Obesity-Linked PPARγ Ser273 Phosphorylation Promotes Beneficial Effects on the Liver, despite Reduced Insulin Sensitivity in Mice.

Since the removal of thiazolidinediones (TZDs) from the market, researchers have been exploring alternative anti-diabetic drugs that target PPARγ without causing adverse effects while promoting insulin sensitization by blocking serine 273 phosphorylation (Ser273 or S273). Nonetheless, the underlying mechanisms of the relationship between insulin resistance and S273 phosphorylation are still largely unknown, except for the involvement of growth differentiation factor (GDF3) regulation in the process. To further investigate potential pathways, we generated a whole organism knockin mouse line with a single S273A mutation (KI) that blocks the occurrence of its phosphorylation. Our observations of KI mice on different diets and feeding schedules revealed that they were hyperglycemic, hypoinsulinemic, presented more body fat at weaning, and presented an altered plasma and hepatic lipid profile, distinctive liver morphology and gene expression. These results suggest that total blockage of S273 phosphorylation may have unforeseen effects that, in addition to promoting insulin sensitivity, could lead to metabolic disturbances, particularly in the liver. Therefore, our findings demonstrate both the beneficial and detrimental effects of PPAR S273 phosphorylation and suggest selective modulation of this post translational modification is a viable strategy to treat type 2 diabetes.

Mass spectrometry-based proteomics of 3D cell culture: A useful tool to validate culture of spheroids and organoids.

Worldwide obesity, defined as abnormal or excessive fat accumulation that may result in different comorbidities, is considered a pandemic condition that has nearly tripled in the last 45 years. Most studies on obesity use animal models or adipocyte monolayer cell culture to investigate adipose tissue. However, besides monolayer cell culture approaches do not fully recapitulate the physiology of living organisms, there is a growing need to reduce or replace animals in research. In this context, the development of 3D self-organized structures has provided models that better reproduce the in vitro aspects of the in vivo physiology in comparison to traditional monolayer cell culture. Besides, recent advances in omics technologies have allowed us to characterize these cultures at the proteome, metabolome, transcription factor, DNA-binding and transcriptomic levels. These two combined approaches, 3D culture and omics, have provided more realistic data about determined conditions. Thereby, here we focused on the development of an obesity study pipeline including proteomic analysis to validate adipocyte-derived spheroids. Through the combination of collected mass spectrometry data from differentiated 3T3-L1 spheroids and from murine white adipose tissue (WAT), we identified 1732 proteins in both samples. By using a comprehensive proteomic analysis, we observed that the in vitro 3D culture of differentiated adipocytes shares important molecular pathways with the WAT, including expression of proteins involved in central metabolic process of the adipose tissue. Together, our results show a combination of an orthogonal method and an image-based analysis that constitutes a useful pipeline to be applied in 3D adipocyte culture.

Multifactorial Basis and Therapeutic Strategies in Metabolism-Related Diseases.

Throughout the 20th and 21st centuries, the incidence of non-communicable diseases (NCDs), also known as chronic diseases, has been increasing worldwide. Changes in dietary and physical activity patterns, along with genetic conditions, are the main factors that modulate the metabolism of individuals, leading to the development of NCDs. Obesity, diabetes, metabolic associated fatty liver disease (MAFLD), and cardiovascular diseases (CVDs) are classified in this group of chronic diseases. Therefore, understanding the underlying molecular mechanisms of these diseases leads us to develop more accurate and effective treatments to reduce or mitigate their prevalence in the population. Given the global relevance of NCDs and ongoing research progress, this article reviews the current understanding about NCDs and their related risk factors, with a focus on obesity, diabetes, MAFLD, and CVDs, summarizing the knowledge about their pathophysiology and highlighting the currently available and emerging therapeutic strategies, especially pharmacological interventions. All of these diseases play an important role in the contamination by the SARS-CoV-2 virus, as well as in the progression and severity of the symptoms of the coronavirus disease 2019 (COVID-19). Therefore, we briefly explore the relationship between NCDs and COVID-19.

