Adipocyte precursor cells from first degree relatives of type 2 diabetic patients feature changes in hsa-mir-23a-5p, -193a-5p, and -193b-5p and insulin-like growth factor 2 expression.

First-degree relatives (FDRs) of type 2 diabetics (T2D) feature dysfunction of subcutaneous adipose tissue (SAT) long before T2D onset. miRNAs have a role in adipocyte precursor cells (APC) differentiation and in adipocyte identity. Thus, impaired miRNA expression may contribute to SAT dysfunction in FDRs. In the present work, we have explored changes in miRNA expression associated with T2D family history which may affect gene expression in SAT APCs from FDRs. Small RNA-seq was performed in APCs from healthy FDRs and matched controls and omics data were validated by qPCR. Integrative analyses of APC miRNome and transcriptome from FDRs revealed down-regulated hsa-miR-23a-5p, -193a-5p and -193b-5p accompanied by up-regulated Insulin-like Growth Factor 2 (IGF2) gene which proved to be their direct target. The expression changes in these marks were associated with SAT adipocyte hypertrophy in FDRs. APCs from FDRs further demonstrated reduced capability to differentiate into adipocytes. Treatment with IGF2 protein decreased APC adipogenesis, while over-expression of hsa-miR-23a-5p, -193a-5p and -193b-5p enhanced adipogenesis by IGF2 targeting. Indeed, IGF2 increased the Wnt Family Member 10B gene expression in APCs. Down-regulation of the three miRNAs and IGF2 up-regulation was also observed in Peripheral Blood Leukocytes (PBLs) from FDRs. In conclusion, APCs from FDRs feature a specific miRNA/gene profile, which associates with SAT adipocyte hypertrophy and appears to contribute to impaired adipogenesis. PBL detection of this profile may help in identifying adipocyte hypertrophy in individuals at high risk of T2D.

Nutritional Factors, DNA Methylation, and Risk of Type 2 Diabetes and Obesity: Perspectives and Challenges.

A healthy diet improves life expectancy and helps to prevent common chronic diseases such as type 2 diabetes (T2D) and obesity. The mechanisms driving these effects are not fully understood, but are likely to involve epigenetics. Epigenetic mechanisms control gene expression, maintaining the DNA sequence, and therefore the full genomic information inherited from our parents, unchanged. An interesting feature of epigenetic changes lies in their dynamic nature and reversibility. Accordingly, they are susceptible to correction through targeted interventions. Here we will review the evidence supporting a role for nutritional factors in mediating metabolic disease risk through DNA methylation changes. Special emphasis will be placed on the potential of using DNA methylation traits as biomarkers to predict risk of obesity and T2D as well as on their response to dietary and pharmacological (epi-drug) interventions.

Epigenetic modifications of the Zfp/ZNF423 gene control murine adipogenic commitment and are dysregulated in human hypertrophic obesity.

