The Male Reproductive System and Endocrine Disruptors.

The male reproductive system is exposed to a great number of chemical substances which can interfere with the normal hormonal milieu and reproductive function; these are called endocrine disruptors (EDs). Despite a growing number of studies evaluating the negative effects of EDs, their production is continuously growing although some of them have been prohibited. The prevalence of poor semen quality, hypospadias, cryptorchidism, and testicular cancer has increased in the last decades, and recently, it has been postulated that these could all be part of a unique syndrome called testicular dysgenesis syndrome. This syndrome could be related to exposure to a number of EDs which cause imbalances in the hormonal milieu and oestrogenic over-exposure during the foetal stage. The same EDs can also impair spermatogenesis in offspring and have epigenetic effects. Although studies on animal and in vitro models have raised concerns, data are conflicting. However, these studies must be considered as the basis for future research to promote male reproductive health.

ASCs and their role in obesity and metabolic diseases.

We describe adipose stromal/stem cells (ASCs) in the structural/functional context of the adipose tissue (AT) stem niche (adiponiche), including cell-cell interactions and the microenvironment, and emphasize findings obtained in humans and in lineage-tracing models. ASCs have distinctive markers, 'colors', and anatomical 'locations' which influence their functions. Each adiponiche component can become impaired, thereby contributing to the pathological AT alterations seen in obesity and metabolic diseases. We discuss adiposopathy with a focus on adiponiche dysfunction, and underline the mechanisms that control AT expansion and energy balance. Better understanding of adiponiche regulation and ASC features could help to identify therapeutic targets that favor weight loss and counteract weight regain, and also contribute to innovative strategies for regenerative medicine.

White Adipose Tissue Expansion in Multiple Symmetric Lipomatosis Is Associated with Upregulation of CK2, AKT and ERK1/2.

Multiple symmetric lipomatosis (MSL) is a rare disorder characterized by overgrowing lipomatous tissue (LT) in the subcutaneous adipose tissue (SAT). What LT is and how it expands are not completely understood; previous data suggested that it could derive from brown AT precursors. In six MSL type I patients, we compared LT morphology by histological and immunohistochemistry (IHC) analysis, gene expression, by qPCR, kinase activity, by Western Blot and in vitro assay to paired-control SAT using AT from patients with pheochromocytoma as a human browning reference. In the stromal vascular fraction (SVF), we quantified adipose stem cells (ASCs) by flow cytometry, the proliferation rate, white and beige adipogenic potential and clonogenicity and adipogenicity by a limiting dilution assay. LT displayed white AT morphology and expression pattern and did not show increased levels of the brown-specific marker UCP1. In LT, we evidenced AKT, CK2 and ERK1/2 hyperactivation. LT-SVF contained increased ASCs, proliferated faster, sprouted clones and differentiated into adipocytes better than the control, displaying enhanced white adipogenic potential but not increased browning compared to SAT. In conclusion, LT is a white AT depot expanding by hyperplasia through increased stemness and enhanced white adipogenesis upregulating AKT, CK2 and ERK1/2, which could represent new targets to counteract MSL.

In silico prediction of blood cholesterol levels from genotype data.

In this work we present a framework for blood cholesterol levels prediction from genotype data. The predictor is based on an algorithm for cholesterol metabolism simulation available in literature, implemented and optimized by our group in the R language. The main weakness of the former simulation algorithm was the need of experimental data to simulate mutations in genes altering the cholesterol metabolism. This caveat strongly limited the application of the model in the clinical practice. In this work we present how this limitation could be bypassed thanks to an optimization of model parameters based on patient cholesterol levels retrieved from literature. Prediction performance has been assessed taking into consideration several scoring indices currently used for performance evaluation of machine learning methods. Our assessment shows how the optimization phase improved model performance, compared to the original version available in literature.

Psychological predictors of poor weight loss following LSG: relevance of general psychopathology and impulsivity.