Ventricular Fibrosis and Coronary Remodeling Following Short-Term Exposure of Healthy and Malnourished Mice to Bisphenol A.

Bisphenol-A (BPA) is an endocrine disruptor associated with higher risk of insulin resistance, type 2 diabetes, and cardiovascular diseases especially in susceptible populations. Because malnutrition is a nutritional disorder associated with high cardiovascular risk, we sought to compare the effects of short-term BPA exposure on cardiovascular parameters of healthy and protein-malnourished mice. Postweaned male mice were fed a normo- (control) or low-protein (LP) diet for 8 weeks and then exposed or not to BPA (50 µg kg-1 day-1) for the last 9 days. Systolic blood pressure was higher in BPA or LP groups compared with the control group. However, diastolic blood pressure was enhanced by BPA only in malnourished mice. Left ventricle (LV) end diastolic pressure (EDP), collagen deposition, and CTGF mRNA expression were higher in the control or malnourished mice exposed to BPA than in the respective nonexposed groups. Nevertheless, mice fed LP diet exposed to BPA exhibited higher angiotensinogen and cardiac TGF-ß1 mRNA expression than mice treated with LP or BPA alone. Wall:lumen ratio and cross-sectional area of intramyocardial arteries were higher either in the LP or BPA group compared with the control mice. Taken together, our data suggest that short-term BPA exposure results in LV diastolic dysfunction and fibrosis, and intramyocardial arteries inward remodeling, besides potentiate protein malnutrition-induced hypertension and cardiovascular risk.

Sexual hormones and diabetes: The impact of estradiol in pancreatic ß cell.

Diabetes is one of the most prevalent metabolic diseases and its incidence is increasing throughout the world. Data from World Health Organization (WHO) point-out that diabetes is a major cause of blindness, kidney failure, heart attacks, stroke and lower limb amputation and estimated 1.6 million deaths were directly caused by it in 2016. Population studies show that the incidence of this disease increases in women after menopause, when the production of estrogen is decreasing in them. Knowing the impact that estrogenic signaling has on insulin-secreting ß cells is key to prevention and design of new therapeutic targets. This chapter explores the role of estrogen and their receptors in the regulation of insulin secretion and biosynthesis, proliferation, regeneration and survival in pancreatic ß cells. In addition, delves into the genetic animal models developed and its application for the specific study of the different estrogen signaling pathways. Finally, discusses the impact of menopause and hormone replacement therapy on pancreatic ß cell function.

Diabetogenic effect of pravastatin is associated with insulin resistance and myotoxicity in hypercholesterolemic mice.

BACKGROUND: HMG-CoA reductase inhibitors (statins) are cholesterol-lowering drugs widely used to treat hypercholesterolemia and prevent cardiovascular disease. Statins are generally well tolerated, but adverse reactions may occur, particularly myopathy and new onset of diabetes. The exact mechanism of statin-induced myopathy and diabetes has not been fully elucidated. We have previously shown that treatment of hypercholesterolemic (LDLr-/-) mice with pravastatin for 2 months decreased pancreatic islet insulin secretion and increased oxidative stress and cell death, but no glucose intolerance was observed. The purpose of the current work was to study long-term pravastatin effects on glucose homeostasis, insulin sensitivity, muscle protein turnover and cell viability. METHODS: LDLr-/- mice were treated with pravastatin for 3, 6 and 10 months. Glucose tolerance, insulin resistance and glucose-stimulated insulin secretion were evaluated. The rates of protein synthesis and degradation were determined in gastrocnemius muscle after 10 months of treatment. Insulin signalling, oxidative stress and cell death were analysed in vitro using C2C12 myotubes. RESULTS: After 6 and 10 months of treatment, these mice became glucose intolerant, and after 10 months, they exhibited marked insulin resistance. Reduced islet glucose-stimulated insulin secretion was observed after the 3rd month of treatment. Mice treated for 10 months showed significantly decreased body weight and increased muscle protein degradation. In addition, muscle chymotrypsin-like proteasomal activity and lysosomal cathepsin were markedly elevated. C2C12 myotubes exposed to increasing concentrations of pravastatin presented dose-dependent impairment of insulin-induced Akt phosphorylation, increased apoptotic markers (Bax protein and cleaved caspase-3) and augmented superoxide anion production. CONCLUSIONS: In addition to reduced insulin secretion, long-term pravastatin treatment induces insulin resistance and muscle wasting. These results suggest that the diabetogenic effect of statins is linked to the appearance of myotoxicity induced by oxidative stress, impaired insulin signalling, proteolysis and apoptosis.

