The serine-threonine protein phosphatase 2A (PP2A) is a heterotrimeric enzyme complex that plays a vital role in regulating male reproductive activities. However, as an essential member of the PP2A family, the physiological functions of PP2A regulatory subunit B55α (PPP2R2A) in testis remain inconclusive. Hu sheep are noted for their reproductive precocity and fertility, and are ideal models for the study of male reproductive physiology. Here, we analyzed the expression patterns of PPP2R2A in the male Hu sheep reproductive tract at different developmental stages and further investigated its role in testosterone secretion and its underlying mechanisms. In this study, we found that there were temporal and spatial differences in PPP2R2A protein expression in the testis and epididymis, especially the expression abundance in the testis at 8 months old (8M) was higher than that at 3 months old (3M). Interestingly, we observed that PPP2R2A interference reduced the testosterone levels in the cell culture medium, which is accompanied by a reduction in Leydig cell proliferation and an elevation in Leydig cell apoptosis. The level of reactive oxygen species in cells increased significantly, while the mitochondrial membrane potential (ΔΨm) decreased significantly after PPP2R2A deletion. Meanwhile, the mitochondrial mitotic protein DNM1L was significantly upregulated, while the mitochondrial fusion proteins MFN1/2 and OPA1 were significantly downregulated after PPP2R2A interference. Furthermore, PPP2R2A interference suppressed the AKT/mTOR signaling pathway. Taken together, our data indicated that PPP2R2A enhanced testosterone secretion, promoted cell proliferation, and inhibited cell apoptosis in vitro, all of which were associated with the AKT/mTOR signaling pathway.

G protein-coupled receptors (GPCRs) have a critical role in energy homeostasis, contributing to food intake, energy expenditure and glycaemic control. Dysregulation of energy expenditure can lead to metabolic syndrome (abdominal obesity, elevated plasma triglyceride, LDL cholesterol and glucose, and high blood pressure), which is associated with an increased risk of developing obesity, diabetes mellitus, non-alcoholic fatty liver disease and cardiovascular complications. As the prevalence of these chronic diseases continues to rise worldwide, there is an increased need to understand the molecular mechanisms by which energy expenditure is regulated to facilitate the development of effective therapeutic strategies to treat and prevent these conditions. In recent years, drugs targeting GPCRs have been the focus of efforts to improve treatments for type-2 diabetes and obesity, with GLP-1R agonists a particular success. In this review, we focus on nine GPCRs with roles in energy homeostasis that are current and emerging targets to treat obesity and diabetes. We discuss findings from pre-clinical models and clinical trials of drugs targeting these receptors and challenges that must be overcome before these drugs can be routinely used in clinics. We also describe new insights into how these receptors signal, including how accessory proteins, biased signalling, and complex spatial signalling could provide unique opportunities to develop more efficacious therapies with fewer side effects. Finally, we describe how combined therapies, in which multiple GPCRs are targeted, may improve clinical outcomes and reduce off-target effects.

Ingestion of nutrients stimulates incretin secretion from enteroendocrine cells (EECs) of the epithelial layer of the gut. Glucagon-like peptide-1 (GLP-1) is one of these incretins that stimulate postprandial insulin release and signal satiety to the brain. Understanding the regulation of incretin secretion might open up new therapeutic options for obesity and type-2 diabetes mellitus. To investigate the inhibitory effect of the ketone body β-hydroxybutyrate (βHB) on glucose-induced GLP-1 secretion from EECs, in vitro cultures of murine GLUTag cells and differentiated human jejunal enteroid monolayers were stimulated with glucose to induce GLP-1 secretion. The effect of βHB on GLP-1 secretion was studied using ELISA and ECLIA methods. GLUTag cells stimulated with glucose and βHB were analysed using global proteomics focusing on cellular signalling pathways and the results were verified by Western blot. Results demonstrated βHB had a significant inhibitory effect on glucose-induced GLP-1 secretion at a dose of 100 mM in GLUTag cells. In differentiated human jejunal enteroid monolayers, glucose-induced secretion of GLP-1 was inhibited at a much lower dose of 10 mM βHB. The addition of βHB to GLUTag cells resulted in decreased phosphorylation of kinase AKT and transcription factor STAT3 and also influenced the expressions of signalling molecule IRS-2, kinase DGKε and receptor FFAR3. In conclusion, βHB displays an inhibitory effect on glucose-induced GLP-1 secretion in vitro in GLUTag cells and in differentiated human jejunal enteroid monolayers. This effect may be mediated through multiple downstream mediators of G-protein coupled receptor activation, such as PI3K signalling.

