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Objective: To examine the association of hemoglobin (Hb) A1c variability with microvascular complications in the large cohort of subjects with type 2 diabetes from the Renal Insufficiency And Cardiovascular Events (RIACE) Italian Multicenter Study.Research Design and Methods: Serial (3-5) HbA1c values collected in a 2-year period before enrollment were available from 8,260 subjects from 9 centers (of 15,773 patients from 19 centers). HbA1c variability was measured as the intraindividual SD of 4.52 ± 0.76 values. Diabetic retinopathy (DR) was assessed by dilated funduscopy. Chronic kidney disease (CKD) was defined based on albuminuria, as measured by immunonephelometry or immunoturbidimetry, and estimated glomerular filtration rate (eGFR) was calculated from serum creatinine.Results: Median and interquartile range of average HbA1c (HbA1c-MEAN) and HbA1c-SD were 7.57% (6.86-8.38) and 0.46% (0.29-0.74), respectively. The highest prevalence of microalbuminuria, macroalbuminuria, reduced eGFR, albuminuric CKD phenotypes, and advanced DR was observed when both HbA1c parameters were above the median and the lowest when both were below the median. Logistic regression analyses showed that HbA1c-SD adds to HbA1c-MEAN as an independent correlate of microalbuminuria and stages 1-2 CKD and is an independent predictor of macroalbuminuria, reduced eGFR, and stages 3-5 albuminuric CKD, whereas HbA1c-MEAN is not. The opposite was found for DR, whereas neither HbA1c-MEAN nor HbA1c-SD affected nonalbuminuric CKD.Conclusions: In patients with type 2 diabetes, HbA1c variability affects (albuminuric) CKD more than average HbA1c, whereas only the latter parameter affects DR, thus suggesting a variable effect of these measures on microvascular complications. [ABSTRACT FROM AUTHOR]

Insulin regulates the development and metabolism of adipose tissue (AT) by activating its tyrosine kinase receptor in this tissue. The insulin receptor (IR) consists of two isoforms, a short isoform (IR-A) with prevalent mitogenic property and a long isoform (IR-B) with prevalent metabolic activity. We investigated the expression of IR-A and IR-B in human AT and adipocyte precursors. Biopsies of abdominal subcutaneous AT (S-AT) and visceral AT (V-AT) were obtained from subjects with different BMI. Adipose-derived stem cells (ASC) were also isolated from both AT depots, and were analyzed before and after differentiation into adipocytes (dASC). mRNA levels of IR-A were 2-fold and 3-fold higher compared to IR-B in S-AT and V-AT, respectively. Total IR and IR-A mRNA levels were also 2-fold higher in S-ASC compared to V-ASC. Accordingly, S-ASC exhibited a greater insulin-induced Akt phosphorylation (4-fold) as compared to V-ASC. IR-A expression displayed a positive correlation with BMI in S-ASC (r = 0.50; p = 0.04) but not in V-ASC, in which an inverse correlation was noted (r = -0.66; p = 0.01). Adipocyte differentiation in the presence of human insulin was associated with increased mRNA expression of IR-A in both S-dASC (3-fold) and V-dASC (2.8-fold), and of total IR (5-fold) and IR-B (2-fold) only in S-dASC. Moreover, S-dASC showed higher IR-A mRNA expression (2.5-fold) when compared to V-dASC, similarly to S-ASC and S-AT. In V-dASC, but not in S-dASC, the extent of lipogenesis assessed by oil-Red-O staining was positively correlated with basal expression of total IR (r = 0.94; p = 0.04), while adipogenesis measured by Nile red fluorescent staining showed a correlation with the pAkt/pErk ratio (r = 0.95; p < 0.01). In conclusion, expression of total IR, IR-A and IR-B differs in relation to the stage of tissue development (ASC, dASC, AT) and in a depot-related manner (S vs. V). IR-A appears to be the prevalent IR isoform in human AT and adipocyte precursors and to be particularly involved in the expansion of S-AT in human obesity. Disclosure: V. Genchi: None. A. Cignarelli: Consultant; Self; Eli Lilly and Company. Speaker's Bureau; Self; Merck Sharp & Dohme Corp., Novo Nordisk A/S. S. Perrini: None. S. Porro: None. C. Caccioppoli: None. A. Natalicchio: Other Relationship; Self; Novo Nordisk Inc., Sanofi-Aventis. L. Laviola: Advisory Panel; Self; Abbott, Boehringer Ingelheim Pharmaceuticals, Inc., Lilly Diabetes, Novo Nordisk Inc. Board Member; Self; AstraZeneca, Roche Diabetes Care, Sanofi-Aventis. Speaker's Bureau; Self; Medtronic, Mundipharma, Takeda Pharmaceutical Company Limited. F. Giorgino: Advisory Panel; Self; Aegerion Pharmaceuticals, AstraZeneca, Boehringer Ingelheim International GmbH, Eli Lilly and Company, MedImmune, Merck Sharp & Dohme Corp., Novo Nordisk A/S, Roche Diabetes Care, Sanofi. Consultant; Self; Roche Diabetes Care, Sanofi. Research Support; Self; Eli Lilly and Company, LifeScan, Inc., Takeda Pharmaceutical Company Limited. Other Relationship; Self; AstraZeneca, Eli Lilly and Company, Sanofi. [ABSTRACT FROM AUTHOR]