AIMS/HYPOTHESIS: Subcutaneous adipocyte hypertrophy is associated with insulin resistance and increased risk of type 2 diabetes, and predicts its future development independent of obesity. In humans, subcutaneous adipose tissue hypertrophy is a consequence of impaired adipocyte precursor cell recruitment into the adipogenic pathway rather than a lack of precursor cells. The zinc finger transcription factor known as zinc finger protein (ZFP) 423 has been identified as a major determinant of pre-adipocyte commitment and maintained white adipose cell function. Although its levels do not change during adipogenesis, ectopic expression of Zfp423 in non-adipogenic murine cells is sufficient to activate expression of the gene encoding peroxisome proliferator-activated receptor γ (Pparγ; also known as Pparg) and increase the adipogenic potential of these cells. We investigated whether the Zfp423 gene is under epigenetic regulation and whether this plays a role in the restricted adipogenesis associated with hypertrophic obesity. METHODS: Murine 3T3-L1 and NIH-3T3 cells were used as fibroblasts committed and uncommitted to the adipocyte lineage, respectively. Human pre-adipocytes were isolated from the stromal vascular fraction of subcutaneous adipose tissue of 20 lean non-diabetic individuals with a wide adipose cell size range. mRNA levels were measured by quantitative real-time PCR, while methylation levels were analysed by bisulphite sequencing. Chromatin structure was analysed by micrococcal nuclease protection assay, and DNA-methyltransferases were chemically inhibited by 5-azacytidine. Adipocyte differentiation rate was evaluated by Oil Red O staining. RESULTS: Comparison of uncommitted (NIH-3T3) and committed (3T3-L1) adipose precursor cells revealed that Zfp423 expression increased (p < 0.01) in parallel with the ability of the cells to differentiate into mature adipocytes owing to both decreased promoter DNA methylation (p < 0.001) and nucleosome occupancy (nucleosome [NUC] 1 p < 0.01; NUC2 p < 0.001) in the 3T3-L1 compared with NIH-3T3 cells. Interestingly, non-adipogenic epigenetic profiles can be reverted in NIH-3T3 cells as 5-azacytidine treatment increased Zfp423 mRNA levels (p < 0.01), reduced DNA methylation at a specific CpG site (p < 0.01), decreased nucleosome occupancy (NUC1, NUC2: p < 0.001) and induced adipocyte differentiation (p < 0.05). These epigenetic modifications can also be initiated in response to changes in the pre-adipose cell microenvironment, in which bone morphogenetic protein 4 (BMP4) plays a key role. We finally showed that, in human adipocyte precursor cells, impaired epigenetic regulation of zinc nuclear factor (ZNF)423 (the human orthologue of murine Zfp423) was associated with inappropriate subcutaneous adipose cell hypertrophy. As in NIH-3T3 cells, the normal ZNF423 epigenetic profile was rescued by 5-azacytidine exposure. CONCLUSIONS/INTERPRETATION: Our results show that epigenetic events regulate the ability of precursor cells to commit and differentiate into mature adipocytes by modulating ZNF423, and indicate that dysregulation of these mechanisms accompanies subcutaneous adipose tissue hypertrophy in humans.

Prep1 deficiency improves metabolic response in white adipose tissue.

Prep1 is a gene encoding for a homeodomain transcription factor which induces hepatic and muscular insulin resistance. In this study, we show that Prep1 hypomorphic heterozygous (Prep1i/+) mice, expressing low levels of protein, featured a 23% and a 25% reduction of total body lipid content and epididymal fat, respectively. The percentage of the small adipocytes (25-75 µm) was 30% higher in Prep1i/+ animals than in the WT, with a reciprocal difference in the large adipose cells (100-150 and >150 µm). Insulin-stimulated insulin receptor tyrosine and Akt serine phosphorylation markedly increased in Prep1i/+ mice, paralleled by 3-fold higher glucose uptake and a significant increase of proadipogenic genes such as C/EBPα, GLUT4, and FABP4. Moreover, T cells infiltration and TNF-α, IFNγ and leptin expression were reduced in adipose tissue from Prep1i/+ mice, while adiponectin levels were 2-fold higher. Furthermore, Prep1i/+ mature adipocytes released lower amounts of pro-inflammatory cytokines and higher amount of adiponectin compared to WT cells. Incubation of murine liver cell line (NMuLi) with conditioned media (CM) from mature adipocytes of Prep1i/+ mice improved glucose metabolism, while those from WT mice had no effect. Consistent with these data, Prep1 overexpression in 3T3-L1 adipocytes impaired adipogenesis and insulin signaling, and increased proinflammatory cytokine secretion. All these findings suggest that Prep1 silencing reduces inflammatory response and increases insulin sensitivity in adipose tissue. In addition, CM from mature adipocytes of Prep1i/+ mice improve metabolism in hepatic cells.

Pathologic endoplasmic reticulum stress induced by glucotoxic insults inhibits adipocyte differentiation and induces an inflammatory phenotype.

