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BACKGROUND: Local vibration has shown promise in improving skin blood flow and wound healing. However, the underlying mechanism of local vibration as a preconditioning intervention to alter plantar skin blood flow after walking is unclear. OBJECTIVE: The objective was to use wavelet analysis of skin blood flow oscillations to investigate the effect of preconditioning local vibration on plantar tissues after walking. METHODS: A double-blind, repeated measures design was tested in 10 healthy participants. The protocol included 10-min baseline, 10-min local vibrations (100 Hz or sham), 10-min walking, and 10-min recovery periods. Skin blood flow was measured over the first metatarsal head of the right foot during the baseline and recovery periods. Wavelet amplitudes after walking were expressed as the ratio of the wavelet amplitude before walking. RESULTS: The results showed the significant difference in the metabolic (vibration 10.06 ± 1.97, sham 5.78 ± 1.53, p

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.

Weight-bearing exercise such as walking may increase risk of foot ulcers in people with diabetes mellitus (DM) because of plantar ischemia due to repetitive, high plantar pressure. Applications of local vibrations on plantar tissues as a preconditioning intervention before walking may reduce plantar tissue ischemia during walking. The objective of this study was to explore whether preconditioning local vibrations reduce reactive hyperemia after walking. A double-blind, repeated-measures, and crossover design was tested in 10 healthy participants without DM. The protocol included 10-minute baseline, 10-minute local vibrations (100 Hz or sham), 10-minute walking, and 10-minute recovery periods. The order of local vibrations was randomly assigned. Skin blood flow (SBF) was measured over the first metatarsal head during baseline and recovery periods. SBF responses were characterized as peak SBF, total SBF, and recovery time of reactive hyperemia. SBF was expressed as a ratio of recovery to baseline SBF to quantify the changes. Peak SBF in the vibration protocol (6.98 ± 0.87) was significantly lower than the sham control (9.26 ± 1.34, P < .01). Total SBF in the vibration protocol ([33.32 ± 7.98] × 103) was significantly lower than the sham control ([48.09 ± 8.9] × 103, P < .05). The recovery time in the vibration protocol (166.08 ± 32.71 seconds) was not significantly different from the sham control (223.53 ± 38.85 seconds, P = .1). Local vibrations at 100 Hz could reduce walking-induced hyperemic response on the first metatarsal head. Our finding indicates that preconditioning local vibrations could be a potential preventive intervention for people at risk for foot ulcers.

The development of muscle in the embryo, which is crucial for postnatal skeletal muscle growth, has been investigated widely. Much has been learned during the past several decades about the role of maternal nutrition in the outcome of pregnancy. Protein and carbohydrate levels during pregnancy have been shown to be important in the development of offspring, especially muscle development. However, the maternal effects of steroids were still not clear. Phytosterol esters (PEs) are produced by the esterification of phytosterols and fatty acids and have many beneficial functions, such as anti-inflammation and hypolipemic functions. Through the effect of regulation on lipid metabolism, can pregnant mice fed with PEs show any programming effect on the muscle development of offspring? In our study, PEs were supplied to the maternal diet, and changes in maternal lipid metabolism and the development of offspring skeletal muscle were detected. As a result, the amniotic fluid total bile acid (TBA) and total cholesterol (TC) levels were decreased; the growth of offspring was significantly faster than that of the control group until 6 weeks of age. Adult offspring had a higher lean mass index and grip strength. In skeletal muscle, the proportion of myosin heavy chain (MHC) 1 was significantly decreased, while the proportion of MHC 2 b was increased. In conclusion, maternal PEs significantly reduced sterols in the amniotic fluid, while skeletal muscle development was promoted in the offspring.