Insulin, acting in an autocrine manner, promotes β-cell survival and growth and its own biosynthesis and secretion. Prolonged exposure of β-cells to high levels of saturated fatty acids (SFAs) impairs insulin signaling, reducing the ability of insulin to promote its release. The p66Shc protein is an inducer of cellular oxidative stress and apoptosis, and its mRNA levels are increased in pancreatic islets from overweight/obese subjects. Here we evaluated the role of p66Shc in pancreatic β-cell insulin resistance (IR) occurring in obesity and lipotoxic conditions. Insulin effects on its own content and C-peptide secretion were studied in pancreatic islets from overweight/obese (BMI ≥25 kg/m2) compared to lean (BMI <25 kg/m2) human donors, and in INS-1E cells, murine pancreatic islets and human pancreatic islets exposed to palmitate. To evaluate the role of p66Shc in lipotoxicity-induced β-cell IR, p66Shc levels were silenced or overexpressed. Pancreatic islets from overweight/obese subjects showed a reduced ability of insulin to increase its own biosynthesis and C-peptide secretion. Similarly, prolonged exposure to palmitate augmented p66Shc levels and induced IR in INS-1E cells, human islets and murine islets. When p66Shc protein levels were reduced, insulin-induced C-peptide secretion was enhanced, and the palmitate-induced reductions of C-peptide levels and insulin content were both reversed. By contrast, p66Shc overexpression resulted in inhibition of insulin-induced C-peptide secretion and increase of insulin content, both in absence and presence of palmitate. These effects of p66Shc were found to require p70S6K (Thr389) and IRS-1 (Ser307) phosphorylation, which inhibited insulin-stimulated Akt phosphorylation. In conclusion, the protein p66Shc exerts an inhibitory effect on β-cell insulin signaling and can mediate the ability of SFAs and excess body fat to cause β-cell IR, which results in both reduced survival and impaired secretory function. Disclosure: L. Dipaola: None. A. Natalicchio: Other Relationship; Self; Novo Nordisk Inc., Sanofi-Aventis. G. Biondi: None. N. Marrano: None. M. Bugliani: None. A. Cignarelli: Consultant; Self; Eli Lilly and Company. Speaker's Bureau; Self; Merck Sharp & Dohme Corp., Novo Nordisk A/S. S. Perrini: None. P. Marchetti: None. L. Laviola: Advisory Panel; Self; Abbott, Boehringer Ingelheim Pharmaceuticals, Inc., Lilly Diabetes, Novo Nordisk Inc. Board Member; Self; AstraZeneca, Roche Diabetes Care, Sanofi-Aventis. Speaker's Bureau; Self; Medtronic, Mundipharma, Takeda Pharmaceutical Company Limited. F. Giorgino: Advisory Panel; Self; Aegerion Pharmaceuticals, AstraZeneca, Boehringer Ingelheim International GmbH, Eli Lilly and Company, MedImmune, Merck Sharp & Dohme Corp., Novo Nordisk A/S, Roche Diabetes Care, Sanofi. Consultant; Self; Roche Diabetes Care, Sanofi. Research Support; Self; Eli Lilly and Company, LifeScan, Inc., Takeda Pharmaceutical Company Limited. Other Relationship; Self; AstraZeneca, Eli Lilly and Company, Sanofi. [ABSTRACT FROM AUTHOR]

