Leveraging Genetics to Address the Role of GALNT2 on Atherogenic Dyslipidemia.

Several studies have shown that downregulation of GALNT2 (Polypeptide N-Acetylgalactosaminyltransferase 2), encoding polypeptide N-acetylgalactosaminyltransferase 2, decreases high-density lipoprotein cholesterol (HDL-C) and increases triglycerides levels by glycosylating key enzymes of lipid metabolism, such as angiopoietin like 3, apolipoprotein C-III, and phospholipid transfer protein. GALNT2 is also a positive modulator of insulin signaling and action, associated with in vivo insulin sensitivity and during adipogenesis strongly upregulates adiponectin. Thus, the hypothesis that GALNT2 affects HDL-C and triglycerides levels also through insulin sensitivity and/or circulating adiponectin, is tested. In 881 normoglycemic individuals the G allele of rs4846914 SNP at the GALNT2 locus, known to associate with GALNT2 downregulation, is associated with low HDL-C and high values of triglycerides, triglycerides/HDL-C ratio, and theHomeostatic Model Assessment of insulin resistance HOMAIR (p-values = 0.01, 0.027, 0.002, and 0.016, respectively). Conversely, no association is observed with serum adiponectin levels (p = 0.091). Importantly, HOMAIR significantly mediates a proportion of the genetic association with HDL-C (21%, 95% CI: 7-35%, p = 0.004) and triglyceride levels (32%, 95% CI: 4-59%, p = 0.023). The results are compatible with the hypothesis that, besides the effect on key lipid metabolism enzymes, GALNT2 alters HDL-C and triglyceride levels also indirectly through a positive effect on insulin sensitivity.

Role of GALNT2 on Insulin Sensitivity, Lipid Metabolism and Fat Homeostasis.

O-linked glycosylation, the greatest form of post-translational modifications, plays a key role in regulating the majority of physiological processes. It is, therefore, not surprising that abnormal O-linked glycosylation has been related to several human diseases. Recently, GALNT2, which encodes the GalNAc-transferase 2 involved in the first step of O-linked glycosylation, has attracted great attention as a possible player in many highly prevalent human metabolic diseases, including atherogenic dyslipidemia, type 2 diabetes and obesity, all clustered on the common ground of insulin resistance. Data available both in human and animal models point to GALNT2 as a molecule that shapes the risk of the aforementioned abnormalities affecting diverse protein functions, which eventually cause clinically distinct phenotypes (a typical example of pleiotropism). Pathways linking GALNT2 to dyslipidemia and insulin resistance have been partly identified, while those for type 2 diabetes and obesity are yet to be understood. Here, we will provide a brief overview on the present knowledge on GALNT2 function and dysfunction and propose novel insights on the complex pathogenesis of the aforementioned metabolic diseases, which all impose a heavy burden for patients, their families and the entire society.

Gain of Function of Malate Dehydrogenase 2 and Familial Hyperglycemia.

CONTEXT: Genes causing familial forms of diabetes mellitus are only partially known. OBJECTIVE: We set out to identify the genetic cause of hyperglycemia in multigenerational families with an apparent autosomal dominant form of adult-onset diabetes not due to mutations in known monogenic diabetes genes. METHODS: Existing whole-exome sequencing (WES) data were used to identify exonic variants segregating with diabetes in 60 families from the United States and Italy. Functional studies were carried out in vitro (transduced MIN6-K8 cells) and in vivo (Caenorhabditis elegans) to assess the diabetogenic potential of 2 variants in the malate dehydrogenase 2 (MDH2) gene linked with hyperglycemia in 2 of the families. RESULTS: A very rare mutation (p.Arg52Cys) in MDH2 strongly segregated with hyperglycemia in 1 family from the United States. An infrequent MDH2 missense variant (p.Val160Met) also showed disease cosegregation in a family from Italy, although with reduced penetrance. In silico, both Arg52Cys and Val160Met were shown to affect MDH2 protein structure and function. In transfected HepG2 cells, both variants significantly increased MDH2 enzymatic activity, thereby decreasing the NAD+/NADH ratio-a change known to affect insulin signaling and secretion. Stable expression of human wild-type MDH2 in MIN6-K8 cell lines enhanced glucose- and GLP-1-stimulated insulin secretion. This effect was blunted by the Cys52 or Met160 substitutions. Nematodes carrying equivalent changes at the orthologous positions of the mdh-2 gene showed impaired glucose-stimulated insulin secretion. CONCLUSION: Our findings suggest a central role of MDH2 in human glucose homeostasis and indicate that gain of function variants in this gene may be involved in the etiology of familial forms of diabetes.

