Elisa Mascolo

Bisphenol A (BPA) is a ubiquitous, synthetic chemical proven to induce reproductive disorders in both men and women. The available studies investigated the effects of BPA on male and female steroidogenesis following long-term exposure to the compound at relatively high environmental concentrations. However, the impact of short-term exposure to BPA on reproduction is poorly studied. We evaluated if 8 and 24 h exposure to 1 nM and 1 µM BPA perturbs luteinizing hormone/choriogonadotropin (LH/hCG)-mediated signalling in two steroidogenic cell models, i.e., the mouse tumour Leydig cell line mLTC1, and human primary granulosa lutein cells (hGLC). Cell signalling studies were performed using a homogeneous time-resolved fluorescence (HTRF) assay and Western blotting, while gene expression analysis was carried out using real-time PCR. Immunostainings and an immunoassay were used for intracellular protein expression and steroidogenesis analyses, respectively. The presence of BPA leads to no significant changes in gonadotropin-induced cAMP accumulation, alongside phosphorylation of downstream molecules, such as ERK1/2, CREB and p38 MAPK, in both the cell models. BPA did not impact STARD1, CYP11A1 and CYP19A1 gene expression in hGLC, nor Stard1 and Cyp17a1 expression in mLTC1 treated with LH/hCG. Additionally, the StAR protein expression was unchanged upon exposure to BPA. Progesterone and oestradiol levels in the culture medium, measured by hGLC, as well as the testosterone and progesterone levels in the culture medium, measured by mLTC1, did not change in the presence of BPA combined with LH/hCG. These data suggest that short-term exposure to environmental concentrations of BPA does not compromise the LH/hCG-induced steroidogenic potential of either human granulosa or mouse Leydig cells.

The insulin signaling pathway controls cell growth and metabolism, thus its deregulation is associated with both cancer and diabetes. Phosphatidylinositol 3‐kinase (PI3K) contributes to the cascade of phosphorylation events occurring in the insulin pathway by activating the protein kinase B (PKB/AKT), which phosphorylates several substrates, including those involved in glucose uptake and storage. PI3K inactivating mutations are associated with insulin resistance while activating mutations are identified in human cancers. Here we show that RNAi‐induced depletion of the Drosophila PI3K catalytic subunit (Dp110) results in diabetic phenotypes such as hyperglycemia, body size reduction, and decreased glycogen content. Interestingly, we found that hyperglycemia produces chromosome aberrations (CABs) triggered by the accumulation of advanced glycation end‐products and reactive oxygen species. Rearing PI3K^RNAi flies in a medium supplemented with pyridoxal 5′‐phosphate (PLP; the catalytically active form of vitamin B6) rescues DNA damage while, in contrast, treating PI3K^RNAi larvae with the PLP inhibitor 4‐deoxypyridoxine strongly enhances CAB frequency. Interestingly, PLP supplementation rescues also diabetic phenotypes. Taken together, our results provide a strong link between impaired PI3K activity and genomic instability, a crucial relationship that needs to be monitored not only in diabetes due to impaired insulin signaling but also in cancer therapies based on PI3K inhibitors. In addition, our findings confirm the notion that vitamin B6 is a good natural remedy to counteract insulin resistance and its complications.

Knowledge of the growth and maturation of human antral follicles is based mainly on concepts and deductions from clinical observations and animal models. To date, new experimental approaches and in vitro data contributed to a deep comprehension of gonadotropin receptors’ functioning and may provide new insights into the mechanisms regulating still unclear physiological events. Among these, the production of androgen in the absence of proper LH levels, the programming of follicular atresia and dominance are some of the most intriguing. Starting from evolutionary issues at the basis of the gonadotropin receptor signal specificity, we draw a new hypothesis explaining the molecular mechanisms of the antral follicular growth, based on the modulation of endocrine signals by receptor-receptor interactions. The “heteromer hypothesis” explains how opposite death and life signals are delivered by gonadotropin receptors and other membrane partners, mediating steroidogenesis, apoptotic events, and the maturation of the dominant follicle.

