Deleting the ribosomal prolyl hydroxylase OGFOD1 protects mice against diet-induced obesity and insulin resistance.

Type 2 diabetes predisposes patients to heart disease, which is the primary cause of death across the globe. Type 2 diabetes often accompanies obesity and is defined by insulin resistance and abnormal glucose handling. Insulin resistance impairs glucose uptake and results in hyperglycemia, which damages tissues such as kidneys, liver, and heart. 2-oxoglutarate (2-OG)- and iron-dependent oxygenases (2-OGDOs), a family of enzymes regulating various aspects of cellular physiology, have been studied for their role in obesity and diet-induced insulin resistance. However, nothing is known of the 2-OGDO family member 2-oxoglutarate and iron-dependent prolyl hydroxylase domain containing protein 1 (OGFOD1) in this setting. OGFOD1 deletion leads to protection in cardiac ischemia-reperfusion injury and cardiac hypertrophy, which are two cardiac events that can lead to heart failure. Considering the remarkable correlation between heart disease and diabetes, the cardioprotection observed in OGFOD1-knockout mice led us to challenge these knockouts with high-fat diet. Wildtype mice fed a high-fat diet developed diet-induced obesity, insulin resistance, and glucose intolerance, but OGFOD1 knockout mice fed this same diet were resistant to diet-induced obesity and insulin resistance. These results support OGFOD1 down-regulation as a strategy for preventing obesity and insulin handling defects.

Liver-specific deletion of de novo DNA methyltransferases protects against glucose intolerance in high-fat diet-fed male mice.

Alterations to gene transcription and DNA methylation are a feature of many liver diseases including fatty liver disease and liver cancer. However, it is unclear whether the DNA methylation changes are a cause or a consequence of the transcriptional changes. It is even possible that the methylation changes are not required for the transcriptional changes. If DNA methylation is just a minor player in, or a consequence of liver transcriptional change, then future studies in this area should focus on other systems such as histone tail modifications. To interrogate the importance of de novo DNA methylation, we generated mice that are homozygous mutants for both Dnmt3a and Dnmt3b in post-natal liver. These mice are viable and fertile with normal sized livers. Males, but not females, showed increased adipose depots, yet paradoxically, improved glucose tolerance on both control diet and high-fat diets (HFD). Comparison of the transcriptome and methylome with RNA sequencing and whole-genome bisulfite sequencing in adult hepatocytes revealed that widespread loss of methylation in CpG-rich regions in the mutant did not induce loss of homeostatic transcriptional regulation. Similarly, extensive transcriptional changes induced by HFD did not require de novo DNA methylation. The improved metabolic phenotype of the Dnmt3a/3b mutant mice may be mediated through the dysregulation of a subset of glucose and fat metabolism genes which increase both glucose uptake and lipid export by the liver. However, further work is needed to confirm this.

Genetics and diet shape the relationship between islet function and whole body metabolism.

Despite the fact that genes and the environment are known to play a central role in islet function, our knowledge of how these parameters interact to modulate insulin secretory function remains relatively poor. Presently, we performed ex vivo glucose-stimulated insulin secretion and insulin content assays in islets of 213 mice from 13 inbred mouse strains on chow, Western diet (WD), and a high-fat, carbohydrate-free (KETO) diet. Strikingly, among these 13 strains, islets from the commonly used C57BL/6J mouse strain were the least glucose responsive. Using matched metabolic phenotyping data, we performed correlation analyses of isolated islet parameters and found a positive correlation between basal and glucose-stimulated insulin secretion, but no relationship between insulin secretion and insulin content. Using in vivo metabolic measures, we found that glucose tolerance determines the relationship between ex vivo islet insulin secretion and plasma insulin levels. Finally, we showed that islet glucose-stimulated insulin secretion decreased with KETO in almost all strains, concomitant with broader phenotypic changes, such as increased adiposity and glucose intolerance. This is an important finding as it should caution against the application of KETO diet for beta-cell health. Together these data offer key insights into the intersection of diet and genetic background on islet function and whole body glucose metabolism.NEW & NOTEWORTHY Thirteen strains of mice on chow, Western diet, and high-fat, carbohydrate-free (KETO), correlating whole body phenotypes to ex vivo pancreatic islet functional measurements, were used. The study finds a huge spectrum of functional islet responses and insulin phenotypes across all strains and diets, with the ubiquitous C57Bl/6J mouse exhibiting the lowest secretory response of all strains, highlighting the overall importance of considering genetic background when investigating islet function. Ex vivo basal and stimulated insulin secretion are correlated in the islet, and KETO imparts widescale downregulation of islet insulin secretion.