In utero exposure to bisphenol-A disrupts key elements of retinoid system in male mice offspring.

The retinoid system controls essential cellular processes including mitosis, differentiation and metabolism among others. Although the retinoid-signalling pathway is a potential target for the action of several endocrine disrupting chemicals (EDCs), the information about the developmental effects of bisphenol-A (BPA) on the hepatic retinoid system is scarce. Herein, male mice were in utero exposed to BPA following maternal subcutaneous doses of 0, 10 and 100 µg/kg bw/day from gestational day 9-16 and they were sacrificed at post-natal day 30. Retinoid concentrations and gene expression of key elements involved in the retinoid system were determined in liver. BPA increased all-trans-retinoic acid concentration and expression of Adh1, Aox1 and Cyp1a2 (biosynthesis of retinoic acid), while reduced Mrp3 (efflux from hepatocyte to blood), increased Bcrp expression (biliary excretion) and changed the retinoid-dependent signalling system after reducing expression of Rxrß and increasing that of Fgf21. Furthermore, we found bivariate associations of Rarγ and Rxrγ expressions with all-trans-retinoic acid concentrations and of Fgf21 expression with that of Rarγ. Those findings occurred in animals which showed altered pancreatic function and impaired glucose metabolism during adulthood. The present information should be useful for enhancing testing methods for the identification of EDCs.

Coenzyme Q10 protects against ß-cell toxicity induced by pravastatin treatment of hypercholesterolemia.

New onset of diabetes is associated with the use of statins. We have recently demonstrated that pravastatin-treated hypercholesterolemic LDL receptor knockout (LDLr-/- ) mice exhibit reductions in insulin secretion and increased islet cell death and oxidative stress. Here, we hypothesized that these diabetogenic effects of pravastatin could be counteracted by treatment with the antioxidant coenzyme Q 10 (CoQ 10 ), an intermediate generated in the cholesterol synthesis pathway. LDLr -/- mice were treated with pravastatin and/or CoQ 10 for 2 months. Pravastatin treatment resulted in a 75% decrease of liver CoQ 10 content. Dietary CoQ 10 supplementation of pravastatin-treated mice reversed fasting hyperglycemia, improved glucose tolerance (20%) and insulin sensitivity (>2-fold), and fully restored islet glucose-stimulated insulin secretion impaired by pravastatin (40%). Pravastatin had no effect on insulin secretion of wild-type mice. In vitro, insulin-secreting INS1E cells cotreated with CoQ 10 were protected from cell death and oxidative stress induced by pravastatin. Simvastatin and atorvastatin were more potent in inducing dose-dependent INS1E cell death (10-15-fold), which were also attenuated by CoQ 10 cotreatment. Together, these results demonstrate that statins impair ß-cell redox balance, function and viability. However, CoQ 10 supplementation can protect the statins detrimental effects on the endocrine pancreas.

Diet-induced glucose homeostasis dysregulation is enhanced by taurine supplementation in ovariectomized mice.

