Browning of Mammary Fat Suppresses Pubertal Mammary Gland Development of Mice via Elevation of Serum Phosphatidylcholine and Inhibition of PI3K/Akt Pathway.

Mammary fat plays a profound role in the postnatal development of mammary glands. However, the specific types (white, brown, or beige) of adipocytes in mammary fat and their potential regulatory effects on modulating mammary gland development remain poorly understood. This study aimed to investigate the role of the browning of mammary fat on pubertal mammary gland development and explore the underlying mechanisms. Thus, the mammary gland development and the serum lipid profile were evaluated in mice treated with CL316243, a ß3-adrenoceptor agonist, to induce mammary fat browning. In addition, the proliferation of HC11 cells co-cultured with brown adipocytes or treated with the altered serum lipid metabolite was determined. Our results showed that the browning of mammary fat by injection of CL316243 suppressed the pubertal development of mice mammary glands, accompanied by the significant elevation of serum dioleoylphosphocholine (DOPC). In addition, the proliferation of HC11 was repressed when co-cultured with brown adipocytes or treated with DOPC. Furthermore, DOPC suppressed the activation of the PI3K/Akt pathway, while the DOPC-inhibited HC11 proliferation was reversed by SC79, an Akt activator, suggesting the involvement of the PI3K/Akt pathway in the DOPC-inhibited proliferation of HC11. Together, the browning of mammary fat suppressed the development of the pubertal mammary gland, which was associated with the elevated serum DOPC and the inhibition of the PI3K/Akt pathway.

Dietary chenodeoxycholic acid improves growth performance and intestinal health by altering serum metabolic profiles and gut bacteria in weaned piglets.

Nutritional diarrhea and subsequent performance degradation in weaned piglets are major challenges for the pig industry. Bile acids (BA) can be added to the diet as emulsifiers. This experiment was conducted to investigate the effects of chenodeoxycholic acid (CDCA), a major primary BA, on growth performance, serum metabolic profiles and gut health in weaned piglets. A total of 72 healthy weaned piglets were randomly assigned to the control (CON) and the CDCA groups, which were feed a basal diet and the basal diet supplemented with 200 mg/kg CDCA for 30 d, respectively. Our results demonstrated that CDCA significantly increased final BW and average daily gain (ADG), decreased feed-to-gain (F:G) ratio and tended to reduce diarrhea incidence. In addition, CDCA increased the villus height-to-crypt depth (V:C) ratio, elevated goblet cell numbers and the expression of tight junction proteins, suggesting the enhancement of intestinal barrier function. As an emulsifier, CDCA increased jejunal lipase activity and the mRNA expression of pancreatic lipases. CDCA supplementation also altered the serum metabolic profiles, including increasing the levels of indole 3-acetic acid, N'-formylkynurenine and theobromine that were beneficial for gut health. Moreover, the relative abundance of 2 beneficial gut bacteria, Prevotella 9 and Prevotellaceae TCG-001, were increased, whereas the relative abundance of a harmful bacteria, Dorea, was decreased in the gut of weaned piglets supplemented with CDCA. Importantly, the altered serum metabolic profiles showed a strong correlation with the changed gut bacteria. In conclusion, CDCA improved the growth performance of weaned piglets by improving intestinal morphology and barrier function, and enhancing lipid digestion, accompanied by alterations of serum metabolic profiles, and changes in relative abundance of certain gut bacteria.

Hyodeoxycholic acid (HDCA) suppresses intestinal epithelial cell proliferation through FXR-PI3K/AKT pathway, accompanied by alteration of bile acids metabolism profiles induced by gut bacteria.

Bile acids (BAs) have been implicated in regulation of intestinal epithelial signaling and function. This study aimed to investigate the effects of hyodeoxycholic acid (HDCA) on intestinal epithelial cell proliferation and explore the underlying mechanisms. IPEC-J2 cells and weaned piglets were treated with HDCA and the contributions of cellular signaling pathways, BAs metabolism profiles and gut bacteria were assessed. In vitro, HDCA suppressed IPEC-J2 proliferation via the BAs receptor FXR but not TGR5. In addition, HDCA inhibited the PI3K/AKT pathway, while knockdown of FXR or constitutive activation of AKT eliminated the inhibitory effects of HDCA, suggesting that FXR-dependent inhibition of PI3K/AKT pathway was involved in HDCA-suppressed IPEC-J2 proliferation. In vivo, dietary HDCA inhibited intestinal expression of proliferative markers and PI3K/AKT pathway in weaned piglets. Meanwhile, HDCA altered the BAs metabolism profiles, with decrease in primary BA and increase in total and secondary BAs in feces, and reduction of conjugated BAs in serum. Furthermore, HDCA increased abundance of the gut bacteria associated with BAs metabolism, and thereby induced BAs profiles alternation, which might indirectly contribute to HDCA-suppressed cell proliferation. Together, HDCA suppressed intestinal epithelial cell proliferation through FXR-PI3K/AKT signaling pathway, accompanied by alteration of BAs metabolism profiles induced by gut bacteria.

cis 9, trans 11, but not trans 10, cis 12 CLA isomer, impairs intestinal epithelial barrier function in IPEC-J2 cells and mice through activation of GPR120-[Ca2+]i and the MLCK signaling pathway.