Adipocyte differentiation is critical in obesity. By controlling new adipocyte recruitment, adipogenesis contrasts adipocyte hypertrophy and its adverse consequences, such as insulin resistance. Contrasting data are present in literature on the effect of endoplasmic reticulum (ER) stress and subsequent unfolded protein response (UPR) on adipocyte differentiation, being reported to be either necessary or inhibitory. In this study, we sought to clarify the effect of ER stress and UPR on adipocyte differentiation. We have used two different cell lines, the widely used pre-adipocyte 3T3-L1 cells and a murine multipotent mesenchymal cell line, W20-17 cells. A strong ER stress activator, thapsigargin, and a pathologically relevant inducer of ER stress, glucosamine (GlcN), induced ER stress and UPR above those occurring in the absence of perturbation and inhibited adipocyte differentiation. Very low concentrations of 4-phenyl butyric acid (PBA, a chemical chaperone) inhibited only the overactivation of ER stress and UPR elicited by GlcN, leaving unaltered the part physiologically activated during differentiation, and reversed the inhibitory effect of GlcN on differentiation. In addition, GlcN stimulated proinflammatory cytokine release and PBA prevented these effects. An inhibitor of NF-kB also reversed the effects of GlcN on cytokine release. These results indicate that while ER stress and UPR activation is "physiologically" activated during adipocyte differentiation, the "pathologic" part of ER stress activation, secondary to a glucotoxic insult, inhibits differentiation. In addition, such a metabolic insult, causes a shift of the preadipocyte/adipocyte population towards a proinflammatory phenotype.

IL-3 synergises with basophil-derived IL-4 and IL-13 to promote the alternative activation of human monocytes.

Basophil-derived IL-4 is involved in the alternative activation of mouse monocytes, as recently shown in vivo. Whether this applies to human basophils and monocytes has not been established yet. Here, we sought to characterise the interaction between basophils and monocytes and identify the molecular determinants. A basophil-monocyte co-culture model revealed that IL-3 and basophil-derived IL-4 and IL-13 induced monocyte production of CCL17, a marker of alternative activation. Critically, IL-3 and IL-4 acted directly on monocytes to induce CCL17 production through histone H3 acetylation, but did not increase the recruitment of STAT5 or STAT6. Although freshly isolated monocytes did not express the IL-3 receptor α chain (CD123), and did not respond to IL-3 (as assessed by STAT5 phosphorylation), the overnight incubation with IL-4 (especially if associated with IL-3) upregulated CD123 expression. IL-3-activated JAK2-STAT5 pathway inhibitors reduced the CCL17 production in response to IL-3 and IL-4, but not to IL-4 alone. Interestingly, monocytes isolated from allergen-sensitised asthmatic patients exhibited a higher expression of CD123. Taken together, our data show that the JAK2-STAT5 pathway modulates both basophil and monocyte effector responses. The coordinated activation of STAT5 and STAT6 may have a major impact on monocyte alternative activation in vitro and in vivo.

Peroxisome proliferator-activated receptor-γ activation enhances insulin-stimulated glucose disposal by reducing ped/pea-15 gene expression in skeletal muscle cells: evidence for involvement of activator protein-1.

The gene network responsible for inflammation-induced insulin resistance remains enigmatic. In this study, we show that, in L6 cells, rosiglitazone- as well as pioglitazone-dependent activation of peroxisome proliferator-activated receptor-γ (PPARγ) represses transcription of the ped/pea-15 gene, whose increased activity impairs glucose tolerance in mice and humans. Rosiglitazone enhanced insulin-induced glucose uptake in L6 cells expressing the endogenous ped/pea-15 gene but not in cells expressing ped/pea-15 under the control of an exogenous promoter. The ability of PPARγ to affect ped/pea-15 expression was also lost in cells and in C57BL/6J transgenic mice expressing ped/pea-15 under the control of an exogenous promoter, suggesting that ped/pea-15 repression may contribute to rosiglitazone action on glucose disposal. Indeed, high fat diet mice showed insulin resistance and increased ped/pea-15 levels, although these effects were reduced by rosiglitazone treatment. Both supershift and ChIP assays revealed the presence of the AP-1 component c-JUN at the PED/PEA-15 promoter upon 12-O-tetradecanoylphorbol-13-acetate stimulation of the cells. In these experiments, rosiglitazone treatment reduced c-JUN presence at the PED/PEA-15 promoter. This effect was not associated with a decrease in c-JUN expression. In addition, c-jun silencing in L6 cells lowered ped/pea-15 expression and caused nonresponsiveness to rosiglitazone, although c-jun overexpression enhanced the binding to the ped/pea-15 promoter and blocked the rosiglitazone effect. These results indicate that PPARγ regulates ped/pea-15 transcription by inhibiting c-JUN binding at the ped/pea-15 promoter. Thus, ped/pea-15 is downstream of a major PPARγ-regulated inflammatory network. Repression of ped/pea-15 transcription might contribute to the PPARγ regulation of muscle sensitivity to insulin.

