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.
Ilitch Aquino Marcondes-de-Castro, Thamiris Ferreira Oliveira, Renata Spezani, Thatiany Souza Marinho, Luiz Eduardo Macedo Cardoso, Marcia Barbosa Aguila, and Carlos Alberto Mandarim-de-Lacerda
Stuart Baker, Ricardo Núñez Miguel, Daniel Thomas, Michael Powell, Jadwiga Furmaniak, and Bernard Rees Smith
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.
Ermelindo C Leal, Tatiana Emanuelli, Diana Santos, João Moura, Ana Catarina RG Fonseca, Ana Burgeiro, and Eugenia Carvalho
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.
Alberto Cascón, Bruna Calsina, María Monteagudo, Sara Mellid, Alberto Díaz-Talavera, Maria Currás-Freixes, and Mercedes Robledo
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.
Caroline M Gorvin, Paul J Newey, and Rajesh V Thakker
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.
Rishel B Vohnoutka, Annapurna Kuppa, Yash Hegde, Yue Chen, Asmita Pant, Maurice E Tohme, Eun-Young (Karen) Choi, Sean M McCarty, Devika P Bagchi, Xiaomeng Du, Yanhua Chen, Vincent L Chen, Hiroyuki Mori, Lawrence F Bielak, Lillias H Maguire, Samuel K Handelman, Jonathan Z Sexton, Thomas L Saunders, Brian D Halligan, and Elizabeth K Speliotes
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.
Sarah Theresa Boyle
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.
Tien-Chun Yang, Mei-Hua Lu, Wei-Jie Wang, and Jang-Yi Chen
The pathogenesis of hypertension is not fully understood; ET1 is involved in developing essential hypertension. ET1 can promote VSMC proliferation or hypertrophy through autocrine and paracrine effects. Proliferating smooth muscle cells in the aorta are "dedifferentiated" cells that cause increased arterial stiffness and remodeling. Male SHRs had higher aortic stiffness than normal control male WKY rats. Male SHR VSMCs expressed high levels of the ET1 gene, but endothelial cells did not. Therefore, it is necessary to understand the molecular mechanism of enhanced ET1 expression in SHR VSMCs. We identified POU2f2 and CEBPB as the main molecules that enhance ET1 expression in male SHR VSMCs. A promoter activity analysis confirmed that the enhancer region of the ET1 promoter in male SHR VSMCs was from -1309 to -1279 bp. POU2f2 and CEBPB exhibited an additive role in the enhancer region of the ET1 promoter. POU2f2 or CEBPB overexpression sufficiently increased ET1 expression, and co-transfection with the CEBPB and POU2f2 expression plasmids had additive effects on the activity of the ET1 promoter and ET1 secretion level of male WKY VSMCs. In addition, knockdown of POU2f2 also revealed that POU2f2 is necessary to enhance ET1 expression in SHR VSMCs. The enhancer region of the ET1 promoter is highly conserved in rats, mice, and humans. POU2f2 and CEBPB mRNA levels were significantly increased in remodeled human VMSCs. In conclusion, the novel regulation of POU2f2 and CEBPB in VSMCs will help us understand the pathogenesis of hypertension and support the development of future treatments for hypertension.
Aqfan Jamaluddin and Caroline M. Gorvin
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 increased risk of developing obesity, diabetes mellitus, non-alcoholic fatty liver disease and cardiovascular complications. As the prevalence of these chronic diseases continue 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.
Hong Chen, Weiyu Li, Suping Zhang, Yunteng Sun, Yiping Shen, and Ruimin Chen
Pathogenic variants in the transcription factor CCCTC-binding factor (CTCF) are associated with mental retardation, autosomal dominant 21 (MRD21, MIM#615502). Current studies supported the strong relationship between CTCF variants and short stature, yet the mechanism of CTCF mutation leading to short stature is not known. Clinical information, treatment regimens and follow-up outcomes of a patient with MRD21 were collected. The possible pathogenic mechanisms of CTCF variants leading to short stature were investigated using immortalized lymphocyte cell lines (LCLs), HEK-293T and immortalized normal human liver cell lines (LO2). This patient received a long-term treatment with recombinant human growth hormone (rhGH) and resulted in an increased height of 1.0 SDS. She had low IGF-1 levels before the treatment and the IGF-1 level was not significantly increased during the treatment (-1.38±0.61 SDS). The finding suggested that the CTCFR567W variant could have impaired the insulin growth factors 1 (IGF-1) signaling pathway. We further demonstrated that the mutant CTCF had a reduced ability binding to the promoter region of IGF-1, consequently significantly reducing the transcriptional activation and expression of IGF-1. Our results demonstrated a direct positive regulation of CTCF on the transcription of the IGF1 promoter for the first time. The impaired IGF-1 expression due to CTCF mutation could explain the poor effect of rhGH treatment on MRD21 patients. This study provided novel insights to the molecular basis of CTCF associated disorder.