Obesity, adipose tissue inflammation, and nonalcoholic fatty liver disease (NAFLD) are associated with insulin resistance and type 2 diabetes (T2D). Cotadutide is a dual agonist GLP-1/glucagon, currently in a preclinical study phase 2 that presents an anti-obesity effect. Diet-induced obese (DIO) C57BL/6 mice were treated for 4 weeks with cotadutide (30 nm/kg once a day at 14:00 h). The study focused on epididymal white adipose tissue (eWAT), liver (NAFLD), inflammation, lipid metabolism, AMP-activated protein kinase (AMPK)/mechanistic target of rapamycin (mTOR) pathways, and the endoplasmic reticulum (ER) stress. As a result, cotadutide controlled weight gain, glucose intolerance, and insulin resistance and showed beneficial effects on plasma markers in DIO mice (triacylglycerol, total cholesterol, alanine aminotransferase, and aspartate aminotransferase, leptin, adiponectin, monocyte chemoattractant protein-1, resistin, interleukin-6, tumor necrosis factor-alpha). Also, cotadutide lessened liver fat accumulation, eWAT proinflammatory markers, and ER stress. In addition, cotadutide improved lipid metabolism genes in eWAT, fatty acid synthase, peroxisome proliferator-activated receptor gamma and mitigates adipocyte hypertrophy and apoptosis. Furthermore, the effects of cotadutide were related to liver AMPK/mTOR pathway and ER stress. In conclusion, cotadutide induces weight loss and treats glucose intolerance and insulin resistance in DIO mice. In addition, cotadutide shows beneficial effects on liver lipid metabolism, mitigating steatosis, inflammation, and ER stress. Besides, in adipocytes, cotadutide decreases hypertrophy and reduces apoptosis. These actions rescuing the AMPK and mTOR pathway, improving lipid metabolism, and lessening NAFLD, inflammation, and ER stress in both eWAT and liver of DIO mice indicate cotadutide as a potentially new pharmacological treatment for T2D and associated obesity.

Determination of the structure of the extracellular domain of human thyroid peroxidase (hTPO) by cryo-electron microscopy (cryo-EM) is described. TPO, purified to homogeneity was complexed with the hTPO monoclonal autoantibody 2G4 Fab and also with a mouse monoclonal TPO antibody 4F5 Fab (which competes with autoantibody binding to TPO). Both complexes were analysed by cryo-EM. The two structures (global resolution 3.92 and 3.4 Å for the 2G4 complex and 4F5 complex, respectively) show TPO as a monomer with four domains; the N-terminal domain, the peroxidase domain (POD), the complement control protein (CCP)-like domain and the epidermal growth factor-like domain which are all visible in the structures. The relative positions of the domains are fixed with a disulphide bond between cysteine residues Cys146 in the POD and Cys756 in the CCP domain preventing significant flexibility of the molecule. The entrance to the enzyme active site, the haem group and the calcium binding site are clearly visible on the opposite side of the TPO molecule from the 2G4 and 4F5 binding sites. Extensive interactions are seen between TPO and the two antibodies which both bind to distinct epitopes on the POD domain, including some residues in the immunodominant region B mainly via different residues. However, the epitopes of the two antibodies contain three shared TPO residues. This is the first high-resolution structure of TPO to be reported and it should help guide the development of new inhibitors of TPO enzyme activity for therapeutic applications.

Dysfunction in key cellular organelles has been linked to diabetic complications. This study intended to investigate the alterations in the unfolded protein response (UPR), autophagy, and mitochondrial function, which are part of the endoplasmic reticulum (ER) stress response, in wound healing (WH) under diabetes conditions. WH mouse models were used to evaluate the UPR, autophagy, mitochondrial fusion, fission, and biogenesis as well as mitophagy in the skin of control and diabetic mice at baseline and 10 days after wounding. The autophagic flux in response to high-glucose conditions was also evaluated in keratinocyte and fibroblast cell cultures. WH was impaired in the diabetic mouse model, and we found that the UPR and autophagy pathways were activated in skin wounds of control mice and in the non-wounded skin of diabetic mice. Moreover, high-glucose conditions induced autophagy in the keratinocyte and fibroblast cell cultures. However, mitophagy did not change in the skin of diabetic mice or the wounded skin. In addition, mitochondrial fusion was activated in control but not in the skin wounds of diabetic mice, while mitochondrial biogenesis is downregulated in the skin of diabetic mice. In conclusion, the activation of the UPR, autophagy, and mitochondrial remodeling are crucial for a proper WH. These results suggest that the increase in ER stress and autophagy in the skin of diabetic mice at baseline significantly escalated to pathological levels after wounding, contributing to impaired WH in diabetes.