Morphological and molecular characterization of GALNT2-mediated adipogenesis.

3T3L1 mouse pre-adipocytes develop into adipocytes differently in response to GALNT2 overexpression or to stimulation with rosiglitazone, a reference inducer of adipogenesis. To investigate the biology of alternative pathways of adipogenesis, we studied lipid droplets (LD) morphology, chromatin organization, and gene expression in GALNT2- versus rosiglitazone-induced 3T3L1 adipogenesis. 3T3L1 overexpressing either GALNT2 (GALNT2) or GFP and treated with rosiglitazone (GFPR) were differentiated into adipocytes. LD and nuclei were profiled measuring their morphological features. The expression of adipogenesis-related genes was measured by RT-PCR. As compared to GFPR, GALNT2 showed smaller and more clustered LD, more nuclei with condensed chromatin and several gene expression changes (P < 0.001 for all). As compared to those stimulated by rosiglitazone, GALNT2 overexpressing cells show differences in the most established readouts of adipogenesis. Characterizing alternative pathways of adipogenesis may help tackle those diseases which are secondary to increased dysfunctional mass of adipose tissue.

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 (T0) were generated. Adipogenesis was induced with (R+) or without (R-) rosiglitazone and investigated after 15 days (T15). 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 T0 to T15R- and furthermore to T15R+. Such increases were significantly higher in GALNT2 than in GFP cells so that, as compared to T15R+GFP, T15R- 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.

Suggestive evidence of a multi-cytokine resistin pathway in humans and its role on cardiovascular events in high-risk individuals.

In cells and tissues resistin affects IL-1ß, IL-6, IL-8, IL-12 and TNF-α expression, thus suggesting the existence of a multi-cytokine "resistin pathway". We investigated whether such pathway does exist in humans and, if so, if it is associated with cardiovascular risk factors and with major adverse cardiovascular events (MACE). Serum cytokines were measured in 280 healthy subjects from the Gargano Study 2 (GS2) whose BMI, waist circumference, HOMAIR, triglycerides, HDL-cholesterol, systolic and diastolic blood pressure data were available and in 353 patients with type 2 diabetes and coronary artery disease from the Gargano Heart Study (GHS)-prospective design (follow-up 5.4 ± 2.5 years; 71 MACE). In GS2, cytokines mRNA levels in white blood cells were also measured. In GS2, resistin mRNA was correlated with all cytokines expression (all p < 0.001), but IL-12B. Consistently, serum resistin was correlated with all serum cytokines (all p < 0.001), but IL-12. Expression (eRPS) and serum (sRPS) resistin pathway scores (excluding IL-12) were each other correlated (p < 0.001) and both associated with cardiovascular risk factors (all p < 0.01). In GHS, sRPS was independently associated with MACE (HR = 1.44, 95% CI = 1.10-1.90). Our data indicate the existence of a resistin pathway, which is associated with cardiovascular risk factors and which strongly and independently predicts MACE.

The PPARγ2 P12A polymorphism is not associated with all-cause mortality in patients with type 2 diabetes mellitus.

The high mortality risk of patients with type 2 diabetes mellitus may well be explained by the several comorbidities and/or complications. Also the intrinsic genetic component predisposing to diabetes might have a role in shaping the risk of diabetes-related mortality. Among type 2 diabetes mellitus SNPs, rs1801282 is of particular interest because (i) it is harbored by peroxisome proliferator-activated receptor-γ2 (PPARγ2), which is the target for thiazolidinediones which are used as antidiabetic drugs, decreasing all-cause mortality in type 2 diabetes mellitus, and (ii) it is associated with insulin resistance and related traits, risk factors for overall mortality in type 2 diabetes mellitus. We investigated the role of PPARγ2 P12A, according to a dominant model (PA + AA vs. PP individuals) on incident all-cause mortality in three cohorts of type 2 diabetes mellitus, comprising a total of 1672 patients (462 deaths) and then performed a meta-analysis of ours and all available published data. In the three cohorts pooled and analyzed together, no association between PPARγ2 P12A and all-cause mortality was observed (HR 1.02, 95 % CI 0.79-1.33). Similar results were observed after adjusting for age, sex, smoking habits, and BMI (HR 1.09, 95 % CI 0.83-1.43). In a meta-analysis of ours and all studies previously published (n = 3241 individuals; 666 events), no association was observed between PPARγ2 P12A and all-cause mortality (HR 1.07, 95 % CI 0.85-1.33). Results from our individual samples as well as from our meta-analysis suggest that the PPARγ2 P12A does not significantly affect all-cause mortality in patients with type 2 diabetes mellitus.