Prostate health index (phi) and prostate cancer antigen 3 (PCA3) significantly improve diagnostic accuracy in patients undergoing prostate biopsy.

BACKGROUND: Prostate health index (phi) and prostate cancer antigen 3 (PCA3) have been recently proposed as novel biomarkers for prostate cancer (PCa). We assessed the diagnostic performance of these biomarkers, alone or in combination, in men undergoing first prostate biopsy for suspicion of PCa. METHODS: One hundred sixty male subjects were enrolled in this prospective observational study. PSA molecular forms, phi index (Beckman coulter immunoassay), PCA3 score (Progensa PCA3 assay), and other established biomarkers (tPSA, fPSA, and %fPSA) were assessed before patients underwent a 18-core first prostate biopsy. The discriminating ability between PCa-negative and PCa-positive biopsies of Beckman coulter phi and PCA3 score and other used biomarkers were determined. RESULTS: One hundred sixty patients met inclusion criteria. %p2PSA (p2PSA/fPSA × 100), phi and PCA3 were significantly higher in patients with PCa compared to PCa-negative group (median values: 1.92 vs. 1.55, 49.97 vs. 36.84, and 50 vs. 32, respectively, P ≤ 0.001). ROC curve analysis showed that %p2PSA, phi, and PCA3 are good indicator of malignancy (AUCs = 0.68, 0.71, and 0.66, respectively). A multivariable logistic regression model consisting of both the phi index and PCA3 score allowed to reach an overall diagnostic accuracy of 0.77. Decision curve analysis revealed that this "combined" marker achieved the highest net benefit over the examined range of the threshold probability. CONCLUSIONS: phi and PCA3 showed no significant difference in the ability to predict PCa diagnosis in men undergoing first prostate biopsy. However, diagnostic performance is significantly improved by combining phi and PCA3.

Increased cell senescence in human metabolic disorders.

Cell senescence (CS) is at the nexus between aging and associated chronic disorders, and aging increases the burden of CS in all major metabolic tissues. However, CS is also increased in adult obesity, type 2 diabetes (T2D), and nonalcoholic fatty liver disease independent of aging. Senescent tissues are characterized by dysfunctional cells and increased inflammation, and both progenitor cells and mature, fully differentiated and nonproliferating cells are afflicted. Recent studies have shown that hyperinsulinemia and associated insulin resistance (IR) promote CS in both human adipose and liver cells. Similarly, increased CS promotes cellular IR, showing their interdependence. Furthermore, the increased adipose CS in T2D is independent of age, BMI, and degree of hyperinsulinemia, suggesting premature aging. These results suggest that senomorphic/senolytic therapy may become important for treating these common metabolic disorders.

The Transcription Factor HOXA5: Novel Insights into Metabolic Diseases and Adipose Tissue Dysfunction.

The transcription factor HOXA5, from the HOX gene family, has long been studied due to its critical role in physiological activities in normal cells, such as organ development and body patterning, and pathological activities in cancer cells. Nonetheless, recent evidence supports the hypothesis of a role for HOXA5 in metabolic diseases, particularly in obesity and type 2 diabetes (T2D). In line with the current opinion that adipocyte and adipose tissue (AT) dysfunction belong to the group of primary defects in obesity, linking this condition to an increased risk of insulin resistance (IR) and T2D, the HOXA5 gene has been shown to regulate adipocyte function and AT remodeling both in humans and mice. Epigenetics adds complexity to HOXA5 gene regulation in metabolic diseases. Indeed, epigenetic mechanisms, specifically DNA methylation, influence the dynamic HOXA5 expression profile. In human AT, the DNA methylation profile at the HOXA5 gene is associated with hypertrophic obesity and an increased risk of developing T2D. Thus, an inappropriate HOXA5 gene expression may be a mechanism causing or maintaining an impaired AT function in obesity and potentially linking obesity to its associated disorders. In this review, we integrate the current evidence about the involvement of HOXA5 in regulating AT function, as well as its association with the pathogenesis of obesity and T2D. We also summarize the current knowledge on the role of DNA methylation in controlling HOXA5 expression. Moreover, considering the susceptibility of epigenetic changes to reversal through targeted interventions, we discuss the potential therapeutic value of targeting HOXA5 DNA methylation changes in the treatment of metabolic diseases.

