GALNT2 as a novel modulator of adipogenesis and adipocyte insulin signaling

Background/objectives: A better understanding of adipose tissue biology is crucial to tackle insulin resistance and eventually coronary heart disease and diabetes, leading causes of morbidity and mortality worldwide. GALNT2, a GalNAc-transferase, positively modulates insulin signaling in human liver cells by down-regulating ENPP1, an insulin signaling inhibitor. GALNT2 expression is increased in adipose tissue of obese as compared to that of non-obese individuals. Whether this association is secondary to a GALNT2-insulin sensitizing effect exerted also in adipocytes is unknown. We then investigated in mouse 3T3-L1 adipocytes the GALNT2 effect on adipogenesis, insulin signaling and expression levels of both Enpp1 and 72 adipogenesis-related genes. Methods: Stable over-expressing GALNT2 and GFP preadipocytes (T 0 ) were generated. Adipogenesis was induced with (R+) or without (R−) rosiglitazone and investigated after 15 days (T 15 ). Lipid accumulation (by Oil Red-O staining) and intracellular triglycerides (by fluorimetric assay) were measured. Lipid droplets (LD) measures were analyzed at confocal microscope. Gene expression was assessed by RT-PCR and insulin-induced insulin receptor (IR), IRS1, JNK and AKT phosphorylation by Western blot. Results: Lipid accumulation, triglycerides and LD measures progressively increased from T 0 to T 15 R- and furthermore to T 15 R+. Such increases were significantly higher in GALNT2 than in GFP cells so that, as compared to T 15 R+GFP, T 15 R- GALNT2 cells showed similar (intracellular lipid and triglycerides accumulation) or even higher (LD measures, p < 0.01) values. In GALNT2 preadipocytes, insulin-induced IR, IRS1 and AKT activation was higher than that in GFP cells. GALNT2 effect was totally abolished during adipocyte maturation and completely reversed at late stage maturation. Such GALNT2 effect trajectory was paralleled by coordinated changes in the expression of Enpp1 and adipocyte-maturation key genes. Conclusions: GALNT2 is a novel modulator of adipogenesis and related cellular phenotypes, thus becoming a potential target for tackling the obesity epidemics and its devastating sequelae

Association of a homozygous GCK missense mutation with mild diabetes

Background: Homozygous inactivating GCK mutations have been repeatedly reported to cause severe hyperglycemia, presenting as permanent neonatal diabetes mellitus (PNDM). Conversely, only two cases of GCK homozygous mutations causing mild hyperglycemia have been so far described. We here report a novel GCK mutation (c.1116G>C, p.E372D), in a family with one homozygous member showing mild hyperglycemia. Methods: GCK mutational screening was carried out by Sanger sequencing. Computational analyses to investigate pathogenicity and molecular dynamics (MD) were performed for GCK-E372D and for previously described homozygous mutations associated with mild (n = 2) or severe (n = 1) hyperglycemia, used as references. Results: Of four mildly hyperglycemic family-members, three were heterozygous and one, diagnosed in the adulthood, was homozygous for GCK-E372D. Two nondiabetic family members carried no mutations. Fasting glucose (p = 0.016) and HbA1c (p = 0.035) correlated with the number of mutated alleles (0–2). In-silico predicted pathogenicity was not correlated with the four mutations’ severity. At MD, GCK-E372D conferred protein structure flexibility intermediate between mild and severe GCK mutations. Conclusions: We present the third case of homozygous GCK mutations associated with mild hyperglycemia, rather than PNDM. Our in-silico analyses support previous evidences suggesting that protein stability plays a role in determining clinical severity of GCK mutations

Role of GALNT2 in the modulation of ENPP1 expression, insulin signaling and action: GALNT2: A novel modulator of insulin signaling

Ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) inhibits insulin signaling and action. Understanding the mechanisms underlying ENPP1 expression may help unravel molecular mechanisms of insulin resistance. Recent data suggest a role of ENPP1-3'untraslated region (UTR), in controlling ENPP1 expression. We sought to identify trans-acting ENPP1-3'UTR binding proteins, and investigate their role on insulin signaling. By RNA pull-down, 49 proteins bound to ENPP1-3'UTR RNA were identified by mass spectrometry (MS). Among these, in silico analysis of genome wide association studies and expression profile datasets pointed to N-acetylgalactosaminyltransferase 2 gene (GALNT2) for subsequent investigations. Gene expression levels were evaluated by RT-PCR. Protein expression levels, IRS-1 and Akt phosphorylation were evaluated by Western blot. Insulin receptor (IR) autophosphorylation was evaluated by ELISA. GALNT2 down-regulation increased while GALNT2 over-expression reduced ENPP1 expression levels. In addition, GALNT2 down-regulation reduced insulin stimulation of IR, IRS-1 and Akt phosphorylation and insulin inhibition of phosphoenolpyruvate carboxykinase (PEPCK) expression, a key neoglucogenetic enzyme. Our data point to GALNT2 as a novel factor involved in the modulation of ENPP1 expression as well as insulin signaling and action in human liver HepG2 cell

The +276 G/T Single Nucleotide Polymorphism of the Adiponectin Gene Is Associated With Coronary Artery Disease in Type 2 Diabetic Patients

OBJECTIVE —Two single nucleotide polymorphisms (SNPs) at the adiponectin locus (+45T>G and +276G>T) have been associated with low circulating adiponectin levels, insulin resistance, and type 2 diabetes. We investigated whether these genetic markers are determinants of coronary artery disease (CAD) in type 2 diabetic patients. RESEARCH DESIGN AND METHODS —A total of 376 consecutive type 2 diabetic patients were studied: 142 case subjects with coronary stenosis >50% or previous myocardial infarction and 234 control subjects with no symptoms, no electrocardiogram (ECG) signs of myocardial ischemia, and a normal ECG stress test ( n = 189) and/or ( n = 45) with coronary stenosis ≤50%. RESULTS —No association with CAD was observed for the +45 SNP ( P = 0.48). By contrast, a significant association was observed for the +276 SNP, with T/T homozygotes having a lower risk of CAD than carriers of other genotypes (adjusted odds ratio [OR] 0.13 [95% CI 0.037–0.46], P = 0.002). A similarly protective effect of the +276 T/T genotype was observed in 110 case and 45 control subjects for whom the CAD status had been determined by angiography (0.04 [0.006–0.30], P = 0.002).  Serum adiponectin, although clearly related to several features of the proatherogenic/insulin-resistant phenotype, was not different between control subjects and CAD patients (26 ± 17 vs. 25 ± 13 μg/ml). CONCLUSIONS —In conclusion, the +276 G>T polymorphism is a determinant of CAD risk in type 2 diabetic patients. This marker may assist in the identification of diabetic individuals at especially high risk of CAD, so that preventive programs can be targeted at these subjects

Spinal Volumetric Bone Mineral Density and Vertebral Fractures in Female Patients with Adrenal Incidentalomas: The Effects of Subclinical Hypercortisolism and Gonadal Status

Although adrenal incidentalomas (AI) are not associated with clinically evident syndromes, some patients display biochemical features of subclinical hypercortisolism (SH). Previous studies indicated a negative effect of SH on bone in AI patients, but the prevalence of vertebral fractures and the roles of SH and gonadal status in volumetric bone mineral density are unknown. In 70 female AI patients and 84 controls, the prevalence of vertebral fractures and spinal bone mineral density (by quantitative computed tomography) were evaluated. Subjects were subdivided according to menopausal status into groups Pre (21 patients and 23 controls) and Post (49 patients and 61 controls); there were 14 and 35 patients without SH (SH-) and 7 and 14 patients with SH (SH+) in groups Pre and Post, respectively. The prevalence of fractures was higher in SH+ than in controls and in SH- subjects in both groups Pre [SH+, 42.9%; controls, 0% (P = 0.001); SH-, 7.1% (P = 0.049)] and post [SH+, 78.6%; controls, 37.7% (P = 0.006); SH- 42.9% (P = 0.024)]. In group Post, the mean z-score quantitative computed tomography values were lower in SH+ patients (-0.78 \ub1 0.29) than in controls (0.06 \ub1 0.14; P = 0.011) and SH- patients (0.02 \ub1 0.19; P = 0.034). Evaluation of spinal bone is indicated in female AI patients with SH