Objective: To assess the effectiveness of physical activity (PA) intervention on attention-deficit/hyperactivity disorder (ADHD)-related symptoms.Method: Studies that investigated PA intervention for ADHD-related symptoms were identified through searching PubMed, Web of Science, Cochrane Library, and Embase databases from inception through June 2021. Standardized mean difference (SMD) with 95% confidence interval (CI) was used to assess the effectiveness of PA intervention on improving ADHD-related symptoms. The meta-analyses were conducted using fixed-effect or random-effect models according to the heterogeneity of the studies.Results: Nine before–after studies (232 participants) and 14 two-group control studies (162 participants/141 controls) were included in this meta-analysis. Combined results for before–after studies indicated significant improvements on all studied ADHD-related symptoms (inattention: SMD = 0.604, 95% CI: 0.374–0.834, p < 0.001; hyperactivity/impulsivity: SMD = 0.676, 95% CI: 0.401–0.950, p < 0.001; emotional problems: SMD = 0.416, 95% CI: 0.283–0.549, p < 0.001; behavioral problems: SMD = 0.347, 95% CI: 0.202–0.492, p < 0.001). Meta-analyses for two-group control studies further confirmed that PA intervention significantly improved the inattentive symptom (SMD = 0.715, 95% CI: 0.105, 1.325, p = 0.022). Subgroup analyses suggested significant beneficial effect on inattention symptoms in children. Moreover, closed motor skills were beneficial for hyperactive/impulsive problems (SMD = 0.671, p < 0.001), while open motor skills were beneficial for attention problems (SMD = 0.455, p = 0.049). When excluding studies with combined medication, the studies in unmedicated participants in before–after studies still showed significant results in all studied ADHD-related symptoms as in the overall analysis. Given the limited sample size, the best frequency and intensity of PA intervention need further investigation.Conclusion: Our results suggested that PA intervention could possibly improve ADHD-related symptoms, especially inattention symptoms. Closed-skill and open-skill activities could be beneficial for hyperactivity/impulsivity and inattention symptoms, respectively. Further high-quality randomized clinical trials with large sample size are needed.

Aim Heparin, a widely used antithrombotic drug has many other anticoagulant‐independent physiological functions. Here, we elucidate a novel role of heparin in glucose homeostasis, suggesting an approach for developing heparin‐targeted therapies for diabetes. Methods For serum heparin levels and correlation analysis, 122 volunteer’s plasma, DIO (4 weeks HFD) and db/db mice serums were collected and used for spectrophotometric determination. OGTT, ITT, 2‐NBDG uptake and muscle GLUT4 immunofluorescence were detected in chronic intraperitoneal injection of heparin or heparinase (16 days) and muscle‐specific loss‐of‐function mice. In 293T cells, the binding of insulin to its receptor was detected by fluorescence resonance energy transfer (FRET), Myc‐GLUT4‐mCherry plasmid was used in GLUT4 translocation. In vitro, C2C12 cells as mouse myoblast cells were further verified the effects of heparin on glucose homeostasis through 2‐NBDG uptake, Western blot and co‐immunoprecipitation. Results Serum concentrations of heparin are positively associated with blood glucose levels in humans and are significantly increased in diet‐induced and db/db obesity mouse models. Consistently, a chronic intraperitoneal injection of heparin results in hyperglycaemia, glucose intolerance and insulin resistance. These effects are independent of heparin’s anticoagulant function and associated with decreases in glucose uptake and translocation of glucose transporter type 4 (GLUT4) in skeletal muscle. By using a muscle‐specific loss‐of‐function mouse model, we further demonstrated that muscle GLUT4 is required for the detrimental effects of heparin on glucose homeostasis. Conclusions Heparin reduced insulin binding to its receptor by interacting with insulin and inhibited insulin‐mediated activation of the PI3K/Akt signalling pathway in skeletal muscle, which leads to impaired glucose uptake and hyperglycaemia., Circulating heparin is positively correlated with blood glucose level in humans and increases in diet‐induced obese and type 2 diabetic db/db mice. Chronic intraperitoneal injection of heparin results in hyperglycemia, glucose intolerance, and insulin resistance. Heparin interacts with insulin to inhibit insulin and insulin receptor binding in skeletal muscle, and by doing so, impairs skeletal muscle GLUT4 translocation and glucose uptake.