Strong evidence of sexual dimorphic effect of adiposity excess on insulin sensitivity.

AIMS: Our aims were to investigate in several large samples, with a wide range of adiposity, whether: (1) the effect of BMI on insulin sensitivity is different between sexes; (2) also waist circumference plays a sex-specific role on insulin sensitivity; and (3) serum adiponectin and resistin are mediators of such sex-dimorphic effect. METHODS: Samples used were: Gargano study 1 (GS1), GS2 and Catania study (CS) comprising 3274 individuals. Adiponectin and resistin were measured by ELISA. Associations between variables were tested by linear models. RESULTS: In all samples, relationship between BMI and HOMAIR was steeper in males than in females (BMI-by-sex interaction p = 0.04-0.0007). No interaction was observed on serum adiponectin and resistin (p = 0.40-059), which are therefore unlikely to mediate the sex-dimorphic effect of BMI on insulin resistance. Relationship between waist circumference and HOMAIR was similar between sexes in GS1 and GS2 but not in CS (waist-by-sex interaction p = 0.01), comprising much heavier individuals. This suggests that a sex-dimorphic effect of abdominal adiposity on insulin resistance is observable only in the context of high BMI. CONCLUSIONS: Our findings represent a proof of concept that BMI and insulin sensitivity are associated in a sex-specific manner. This may explain why females are protected from diabetes and cardiovascular disease, compared to males of similar BMI.

Loss-of-Function Mutations in APPL1 in Familial Diabetes Mellitus.

Diabetes mellitus is a highly heterogeneous disorder encompassing several distinct forms with different clinical manifestations including a wide spectrum of age at onset. Despite many advances, the causal genetic defect remains unknown for many subtypes of the disease, including some of those forms with an apparent Mendelian mode of inheritance. Here we report two loss-of-function mutations (c.1655T>A [p.Leu552(∗)] and c.280G>A [p.Asp94Asn]) in the gene for the Adaptor Protein, Phosphotyrosine Interaction, PH domain, and leucine zipper containing 1 (APPL1) that were identified by means of whole-exome sequencing in two large families with a high prevalence of diabetes not due to mutations in known genes involved in maturity onset diabetes of the young (MODY). APPL1 binds to AKT2, a key molecule in the insulin signaling pathway, thereby enhancing insulin-induced AKT2 activation and downstream signaling leading to insulin action and secretion. Both mutations cause APPL1 loss of function. The p.Leu552(∗) alteration totally abolishes APPL1 protein expression in HepG2 transfected cells and the p.Asp94Asn alteration causes significant reduction in the enhancement of the insulin-stimulated AKT2 and GSK3ß phosphorylation that is observed after wild-type APPL1 transfection. These findings-linking APPL1 mutations to familial forms of diabetes-reaffirm the critical role of APPL1 in glucose homeostasis.

Serum resistin, cardiovascular disease and all-cause mortality in patients with type 2 diabetes.

BACKGROUND: High serum resistin has been associated with increased risk of cardiovascular disease in the general population, Only sparse and conflicting results, limited to Asian individuals, have been reported, so far, in type 2 diabetes. We studied the role of serum resistin on coronary artery disease, major cardiovascular events and all-cause mortality in type 2 diabetes. METHODS: We tested the association of circulating resistin concentrations with coronary artery disease, major cardiovascular events (cardiovascular death, non-fatal myocardial infarction and non-fatal stroke) and all-cause mortality in 2,313 diabetic patients of European ancestry from two cross-sectional and two prospective studies. In addition, the expression of resistin gene (RETN) was measured in blood cells of 68 diabetic patients and correlated with their serum resistin levels. RESULTS: In a model comprising age, sex, smoking habits, BMI, HbA1c, and insulin, antihypertensive and antidyslipidemic therapies, serum resistin was associated with coronary artery disease in both cross-sectional studies: OR (95%CI) per SD increment = 1.35 (1.10-1.64) and 1.99 (1.55-2.55). Additionally, serum resistin predicted incident major cardiovascular events (HR per SD increment = 1.31; 1.10-1.56) and all-cause mortality (HR per SD increment = 1.16; 1.06-1.26). Adjusting also for fibrinogen levels affected the association with coronary artery disease and incident cardiovascular events, but not that with all cause-mortality. Finally, serum resistin was positively correlated with RETN mRNA expression (rho = 0.343). CONCLUSIONS: This is the first study showing that high serum resistin (a likely consequence, at least partly, of increased RETN expression) is a risk factor for cardiovascular disease and all-cause mortality in diabetic patients of European ancestry.