Altered H3K4me3 profile at the TFAM promoter causes mitochondrial alterations in preadipocytes from first-degree relatives of type 2 diabetics.

BACKGROUND: First-degree relatives of type 2 diabetics (FDR) exhibit a high risk of developing type 2 diabetes (T2D) and feature subcutaneous adipocyte hypertrophy, independent of obesity. In FDR, adipose cell abnormalities contribute to early insulin-resistance and are determined by adipocyte precursor cells (APCs) early senescence and impaired recruitment into the adipogenic pathway. Epigenetic mechanisms signal adipocyte differentiation, leading us to hypothesize that abnormal epigenetic modifications cause adipocyte dysfunction and enhance T2D risk. To test this hypothesis, we examined the genome-wide histone profile in APCs from the subcutaneous adipose tissue of healthy FDR. RESULTS: Sequencing-data analysis revealed 2644 regions differentially enriched in lysine 4 tri-methylated H3-histone (H3K4me3) in FDR compared to controls (CTRL) with significant enrichment in mitochondrial-related genes. These included TFAM, which regulates mitochondrial DNA (mtDNA) content and stability. In FDR APCs, a significant reduction in H3K4me3 abundance at the TFAM promoter was accompanied by a reduction in TFAM mRNA and protein levels. FDR APCs also exhibited reduced mtDNA content and mitochondrial-genome transcription. In parallel, FDR APCs exhibited impaired differentiation and TFAM induction during adipogenesis. In CTRL APCs, TFAM-siRNA reduced mtDNA content, mitochondrial transcription and adipocyte differentiation in parallel with upregulation of the CDKN1A and ZMAT3 senescence genes. Furthermore, TFAM-siRNA significantly expanded hydrogen peroxide (H2O2)-induced senescence, while H2O2 did not affect TFAM expression. CONCLUSIONS: Histone modifications regulate APCs ability to differentiate in mature cells, at least in part by modulating TFAM expression and affecting mitochondrial function. Reduced H3K4me3 enrichment at the TFAM promoter renders human APCs senescent and dysfunctional, increasing T2D risk.

Methylglyoxal Impairs the Pro-Angiogenic Ability of Mouse Adipose-Derived Stem Cells (mADSCs) via a Senescence-Associated Mechanism.

Adipose-derived stem cells (ADSCs) play a crucial role in angiogenesis and repair of damaged tissues. However, in pathological conditions including diabetes, ADSC function is compromised. This work aims at evaluating the effect of Methylglyoxal (MGO), a product of chronic hyperglycemia, on mouse ADSCs' (mADSCs) pro-angiogenic function and the molecular mediators involved. The mADSCs were isolated from C57bl6 mice. MGO-adducts and p-p38 MAPK protein levels were evaluated by Western Blot. Human retinal endothelial cell (hREC) migration was analyzed by transwell assays. Gene expression was measured by qRT-PCR, and SA-ßGal activity by cytofluorimetry. Soluble factor release was evaluated by multiplex assay. MGO treatment does not impair mADSC viability and induces MGO-adduct accumulation. hREC migration is reduced in response to both MGO-treated mADSCs and conditioned media from MGO-treated mADSCs, compared to untreated cells. This is associated with an increase of SA-ßGal activity, SASP factor release and p53 and p21 expression, together with a VEGF- and PDGF-reduced release from MGO-treated mADSCs and a reduced p38-MAPK activation in hRECs. The MGO-induced impairment of mADSC function is reverted by senolytics. In conclusion, MGO impairs mADSCs' pro-angiogenic function through the induction of a senescent phenotype, associated with the reduced secretion of growth factors crucial for hREC migration.

Type 2 Diabetes, Independent of Obesity and Age, Is Characterized by Senescent and Dysfunctional Mature Human Adipose Cells.