Osteocalcin improves glucose tolerance, insulin sensitivity and secretion in older male mice.

Osteocalcin deficient mice (OC-/-), on a mixed 129/BL6J background, were reported to show glucose intolerance, insulin insensitivity and reduced insulin secretion at 1-6 mos of age. This is controversial as two studies in OC-/- mice on different backgrounds (C3H/BL6 (5-6 mos.) and C57BL/6N (5 and 9 mos.)) found no effect on glucose metabolism. To determine the role of OC in glucose metabolism we conducted glucose tolerance tests (GTT), insulin tolerances tests (ITT) and glucose stimulated insulin secretion (GSIS) on 6 and 9.5 month-old male OC-/- and OC+/+ mice on a pure C57BL/6J background and fed a normal chow diet. All results were analyzed with a two-way repeated measures ANOVA. The GTT results showed no effect on males at 6 months of age but glucose intolerance was significantly increased (p < 0.05) in male OC-/- mice at 9.5 months of age. The ITT results indicated significantly increased insulin resistance in male OC-/- mice. Glucose stimulated insulin secretion (GSIS) showed insulin significantly (p < 0.05) reduced in OC-/- at several time points. Mouse Osteocalcin injected into OC-/- mice decreased the glucose level. Our results confirm the role of OC in glucose metabolism and insulin sensitivity and demonstrate a role in insulin secretion in older male mice on a C57BL/6J background. Differences in background, age, or experimental procedures could explain controversial results. A delayed onset of the effect of OC on glucose metabolism at 9.5 months in male C57BL/6J mice highlights the importance of background on phenotype. Consideration of genetic background and age may be beneficial for human studies on osteocalcin and glucose homeostasis and may be relevant to the elderly where osteocalcin is reduced.

Altered expression of long noncoding RNA MEG3 in the offspring of gestational diabetes mellitus induces impaired glucose tolerance in adulthood.

AIM: Gestational diabetes mellitus (GDM) affects a significant number of women worldwide and has been associated with lifelong health consequences for their offspring, including increased susceptibility to obesity, insulin resistance, and type II diabetes. Recent studies have suggested that aberrant expression of the long non-coding RNA Meg3 in the liver may contribute to impaired glucose metabolism in individuals. In this study, we aimed to investigate whether intrauterine exposure to hyperglycemia affects glucose intolerance in puberty by mediating the overexpression of LncMeg3 in the liver. METHODS: To test our hypothesis, we established an animal model of intrauterine hyperglycemia to mimic GDM. The progeny was observed for phenotypic changes, and intraperitoneal glucose tolerance tests, insulin tolerance tests, and pyruvate tolerance tests were conducted to assess glucose and insulin tolerance. We also measured LncMeg3 expression in the liver using real-time quantitative PCR and examined differential methylation areas (DMRs) in the Meg3 gene using pyrophosphoric sequencing. To investigate the role of LncMeg3 in glucose tolerance, we conducted Meg3 intervention by vein tail and analyzed the changes in the phenotype and transcriptome of the progeny using bioinformatics analysis. RESULTS: We found that intrauterine exposure to hyperglycemia led to impaired glucose and insulin tolerance in the progeny, with a tendency toward increased fasting blood glucose in fat offspring at 16 weeks (P = 0.0004). LncMeg3 expression was significantly upregulated (P = 0.0061), DNMT3B expression downregulated (P = 0.0226), and DNMT3A (P = 0.0026), TET2 (P = 0.0180) expression upregulated in the liver. Pyrophosphoric sequencing showed hypomethylation in Meg3-DMRs (P = 0.0005). Meg3 intervention by vein tail led to a decrease in the percentage of obese and emaciated offspring (emaciation: 44% vs. 23%; obesity: 25% vs. 15%) and attenuated glucose intolerance. Bioinformatics analysis revealed significant differences in the transcriptome of the progeny, particularly in circadian rhythm and PPAR signaling pathways. CONCLUSION: In conclusion, our study suggests that hypomethylation of Meg3-DMRs increases the expression of the imprinted gene Meg3 in the liver of males, which is associated with impaired glucose tolerance in GDM-F1. MEG3 interference may attenuate glucose intolerance, which may be related to transcriptional changes. Our findings provide new insights into the mechanisms underlying the long-term effects of intrauterine hyperglycemia on progeny health and highlight the potential of Meg3 as an intervention target for glucose intolerance.