PURPOSE: After bariatric surgery (BS) a significant minority of patients do not reach successful weight loss or tend to regain weight. In recent years, interest for the psychological factors that predict post-surgical weight loss has increased with the objective of developing interventions aimed to ameliorate post-surgical outcomes. In the present study, predictive models of successful or poor weight loss 12 months after BS were investigated considering pre-surgery level of psychopathological symptoms, dysfunctional eating behaviors and trait impulsivity at baseline (pre-surgery). METHODS: Sixty-nine patients with morbid obesity canditates for laparoscopic sleeve gastrectomy were assessed regarding metabolic and psychological dimensions. Successful post-surgery weight loss was defined as losing at least 50% of excess body weight (%EWL). RESULTS: Logistic models adjusted for patient sex, age and presence of metabolic diseases showed that the baseline presence of intense psychopathological symptoms and low attentional impulsivity predict poor %EWL (< 50%), as assessed 12-month post-surgery. CONCLUSIONS: The present findings suggest that intensity of general psychopathology and impulsivity, among other psychological factors, might affect post-surgery %EWL. Conducting adequate psychological assessment at baseline of patients candidates for BS seems to be crucial to orient specific therapeutic interventions. LEVEL OF EVIDENCE: Level III, case-control analytic study.

Resting Energy Expenditure, Insulin Resistance and UCP1 Expression in Human Subcutaneous and Visceral Adipose Tissue of Patients With Obesity.

Determinants of resting energy expenditure (REE) in humans are still under investigation, especially the association with insulin resistance. Brown adipose tissue (AT) regulates energy expenditure through the activity of the uncoupling protein 1 (UCP1). White AT browning is the process by which some adipocytes within AT depots acquire properties of brown adipocytes ("brite" adipocytes) and it correlates with metabolic improvement. We analyzed determinants of REE in patients with obesity and assessed UCP1 expression as a "brite" marker in abdominal subcutaneous AT (SAT) and visceral omental AT (VAT). Clinical data, REE, free fat mass (FFM), and fat mass (FM) were determined in 209 patients with obesity. UCP1, PPARG coactivator 1 alpha (PPARGC1A), transcription factor A, mitochondrial (TFAM), T-box transcription factor 1 (TBX1), and solute carrier family 27 member 1 (SLC27A1) expression was assayed in SAT and VAT samples, obtained during sleeve gastrectomy from 62 patients with obesity. REE and body composition data were also available for a subgroup of 35 of whom. In 209 patients with obesity a multiple regression model was computed with REE as the dependent variable and sex, waist, FFM, FM, homeostasis model assessment-insulin resistance (HOMA), interleukin-6 and High Density Lipoprotein-cholesterol as the independent variables. Only FFM, FM and HOMA were independently correlated with REE (r = 0.787, AdjRsqr = 0.602). In each patient VAT displayed a higher UCP1, PPARGC1A, TFAM, TBX1, and SLC27A1 expression than SAT and UCP1 expression in VAT (UCP1-VAT) correlated with Body Mass Index (BMI) (r = 0.287, p < 0.05). Introducing UCP1-VAT in the multivariate model, we showed that FFM, HOMA, interleukin-6, High Density Lipoprotein-cholesterol, and UCP1-VAT were independent factors correlated with REE (r = 0.736, AdjRsqr = 0.612). We confirmed that REE correlates with FFM, FM and HOMA in a large cohort of patients. Our results clearly showed that UCP1-VAT expression was significantly increased in severe human obesity (BMI > 50 kg/m2) and that it behaved as an independent predictor of REE. Lastly, we suggest that an increased REE and browning in metabolically complicated severe obesity could represent an effort to counteract further weight gain.

Characterization of subcutaneous and omental adipose tissue in patients with obesity and with different degrees of glucose impairment.

Although obesity represents a risk factor for the development of type 2 diabetes mellitus (T2DM), the link between these pathological conditions is not so clear. The manner in which the different elements of adipose tissue (AT) interplay in order to grow has been suggested to have a role in the genesis of metabolic complications, but this has not yet been fully addressed in humans. Through IHC, transmission electron microscopy, cytometry, and in vitro cultures, we described the morphological and functional changes of subcutaneous and visceral AT (SAT and VAT) in normoglycemic, prediabetic and T2DM patients with obesity compared to lean subjects. In both SAT and VAT we measured a hypertrophic and hyperplastic expansion, causing similar vascular rarefaction in obese patients with different degrees of metabolic complications. Capillaries display dysfunctional basement membrane thickening only in T2DM patients evidencing VAT as a new target of T2DM microangiopathy. The largest increase in adipocyte size and decrease in adipose stem cell number and adipogenic potential occur both in T2DM and in prediabetes. We showed that SAT and VAT remodeling with stemness deficit is associated with early glucose metabolism impairment suggesting the benefit of an AT-target therapy controlling hypertrophy and hyperplasia already in prediabetic obese patients.