Obesity with dysfunctional adipose cells is the major cause of the current epidemic of type 2 diabetes (T2D). We examined senescence in human adipose tissue cells from age- and BMI-matched individuals who were lean, obese, and obese with T2D. In obese individuals and, more pronounced, those with T2D, we found mature and fully differentiated adipose cells to exhibit increased senescence similar to what we previously have shown in the progenitor cells. The degree of adipose cell senescence was positively correlated with whole-body insulin resistance and adipose cell size. Adipose cell protein analysis revealed dysfunctional cells in T2D with increased senescence markers reduced PPAR-γ, GLUT4, and pS473AKT. Consistent with a recent study, we found the cell cycle regulator cyclin D1 to be increased in obese cells and further elevated in T2D cells, closely correlating with senescence markers, ambient donor glucose, and, more inconsistently, plasma insulin levels. Furthermore, fully differentiated adipose cells were susceptible to experimentally induced senescence and to conditioned medium increasing cyclin D1 and responsive to senolytic agents. Thus, fully mature human adipose cells from obese individuals, particularly those with T2D become senescent, and SASP secretion by senescent progenitor cells can play an important role in addition to donor hyperinsulinemia.

Chronic hyperinsulinemia promotes human hepatocyte senescence.

OBJECTIVE: Cellular senescence, an irreversible proliferative cell arrest, is caused by excessive intracellular or extracellular stress/damage. Increased senescent cells have been identified in multiple tissues in different metabolic and other aging-related diseases. Recently, several human and mouse studies emphasized the involvement of senescence in development and progression of NAFLD. Hyperinsulinemia, seen in obesity, metabolic syndrome, and other conditions of insulin resistance, has been linked to senescence in adipocytes and neurons. Here, we investigate the possible direct role of chronic hyperinsulinemia in the development of senescence in human hepatocytes. METHODS: Using fluorescence microscopy, immunoblotting, and gene expression, we tested senescence markers in human hepatocytes subjected to chronic hyperinsulinemia in vitro and validated the data in vivo by using liver-specific insulin receptor knockout (LIRKO) mice. The consequences of hyperinsulinemia were also studied in senescent hepatocytes following doxorubicin as a model of stress-induced senescence. Furthermore, the effects of senolytic agents in insulin- and doxorubicin-treated cells were analyzed. RESULTS: Results showed that exposing the hepatocytes to prolonged hyperinsulinemia promotes the onset of senescence by increasing the expression of p53 and p21. It also further enhanced the senescent phenotype in already senescent hepatocytes. Addition of insulin signaling pathway inhibitors prevented the increase in cell senescence, supporting the direct contribution of insulin. Furthermore, LIRKO mice, in which insulin signaling in the liver is abolished due to deletion of the insulin receptor gene, showed no differences in senescence compared to their wild-type counterparts despite having marked hyperinsulinemia indicating these are receptor-mediated effects. In contrast, the persistent hyperinsulinemia in LIRKO mice enhanced senescence in white adipose tissue. In vitro, senolytic agents dasatinib and quercetin reduced the prosenescent effects of hyperinsulinemia in hepatocytes. CONCLUSION: Our findings demonstrate a direct link between chronic hyperinsulinemia and hepatocyte senescence. This effect can be blocked by reducing the levels of insulin receptors or administration of senolytic drugs, such as dasatinib and quercetin.

Epigenetic Dysregulation of the Homeobox A5 (HOXA5) Gene Associates with Subcutaneous Adipocyte Hypertrophy in Human Obesity.

Along with insulin resistance and increased risk of type 2 diabetes (T2D), lean first-degree relatives of T2D subjects (FDR) feature impaired adipogenesis in subcutaneous adipose tissue (SAT) and subcutaneous adipocyte hypertrophy well before diabetes onset. The molecular mechanisms linking these events have only partially been clarified. In the present report, we show that silencing of the transcription factor Homeobox A5 (HOXA5) in human preadipocytes impaired differentiation in mature adipose cells in vitro. The reduced adipogenesis was accompanied by inappropriate WNT-signaling activation. Importantly, in preadipocytes from FDR individuals, HOXA5 expression was attenuated, with hypermethylation of the HOXA5 promoter region found responsible for its downregulation, as revealed by luciferase assay. Both HOXA5 gene expression and DNA methylation were significantly correlated with SAT adipose cell hypertrophy in FDR, whose increased adipocyte size marks impaired adipogenesis. In preadipocytes from FDR, the low HOXA5 expression negatively correlated with enhanced transcription of the WNT signaling downstream genes NFATC1 and WNT2B. In silico evidence indicated that NFATC1 and WNT2B were directly controlled by HOXA5. The HOXA5 promoter region also was hypermethylated in peripheral blood leukocytes from these same FDR individuals, which was further revealed in peripheral blood leukocytes from an independent group of obese subjects. Thus, HOXA5 controlled adipogenesis in humans by suppressing WNT signaling. Altered DNA methylation of the HOXA5 promoter contributed to restricted adipogenesis in the SAT of lean subjects who were FDR of type 2 diabetics and in obese individuals.

