Oral intake of Lactobacillus plantarum L‐14 extract alleviates TLR2‐ and AMPK‐mediated obesity‐associated disorders in high‐fat‐diet‐induced obese C57BL/6J mice

ObjectivesWhether periodic oral intake of postbiotics positively affects weight regulation and prevents obesity‐associated diseases in vivo is unclear. This study evaluated the action mechanism of Lactobacillus plantarum L‐14 (KTCT13497BP) extract and the effects of its periodic oral intake in a high‐fat‐diet (HFD) mouse model.Materials and methodsMouse pre‐adipocyte 3T3‐L1 cells and human bone marrow mesenchymal stem cells (hBM‐MSC) were treated with L‐14 extract every 2 days during adipogenic differentiation, and the mechanism underlying anti‐adipogenic effects was analysed at cellular and molecular levels. L‐14 extract was orally administrated to HFD‐feeding C57BL/6J mice every 2 days for 7 weeks. White adipose tissue was collected and weighed, and liver and blood serum were analysed. The anti‐adipogenic mechanism of exopolysaccharide (EPS) isolated from L‐14 extract was also analysed using Toll‐like receptor 2 (TLR2) inhibitor C29.ResultsL‐14 extract inhibited 3T3‐L1 and hBM‐MSC differentiation into mature adipocytes by upregulating AMPK signalling pathway in the early stage of adipogenic differentiation. The weight of the HFD + L‐14 group (31.51 ± 1.96 g) was significantly different from that of the HFD group (35.14 ± 3.18 g). L‐14 extract also significantly decreased the serum triacylglycerol/high‐density lipoprotein cholesterol ratio (an insulin resistance marker) and steatohepatitis. In addition, EPS activated the AMPK signalling pathway by interacting with TLR2, consequently inhibiting adipogenesis.ConclusionsEPS from L‐14 extract inhibits adipogenesis via TLR2 and AMPK signalling pathways, and oral intake of L‐14 extract improves obesity and obesity‐associated diseases in vivo. Therefore, EPS can be used to prevent and treat obesity and metabolic disorders.     

Cyclooxygenase-2 dependent metabolism of 20-HETE increases adiposity and adipocyte enlargement in mesenchymal stem cell-derived adipocytes

20-Hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE), a product of the cytochrome P450 (CYP)-catalyzed ω-hydroxylation of arachidonic acid, induces oxidative stress and, in clinical studies, is associated with increased body mass index (BMI) and the metabolic syndrome. This study was designed to examine the effects of exogenous 20-HETE on mesenchymal stem cell (MSC)-derived adipocytes. The expression levels of CYP4A11 and CYP4F2 (major 20-HETE synthases in humans) in MSCs decreased during adipocyte differentiation; however, exogenous administration of 20-HETE (0.1–1 μM) increased adipogenesis in a dose-dependent manner in these cells (P < 0.05). The inability of a 20-HETE analog to reproduce these effects suggested the involvement of a metabolic product of 20-HETE in mediating its pro-adipogenic effects. A cyclooxygenase (COX)-1 selective inhibitor enhanced, whereas a COX-2 selective or a dual COX-1/2 inhibitor attenuated adipogenesis induced by 20-HETE. The COX-derived metabolite of 20-HETE, 20-OH-PGE₂, enhanced adipogenesis and lipid accumulation in MSCs. The pro-adipogenic effects of 20-HETE and 20-OH-PGE₂ resulted in the increased expression of the adipogenic regulators PPARγ and β-catenin in MSC-derived adipocytes. Taken together we show for the first time that 20-HETE-derived COX-2-dependent 20-OH-PGE₂ enhances mature inflamed adipocyte hypertrophy in MSC undergoing adipogenic differentiation.     

Inhibition of cyclooxygenase‐2 enhanced intestinal epithelial homeostasis via suppressing β‐catenin signalling pathway in experimental liver fibrosis