GALNT2 expression is reduced in patients with Type 2 diabetes: possible role of hyperglycemia.

Impaired insulin action plays a major role in the pathogenesis of type 2 diabetes, a chronic metabolic disorder which imposes a tremendous burden to morbidity and mortality worldwide. Unraveling the molecular mechanisms underlying insulin resistance would improve setting up preventive and treatment strategies of type 2 diabetes. Down-regulation of GALNT2, an UDPN-acetyl-alpha-D-galactosamine polypeptideN-acetylgalactosaminyltransferase-2 (ppGalNAc-T2), causes impaired insulin signaling and action in cultured human liver cells. In addition, GALNT2 mRNA levels are down-regulated in liver of spontaneously insulin resistant, diabetic Goto-Kakizaki rats. To investigate the role of GALNT2 in human hyperglycemia, we measured GALNT2 mRNA expression levels in peripheral whole blood cells of 84 non-obese and 46 obese non-diabetic individuals as well as of 98 obese patients with type 2 diabetes. We also measured GALNT2 mRNA expression in human U937 cells cultured under different glucose concentrations. In vivo studies indicated that GALNT2 mRNA levels were significantly reduced from non obese control to obese non diabetic and to obese diabetic individuals (p<0.001). In vitro studies showed that GALNT2 mRNA levels was reduced in U937 cells exposed to high glucose concentrations (i.e. 25 mmol/l glucose) as compared to cells exposed to low glucose concentration (i.e. 5.5 mmol/l glucose +19.5 mmol/l mannitol). In conclusion, our data indicate that GALNT2 is down-regulated in patients with type 2 diabetes and suggest that this association is, at least partly, secondary to hyperglycemia. Further studies are needed to understand whether GALNT2 down-regulation plays a pathogenic role in maintaining and/or aggravating the metabolic abnormalities of diabetic milieu.

Role of GALNT2 in the modulation of ENPP1 expression, and 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 cells.

Role of somatomedin-B-like domains on ENPP1 inhibition of insulin signaling.

The exact mechanism by which ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) inhibits insulin signaling is not known. ENPP1 contains two somatomedin-B-like domains (i.e. SMB 1 and 2) involved in ENPP1 dimerization in animal cells. The aim of the present study was to investigate if these domains modulate ENPP1 inhibitory activity on insulin signaling in human insulin target cells (HepG2). ENPP1 (ENPP1-3'myc), ENPP1 deleted of SMB 1 (ENPP1-ΔI-3'myc) or of SMB 2 (ENPP1-ΔII-3'myc) domain were cloned in frame with myc tag in mammalian expression vector pRK5. Plasmids were transiently transfected in human liver HepG2 cells. ENPP1 inhibitory activity on insulin signaling, dimerization and protein-protein interaction with insulin receptor (IR), reported to mediate the modulation of ENPP1 inhibitory activity, were studied. As compared to untransfected cells, a progressive increase of ENPP1 inhibitory activity on insulin-induced IR ß-subunit autophosphorylation and on Akt-S(473) phosphorylation was observed in ENPP1-3'myc, ENPP1-ΔI-3'myc and ENPP1-ΔII-3'myc cells. Under non reducing conditions a 260 kDa homodimer, indicating ENPP1 dimerization, was observed. The ratio of non reduced (260 kDa) to reduced (130 kDa) ENPP1 was significantly decreased by two thirds in ENPP1-ΔII-3'myc vs. ENPP1-3'myc but not in ENPP1-ΔI-3'myc. A similar ENPP1/IR interaction was detectable by co-immunoprecipitation in ENPP1-3'myc, ENPP1-ΔI-3'myc and ENPP1-ΔII-3'myc cells. In conclusion, SMB 1 and SMB 2 are negative modulators of ENPP1 inhibitory activity on insulin signaling. For SMB 2 such effect might be mediated by a positive role on protein dimerization.