Overexpression of UBE2E2 in Mouse Pancreatic ß-Cells Leads to Glucose Intolerance via Reduction of ß-Cell Mass.

Genome-wide association studies have identified several gene polymorphisms, including UBE2E2, associated with type 2 diabetes. Although UBE2E2 is one of the ubiquitin-conjugating enzymes involved in the process of ubiquitin modifications, the pathophysiological roles of UBE2E2 in metabolic dysfunction are not yet understood. Here, we showed upregulated UBE2E2 expression in the islets of a mouse model of diet-induced obesity. The diabetes risk allele of UBE2E2 (rs13094957) in noncoding regions was associated with upregulation of UBE2E2 mRNA in the human pancreas. Although glucose-stimulated insulin secretion was intact in the isolated islets, pancreatic ß-cell-specific UBE2E2-transgenic (TG) mice exhibited reduced insulin secretion and decreased ß-cell mass. In TG mice, suppressed proliferation of ß-cells before the weaning period and while receiving a high-fat diet was accompanied by elevated gene expression levels of p21, resulting in decreased postnatal ß-cell mass expansion and compensatory ß-cell hyperplasia, respectively. In TG islets, proteomic analysis identified enhanced formation of various types of polyubiquitin chains, accompanied by increased expression of Nedd4 E3 ubiquitin protein ligase. Ubiquitination assays showed that UBE2E2 mediated the elongation of ubiquitin chains by Nedd4. The data suggest that UBE2E2-mediated ubiquitin modifications in ß-cells play an important role in regulating glucose homeostasis and ß-cell mass.

Periportal hepatocyte proliferation at midgestation governs maternal glucose homeostasis in mice.

The maternal liver is challenged by metabolic demands throughout pregnancy. However, hepatocyte dynamics and their physiological significance in pregnancy remain unclear. Here, we show in mice that hepatocyte proliferation is spatiotemporally regulated in each liver lobular zone during pregnancy, with transient proliferation of periportal and pericentral hepatocytes during mid and late gestation, respectively. Using adeno-associated virus (AAV)-8-mediated expression of the cell cycle inhibitor p21 in hepatocytes, we show that inhibition of hepatocyte proliferation during mid, but not late, gestation impairs liver growth. Transcriptionally, genes involved in glucose/glycogen metabolism are downregulated in late pregnancy when midgestational hepatocyte proliferation is attenuated. In addition, hepatic glycogen storage is abolished, with concomitant elevated blood glucose concentrations, glucose intolerance, placental glycogen deposition, and fetal overgrowth. Laser capture microdissection and RNA-seq analysis of each liver lobular zone show zone-specific changes in the transcriptome during pregnancy and identify genes that are periportally expressed at midgestation, including the hyaluronan-mediated motility receptor (Hmmr). Knockdown of Hmmr in hepatocytes by AAV8-shHmmr suppresses periportal hepatocyte proliferation at midgestation and induces impaired hepatic glycogen storage, glucose intolerance, placental glycogen deposition and fetal overgrowth. Our results suggest that periportal hepatocyte proliferation during midgestation is critical for maternal glycogen metabolism and fetal size.