The intestinal barrier dysfunction is crucial for the development of liver fibrosis but can be disturbed by intestinal chronic inflammation characterized with cyclooxygenase‐2 (COX‐2) expression. This study focused on the unknown mechanism by which COX‐2 regulates intestinal epithelial homeostasis in liver fibrosis. The animal models of liver fibrosis induced with TAA were established in rats and in intestinal epithelial–specific COX‐2 knockout mice. The impacts of COX‐2 on intestinal epithelial homeostasis via suppressing β‐catenin signalling pathway were verified pharmacologically and genetically in vivo. A similar assumption was tested in Ls174T cells with goblet cell phenotype in vitro. Firstly, disruption of intestinal epithelial homeostasis in cirrhotic rats was ameliorated by celecoxib, a selective COX‐2 inhibitor. Then, β‐catenin signalling pathway in cirrhotic rats was associated with the activation of COX‐2. Furthermore, intestinal epithelial–specific COX‐2 knockout could suppress β‐catenin signalling pathway and restore the disruption of ileal epithelial homeostasis in cirrhotic mice. Moreover, the effect of COX‐2/PGE2 was dependent on the β‐catenin signalling pathway in Ls174T cells. Therefore, inhibition of COX‐2 may enhance intestinal epithelial homeostasis via suppression of the β‐catenin signalling pathway in liver fibrosis.     

Pygopus2 ameliorates mesenteric adipocyte poor differentiation to alleviate Crohn's disease ‐like colitis via the Axin2/GSK3β pathway

ObjectivesCrohn''s disease (CD) mesenteric adipose tissue (MAT) inflammation affects enteritis through the interaction between the mesentery and intestine, and we previously found that poorly differentiated mesenteric adipocytes were related to its inflammatory features. Pygopus2 (Pygo2) is a key negative regulator of adipocyte differentiation. We aimed to determine whether Pygo2 participates in CD mesenteric lesions and whether Pygo2 knockdown would be beneficial in a CD model (Il‐10 ^−/− mice).MethodsPygo2 expression in MAT from control and CD patients and Il‐10 ^−/− mice was measured by immunohistochemistry. Lentiviral transfection was used to regulate Pygo2 expression in Il‐10 ^−/− mice, and the effects on mesenteric adipocyte differentiation, inflammation, and dysfunction during spontaneous colitis, as well as the possible mechanism, were investigated.ResultsPygo2 expression was increased in MAT from CD patients and Il‐10 ^−/− mice, and its expression correlated with poor adipocyte differentiation and inflammation. Pygo2 knockdown significantly ameliorated colitis in Il‐10 ^−/− mice. Moreover, the downregulation of Pygo2 gene expression could promote adipocyte differentiation and inhibit adipocyte inflammation in vivo and in vitro, and the effects were at least partly mediated by the Axis inhibition protein 2 (Axin2)/glycogen synthase kinase 3 beta (GSK3β) pathway.ConclusionsThe increase in Pygo2 may be related to mesenteric adipocyte poor differentiation and inflammatory features of CD, and Pygo2 inhibition could alleviate CD‐like colitis by improving mesenteric lesions by regulating the Axin2/GSK3β pathway.

The increase in Pygopus2 (Pygo2) may be related to mesenteric adipocyte poor differentiation and inflammatory features of CD, and Pygo2 inhibition could alleviate adipocyte differentiation and mesenteric lesions by regulating the Axin2/GSK3β pathway.     

Induction of beige‐like adipocyte markers and functions in 3T3‐L1 cells by Clk1 and PKCβII inhibitory molecules

Excessive dietary intake of fat results in its storage in white adipose tissue (WAT). Energy expenditure through lipid oxidation occurs in brown adipose tissue (BAT). Certain WAT depots can undergo a change termed beiging where markers that BAT express are induced. Little is known about signalling pathways inducing beiging. Here, inhibition of a signalling pathway regulating alternative pre‐mRNA splicing is involved in adipocyte beiging. Clk1/2/4 kinases regulate splicing by phosphorylating factors that process pre‐mRNA. Clk1 inhibition by TG003 results in beige‐like adipocytes highly expressing PGC1α and UCP1. SiRNA for Clk1, 2 and 4, demonstrated that Clk1 depletion increased UCP1 and PGC1α expression, whereas Clk2/4 siRNA did not. TG003‐treated adipocytes contained fewer lipid droplets, are smaller, and contain more mitochondria, resulting in proton leak increases. Additionally, inhibition of PKCβII activity, a splice variant regulated by Clk1, increased beiging. PGC1α is a substrate for both Clk1 and PKCβII kinases, and we surmised that inhibition of PGC1α phosphorylation resulted in beiging of adipocytes. We show that TG003 binds Clk1 more than Clk2/4 through direct binding, and PGC1α binds to Clk1 at a site close to TG003. Furthermore, we show that TG003 is highly specific for Clk1 across hundreds of kinases in our activity screen. Hence, Clk1 inhibition becomes a target for induction of beige adipocytes.     