Low levels of estrogens are associated with obesity-related comorbidities. Mice with lower levels of estrogens are thereby more sensitive to the effects of a high-fat-diet (HFD) for the development of glucose intolerance and insulin resistance. Studies in vivo have demonstrated that taurine (TAU) supplementation prevents glucose and insulin resistance. Thus, we aimed to investigate the potential beneficial effects of TAU supplementation on glucose homeostasis of mice with low levels of estrogens fed with a HFD. 3-month-old female C57BL/6J mice underwent bilateral ovariectomy (OVX). After 1 week of recovery, mice were divided into 4 groups and either received: a standard chow diet (OVXC), chow diet plus drinking water enriched with 3% of TAU (OVXCT), HFD (OVXH), and HFD plus supplementation of TAU (OVXHT) for 14 weeks. Exposure to the HFD increased adiposity and plasma levels of glucose and insulin. Contrary to our prediction, the addition of TAU enhanced the deleterious effects of the HFD. Glucose and insulin tolerance tests (ipGTT and ipITT) indicated that mice maintained on the HFD + TAU had worse glucose intolerance and insulin resistance that was linked to lower insulin signaling in skeletal muscle and liver. Insulin secretion of isolated pancreatic islets of OVXH mice was higher than OVXC, and the addition of TAU associated with a HFD did not modulate insulin secretion, suggesting a failure of pancreatic ß cells of OVXHT mice. These results suggest that despite the beneficial reports of TAU, it should be used cautiously in situations where the levels of estrogens are low.

Maternal Exposure to Bisphenol-A During Pregnancy Increases Pancreatic ß-Cell Growth During Early Life in Male Mice Offspring.

Alterations during development of metabolic key organs such as the endocrine pancreas affect the phenotype later in life. There is evidence that in utero or perinatal exposure to bisphenol-A (BPA) leads to impaired glucose metabolism during adulthood. However, how BPA exposure during pregnancy affects pancreatic ß-cell growth and function in offspring during early life has not been explored. We exposed pregnant mice to either vehicle (control) or BPA (10 and 100 µg/kg·d, BPA10 and BPA100) and examined offspring on postnatal days (P) P0, P21, P30, and P120. BPA10 and BPA100 mice presented lower birth weight than control and subsequently gained weight until day 30. At that age, concentration of plasma insulin, C-peptide, and leptin were increased in BPA-exposed animals in the nonfasting state. Insulin secretion and content were diminished in BPA10 and maintained in BPA100 compared with control. A global gene expression analysis indicated that genes related with cell division were increased in islets from BPA-treated animals. This was associated with an increase in pancreatic ß-cell mass at P0, P21, and P30 together with increased ß-cell proliferation and decreased apoptosis. On the contrary, at P120, BPA-treated animals presented either equal or decreased ß-cell mass compared with control and altered fasting glucose levels. These data suggest that in utero exposure to environmentally relevant doses of BPA alters the expression of genes involved in ß-cell growth regulation, incrementing ß-cell mass/area, and ß-cell proliferation during early life. An excess of insulin signaling during early life may contribute to impaired glucose tolerance during adulthood.

Pancreatic alpha-cells from female mice undergo morphofunctional changes during compensatory adaptations of the endocrine pancreas to diet-induced obesity.

Obesity is frequently associated with insulin resistance. To compensate for this situation and maintain normoglycaemia, pancreatic beta-cells undergo several morphofunctional adaptations, which result in insulin hypersecretion and hyperinsulinaemia. However, no information exists about pancreatic alpha-cells during this compensatory stage of obesity. Here, we studied alpha-cells in mice fed a high-fat diet (HFD) for 12 weeks. These animals exhibited hyperinsulinaemia and normoglycaemia compared with control animals in addition to hypoglucagonaemia. While the in vivo response of glucagon to hypoglycaemia was preserved in the obese mice, the suppression of glucagon secretion during hyperglycaemia was impaired. Additionally, in vitro glucagon release at low glucose levels and glucagon content in isolated islets were decreased, while alpha-cell exocytosis remained unchanged. Assessment of morphological parameters revealed that alpha-cell area was reduced in the pancreas of the obese mice in association with alpha-cell hypotrophy, increased apoptosis and decreased proliferation. HFD feeding for 24 weeks led to significant deterioration in beta-cell function and glucose homeostasis. Under these conditions, the majority of alpha-cell changes were reversed and became comparable to controls. These findings indicate that pancreatic compensatory adaptations during obesity may also involve pancreatic alpha-cells. Additionally, defects in alpha-cell function during obesity may be implicated in progression to diabetes.