Adipocyte Subpopulations Mediate Growth Hormone-induced Lipolysis and Glucose Tolerance in Male Mice.

In adipose tissue, growth hormone (GH) stimulates lipolysis, leading to an increase in plasma free fatty acid levels and a reduction in insulin sensitivity. In our previous studies, we have found that GH increases lipolysis by reducing peroxisome proliferator-activated receptor γ (PPARγ) transcription activity, leading to a reduction of tat-specific protein 27 (FSP27, also known as CIDEC) expression. In previous studies, our laboratory uncovered 3 developmentally distinct subpopulations of white adipocytes. In this manuscript, we show that one of the subpopulations, termed type 2 adipocytes, has increased GH-induced signaling and lipolysis compared to other adipocyte subtypes. To assess the physiological role of GH-mediated lipolysis mediated by this adipocyte subpopulation, we specifically expressed human FSP27 (hFSP27) transgene in type 2 adipocytes (type2Ad-hFSP27tg mice). Systemically, male type2Ad-hFSP27tg mice displayed reduced serum glycerol release and nonesterified fatty acids levels after acute GH treatment, and improvement in acute, but not chronic, GH-induced glucose intolerance. Furthermore, we demonstrate that type2Ad-hFSP27tg mice displayed improved hepatic insulin signaling. Taken together, these results indicate that this adipocyte subpopulation is a critical regulator of the GH-mediated lipolytic and metabolic response. Thus, further investigation of adipocyte subpopulations may provide novel treatment strategies to regulate GH-induced glucose intolerance in patients with growth and metabolic disorders.

Akkermansia muciniphila supplementation improves glucose tolerance in intestinal Ffar4 knockout mice during the daily light to dark transition.

IMPORTANCE: Alterations in the intestinal environment are associated with various diseases, and FFAR4 is abundantly enriched in the intestine, where it has been shown to have the ability to regulate intestinal hormone secretion and intestinal microbiota; here, we confirmed previous reports. Meanwhile, we found that intestinal FFAR4 regulates glucagon-like peptide 1 secretion by decreasing Akkermansia muciniphila abundance and show that such change is associated with the level of glucose utilization at ZT12 in mice. Intestinal FFAR4 deficiency leads to severely impaired glucose tolerance at the ZT12 moment in mice, and Akkermansia muciniphila supplementation ameliorates the abnormal glucose utilization at the ZT12 moment caused by FFAR4 deficiency, which is very similar to the dawn phenomenon in diabetic patients. Collectively, our data suggest that intestinal Ffar4 deteriorates glucose tolerance at the daily light to dark transition by affecting Akkermansia muciniphila.

Changes in subcutaneous white adipose tissue cellular composition and molecular programs underlie glucose intolerance in persons with HIV.

Introduction: Subcutaneous adipose tissue (SAT) is a critical regulator of systemic metabolic homeostasis. Persons with HIV (PWH) have an increased risk of metabolic diseases and significant alterations in the SAT immune environment compared with the general population. Methods: We generated a comprehensive single-cell multi-omic SAT atlas to characterize cellular compositional and transcriptional changes in 59 PWH across a spectrum of metabolic health. Results: Glucose intolerance was associated with increased lipid-associated macrophages, CD4+ and CD8+ T effector memory cells, and decreased perivascular macrophages. We observed a coordinated intercellular regulatory program which enriched for genes related to inflammation and lipid-processing across multiple cell types as glucose intolerance increased. Increased CD4+ effector memory tissue-resident cells most strongly associated with altered expression of adipocyte genes critical for lipid metabolism and cellular regulation. Intercellular communication analysis demonstrated enhanced pro-inflammatory and pro-fibrotic signaling between immune cells and stromal cells in PWH with glucose intolerance compared with non-diabetic PWH. Lastly, while cell type-specific gene expression among PWH with diabetes was globally similar to HIV-negative individuals with diabetes, we observed substantially divergent intercellular communication pathways. Discussion: These findings suggest a central role of tissue-resident immune cells in regulating SAT inflammation among PWH with metabolic disease, and underscore unique mechanisms that may converge to promote metabolic disease.