This study aimed to investigate the effects of conjugated linoleic acid (CLA) on intestinal epithelial barrier function and explore the underlying mechanisms. IPEC-J2 cells and mice were treated with different CLA isomers. The intestinal epithelial barrier function determined by transepithelial electrical resistance (TEER), the expression of tight junction proteins, and the involvement of G-protein coupled receptor 120 (GPR120), intracellular calcium ([Ca2+]i) and myosin light chain kinase (MLCK) were assessed. In vitro, c9, t11-CLA, but not t10, c12-CLA isomer, impaired epithelial barrier function in IPEC-J2 by downregulating the expression of tight junction proteins. Meanwhile, c9, t11-CLA isomer enhanced GPR120 expression, while knockdown of GPR120 eliminated the impaired epithelial barrier function induced by c9, t11-CLA isomer. In addition, c9, t11-CLA isomer increased [Ca2+]i and activated the MLCK signaling pathway in a GPR120-dependent manner. However, chelation of [Ca2+]i reversed c9, t11-CLA isomer-induced MLCK activation and the epithelial barrier function impairment of IPEC-J2. Furthermore, inhibition of MLCK totally abolished the impairment of epithelial barrier function induced by c9, t11-CLA. In vivo, dietary supplementation of c9, t11-CLA rather than t10, c12-CLA isomer decreased the expression of intestinal tight junction proteins and GPR120, increased intestinal permeability, and activated the MLCK signaling pathway in mice. Taken together, our findings showed that c9, t11-CLA, but not t10, c12-CLA isomer, impaired intestinal epithelial barrier function in IPEC-J2 cells and mice through activation of GPR120-[Ca2+]i and the MLCK signaling pathway. These data provided new insight into the regulation of the intestinal epithelial barrier by different CLA isomers and more references for CLA application in humans and animals.

Calcium Supplementation Alleviates High-Fat Diet-Induced Estrous Cycle Irregularity and Subfertility Associated with Concomitantly Enhanced Thermogenesis of Brown Adipose Tissue and Browning of White Adipose Tissue.

Obesity has been demonstrated as a disruptor of female fertility. Our previous study showed the antiobesity effects of calcium on HFD-fed male mice. However, the role of calcium in alleviating reproductive dysfunction of HFD-fed female mice remains unclear. Here, we found that HFD led to estrus cycle irregularity (longer cycle duration and shorter estrus period) and subfertility (longer conception time, lower fertility index, and less implantations) in mice. However, the HFD-induced reproductive abnormality was alleviated by calcium supplementation. Additionally, calcium supplementation enhanced activation/thermogenesis of BAT and browning of WAT in HFD-fed mice. Consequently, the abnormality of energy metabolism and glucose homeostasis induced by HFD were improved by calcium supplementation, with elevated metabolic rates and core temperature. In conclusion, these data showed that calcium supplementation alleviated HFD-induced estrous cycle irregularity and subfertility associated with concomitantly enhanced BAT thermogenesis and WAT browning, suggesting the potential application of calcium in improving obesity-related reproductive disorders.

Calcium Supplementation Enhanced Adipogenesis and Improved Glucose Homeostasis Through Activation of Camkii and PI3K/Akt Signaling Pathway in Porcine Bone Marrow Mesenchymal Stem Cells (pBMSCs) and Mice Fed High Fat Diet (HFD).

BACKGROUND/AIMS: It has been implicated that calcium supplementation is involved in reducing body weight/fat and improving glucose homeostasis. However, the underlying mechanisms are still not fully understood. Here, we investigated the effects of calcium supplementation on adipogenesis and glucose homeostasis in porcine bone marrow mesenchymal stem cells (pBMSCs) and high fat diet (HFD)-fed mice and explored the involved signaling pathways. METHODS: In vitro, pBMSCs were treated with 4 mM extracellular calcium ([Ca2+]o) and/or 1 µM nifedipine, 0.1 µM BAPTA-AM, 1 µM KN-93, 50 nM wortmannin for 10 days. The intracellular calcium ([Ca2+]i) levels were measured using Fluo 3-AM by flow cytometry. The adipogenic differentiation of pBMSCs was determined by Oil Red-O staining and triglyceride assay. The expression of marker genes involved in adipogenesis (peroxisome proliferator activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα)) and glucose uptake (glucose transporter 4 (GLUT4)), as well as the activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and PI3K/Akt-FoxO1/AS160 signaling pathways were determined by Western blotting. Glucose uptake and utilization were examined using 2-NBDG assay and glucose content assay, respectively. In vivo, C57BL/6J male mice were fed a HFD (containing 1.2% calcium) without or with 0.6% (w/w) calcium chloride in drinking water for 13 weeks. The adipogenesis, glucose homeostasis and the involvement of CaMKII and PI3K/Akt signaling pathway were also assessed. RESULTS: In vitro, [Ca2+]o stimulated pBMSCs adipogenesis by increasing [Ca2+]i level and activating CaMKII and PI3K/Akt-FoxO1 pathways. In addition, [Ca2+]o promoted glucose uptake/utilization by enhancing AS160 phosphorylation, GLUT4 expression and translocation. However, the stimulating effects of [Ca2+]o on pBMSCs adipogenesis and glucose uptake/utilization were abolished by L-VGCC blocker Nifedipine, [Ca2+]i chelator BAPTA-AM, CaMKII inhibitor KN-93, or PI3K inhibitor Wortmannin. In vivo, calcium supplementation decreased body weight and fat content, increased adipocyte number, and improved glucose homeostasis, with elevated PPARγ and GLUT4 expression and PI3K/Akt activation in iWAT. CONCLUSION: calcium supplementation enhanced adipogenesis and glucose uptake in pBMSCs, which was coincident with the increased adipocyte number and improved glucose homeostasis in HFD-fed mice, and was associated with activation of CaMKII and PI3K/Akt-FoxO1/AS160 pathways. These data provided a broader understanding of the mechanisms underlying calcium-induced body weight/fat loss and glycemic control.