulimic Investigation Test, Edinburgh (BITE) and the Binge Eating Scale (BES). The Beck Depression Inventory (BDI) and Spielbergs State-Trait Anxiety Inventory (STAI) were also applied. The prevalence of BED was 7.5%. Patients with BED had a higher BMI compared with obese patients without BED. Differences in the lifetime prevalence of mental disorders in patients with and without BED were not statistically significant. Using the BES as a screening instrument for BED with a threshold of 17, the sensitivity was 84.8%, specificity 74.6%, positive predictive value 26.2%, and negative predictive value 97.9%. Using the BITE with a threshold of at least 10, the sensitivity was 91%, specificity 51.4%, positive predictive value 71.8%, and negative predictive value 98.2%. The BITE can be a valid alternative to the BES as a screening method for BED in obese patients.

Mechanisms of glucose-enhanced extracellular matrix accumulation in rat glomerular mesangial cells

In view of the importance of mesangial extracellular matrix (ECM) accumulation in the pathogenesis of diabetic glomerulosclerosis, we investigated 1) the effects of high glucose on ECM production by rat glomerular mesangial cells in culture (study A) and 2) the mechanisms underlying these effects, particularly the role of high sugar levels irrespective of intracellular metabolism (study B1) and of excess glucose disposal via the polyol pathway and associated biochemical alterations (study B2). Cells were cultured for 4 weeks, through six to eight passages, under the experimental conditions indicated below and, at each passage, the levels of fibronectin (FN), laminin (LAM), and collagen types I (C-I), III (C-III), IV (C-IV), and VI (C-VI) in media and cell extracts were quantified by an enzyme immunoassay. In study A, medium and cell content of matrix were assessed, together with [3H]leucine and [3H]thymidine incorporation into monolayers, polyol, fructose, and myo-inositol levels and the cytosolic redox state, in cells grown in high (30 mM) D-glucose or iso-osmolar mannitol versus cells cultured in normal (5.5 mM) D-glucose. FN, LAM, C-IV, and C-VI accumulation, but not C-I and C-III accumulation, was increased by 30 mM glucose, but not by iso-osmolar mannitol, when compared with 5.5 mM glucose, starting at week 2 and, except for C-VI, persisting throughout the remaining 2 weeks, whereas no change was observed in the measured indexes of total protein synthesis and DNA synthesis/cell proliferation. At any time point, polyol levels were increased, whereas myo-inositol was reduced by high glucose; in cells grown under elevated glucose concentrations, the lactate/pyruvate (L/P) ratio, an index of the cytosolic redox state, progressively increased. In study B1, the effects of high D-glucose were compared with those of iso-osmolar concentrations of sugars that are partly or not metabolized but are capable of inducing nonenzymatic glycosylation, such as D-galactose and L-glucose, and of mannitol, which does not enter the cell. Both D-galactose and L-glucose, but not mannitol, partly mimicked D-glucose-induced ECM overproduction. Although D-galactose is metabolized via the polyol pathway and alters the cytosolic redox state, ECM changes induced by high galactose were not prevented by the use of an aldose reductase inhibitor (ARI), Alcon 1576 (14 μM). In study B2, agents interfering with intracellular metabolism of excess glucose via the polyol pathway (14 μM Alcon 1576) and associated changes in myo-inositol metabolism (1 mM myo-inositol) and cytosolic redox state (1 mM sodium pyruvate, which corrects glucose-induced polyol pathway-dependent increased NADH/NAD+) were added to cells cultured in 30 and 5.5 mM D-glucose. Alcon 1576 inhibited polyol pathway activity with decreasing efficacy during the 4-week period, whereas myo-inositol and pyruvate produced complete and persistent prevention of reduced myo-inositol levels and increased L/P ratio, respectively. High glucose-induced ECM overproduction was transiently reduced by pyruvate and, to a lesser extent, by the ARI and myo-inositol. These results suggest that 1) high glucose selectively increases accumulation of basement membrane components and 2) multiple mechanisms seem to be operating in the pathogenesis of glucose-induced ECM overproduction, including elevated sugar levels per se, possibly via nonenzymatic glycosylation, and to a lesser extent, intracellular glucose metabolism via the polyol pathway and associated changes in myo-inositol metabolism and cytosolic redox state.

The diabetic cardiomyopathy.