The inhibition of enterocyte proliferation by lithocholic acid exacerbates necrotizing enterocolitis through downregulating the Wnt/β‐catenin signalling pathway

ObjectivesNecrotizing enterocolitis (NEC) is a catastrophic gastrointestinal emergency in preterm infants, whose exact aetiology remains unknown. The role of lithocholic acid (LCA), a key component of secondary bile acids (BAs), in NEC is unclear.MethodsClinical data were collected to analyse the changes of BAs in NEC patients. In vitro studies, the cell proliferation and cell death were assessed. In vivo experiments, the newborn rats were administered with low or high dose of LCA and further induced NEC.ResultsClinically, compared with control group, total BAs in the NEC patients were significantly higher when NEC occurred. In vitro, LCA treatment significantly inhibited the cell proliferation through arresting cell cycle at G1/S phase without inducing apoptosis or necroptosis. Mechanistically, the Wnt/β‐catenin pathway was involved. In vivo, LCA inhibited intestinal cell proliferation leading to disruption of intestinal barrier, and thereby increased the severity of NEC. Specifically, LCA supplementation caused higher levels of FITC‐labelled dextran in serum, reduced PCNA expression and inhibited the activity of Wnt/β‐catenin pathway in enterocytes. The LC–MS/MS test found that LCA was significantly higher in intestinal tissue of NEC group, and more obviously in the NEC‐L and NEC‐H group compared with the DM group.ConclusionLCA exacerbates NEC by inhibiting intestinal cell proliferation through downregulating the Wnt/β‐catenin pathway.

The role of lithocholic acid (LCA), a key component of bile acids, in necrotizing enterocolitis(NEC) remains unclear. Our data indicate that LCA exacerbates NEC by inhibiting enterocyte proliferation through downregulating the activity of Wnt/β‐catenin pathway.     

Dynamic folding modulation generates FGF21 variant against diabetes

Fibroblast growth factor 21 (FGF21) is a regulator of glucose and lipid metabolism. It has been widely considered as a promising candidate for the treatment of type 2 diabetes mellitus (T2DM) and other related metabolic disorders. However, lack of structural and dynamic information has limited FGF21‐based drug development. Here, using nuclear magnetic resonance (NMR) spectroscopy, we determine the structure of FGF21 and find that its non‐canonical flexible β‐trefoil conformation affects the folding of β2‐β3 hairpin and further overall protein stability. To modulate folding dynamics, we designed an FGF21‐FGF19 chimera, FGF21^SS. As expected, FGF21^SS shows better thermostability without inducing hepatocyte proliferation. Functional characterization of FGF21^SS shows its better insulin sensitivity, reduced inflammation in 3T3‐L1 adipocytes, and lower blood glucose and insulin levels in ob/ob mice compared with wild type. Our dynamics‐based rational design provides a promising approach for FGF21‐based therapeutic development against T2DM.     

Phosphorylation of β‐catenin Ser60 by polo‐like kinase 1 drives the completion of cytokinesis

β‐Catenin is a multifunctional protein and participates in numerous processes required for embryonic development, cell proliferation, and homeostasis through various molecular interactions and signaling pathways. To date, however, there is no direct evidence that β‐catenin contributes to cytokinesis. Here, we identify a novel p‐S60 epitope on β‐catenin generated by Plk1 kinase activity, which can be found at the actomyosin contractile ring of early telophase cells and at the midbody of late telophase cells. Depletion of β‐catenin leads to cytokinesis‐defective phenotypes, which eventually result in apoptotic cell death. In addition, phosphorylation of β‐catenin Ser60 by Plk1 is essential for the recruitment of Ect2 to the midbody, activation of RhoA, and interaction between β‐catenin, Plk1, and Ect2. Time‐lapse image analysis confirmed the importance of β‐catenin phospho‐Ser60 in furrow ingression and the completion of cytokinesis. Taken together, we propose that phosphorylation of β‐catenin Ser60 by Plk1 in cooperation with Ect2 is essential for the completion of cytokinesis. These findings may provide fundamental knowledge for the research of cytokinesis failure‐derived human diseases.     