ZMAT3 hypomethylation contributes to early senescence of preadipocytes from healthy first-degree relatives of type 2 diabetics.

Senescence of adipose precursor cells (APC) impairs adipogenesis, contributes to the age-related subcutaneous adipose tissue (SAT) dysfunction, and increases risk of type 2 diabetes (T2D). First-degree relatives of T2D individuals (FDR) feature restricted adipogenesis, reflecting the detrimental effects of APC senescence earlier in life and rendering FDR more vulnerable to T2D. Epigenetics may contribute to these abnormalities but the underlying mechanisms remain unclear. In previous methylome comparison in APC from FDR and individuals with no diabetes familiarity (CTRL), ZMAT3 emerged as one of the top-ranked senescence-related genes featuring hypomethylation in FDR and associated with T2D risk. Here, we investigated whether and how DNA methylation changes at ZMAT3 promote early APC senescence. APC from FDR individuals revealed increases in multiple senescence markers compared to CTRL. Senescence in these cells was accompanied by ZMAT3 hypomethylation, which caused ZMAT3 upregulation. Demethylation at this gene in CTRL APC led to increased ZMAT3 expression and premature senescence, which were reverted by ZMAT3 siRNA. Furthermore, ZMAT3 overexpression in APC determined senescence and activation of the p53/p21 pathway, as observed in FDR APC. Adipogenesis was also inhibited in ZMAT3-overexpressing APC. In FDR APC, rescue of ZMAT3 methylation through senolytic exposure simultaneously downregulated ZMAT3 expression and improved adipogenesis. Interestingly, in human SAT, aging and T2D were associated with significantly increased expression of both ZMAT3 and the P53 senescence marker. Thus, DNA hypomethylation causes ZMAT3 upregulation in FDR APC accompanied by acquisition of the senescence phenotype and impaired adipogenesis, which may contribute to FDR predisposition for T2D.

Altered PTPRD DNA methylation associates with restricted adipogenesis in healthy first-degree relatives of Type 2 diabetes subjects.

Aim: First-degree relatives (FDR) of individuals with Type 2 diabetes (T2D) feature restricted adipogenesis, which render them more vulnerable to T2D. Epigenetics may contribute to these abnormalities. Methods: FDR pre-adipocyte Methylome and Transcriptome were investigated by MeDIP- and RNA-Seq, respectively. Results:Methylome analysis revealed 2841 differentially methylated regions (DMR) in FDR. Most DMR localized into gene-body and were hypomethylated. The strongest hypomethylation signal was identified in an intronic-DMR at the PTPRD gene. PTPRD hypomethylation in FDR was confirmed by bisulphite sequencing and was responsible for its upregulation. Interestingly, Ptprd-overexpression in 3T3-L1 pre-adipocytes inhibited adipogenesis. Notably, the validated PTPRD-associated DMR was significantly hypomethylated in peripheral blood leukocytes from the same FDR individuals. Finally, PTPRD methylation pattern was also replicated in obese individuals. Conclusion: Our findings indicated a previously unrecognized role of PTPRD in restraining adipogenesis. This abnormality may contribute to increase FDR proclivity toward T2D.

KCTD1: A novel modulator of adipogenesis through the interaction with the transcription factor AP2α.