SCCA-IgM as a Potential Biomarker of Non-Alcoholic Fatty Liver Disease in Patients with Obesity, Prediabetes and Diabetes Undergoing Sleeve Gastrectomy.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) has a high prevalence in obesity and its presence should be screened. Laparoscopic sleeve gastrectomy (LSG) is an effective treatment for obesity, but its effects on NAFLD are still to be firmly established. The diagnosis of non-alcoholic steatohepatitis (NASH) is currently performed by liver biopsy, a costly and invasive procedure. Squamous cell carcinoma antigen-IgM (SCCA-IgM) is a biomarker of viral hepatitis to hepatocellular carcinoma development and its role in NAFLD to NASH progression has not yet been investigated. OBJECTIVE: The aim of this study was to evaluate SCCA-IgM as a non-invasive biomarker of NAFLD/NASH in patients with different degrees of metabolic-complicated obesity before and after LSG. METHOD: Fifty-six patients with obesity were studied before and 12 months after LSG; anthropometric, biochemical, clinical, and imaging data were collected. RESULTS: At baseline steatosis was strongly associated with the glycaemic profile (p = 0.016) and was already present in prediabetic patients with obesity (82%). Only 3 patients had an SCCA-IgM level above the normal cut-off. SCCA-IgM titre did not change according to glycaemic profile or steatosis. Metabolic and inflammatory factors and transaminases significantly reduced after LSG-induced weight loss, except for SCCA-IgM. The ALT/AST ratio decreased post-LSG correlated with BMI (r = 0.297, p = 0.031), insulin (r = 0.354, p = 0.014), and triglycerides (r = 0.355, p = 0.009) reduction. CONCLUSIONS: Our results confirm the tight link between NAFLD and metabolic complications, suggesting prediabetes as a new risk factor of steatosis. SCCA-IgM does not seem to have a role in the identification and prognosis of NAFLD.

Modifications of Resting Energy Expenditure After Sleeve Gastrectomy.

OBJECTIVES: Resting energy expenditure (REE) declines more than what is expected according to body composition changes after caloric restriction. This metabolic adaptation is considered one of the factors favoring weight regain. The aim of this study is to evaluate the changes of REE and calculate the degree of metabolic adaptation occurring after laparoscopic sleeve gastrectomy (LSG). METHODS: REE (by indirect calorimetry) and body composition (fat-free mass or FFM, fat mass or FM by bioelectrical impedance analysis) were determined before and after 12 months in 154 patients with obesity treated with laparoscopic sleeve gastrectomy (LSG). RESULTS: Weight loss was 29.8 ± 10.6%, with corresponding relative reductions in FM (44.5 ± 22.8%), FFM (13.7 ± 9.9%), and REE (27.3 ± 12.9%). A predictive equation for REE was computed by using the baseline FFM and FM values to account for body composition changes. A predicted post-weight loss REE was calculated by using this equation and entering post-weight loss body composition values. Observed post-surgery REE was significantly lower than predicted one (1410 ± 312 vs 1611 ± 340 kcal/day, P < 0.001) and metabolic adaptation, calculated as the difference between observed and predicted post-weight loss REE, was - 199 ± 238 kcal/day. The post-surgery level of metabolic adaptation was inversely related to postoperative percent weight loss (r = - 0.170; P < 0.05) and FM loss (r = - 0.245; P < 0.01). CONCLUSIONS: A significant reduction of resting energy expenditure and a significant degree of metabolic adaptation both occur after sleeve gastrectomy. A greater metabolic adaptation could be partly responsible for a lower weight loss after surgery.

CK2 modulates adipocyte insulin-signaling and is up-regulated in human obesity.

Insulin plays a major role in glucose metabolism and insulin-signaling defects are present in obesity and diabetes. CK2 is a pleiotropic protein kinase implicated in fundamental cellular pathways and abnormally elevated in tumors. Here we report that in human and murine adipocytes CK2-inhibition decreases the insulin-induced glucose-uptake by counteracting Akt-signaling and GLUT4-translocation to the plasma membrane. In mice CK2 acts on insulin-signaling in adipose tissue, liver and skeletal muscle and its acute inhibition impairs glucose tolerance. Notably, CK2 protein-level and activity are greatly up-regulated in white adipose tissue from ob/ob and db/db mice as well as from obese patients, regardless the severity of their insulin-resistance and the presence of pre-diabetes or overt type 2 diabetes. Weight loss obtained by both bariatric surgery or hypocaloric diet reverts CK2 hyper-activation to normal level. Our data suggest a central role of CK2 in insulin-sensitivity, glucose homeostasis and adipose tissue remodeling. CK2 up-regulation is identified as a hallmark of adipose tissue pathological expansion, suggesting a new potential therapeutic target for human obesity.