Diabetic cardiomyopathy has been defined as “a distinct entity characterized by the presence of abnormal myocardial performance or structure in the absence of epicardial coronary artery disease, hypertension, and significant valvular disease”. The diagnosis stems from the detection of myocardial abnormalities and the exclusion of other contributory causes of cardiomyopathy. It rests on non-invasive imaging techniques which can demonstrate myocardial dysfunction across the spectra of clinical presentation. The presence of diabetes is associated with an increased risk of developing heart failure, and the 75% of patients with unexplained idiopathic dilated cardiomyopathy were found to be diabetic. Diabetic patients with microvascular complications show the strongest association between diabetes and cardiomyopathy, an association that parallels the duration and severity of hyperglycemia. Metabolic abnormalities (that is hyperglycemia, hyperinsulinemia, and hyperlipemia) can lead to the cellular alterations characterizing diabetic cardiomyopathy (that is myocardial fibrosis and/or myocardial hypertrophy) directly or indirectly (that is by means of renin-angiotensin system activation, cardiac autonomic neuropathy, alterations in calcium homeostasis). Moreover, metabolic abnormalities represent, on a clinical ground, the main therapeutic target in the patients with diabetes since the diagnosis of diabetes is made. Since diabetic cardiomyopathy is highly prevalent in the asymptomatic type 2 diabetic patients, screening for its presence at the earliest stage of development can lead to prevent the progression to chronic heart failure. The most sensitive test is standard echocardiogram, while a less expensive pre-screening method is the detection of microalbuminuria.

BONE FRACTURES AND HYPOGLYCAEMIC TREATMENT IN TYPE 2 DIABETIC PATIENTS: A CASE-CONTROL STUDY.

OBJECTIVE—Hypoglycemic treatments could modulate the risk for fractures in many ways. Most studies have not explored the effect on the incidence of bone fractures of individual oral hypoglycemic agents, rather all oral treatments as a whole. The aim of this case-control study, nested within a retrospective cohort, is the assessment of the risk for bone fractures associated with exposure to insulin or different oral hypoglycemic agents. RESEARCH DESIGN AND METHODS—A case-control study nested within a cohort of 1,945 diabetic outpatients with a follow-up of 4.1 ± 2.3 years was performed, comparing 83 case subjects of bone fractures and 249 control subjects matched for age, sex, duration of diabetes, BMI, A1C, comorbidity, smoking, and alcohol abuse. Exposure to hypoglycemic drugs during the 10 years preceding the event (or matching index date) was assessed. RESULTS—In a model including treatment with insulin secretagogues metformin and insulin for at least 36 months during the previous 10 years, no significant association was observed between bone fractures and medications. In an alternative model considering treatments at the time of fracture, insulin treatment was significantly associated with bone fractures in men (OR 3.20 [95% CI 1.32–7.74]) but not in women (1.41 [0.73–2.73]). CONCLUSIONS—Insulin-sensitizing treatment with metformin is not associated with a higher incidence of bone fractures, suggesting that the negative effect of thiazolidinediones is due to a specific action on bone metabolism rather a reduction of insulinemia. Conversely, current treatment with insulin increases the risk of fractures; at the same time, exposure to this agent in the longer term does not appear to affect bone frailty.

Diagnostic crossover and outcome predictors in eating disorders according to DSM-IV and DSM-V proposed criteria: a 6-year follow-up study.

Objective: To evaluate in a 6-year follow-up study the course of a large clinical sample of patients with eating disorders (EDs) who were treated with individual cognitive behavior therapy. The diagnostic crossover, recovery, and relapses were assessed, applying both Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV) and the DSM-V proposed criteria. Patients with EDs move in and out of illness states over time, display frequent relapses, show a relevant lifetime psychiatric comorbidity, and migrate between different diagnoses. Method: A total of 793 patients (including anorexia nervosa, bulimia nervosa, binge eating disorder, and EDs not otherwise specified) were evaluated on the first day of admission, at the end of treatment, 3 years after the end of treatment, and 3 years after the first follow-up. Clinical data were collected through a face-to-face interview; diagnosis was performed by means of the Structured Clinical Interview for DSM-IV and the Eating Disorder Examination Questionnaire was applied. Results: A consistent rate of relapse and crossover between the different diagnoses over time was observed. Mood disorders comorbidity has been found to be an important determinant of diagnostic instability, whereas the severity of shape concern represented a relevant outcome modifier. Using the DSM-V proposed criteria, most patients of EDs not otherwise specified were reclassified, so that the large majority of ED patients seeking treatment would be included in full-blown diagnoses. Conclusions: Among EDs, there are different subgroups of patients displaying various courses and outcomes. The diagnostic instability involves the large majority of patients. An integration of categorical and dimensional approaches could improve the psychopathological investigation and the treatment choices. AN = anorexia nervosa; BED = binge eating disorder; BMI = body mass index; BN = bulimia nervosa; CBT = cognitive behavior therapy; DSM = Diagnostic and Statistical Manual of Mental Disorders; EDs = eating disorders; EDE 12.0D = Eating Disorder Examination Interview; EDNOS = eating disorders not otherwise specified; EDNOS-A = eating disorders not otherwise specified Anorectic type; EDNOS-B = eating disorders not otherwise specified Bulimic type; OBEs = objective binge episodes; SBEs = subjective binge episodes; s-BED = subthreshold BED.