Bisphenol-A treatment during pregnancy in mice: a new window of susceptibility for the development of diabetes in mothers later in life.

Evidence now exists supporting the hypothesis that endocrine-disrupting chemicals (EDCs) can harmfully impact glucose metabolism. Thus, EDCs are beginning to be considered important contributors to the increased incidence of diabetes, obesity, or both. The possible effect of exposure to EDCs during pregnancy on glucose homeostasis in mothers later in life is presently unknown. Here we show that several months after delivery, mothers treated with the widespread EDC bisphenol-A (BPA) during gestation, at environmentally relevant doses, exhibit profound glucose intolerance and altered insulin sensitivity as well as increased body weight. These mice presented a decreased insulin secretion both in vivo and in vitro together with reduced pancreatic ß-cell mass. The proliferation capacity was decreased in association with a diminished expression of the cell cycle activators: cyclin D2 and cyclin-dependent kinase-4. In addition, the rate of ß-cells apoptosis was increased as well as the expression of the cell cycle inhibitors p16 and p53. Conversely, no effects on glucose metabolism or insulin sensitivity were observed when female nonpregnant mice were treated with BPA at the same doses. Taken together, these findings reveal that BPA exposure during gestation has harmful long-term implications in glucose metabolism for the mother. This finding highlights a new window of susceptibility for EDC exposure that may be important for the development of type 2 diabetes.

Exposure to bisphenol-A during pregnancy partially mimics the effects of a high-fat diet altering glucose homeostasis and gene expression in adult male mice.

Bisphenol-A (BPA) is one of the most widespread EDCs used as a base compound in the manufacture of polycarbonate plastics. The aim of our research has been to study how the exposure to BPA during pregnancy affects weight, glucose homeostasis, pancreatic ß-cell function and gene expression in the major peripheral organs that control energy flux: white adipose tissue (WAT), the liver and skeletal muscle, in male offspring 17 and 28 weeks old. Pregnant mice were treated with a subcutaneous injection of 10 µg/kg/day of BPA or a vehicle from day 9 to 16 of pregnancy. One month old offspring were divided into four different groups: vehicle treated mice that ate a normal chow diet (Control group); BPA treated mice that also ate a normal chow diet (BPA); vehicle treated animals that had a high fat diet (HFD) and BPA treated animals that were fed HFD (HFD-BPA). The BPA group started to gain weight at 18 weeks old and caught up to the HFD group before week 28. The BPA group as well as the HFD and HFD-BPA ones presented fasting hyperglycemia, glucose intolerance and high levels of non-esterified fatty acids (NEFA) in plasma compared with the Control one. Glucose stimulated insulin release was disrupted, particularly in the HFD-BPA group. In WAT, the mRNA expression of the genes involved in fatty acid metabolism, Srebpc1, Pparα and Cpt1ß was decreased by BPA to the same extent as with the HFD treatment. BPA treatment upregulated Pparγ and Prkaa1 genes in the liver; yet it diminished the expression of Cd36. Hepatic triglyceride levels were increased in all groups compared to control. In conclusion, male offspring from BPA-treated mothers presented symptoms of diabesity. This term refers to a form of diabetes which typically develops in later life and is associated with obesity.

Antidiabetic actions of an estrogen receptor ß selective agonist.

The estrogen receptor ß (ERß) is emerging as an important player in the physiology of the endocrine pancreas. We evaluated the role and antidiabetic actions of the ERß selective agonist WAY200070 as an insulinotropic molecule. We demonstrate that WAY200070 enhances glucose-stimulated insulin secretion both in mouse and human islets. In vivo experiments showed that a single administration of WAY200070 leads to an increase in plasma insulin levels with a concomitant improved response to a glucose load. Two-week treatment administration increased glucose-induced insulin release and pancreatic ß-cell mass and improved glucose and insulin sensitivity. In addition, streptozotocin-nicotinamide-induced diabetic mice treated with WAY200070 exhibited a significant improvement in plasma insulin levels and glucose tolerance as well as a regeneration of pancreatic ß-cell mass. Studies performed in db/db mice demonstrated that this compound restored first-phase insulin secretion and enhanced pancreatic ß-cell mass. We conclude that ERß agonists should be considered as new targets for the treatment of diabetes.