Adipogenesis has an important role in regulating energy balance, tissue homeostasis and disease pathogenesis. 3T3-L1 preadipocytes have been widely used as an in vitro model for studying adipocyte differentiation. We here show that KCTD1, a member of the potassium channel containing tetramerization domain proteins, plays an active role in adipogenesis. In particular, we show KCTD1 expression 3T3-L1 cells increases upon adipogenesis induction. Treatment of 3T3-L1 preadipocytes with Kctd1-specific siRNA inhibited the differentiation, as indicated by reduction of expression of the specific adipogenic markers C/ebpα, Pparγ2, Glut4, and Adiponectin. Moreover, we also show that the protein physically interacts with the transcription factor AP2α, a known inhibitor of adipogenesis, both in vitro and in cells. Interestingly, our data indicate that KCTD1 promotes adipogenesis through the interaction with AP2α and by removing it from the nucleus. Collectively, these findings disclose a novel role for KCTD1 and pave the way for novel strategies aimed at modulating adipogenesis.

Methylglyoxal accumulation de-regulates HoxA5 expression, thereby impairing angiogenesis in glyoxalase 1 knock-down mouse aortic endothelial cells.

Impaired angiogenesis leads to long-term complications and is a major contributor of the high morbidity in patients with Diabetes Mellitus (DM). Methylglyoxal (MGO) is a glycolysis byproduct that accumulates in DM and is detoxified by the Glyoxalase 1 (Glo1). Several studies suggest that MGO contributes to vascular complications through mechanisms that remain to be elucidated. In this study we have clarified for the first time the molecular mechanism involved in the impairment of angiogenesis induced by MGO accumulation. Angiogenesis was evaluated in mouse aortic endothelial cells isolated from Glo1-knockdown mice (Glo1KD MAECs) and their wild-type littermates (WT MAECs). Reduction in Glo1 expression led to an accumulation of MGO and MGO-modified proteins and impaired angiogenesis of Glo1KD MAECs. Both mRNA and protein levels of the anti-angiogenic HoxA5 gene were increased in Glo1KD MAECs and its silencing improved both their migration and invasion. Nuclear NF-ĸB-p65 was increased 2.5-fold in the Glo1KD as compared to WT MAECs. Interestingly, NF-ĸB-p65 binding to HoxA5 promoter was also 2-fold higher in Glo1KD MAECs and positively regulated HoxA5 expression in MAECs. Consistent with these data, both the exposure to a chemical inhibitor of Glo1 "SpBrBzGSHCp2" (GI) and to exogenous MGO led to the impairment of migration and the increase of HoxA5 mRNA and NF-ĸB-p65 protein levels in microvascular mouse coronary endothelial cells (MCECs). This study demonstrates, for the first time, that MGO accumulation increases the antiangiogenic factor HoxA5 via NF-ĸB-p65, thereby impairing the angiogenic ability of endothelial cells.

Epigenetic silencing of the ANKRD26 gene correlates to the pro-inflammatory profile and increased cardio-metabolic risk factors in human obesity.

BACKGROUND: Obesity is a major worldwide threat to human health. Increasing evidence indicates that epigenetic modifications have a major impact on the natural history of this disorder. Ankyrin Repeat Domain 26 (Ankrd26) is involved in the development of both obesity and diabetes in mice and is modulated by environmentally induced epigenetic modifications. This study aims at investigating whether impaired ANKRD26 gene expression and methylation occur in human obesity and whether they correlate to the phenotype of these subjects. RESULTS: We found that downregulation of ANKRD26 mRNA and hyper-methylation of a specific region of the ANKRD26 promoter, embedding the CpG dinucleotides - 689, - 659, and - 651 bp, occur in peripheral blood leukocytes from obese compared with the lean subjects. ANKRD26 gene expression correlates inversely to the percentage of DNA methylation at these 3 CpG sites. Luciferase assays reveal a cause-effect relationship between DNA methylation at the 3 CpG sites and ANKRD26 gene expression. Finally, both ANKRD26 mRNA levels and CpG methylation correlate to body mass index and to the pro-inflammatory status and the increased cardio-metabolic risk factors of these same subjects. CONCLUSION: Downregulation of the ANKRD26 gene and hyper-methylation at specific CpGs of its promoter are common abnormalities in obese patients. These changes correlate to the pro-inflammatory profile and the cardio-metabolic risk factors of the obese individuals, indicating that, in humans, they mark adverse health outcomes.