Increased mitochondrial calcium uniporter in adipocytes underlies mitochondrial alterations associated with insulin resistance.

Intracellular calcium influences an array of pathways and affects cellular processes. With the rapidly progressing research investigating the molecular identity and the physiological roles of the mitochondrial calcium uniporter (MCU) complex, we now have the tools to understand the functions of mitochondrial Ca2+ in the regulation of pathophysiological processes. Herein, we describe the role of key MCU complex components in insulin resistance in mouse and human adipose tissue. Adipose tissue gene expression was analyzed from several models of obese and diabetic rodents and in 72 patients with obesity as well as in vitro insulin-resistant adipocytes. Genetic manipulation of MCU activity in 3T3-L1 adipocytes allowed the investigation of the role of mitochondrial calcium uptake. In insulin-resistant adipocytes, mitochondrial calcium uptake increased and several MCU components were upregulated. Similar results were observed in mouse and human visceral adipose tissue (VAT) during the progression of obesity and diabetes. Intriguingly, subcutaneous adipose tissue (SAT) was spared from overt MCU fluctuations. Furthermore, MCU expression returned to physiological levels in VAT of patients after weight loss by bariatric surgery. Genetic manipulation of mitochondrial calcium uptake in 3T3-L1 adipocytes demonstrated that changes in mitochondrial calcium concentration ([Ca2+]mt) can affect mitochondrial metabolism, including oxidative enzyme activity, mitochondrial respiration, membrane potential, and reactive oxygen species formation. Finally, our data suggest a strong relationship between [Ca2+]mt and the release of IL-6 and TNFα in adipocytes. Altered mitochondrial calcium flux in fat cells may play a role in obesity and diabetes and may be associated with the differential metabolic profiles of VAT and SAT.

Incidence and Predictors of Hypoglycemia 1 Year After Laparoscopic Sleeve Gastrectomy.

INTRODUCTION: Hypoglycemia is a known adverse event following gastric bypass. The incidence of hypoglycemia after laparoscopic sleeve gastrectomy (LSG) is still under investigation. The aim of our study was to verify the presence of oral glucose tolerance test (OGTT)-related hypoglycemia after LSG and to identify any baseline predictors of its occurrence. METHODS: We analyzed 197 consecutive non-diabetic morbid obese patients that underwent LSG. All patients were studied before and 12 months after LSG. Evaluation included anthropometric parameters, 3-h OGTT for blood glucose (BG), insulin and c-peptide, lipid profile, interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), highly sensitive C-reactive protein (hsCRP), and leptin. Hypoglycemia was defined as BG ≤ 2.7 mmol/l. RESULTS: After surgery, 180 patients completed the OGTT. Eleven patients did not complete the test for gastric intolerance, and in six patients, the test was stopped earlier for the onset of severe symptomatic hypoglycemia. Of the patients, 61/186 (32.8%) had at least one OGTT-related hypoglycemia. The highest frequency of hypoglycemic events occurred 150' after glucose load (20.2%). At baseline, patients with hypoglycemic events after surgery (Hypo) were younger (40 ± 11 vs 46 ± 10 years; p < 0.001), less obese (BMI 46 ± 5.7 vs 48.4 ± 7.9 kg/m2; p < 0.05), and had a worse lipid profile as compared to patients without hypoglycemic events (N-Hypo). Moreover, after LSG, Hypo patients compared with N-Hypo presented a higher weight loss (%EBMIL 80 ± 20 vs 62 ± 21%; p < 0.001). Low age, low fasting glucose, and high triglyceride levels before LSG were independent predictors of hypoglycemia development after surgery (r 2 = 0.131). CONCLUSION: These findings confirm the high incidence of post-prandial hypoglycemia 1 year after LSG. Hypoglycemia is more frequent in younger patients with lower fasting glucose and higher triglyceride levels before surgery.

The rs2274911 polymorphism in GPRC6A gene is associated with insulin resistance in normal weight and obese subjects.