Downregulation of STRA6 in Adipocytes and Adipose Stromovascular Fraction in Obesity and Effects of Adipocyte-Specific STRA6 Knockdown In Vivo

To investigate the mechanisms by which elevated retinol-binding protein 4 (RBP4) causes insulin resistance, we studied the role of the high-affinity receptor for RBP4, STRA6 (stimulated by retinoic acid), in insulin resistance and obesity. In high-fat-diet-fed and ob/ob mice, STRA6 expression was decreased 70 to 95% in perigonadal adipocytes and both perigonadal and subcutaneous adipose stromovascular cells. To determine whether downregulation of STRA6 in adipocytes contributes to insulin resistance, we generated adipose-Stra6^−/− mice. Adipose-Stra6^−/− mice fed chow had decreased body weight, fat mass, leptin levels, insulin levels, and adipocyte number and increased expression of brown fat-selective markers in white adipose tissue. When fed a high-fat diet, these mice had a mild improvement in insulin sensitivity at an age when adiposity was unchanged. STRA6 has been implicated in retinol uptake, but retinol uptake and the expression of retinoid homeostatic genes (encoding retinoic acid receptor β [RARβ], CYP26A1, and lecithin retinol acyltransferase) were not altered in adipocytes from adipose-Stra6^−/− mice, indicating that retinoid homeostasis was maintained with STRA6 knockdown. Thus, STRA6 reduction in adipocytes in adipose-Stra6^−/− mice fed chow resulted in leanness, which may contribute to their increased insulin sensitivity. However, in wild-type mice with high-fat-diet-induced obesity and in ob/ob mice, the marked downregulation of STRA6 in adipocytes and adipose stromovascular cells does not compensate for obesity-associated insulin resistance.     

Zinc finger protein 91 accelerates tumour progression by activating β‐catenin signalling in pancreatic cancer

ObjectivesZFP91, an E3 ligase, has been reported to possess cancer‐promoting functions. This study aimed to elucidate the exact role of ZFP91 in tumour progression of pancreatic cancer and underlying mechanisms.Materials and MethodsWe analysed the correlation between ZFP91 expression and pancreatic cancer through TCGA and GEO data sets. Growth curve, colony formation, wound healing and transwell invasion assays were conducted to evaluate proliferation, migration and invasion of lentivirus transfected pancreatic cancer cells. GSEA and Western blot analysis were performed to validate the regulatory effect of ZFP91 on β‐catenin. Drug response curve and orthotopic implantation model reflected the sensitivity of chemotherapies.ResultsZFP91 overexpression is prevalent in pancreatic cancer and negatively correlated with overall survival. ZFP91 knock‐down attenuated proliferation, migration and invasion of pancreatic cancer cells. β‐catenin was a downstream gene of ZFP91, and its agonist could reverse the phenotype. ZFP91 promoted EMT and chemoresistance in pancreatic cancer.ConclusionsWe demonstrated that ZFP91 promoted pancreatic cancer proliferation, migration and invasion through activating β‐catenin signalling. EMT and chemoresistance were also regulated by ZFP91. ZFP91 might be a potential therapeutic target for pancreatic cancer.     

An adiponectin receptor agonist promote osteogenesis via regulating bone‐fat balance

ObjectivesAdiponectin signalling has been considered to be a promising target to treat diabetes‐related osteoporosis. However, contradictory results regarding bone formation were observed due to the various isoforms of adiponectin. Therefore, it would be necessary to investigate the effect of adiponectin receptor signals in regulating bone‐fat balance.Materials and MethodsWe primarily applied a newly found specific activator for adiponectin receptor, AdipoRon, to treat bone metabolism‐related cells to investigate the role of Adiponectin receptor signals on bone‐fat balance. We then established femur defect mouse model and treated them with AdipoRon to see whether adiponectin receptor activation could promote bone regeneration.ResultsWe found that AdipoRon could slightly inhibit the proliferation of pre‐osteoblast and pre‐osteoclast, but AdipoRon showed no effect on the viability of mesenchymal stromal cells. AdipoRon could remarkably promote cell migration of mesenchymal stromal cells. Additionally, AdipoRon promoted osteogenesis in both pre‐osteoblasts and mesenchymal cells. Besides, AdipoRon significantly inhibited osteoclastogenesis via its direct impact on pre‐osteoclast and its indirect inhibition of RANKL in osteoblast. Moreover, mesenchymal stromal stems cells showed obviously decreased adipogenesis when treated with AdipoRon. Consistently, AdipoRon‐treated mice showed faster bone regeneration and repressed adipogenesis.ConclusionsOur study demonstrated a pro‐osteogenic, anti‐adipogenic and anti‐osteoclastogenic effect of adiponectin receptor activation in young mice, which suggested adiponectin receptor signalling was involved in bone regeneration and bone‐fat balance regulation.     