Deletion of complement factor 5 amplifies glucose intolerance in obese male but not female mice.

Complement factor 5 of the innate immune system generates C5a and C5b ligands, which initiate inflammatory and cell lysis events, respectively. C5 activation has been linked with obesity-associated metabolic disorders; however, whether it has a causative role is unclear. We generated a C5 null (C5-/-) mouse using CRISPR-Cas9 gene editing to determine whether loss of C5 improves obesity-linked metabolic dysfunction. Generation of a new mouse model was prompted in part by the observation of off-target gene mutations in commercially available C5-/- lines. Male and female wild-type (WT), heterozygous (Het), and C5-/- mice were fed low-fat diet (LFD) or high-fat diet (HFD) for 22 wk. Body weight gain did not differ between genotypes on LFD or HFD. In lean animals, male C5-/- mice had similar glucose tolerance compared with WT controls; however, in obese conditions, glucose tolerance was worsened in C5-/- compared with controls. In contrast, female mice did not exhibit differences in glucose tolerance between genotypes under either dietary paradigm. Fasting insulin was not different between genotypes, whereas diet-induced obese male C5-/- mice had lower fed insulin concentrations compared with WT controls. No differences in adipose tissue inflammation or adipocyte size were identified between groups. Similarly, susceptibility to fatty liver and hepatic inflammation was similar between WT and C5-/- mice. However, the systemic cytokine response to acute endotoxin exposure was decreased in C5-/- mice. Together, these data suggest that loss of C5 worsens glucose tolerance in obese male but not female mice. Additional work is required to pinpoint the mechanisms by which loss of C5 amplifies glucose intolerance in obesity.NEW & NOTEWORTHY We generated a new mouse model of complement factor 5 deficiency. This work was prompted by a need for improved transgenic mouse lines of C5, due to off-target gene mutations. We find that loss of C5 worsens glucose tolerance in a sex-dependent manner. Though the mechanisms evoking glucose intolerance are not clear, we are confident this model will be useful in interrogating complement activation in obesity-associated diseases.

Chronic Glucocorticoid Exposure Induced an S1PR2-RORγ Axis to Enhance Hepatic Gluconeogenesis in Male Mice.

It is well established that chronic glucocorticoid exposure causes hyperglycemia. While glucocorticoid receptor (GR) stimulates hepatic gluconeogenic gene transcription, additional mechanisms are activated by chronic glucocorticoid exposure to enhance gluconeogenesis. We found that chronic glucocorticoid treatment activated sphingosine-1-phosphate (S1P)-mediated signaling. Hepatic knockdown of hepatic S1P receptor 1 (S1PR1) had no effect on chronic glucocorticoid-induced glucose intolerance but elevated fasting plasma insulin levels. In contrast, hepatic S1PR3 knockdown exacerbated chronic glucocorticoid-induced glucose intolerance without affecting fasting plasma insulin levels. Finally, hepatic S1PR2 knockdown attenuated chronic glucocorticoid-induced glucose intolerance and reduced fasting plasma insulin levels. Here, we focused on dissecting the role of S1PR2 signaling in chronic glucocorticoid response on glucose homeostasis. We found that chronic glucocorticoid-induced hepatic gluconeogenesis, gluconeogenic gene expression, and GR recruitment to the glucocorticoid response elements (GREs) of gluconeogenic genes were all reduced in hepatic S1PR2 knockdown male mice. Hepatic S1PR2 knockdown also enhanced glucocorticoid suppression of RAR-related orphan receptor γ (RORγ) expression. Hepatic RORγ overexpression in hepatic S1PR2 knockdown mice restored glucocorticoid-induced glucose intolerance, gluconeogenic gene expression, and GR recruitment to their GREs. Conversely, RORγ antagonist and the reduction of hepatic RORγ expression attenuated such glucocorticoid effects. Thus, chronic glucocorticoid exposure induces an S1PR2-RORγ axis to cooperate with GR to enhance hepatic gluconeogenesis. Overall, this work provides novel mechanisms of and pharmaceutical targets against steroid-induced hyperglycemia.