The genetics of pheochromocytoma and paraganglioma (PPGL) has become increasingly complex over the last two decades. The list of genes involved in the development of these tumors has grown steadily, and there are currently more than 20 driver genes implicated in either the hereditary or the sporadic nature of the disease. Although genetic diagnosis is achieved in about 75–80% of patients, genetic etiology remains unexplained in a significant percentage of cases. Patients lacking a genetic diagnosis include not only those with apparently sporadic PPGL but also patients with a family history of the disease or with multiple tumors, that meet the criteria to be considered as candidates for carrying germline mutations in yet undiscovered genes. Mutations in known PPGL genes deregulate three main signaling pathways (hypoxia, kinase signaling, and Wnt-signaling pathways), which could be the starting point for the development of personalized treatment for PPGL patients. Furthermore, the integration of results from several genomic high-throughput platforms enables the discovery of regulatory mechanisms that cannot be identified by analyzing each piece of information separately. These strategies are powerful tools for elucidating optimal therapeutic options based on molecular biomarkers in PPGL and represent an important step toward the achievement of precision medicine for patients with metastatic PPGL.

The prolactin receptor (PRLR) signals predominantly through the JAK2-STAT5 pathway regulating multiple physiological functions relating to fertility, lactation, and metabolism. However, the molecular pathology and role of PRLR mutations and signalling are incompletely defined, with progress hampered by a lack of reported disease-associated PRLR variants. To date, two common germline PRLR variants are reported to demonstrate constitutive activity, with one, Ile146Leu, overrepresented in benign breast disease, while a rare activating variant, Asn492Ile, is reported to be associated with an increased incidence of prolactinoma. In contrast, an inactivating germline heterozygous PRLR variant (His188Arg) was reported in a kindred with hyperprolactinaemia, while an inactivating compound heterozygous PRLR variant (Pro269Leu/Arg171Stop) was identified in an individual with hyperprolactinaemia and agalactia. We hypothesised that additional rare germline PRLR variants, identified from large-scale sequencing projects (ExAC and GnomAD), may be associated with altered in vitro PRLR signalling activity. We therefore evaluated >300 previously uncharacterised non-synonymous, germline PRLR variants and selected 10 variants for in vitro analysis based on protein prediction algorithms, proximity to known functional domains and structural modelling. Five variants, including extracellular and intracellular domain variants, were associated with altered responses when compared to the wild-type receptor. These altered responses included loss- and gain-of-function activities related to STAT5 signalling, Akt and FOXO1 activity, as well as cell viability and apoptosis. These studies provide further insight into PRLR structure–function and indicate that rare germline PRLR variants may have diverse modulating effects on PRLR signalling, although the pathophysiologic relevance of such alterations remains to be defined.

Human genome-wide association studies found single-nucleotide polymorphisms (SNPs) near LYPLAL1 (Lysophospholipase-like protein 1) that have sex-specific effects on fat distribution and metabolic traits. To determine whether altering LYPLAL1 affects obesity and metabolic disease, we created and characterized a mouse knockout (KO) of Lyplal1. We fed the experimental group of mice a high-fat, high-sucrose (HFHS) diet for 23 weeks, and the controls were fed regular chow diet. Here, we show that CRISPR-Cas9 whole-body Lyplal1 KO mice fed an HFHS diet showed sex-specific differences in weight gain and fat accumulation as compared to chow diet. Female, not male, KO mice weighed less than WT mice, had reduced body fat percentage, had white fat mass, and had adipocyte diameter not accounted for by changes in the metabolic rate. Female, but not male, KO mice had increased serum triglycerides, decreased aspartate, and decreased alanine aminotransferase. Lyplal1 KO mice of both sexes have reduced liver triglycerides and steatosis. These diet-specific effects resemble the effects of SNPs near LYPLAL1 in humans, suggesting that LYPLAL1 has an evolutionary conserved sex-specific effect on adiposity. This murine model can be used to study this novel gene-by-sex-by-diet interaction to elucidate the metabolic effects of LYPLAL1 on human obesity.

The tumor microenvironment is a dynamic ecosystem of stromal and immune cells that, under the influence of cancer cells, govern biochemical signaling, mechanical signaling via production and remodeling of the extracellular matrix (ECM), formation of vascular networks, and ultimately promotion of tumor growth. In breast cancer, hormone receptor-mediated signaling is a key coordinator of cancer cell proliferation and invasiveness not only through cell-autonomous means but also via cancer cell–stroma cross-talk. In the absence of hormone receptors, a different microenvironment landscape emerges, which comes with its own challenges for therapy. This review summarizes the current knowledge regarding the associations of hormone receptor profiles with composition of the microenvironment, how hormones directly influence stromal cells, immune cells and cells associated with the vasculature, and the paracrine mechanisms that lead to the formation of a tumor-promoting ECM.