Direct interaction of β‐catenin with nuclear ESM1 supports stemness of metastatic prostate cancer

Wnt/β‐catenin signaling is frequently activated in advanced prostate cancer and contributes to therapy resistance and metastasis. However, activating mutations in the Wnt/β‐catenin pathway are not common in prostate cancer, suggesting alternative regulations may exist. Here, we report that the expression of endothelial cell‐specific molecule 1 (ESM1), a secretory proteoglycan, is positively associated with prostate cancer stemness and progression by promoting Wnt/β‐catenin signaling. Elevated ESM1 expression correlates with poor overall survival and metastasis. Accumulation of nuclear ESM1, instead of cytosolic or secretory ESM1, supports prostate cancer stemness by interacting with the ARM domain of β‐catenin to stabilize β‐catenin–TCF4 complex and facilitate the transactivation of Wnt/β‐catenin signaling targets. Accordingly, activated β‐catenin in turn mediates the nuclear entry of ESM1. Our results establish the significance of mislocalized ESM1 in driving metastasis in prostate cancer by coordinating the Wnt/β‐catenin pathway, with implications for its potential use as a diagnostic or prognostic biomarker and as a candidate therapeutic target in prostate cancer.     

The orphan nuclear receptor small heterodimer partner is required for thiazolidinedione effects in leptin-deficient mice

Background

Small heterodimer partner (SHP, NR0B2) is involved in diverse metabolic pathways, including hepatic bile acid, lipid and glucose homeostasis, and has been implicated in effects on the peroxisome proliferator-activated receptor γ (PPARγ), a master regulator of adipogenesis and the receptor for antidiabetic drugs thiazolidinediones (TZDs). In this study, we aim to investigate the role of SHP in TZD response by comparing TZD-treated leptin-deficient (ob/ob) and leptin-, SHP-deficient (ob/ob;Shp^−/−) double mutant mice.

Results

Both ob/ob and double mutant ob/ob;Shp^−/− mice developed hyperglycemia, insulin resistance, and hyperlipidemia, but hepatic fat accumulation was decreased in the double mutant ob/ob;Shp^−/− mice. PPARγ2 mRNA levels were markedly lower in ob/ob;Shp^−/− liver and decreased to a lesser extent in adipose tissue. The TZD troglitazone did not reduce glucose or circulating triglyceride levels in ob/ob;Shp^−/− mice. Expression of the adipocytokines, such as adiponectin and resistin, was not stimulated by troglitazone treatment. Expression of hepatic lipogenic genes was also reduced in ob/ob;Shp^−/− mice. Moreover, overexpression of SHP by adenovirus infection increased PPARγ2 mRNA levels in mouse primary hepatocytes.

Conclusions

Our results suggest that SHP is required for both antidiabetic and hypolipidemic effects of TZDs in ob/ob mice through regulation of PPARγ expression.     

Cask methylation involved in the injury of insulin secretion function caused by interleukin1‐β

Islet inflammation severely impairs pancreatic β‐cell function, but the specific mechanisms are still unclear. Interleukin1‐β (IL‐1β), an essential inflammatory factor, exerts a vital role in multiple physio‐pathologic processes, including diabetes. Calcium/calmodulin‐dependent serine protein kinase (CASK) is an important regulator especially in insulin secretion process. This study aims to unveil the function of CASK in IL‐1β–induced insulin secretion dysfunction and the possible mechanism thereof. Islets of Sprague‐Dawley (SD) rats and INS‐1 cells stimulated with IL‐1β were utilized as models of chronic inflammation. Insulin secretion function associated with Cask and DNA methyltransferases (DNMT) expression were assessed. The possible mechanisms of IL‐1β‐induced pancreatic β‐cell dysfunction were also explored. In this study, CASK overexpression effectively improved IL‐1β‐induced islet β‐cells dysfunction, increased insulin secretion. DNA methyltransferases and the level of methylation in the promoter region of Cask were elevated after IL‐1β administration. Methyltransferase inhibitor 5‐Aza‐2’‐deoxycytidine (5‐Aza‐dC) and si‐DNMTs partially up‐regulated CASK expression and reversed potassium stimulated insulin secretion (KSIS) and glucose‐stimulated insulin secretion (GSIS) function under IL‐1β treatment in INS‐1 and rat islets. These results reveal a previously unknown effect of IL‐1β on insulin secretion dysfunction and demonstrate a novel pathway for Cask silencing based on activation of DNA methyltransferases via inducible nitric oxide synthase (iNOS) and modification of gene promoter methylation.