OBJECTIVE: Identification of the novel endocrine role of osteocalcin (OC) and its receptor GPRC6A has given rise to a new branch of research in OC/GPRC6A axis related to glucose metabolism. GPRC6A- and OC-deficient mice share features of the metabolic syndrome, in addition to male infertility. Recently, the polymorphism rs2274911 in GPRC6A was shown to be associated with testicular impairment. We aimed to investigate the role of rs2274911 polymorphism in glucose and lipid metabolism in a cohort of normal weight and obese subjects DESIGN, PATIENTS, SETTINGS: A total of 392 male and females, including 218 obese patients and 174 age-matched normal weight controls, were retrospectively selected. RESULTS: The distribution of rs2274911 alleles and genotypes did not differ either between normal weight and obese subjects or sexes (all P > 0·05). Age- and OC-adjusted multivariate analysis revealed that, in the normal weight group, fasting insulin and HOMA-IR increased in GA (P = 0·016 and P = 0·025) and AA genotypes (P = 0·033 and P = 0·040) compared with GG homozygotes. In the obese group, AA homozygotes had increased fasting glucose (P = 0·041 vs GG). Triglycerides, fasting insulin and HOMA-IR increased in both GA (P = 0·020, P < 0·001 and P = 0·001) and AA genotype (P = 0·021, P = 0·013 and P = 0·013). CONCLUSION: In a cohort of normal weight and obese subjects, we found that the nonrare polymorphism rs2274911 in the GPRC6A gene was associated with insulin resistance features, independently of the metabolic phenotype and OC levels.

Clonal characterization of rat muscle satellite cells: proliferation, metabolism and differentiation define an intrinsic heterogeneity.

Satellite cells (SCs) represent a distinct lineage of myogenic progenitors responsible for the postnatal growth, repair and maintenance of skeletal muscle. Distinguished on the basis of their unique position in mature skeletal muscle, SCs were considered unipotent stem cells with the ability of generating a unique specialized phenotype. Subsequently, it was demonstrated in mice that opposite differentiation towards osteogenic and adipogenic pathways was also possible. Even though the pool of SCs is accepted as the major, and possibly the only, source of myonuclei in postnatal muscle, it is likely that SCs are not all multipotent stem cells and evidences for diversities within the myogenic compartment have been described both in vitro and in vivo. Here, by isolating single fibers from rat flexor digitorum brevis (FDB) muscle we were able to identify and clonally characterize two main subpopulations of SCs: the low proliferative clones (LPC) present in major proportion (approximately 75%) and the high proliferative clones (HPC), present instead in minor amount (approximately 25%). LPC spontaneously generate myotubes whilst HPC differentiate into adipocytes even though they may skip the adipogenic program if co-cultured with LPC. LPC and HPC differ also for mitochondrial membrane potential (DeltaPsi(m)), ATP balance and Reactive Oxygen Species (ROS) generation underlying diversities in metabolism that precede differentiation. Notably, SCs heterogeneity is retained in vivo. SCs may therefore be comprised of two distinct, though not irreversibly committed, populations of cells distinguishable for prominent differences in basal biological features such as proliferation, metabolism and differentiation. By these means, novel insights on SCs heterogeneity are provided and evidences for biological readouts potentially relevant for diagnostic purposes described.

The origin of intermuscular adipose tissue and its pathophysiological implications.

The intermuscular adipose tissue (IMAT) is a depot of adipocytes located between muscle bundles. Several investigations have recently been carried out to define the phenotype, the functional characteristics, and the origin of the adipocytes present in this depot. Among the different mechanisms that could be responsible for the accumulation of fat in this site, the dysdifferentiation of muscle-derived stem cells or other mesenchymal progenitors has been postulated, turning them into cells with an adipocyte phenotype. In particular, muscle satellite cells (SCs), a heterogeneous stem cell population characterized by plasticity and self-renewal that allow muscular growth and regeneration, can acquire features of adipocytes, including the abilities to express adipocyte-specific genes and accumulate lipids. Failure to express the transcription factors that direct mesenchymal precursors into fully differentiated functionally specialized cells may be responsible for their phenotypic switch into the adipogenic lineage. We proved that human SCs also possess a clear adipogenic potential that could explain the presence of mature adipocytes within skeletal muscle. This occurs under some pathological conditions (i.e., primary myodystrophies, obesity, hyperglycemia, high plasma free fatty acids, hypoxia, etc.) or as a consequence of thiazolidinedione treatment or simply because of a sedentary lifestyle or during aging. Several pathways and factors (PPARs, WNT growth factors, myokines, GEF-GAP-Rho, p66(shc), mitochondrial ROS production, PKCß) could be implicated in the adipogenic conversion of SCs. The understanding of the molecular pathways that regulate muscle-to-fat conversion and SC behavior could explain the increase in IMAT depots that characterize many metabolic diseases and age-related sarcopenia.