Visceral (V) and subcutaneous (SC) fat depots possess different metabolic properties that can be demonstrated both in whole-tissue specimens and isolated adipocytes. In this study, we investigated whether human adipocytes obtained from V or SC fat depots retain distinct properties also when they are differentiated in vitro from precursor stromal cells. Paired V and SC fat precursor cells from 10 non-diabetic subjects (4F/6M, age 45 ± 1 yrs, BMI 23.1 ± 1.4) were differentiated in vitro with dexamethasone, IBMX, insulin and rosiglitazone. Upon differentiation, mRNA levels of GLUT4, PPARgamma, C/EBPalpha, aP2, and adiponectin, measured by real-time RT-PCR, were markedly up-regulated (p<0.05), whereas GLUT1 expression did not change. However, mRNA expression levels of C/EBPalpha, adiponectin and aP2 were higher in V than in SC adipocytes (p<0.05). Adiponectin Secretion in the culture medium was also ∼ 3-fold higher in V than in SC adipocytes (p<0.05). Intracellular insulin signaling was next evaluated by analyzing the phosphorylation levels of various proteins after stimulation with 10 nM insulin for 5 to 120 min. Insulin-induced phosphorylation of the insulin receptor, IRS-1/2, Akt and Erk-1/2 appeared to be more rapid and tended to decrease at earlier time points in V as compared to SC adipocytes, in which more persistent protein phosphorylation in response to insulin was observed. When compared to SC adipocytes, V adipocytes also showed approx. 80% higher rates of insulin-induced 2-deoxy-D-glucose uptake. In conclusion, SC and V adipocytes, even when differentiated in vitro from precursor stromal cells, retain differences in gene expression, adiponectin secretion, and insulin action and signaling. This suggests that the precursor cells resident in V and SC fat depots possess inherent and specific metabolic characteristics that are expressed upon completion of the differentiation programme. [ABSTRACT FROM AUTHOR]

TNFα represents an important mediator of beta-cell dysfunction and death in diabetes. To evaluate the mechanisms of TNFα induced beta-cell damage, INS-1 beta-cells were stimulated with 20 ng/ml TNFα for several hours. Treatment with TNFα increased the phosphorylation levels of the serine-kinase INK 2-fold (p<0.05) and reduced Iκ-Bα protein content 50% (p<0.05). Furthermore, TNFα stimulation resulted in enhanced serine phosphorylation of IRS proteins (p<0.05), but did not change the total levels of IRS-1 or IRS-2. Sequential immunoprecipitation with anti-IRS-1 or anti-IRS-2 antibodies, followed by immunoblotting with phosphoserine antibodies, demonstrated that IRS-2 was the most important substrate affected by TNFα, whereas serine phosphorylation of IRS-1 could be barely detected. Moreover, TNFα reduced basal AM phosphorylation 30% and increased beta-cell apoptosis 7-fold (p<0.05). Since the incretin hormone GLP-1 has been shown to antagonize beta-cell damage induced by various cytokines, we next investigated potential protective effects of GLP-1 receptor agonists on TNFα-induced beta-cell damage. Pre-incubation of INS-1 cells with GLP-1 (10 nM) or Exenatide (Ex, 10 nM) for 18 h prevented TNFα-induced JNK phosphorylation and IRS-2 serine phosphorylation, and restored AM phosphorylation, without affecting p38 phosphorylation or protein content. In addition, Ex treatment resulted in a 3-fold increase in IRS-2 protein expression. Finally, both GLP-1 and Ex markedly inhibited TNFα-induced apoptosis by 50% (p<0.05). In conclusion, TNFα induces apoptosis in INS-1 beta-cells, and this likely occurs by induction of JNK phosphorylation and impairment of IRS-2 and AM signalling. GLP-1 and Ex prevent the signaling abnormalities and cell apoptosis induced by TNFα. Thus, GLP-1 and Exenatide may promote beta-cell survival by directly antagonizing cytokine-dependent signaling reactions mediating beta-cell death. [ABSTRACT FROM AUTHOR]

SUMO-ylation has emerged as a key modification step in regulating adipocyte transcription factors such as C/EBPα/β and PPARγ. The objective of this study was to explore the role of Ubc9, the only enzyme able to conjugate SUMO to other substrates, on adipocyte differentiation, using a siRNA-based strategy. Ubc9-specific siRNA reduced Ubc9 levels in 3T3-L1 preadipocytes of about 60% up to 3-4 days after exposure to the differentiation-inducing mixture. PPARTγ and C/EBPβ expression was markedly increased in control cells 2 to 6 days after the induction of differentiation. By contrast, the expression of these factors was delayed and significantly lower (p<0.05 at day 2 vs control) in Ubc9 siRNA-treated cells. Oil Red O staining demonstrated cell triglycerides as minute lipid droplets in only 30% of Ubc9-siRNA treated cells, as compared to larger droplets evident in 100% of control cells. To confirm the importance of Ubc9 in regulating adipogenesis, we next investigated the time-course and subcellular localization of this enzyme during the differentiation process. Ubc9 mRNA and protein levels peaked at day 2 (2.5 fold, p<0.05) and then gradually declined to basal levels at day 8. Ubc9 protein, revealed by immunoblotting of cellular fractions and immunofluorescence by confocal microscopy, was confined mostly in the nucleus of preadipocytes, whereas was localized also in specific cytoplasmic compartments, beginning on day 4 after differentiation induction, partially co-localizing with GLUT4. In conclusion, these results indicate that Ubc9 expression and localization is regulated during the differentiation process, and suggest that Ubc9 participates in adipogenesis at various stages of the differentiation program. [ABSTRACT FROM AUTHOR]