Implantation of CPT1AM-expressing adipocytes reduces obesity and glucose intolerance in mice.

Obesity and its associated metabolic comorbidities are a rising global health and social issue, with novel therapeutic approaches urgently needed. Adipose tissue plays a key role in the regulation of energy balance and adipose tissue-derived mesenchymal stem cells (AT-MSCs) have gained great interest in cell therapy. Carnitine palmitoyltransferase 1A (CPT1A) is the gatekeeper enzyme for mitochondrial fatty acid oxidation. Here, we aimed to generate adipocytes expressing a constitutively active CPT1A form (CPT1AM) that can improve the obese phenotype in mice after their implantation. AT-MSCs were differentiated into mature adipocytes, subjected to lentivirus-mediated expression of CPT1AM or the GFP control, and subcutaneously implanted into mice fed a high-fat diet (HFD). CPT1AM-implanted mice showed lower body weight, hepatic steatosis and serum insulin and cholesterol levels alongside improved glucose tolerance. HFD-induced increases in adipose tissue hypertrophy, fibrosis, inflammation, endoplasmic reticulum stress and apoptosis were reduced in CPT1AM-implanted mice. In addition, the expression of mitochondrial respiratory chain complexes was enhanced in the adipose tissue of CPT1AM-implanted mice. Our results demonstrate that implantation of CPT1AM-expressing AT-MSC-derived adipocytes into HFD-fed mice improves the obese metabolic phenotype, supporting the future clinical use of this ex vivo gene therapy approach.

Short-term tamoxifen administration improves hepatic steatosis and glucose intolerance through JNK/MAPK in mice.

Nonalcoholic fatty liver disease (NAFLD) which is a leading cause of chronic liver diseases lacks effective treatment. Tamoxifen has been proven to be the first-line chemotherapy for several solid tumors in clinics, however, its therapeutic role in NAFLD has never been elucidated before. In vitro experiments, tamoxifen protected hepatocytes against sodium palmitate-induced lipotoxicity. In male and female mice fed with normal diets, continuous tamoxifen administration inhibited lipid accumulation in liver, and improved glucose and insulin intolerance. Short-term tamoxifen administration largely improved hepatic steatosis and insulin resistance, however, the phenotypes manifesting inflammation and fibrosis remained unchanged in abovementioned models. In addition, mRNA expressions of genes related to lipogenesis, inflammation, and fibrosis were downregulated by tamoxifen treatment. Moreover, the therapeutic effect of tamoxifen on NAFLD was not gender or ER dependent, as male and female mice with metabolic disorders shared no difference in response to tamoxifen and ER antagonist (fulvestrant) did not abolish its therapeutic effect as well. Mechanistically, RNA sequence of hepatocytes isolated from fatty liver revealed that JNK/MAPK signaling pathway was inactivated by tamoxifen. Pharmacological JNK activator (anisomycin) partially deprived the therapeutic role of tamoxifen in treating hepatic steatosis, proving tamoxifen improved NAFLD in a JNK/MAPK signaling-dependent manner.

Mouse Models of Gestational Diabetes Mellitus and Its Subtypes: Recent Insights and Pitfalls.