A Serum Resistin and Multicytokine Inflammatory Pathway Is Linked With and Helps Predict All-cause Death in Diabetes

Context: Type 2 diabetes (T2D) shows a high mortality rate, partly mediated by atherosclerotic plaque instability. Discovering novel biomarkers may help identify high-risk patients who would benefit from more aggressive and specific managements. We recently described a serum resistin and multicytokine inflammatory pathway (REMAP), including resistin, interleukin (IL)-1 beta, IL-6, IL-8, and TNF-alpha, that is associated with cardiovascular disease.Objective: We investigated whether REMAP is associated with and improves the prediction of mortality in T2D.Methods: A REMAP score was investigated in 3 cohorts comprising 1528 patients with T2D (409 incident deaths) and in 59 patients who underwent carotid endarterectomy (CEA; 24 deaths). Plaques were classified as unstable/stable according to the modified American Heart Association atherosclerosis classification.Results: REMAP was associated with all-cause mortality in each cohort and in all 1528 individuals (fully adjusted hazard ratio [HR] for 1 SD increase=1.34, P<.001). In CEA patients, REMAP was associated with mortality (HR=1.64, P=.04) and a modest change was observed when plaque stability was taken into account (HR=1.58; P=.07). REMAP improved discrimination and reclassification measures of both Estimation of Mortality Risk in Type 2 Diabetic Patients and Risk Equations for Complications of Type 2 Diabetes, well-established prediction models of mortality in T2D (P<.05-<.001).Conclusion: REMAP is independently associated with and improves predict all-cause mortality in T2D; it can therefore be used to identify high-risk individuals to be targeted with more aggressive management. Whether REMAP can also identify patients who are more responsive to IL-6 and IL-1 beta monoclonal antibodies that reduce cardiovascular burden and total mortality is an intriguing possibility to be tested

Altered bone mass and turnover in female patients with adrenal incidentaloma: The effect of subclinical hypercortisolism

The strategy of treatment for patients with adrenal incidentalomas (AI) may depend upon the presence of hormonal hypersecretion. Although alterations of bone turnover have been recently reported, data on bone mineral density (BMD) are not available in AI patients. We evaluated bone turnover and BMD in 32 female AI patients and 64 matched controls. Spinal and femoral BMD were similar in patients and controls. Serum bone GLA protein (6.8 \ub1 3.5 vs. 8.8 \ub1 3.2 ng/mL; P < 0.005) and PTH (48.8 \ub1 15.1 vs. 37.2 \ub1 10.9 pg/mL; P < 0.0001) were different in patients and controls. Patients were then subdivided into 2 groups: with (n = 8; group A) or without (n = 24; group B) subclinical hypercortisolism. PTH was higher (P < 0.05) in group A than in group B and in both groups than in controls (57.1 \ub1 13.6, 46.0 \ub1 14.8, and 37.2 \ub1 10.9 pg/mL, respectively), and bone GLA protein was lower in group A than in group B and controls (3.8 \ub1 2.3, 7.5 \ub1 3.1, and 8.8 \ub1 3.2 ng/mL, respectively; P < 0.05). Serum type I cross-linked C telopeptide and fasting urinary deoxypyridinoline/creatinine were not different in the three groups. BMD at each site was lower (P < 0.05) in group A than in group B and controls. Bone mass and metabolism are altered in AI patients with subclinical hypercortisolism and should be taken into account, therefore, when addressing the treatment of choice for these patients