An increasing number of pollutants with endocrine disrupting potential are accumulating in the environment, increasing the exposure risk for humans. Several of them are known or suspected to interfere with endocrine signals, impairing reproductive functions. Follicle-stimulating hormone (FSH) is a glycoprotein playing an essential role in supporting antral follicle maturation and may be a target of disrupting chemicals (EDs) likely impacting female fertility. EDs may interfere with FSH-mediated signals at different levels, since they may modulate the mRNA or protein levels of both the hormone and its receptor (FSHR), perturb the functioning of partner membrane molecules, modify intracellular signal transduction pathways and gene expression. In vitro studies and animal models provided results helpful to understand ED modes of action and suggest that they could effectively play a role as molecules interfering with the female reproductive system. However, most of these data are potentially subjected to experimental limitations and need to be confirmed by long-term observations in human.

Diabetes mellitus is a heterogeneous disease characterized by hyperglycemia due to impaired insulin secretion and/or action. All diabetes types have a strong genetic component. The most frequent forms, type 1 diabetes (T1D), type 2 diabetes (T2D) and gestational diabetes mellitus (GDM), are multifactorial syndromes associated with several genes’ effects together with environmental factors. Conversely, rare forms, neonatal diabetes mellitus (NDM) and maturity onset diabetes of the young (MODY), are caused by mutations in single genes. Large scale genome screenings led to the identification of hundreds of putative causative genes for multigenic diabetes, but all the loci identified so far explain only a small proportion of heritability. Nevertheless, several recent studies allowed not only the identification of some genes as causative, but also as putative targets of new drugs. Although monogenic forms of diabetes are the most suited to perform a precision approach and allow an accurate diagnosis, at least 80% of all monogenic cases remain still undiagnosed. The knowledge acquired so far addresses the future work towards a study more focused on the identification of diabetes causal variants; this aim will be reached only by combining expertise from different areas. In this perspective, model organism research is crucial. This review traces an overview of the genetics of diabetes and mainly focuses on Drosophila as a model system, describing how flies can contribute to diabetes knowledge advancement.

Background: Elevated human epididymis secretory protein 4 (HE4) serum levels have been widely investigated in patients with ovarian cancer. However, high levels of HE4 can be also found in other tumors and in renal fibrosis. To date, the HE4 assay manufacturer features the reference value only for the female pre- and post-menopausal population. The aim of this study was to determine the upper reference limit (URL) of HE4 in a well-defined and large cohort of healthy male individuals and investigate potential factors influencing HE4 levels in healthy subjects. Methods: The study included 307 Italian healthy male individuals. HE4 was measured using a chemiluminescent assay (Abbott Laboratories, Wiesbaden, Germany). The URL was calculated using the non-parametric percentile method. Differences in HE4 concentrations according to age, estimated glomerular filtration rate (eGFR), free and bioavailable testosterone were also evaluated. Results: The 97.5th percentile URL of serum HE4 in our study population was 57 pmol/L (90% CI). After stratifying subjects according to age, we found that the URL of HE4 was higher in older (> 50 years) than in younger subjects (18-30 years old), and overlapping with the URL in males from 31 to 50 years old (P=4.769e-16, r=0.44). A strong negative correlation between HE4 and eGFR was observed (P=8.412e-12, r=- 0.38). Moreover, a statistically significant negative correlation was also found between HE4 and free and bioavailable testosterone. Conclusion: In this study was determined the URL of HE4 in a large cohort of healthy male subjects. Our findings indicate that the HE4 age-dependent differences in males need to be taken into account. The definition of the HE4 URL in males and the correlation observed with eGFR and testosterone should foster the clinical use of HE4 beyond gynecologic cancer.