ENPP1 affects insulin action and secretion: evidences from in vitro studies.

The aim of this study was to deeper investigate the mechanisms through which ENPP1, a negative modulator of insulin receptor (IR) activation, plays a role on insulin signaling, insulin secretion and eventually glucose metabolism. ENPP1 cDNA (carrying either K121 or Q121 variant) was transfected in HepG2 liver-, L6 skeletal muscle- and INS1E beta-cells. Insulin-induced IR-autophosphorylation (HepG2, L6, INS1E), Akt-Ser(473), ERK1/2-Thr(202)/Tyr(204) and GSK3-beta Ser(9) phosphorylation (HepG2, L6), PEPCK mRNA levels (HepG2) and 2-deoxy-D-glucose uptake (L6) was studied. GLUT 4 mRNA (L6), insulin secretion and caspase-3 activation (INS1E) were also investigated. Insulin-induced IR-autophosphorylation was decreased in HepG2-K, L6-K, INS1E-K (20%, 52% and 11% reduction vs. untransfected cells) and twice as much in HepG2-Q, L6-Q, INS1E-Q (44%, 92% and 30%). Similar data were obtained with Akt-Ser(473), ERK1/2-Thr(202)/Tyr(204) and GSK3-beta Ser(9) in HepG2 and L6. Insulin-induced reduction of PEPCK mRNA was progressively lower in untransfected, HepG2-K and HepG2-Q cells (65%, 54%, 23%). Insulin-induced glucose uptake in untransfected L6 (60% increase over basal), was totally abolished in L6-K and L6-Q cells. GLUT 4 mRNA was slightly reduced in L6-K and twice as much in L6-Q (13% and 25% reduction vs. untransfected cells). Glucose-induced insulin secretion was 60% reduced in INS1E-K and almost abolished in INS1E-Q. Serum deficiency activated caspase-3 by two, three and four folds in untransfected INS1E, INS1E-K and INS1E-Q. Glyburide-induced insulin secretion was reduced by 50% in isolated human islets from homozygous QQ donors as compared to those from KK and KQ individuals. Our data clearly indicate that ENPP1, especially when the Q121 variant is operating, affects insulin signaling and glucose metabolism in skeletal muscle- and liver-cells and both function and survival of insulin secreting beta-cells, thus representing a strong pathogenic factor predisposing to insulin resistance, defective insulin secretion and glucose metabolism abnormalities.

ENPP1 Q121 variant, increased pulse pressure and reduced insulin signaling, and nitric oxide synthase activity in endothelial cells.

OBJECTIVE: Insulin resistance induces increased pulse pressure (PP), endothelial dysfunction (ED), and reduced bioavailability of endothelium-derived nitric oxide (NO). The genetic background of these 3 cardiovascular risk factors might be partly common. The ENPP1 K121Q polymorphism is associated with insulin resistance and cardiovascular risk. METHODS AND RESULTS: We investigated whether the K121Q polymorphism is associated with increased PP in white Caucasians and with ED in vitro. In 985 individuals, (390 unrelated and 595 from 248 families), the K121Q polymorphism was associated with PP (P=8.0 x 10(-4)). In the families, the Q121 variant accounted for 0.08 of PP heritability (P=9.4 x 10(-4)). This association was formally replicated in a second sample of 475 individuals (P=2.6 x 10(-2)) but not in 2 smaller samples of 289 and 236 individuals (P=0.49 and 0.21, respectively). In the individual patients' data meta-analysis, comprising 1985 individuals, PP was associated with the Q121 variant (P=1.2 x 10(-3)). Human endothelial cells carrying the KQ genotype showed, as compared to KK cells, reduced insulin-mediated insulin receptor autophosphorylation (P=0.03), Ser(473)-Akt phosphorylation (P=0.03), and NO synthase activity (P=0.003). CONCLUSIONS: Our data suggest that the ENPP1 Q121 variant is associated with increased PP in vivo and reduced insulin signaling and ED in vitro, thus indicating a possible pathogenic mechanism for the increased cardiovascular risk observed in ENPP1 Q121 carriers.