Gestational diabetes mellitus (GDM) is currently the most common complication of pregnancy and is defined as a glucose intolerance disorder with recognition during pregnancy. GDM is considered a uniform group of patients in conventional guidelines. In recent years, evidence of the disease's heterogeneity has led to a growing understanding of the value of dividing patients into different subpopulations. Furthermore, in view of the increasing incidence of hyperglycemia outside pregnancy, it is likely that many cases diagnosed as GDM are in fact patients with undiagnosed pre-pregnancy impaired glucose tolerance (IGT). Experimental models contribute significantly to the understanding of the pathogenesis of GDM and numerous animal models have been described in the literature. The aim of this review is to provide an overview of the existing mouse models of GDM, in particular those that have been obtained by genetic manipulation. However, these commonly used models have certain limitations in the study of the pathogenesis of GDM and cannot fully describe the heterogeneous spectrum of this polygenic disease. The polygenic New Zealand obese (NZO) mouse is introduced as a recently emerged model of a subpopulation of GDM. Although this strain lacks conventional GDM, it exhibits prediabetes and an IGT both preconceptionally and during gestation. In addition, it should be emphasized that the choice of an appropriate control strain is of great importance in metabolic studies. The commonly used control strain C57BL/6N, which exhibits IGT during gestation, is discussed in this review as a potential model of GDM.

Paternal sleep deprivation induces metabolic perturbations in male offspring via altered LRP5 DNA methylation of pancreatic islets.

Diabetes and metabolic perturbation are global health challenges. Sleep insufficiency may trigger metabolic dysregulation leading to diabetes. However, the intergenerational transmission of this environmental information is not clearly understood. The research objective was to determine the possible effect of paternal sleep deprivation on the metabolic phenotype of the offspring and to investigate the underlying mechanism of epigenetic inheritance. Male offspring of sleep-deprived fathers exhibit glucose intolerance, insulin resistance, and impaired insulin secretion. In these SD-F1 offspring, a reduction in beta cell mass and proliferation of beta cells were observed. Mechanistically, in pancreatic islets of SD-F1 offspring, we identified alterations in DNA methylation at the promoter region of the LRP5 (LDL receptor related protein 5) gene, a coreceptor of Wnt signaling, resulting in downregulation of downstream effectors cyclin D1, cyclin D2, and Ctnnb1. Restoration of Lrp5 in the pancreas of SD-F1 male mice could improve impaired glucose tolerance and expression of cyclin D1, cyclin D2, and Ctnnb1. This study might significantly contribute to our understanding of the effects of sleeplessness on health and metabolic disease risk from the perspective of the heritable epigenome.

Identification of FGF13 as a Potential Biomarker and Target for Diagnosis of Impaired Glucose Tolerance.

Early identification of pre-diabetes provides an opportunity for intervention and treatment to delay its progression to type 2 diabetes mellitus (T2DM). We aimed to identify the biomarkers of impaired glucose tolerance (IGT) through bioinformatics analysis. The GSE76896 dataset, including non-diabetic (ND), IGT, and T2DM clinical samples, was deeply analyzed to identify 309 Co-DEGs for IGT and T2DM. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses indicated that inflammatory responses and the PI3K-AKT signaling pathway are important patho-physiological features of IGT and T2DM. Protein-protein interaction (PPI) network analysis and cytoHubba technolgy identified seven hub genes: namely, CCL2, CXCL1, CXCL8, EDN1, FGF13, MMP1, and NGF. The expression and ROC curves of these hub genes were validated using the GSE38642 dataset. Through an immunofluorescence assay, we found that the expression of FGF13 in islets of mice in the HFD and T2DM groups was significantly lower than in the control group. Similarly, the level of FGF13 in the sera of IGT and T2DM patients was lower than that in the healthy group. Together, these results suggest that FGF13 can be treated as a novel biomarker of IGT, which may provide new targets for the diagnosis and treatment of pre-diabetes and T2DM.

miR-24-3p and Body Mass Index as Type 2 Diabetes Risk Factors in Spanish Women 15 Years after Gestational Diabetes Mellitus Diagnosis.