Rapid insulinotropic action of low doses of bisphenol-A on mouse and human islets of Langerhans: role of estrogen receptor ß.

Bisphenol-A (BPA) is a widespread endocrine-disrupting chemical (EDC) used as the base compound in the manufacture of polycarbonate plastics. It alters pancreatic ß-cell function and can be considered a risk factor for type 2 diabetes in rodents. Here we used ERß-/- mice to study whether ERß is involved in the rapid regulation of K(ATP) channel activity, calcium signals and insulin release elicited by environmentally relevant doses of BPA (1 nM). We also investigated these effects of BPA in ß-cells and whole islets of Langerhans from humans. 1 nM BPA rapidly decreased K(ATP) channel activity, increased glucose-induced [Ca(2+)](i) signals and insulin release in ß-cells from WT mice but not in cells from ERß-/- mice. The rapid reduction in the K(ATP) channel activity and the insulinotropic effect was seen in human cells and islets. BPA actions were stronger in human islets compared to mouse islets when the same BPA concentration was used. Our findings suggest that BPA behaves as a strong estrogen via nuclear ERß and indicate that results obtained with BPA in mouse ß-cells may be extrapolated to humans. This supports that BPA should be considered as a risk factor for metabolic disorders in humans.

Bisphenol-A acts as a potent estrogen via non-classical estrogen triggered pathways.

Bisphenol-A (BPA) is an estrogenic monomer commonly used in the manufacture of numerous consumer products such as food and beverage containers. Widespread human exposure to significant doses of this compound has been reported. Traditionally, BPA has been considered a weak estrogen, based on its lower binding affinity to the nuclear estrogen receptors (ERs) compared to 17-ß estradiol (E2) as well as its low transcriptional activity after ERs activation. However, in vivo animal studies have demonstrated that it can interfere with endocrine signaling pathways at low doses during fetal, neonatal or perinatal periods as well as in adulthood. In addition, mounting evidence suggests a variety of pathways through which BPA can elicit cellular responses at very low concentrations with the same or even higher efficiency than E2. Thus, the purpose of the present review is to analyze with substantiated scientific evidence the strong estrogenic activity of BPA when it acts through alternative mechanisms of action at least in certain cell types.

Changes in the expression profile of the meiosis-involved mismatch repair genes in impaired human spermatogenesis.

DNA mismatch repair (MMR) genes have been described to participate in crossover events during meiotic recombination, which is, in turn, a key step of spermatogenesis. This evidence suggests that MMR family gene expression may be altered in infertile men with defective sperm production. In order to determine the expression profile of MMR genes in impaired human spermatogenesis, we performed transcript levels analysis of MMR genes (MLH1, MLH3, PMS2, MSH4, and MSH5), and other meiosis-involved genes (ATR, HSPA2, and SYCP3) as controls, by real-time reverse transcription-polymerase chain reaction in testis from 13 patients with spermatogenic failure, 5 patients with primary germ cell tumors, and 10 controls with conserved spermatogenesis. Correlation of the expression values with the histological findings was also performed. The MMR gene expression values, with the exception of PMS2, are significantly decreased in men with spermatogenic failure. The pattern of MMR reduction correlates with the severity of damage, being maximum in maturation arrest. Specifically, expression of the testicular MSH4 gene could be useful as a surrogate marker for the presence of intratesticular elongated spermatid in patients with nonobstructive azoospermia, contributing to predict the viability of assisted reproduction. Interestingly, a reduction in the MSH4 and MSH5 transcript concentration per spermatocyte was also observed. The decreased expression level of other meiosis-specific genes, such as HSPA2 and SYCP3, suggests that the spermatocyte capacity to express meiosis-related genes is markedly reduced in spermatogenic failure, contributing to meiosis impairment and spermatogenic blockade.