Maturity-onset diabetes of the young (MODY) type 2 is caused by heterozygous inactivating mutations in the gene encoding glucokinase (GCK), a pivotal enzyme for glucose homeostasis. In the pancreas GCK regulates insulin secretion, while in the liver it promotes glucose utilization and storage. We showed that silencing the DrosophilaGCK orthologs Hex-A and Hex-C results in a MODY-2-like hyperglycemia. Targeted knock-down revealed that Hex-A is expressed in insulin producing cells (IPCs) whereas Hex-C is specifically expressed in the fat body. We showed that Hex-A is essential for insulin secretion and it is required for Hex-C expression. Reduced levels of either Hex-A or Hex-C resulted in chromosome aberrations (CABs), together with an increased production of advanced glycation end-products (AGEs) and reactive oxygen species (ROS). This result suggests that CABs, in GCK depleted cells, are likely due to hyperglycemia, which produces oxidative stress through AGE metabolism. In agreement with this hypothesis, treating GCK-depleted larvae with the antioxidant vitamin B6 rescued CABs, whereas the treatment with a B6 inhibitor enhanced genomic instability. Although MODY-2 rarely produces complications, our data revealed the possibility that MODY-2 impacts genome integrity.

Vitamin B6 is a cofactor for approximately 150 reactions that regulate the metabolism of glucose, lipids, amino acids, DNA, and neurotransmitters. In addition, it plays the role of antioxidant by counteracting the formation of reactive oxygen species (ROS) and advanced glycation end-products (AGEs). Epidemiological and experimental studies indicated an evident inverse association between vitamin B6 levels and diabetes, as well as a clear protective effect of vitamin B6 on diabetic complications. Interestingly, by exploring the mechanisms that govern the relationship between this vitamin and diabetes, vitamin B6 can be considered both a cause and effect of diabetes. This review aims to report the main evidence concerning the role of vitamin B6 in diabetes and to examine the underlying molecular and cellular mechanisms. In addition, the relationship between vitamin B6, genome integrity, and diabetes is examined. The protective role of this vitamin against diabetes and cancer is discussed.

In eukaryotes, pyridoxal kinase (PDXK) acts in vitamin B₆salvage pathway to produce pyridoxal 5′-phosphate (PLP), the active form of the vitamin, which is implicated in numerous crucial metabolic reactions. In Drosophila, mutations in the dPdxk gene cause chromosome aberrations (CABs) and increase glucose content in larval hemolymph. Both phenotypes are rescued by the expression of the wild type human PDXK counterpart. Here we expressed, in dPdxk¹ mutant flies, four PDXK human variants: three (D87H, V128I and H246Q) listed in databases, and one (A243G) found in a genetic screening in patients with diabetes. Differently from human wild type PDXK, none of the variants was able to completely rescue CABs and glucose content elicited by dPdxk¹ mutation. Biochemical analysis of D87H, V128I, H246Q and A243G proteins revealed reduced catalytic activity and/or reduced affinity for PLP precursors which justify this behavior. Although these variants are rare in population and carried in heterozygous condition, our findings suggest that in certain metabolic contexts and diseases in which PLP levels are reduced, the presence of these PDXK variants could threaten genome integrity and increase cancer risk.

Pyridoxine/pyridoxamine 5′‐phosphate oxidase (PNPO) and pyridoxal kinase (PDXK) cooperate to produce pyridoxal 5′‐phosphate (PLP), the active form of vitamin B6. PDXK phosphorylates pyridoxine, pyridoxamine, and pyridoxal by producing PNP, PMP, and PLP, whereas PNPO oxidizes PNP, PMP, into PLP. We previously demonstrated that PDXK depletion in Drosophila and human cells impacts on glucose metabolism and DNA integrity. Here we characterized sgll, the Drosophila ortholog of PNPO gene, showing that its silencing by RNA interference elicits chromosome aberrations (CABs) in brains and induces diabetic hallmarks such as hyperglycemia and small body size. We showed that in sgll^RNAi neuroblasts CABs are largely produced by the genotoxic effect of the advanced glycation end products triggered by high glucose. As in sgll^RNAi cells, part of PLP is still produced by PDXK activity, these data suggest that PLP dosage need to be tightly regulated to guarantee glucose homeostasis and DNA integrity.