Gestational diabetes mellitus (GDM) is defined as any degree of glucose intolerance that is diagnosed for the first time during pregnancy. The objective of this study is to know the glucose tolerance status after 15 years of pregnancy in patients diagnosed with gestational diabetes and to assess the long-term effect of GDM on the circulating miRNA profile of these women. To answer these, 30 randomly selected women diagnosed with GDM during 2005-2006 were included in the study, and glucose tolerance was measured using the National Diabetes Data Group criteria. Additionally, four miRNAs (hsa-miR-1-3p, hsa-miR-24-3p, hsa-miR-329-3p, hsa-miR-543) were selected for their analysis in the plasma of women 15 years after the diagnosis of GDM. In our study we discovered that, fifteen years after the diagnosis of GDM, 50% of women have some degree of glucose intolerance directly related to body weight and body mass index during pregnancy. Dysglycemic women also showed a significantly increased level of circulating hsa-miR-24-3p. Thus, we can conclude that initial weight and BMI, together with circulating expression levels of hsa-miR-24-3p, could be good predictors of the future development of dysglycemia in women with a previous diagnosis of GDM.

The Efficacy of HGF/VEGF Gene Therapy for Limb Ischemia in Mice with Impaired Glucose Tolerance: Shift from Angiogenesis to Axonal Growth and Oxidative Potential in Skeletal Muscle.

BACKGROUND: Combined non-viral gene therapy (GT) of ischemia and cardiovascular disease is a promising tool for potential clinical translation. In previous studies our group has developed combined gene therapy by vascular endothelial growth factor 165 (VEGF165) + hepatocyte growth factor (HGF). Our recent works have demonstrated that a bicistronic pDNA that carries both human HGF and VEGF165 coding sequences has a potential for clinical application in peripheral artery disease (PAD). The present study aimed to test HGF/VEGF combined plasmid efficacy in ischemic skeletal muscle comorbid with predominant complications of PAD-impaired glucose tolerance and type 2 diabetes mellitus (T2DM). METHODS: Male C57BL mice were housed on low-fat (LFD) or high-fat diet (HFD) for 10 weeks and metabolic parameters including FBG level, ITT, and GTT were evaluated. Hindlimb ischemia induction and plasmid administration were performed at 10 weeks with 3 weeks for post-surgical follow-up. Limb blood flow was assessed by laser Doppler scanning at 7, 14, and 21 days after ischemia induction. The necrotic area of m.tibialis anterior, macrophage infiltration, angio- and neuritogenesis were evaluated in tissue sections. The mitochondrial status of skeletal muscle (total mitochondria content, ETC proteins content) was assessed by Western blotting of muscle lysates. RESULTS: At 10 weeks, the HFD group demonstrated impaired glucose tolerance in comparison with the LFD group. HGF/VEGF plasmid injection aggravated glucose intolerance in HFD conditions. Blood flow recovery was not changed by HGF/VEGF plasmid injection either in LFD or HFD conditions. GT in LFD, but not in HFD conditions, enlarged the necrotic area and CD68+ cells infiltration. However, HGF/VEGF plasmid enhanced neuritogenesis and enlarged NF200+ area on muscle sections. In HFD conditions, HGF/VEGF plasmid injection significantly increased mitochondria content and ETC proteins content. CONCLUSIONS: The current study demonstrated a significant role of dietary conditions in pre-clinical testing of non-viral GT drugs. HGF/VEGF combined plasmid demonstrated a novel aspect of potential participation in ischemic skeletal muscle regeneration, through regulation of innervation and bioenergetics of muscle. The obtained results made HGF/VEGF combined plasmid a very promising tool for PAD therapy in impaired glucose tolerance conditions.