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.

Thymol is a major component of thyme, and it has been reported that thymol administration reduces body weight, plasma insulin and blood glucose in type-2 diabetes. Skeletal muscle is the most important metabolism organs in the body; however, to date, there is no report on the effect of thymol on skeletal muscle. Our goal was to determine whether thymol has an effect on the different types of skeletal muscle fibers and their metabolism characteristics. Hence, we performed in vivo and in vitro experiments. In vivo, SD rats (4 weeks old) were fed with different concentrations of thymol for 4 weeks, and in vitro C2C12 myotubes were directly treated with thymol for 2 days. The rats fed with 0.025% thymol showed a significantly lower body weight, subcutaneous white adipose tissue index and gastrocnemius muscle index (P < 0.05), while their proportion of brown adipose tissue significantly increased (P < 0.05). The protein and mRNA expression of MyHC I and MyHC IIa in the gastrocnemius muscle of the rats significantly increased (P < 0.05), while the protein level of MyHC II and mRNA expression of MyHC IIb decreased (P < 0.05). Furthermore, 0.025% thymol supplement significantly reduced (P < 0.05) the activity of lactate dehydrogenase (LDH) in the gastrocnemius muscle of the rats, but their succinate dehydrogenase (SDH) and hexokinase (HK) activities increased (P < 0.05). Also, the expression of the fatty acid oxidation-related genes in the gastrocnemius muscle of the rats decreased with the thymol supplement (P < 0.05). In vitro, similar results were obtained. Furthermore, the Ca2+-calcineurin-NFAT pathway, which is an important pathway to regulate the transformation of skeletal muscle fiber type, was studied. We found that the effects of thymol on the myosin heavy chain isoforms, genes related to metabolism and the activation of the Ca2+-calcineurin-NFAT pathway were all reversed by a Ca2+ chelator (P < 0.05). Thus, thymol can promote the oxidative metabolism and fiber type switch in skeletal muscle, and the Ca2+-calcineurin-NFAT pathway plays an important role in it.

Skeletal muscle is the largest organ of the body, the development of skeletal muscle is very important for the health of the animal body. Prolyl hydroxylases (PHDs) are the classical regulator of the hypoxia inducible factor (HIF) signal pathway, many researchers found that PHDs are involved in the muscle fiber type transformation, muscle regeneration, and myocyte differentiation. However, whether PHDs can impact the protein turnover of skeletal muscle is poorly understood. In this study, we constructed denervated muscle atrophy mouse model and found PHD3 was highly expressed in the atrophic muscles and there was a significant correlation between the expression level of PHD3 and skeletal muscle weight which was distinct from PHD1 and PHD2. Then, the similar results were getting from the different weight muscles of normal mice. To further verify the relationship between PHD3 and skeletal muscle protein turnover, we established a PHD3 interference model by injecting PHD3 sgRNA virus into tibialis anterior muscle (TA) muscle of MCK-Cre-cas9 mice and transfecting PHD3 shRNA lentivirus into primary satellite cells. It was found that the Knock-out of PHD3 in vivo led to a significant increase in muscle weight and muscle fiber area (P < .05). Besides, the activity of protein synthesis signal pathway increased significantly, while the protein degradation pathway was inhibited evidently (P < .05). In vitro, the results of 5-ethynyl-2'-deoxyuridine (EdU) and tetramethylrhodamine ethyl ester (TMRE) fluorescence detection showed that PHD3 interference could lead to a decrease in cell proliferation and an increase of cell apoptosis. After the differentiation of satellite cells, the production of puromycin in the interference group was higher than that in the control group, and the content of 3-methylhistidine in the interference group was lower than that in the control group (P < .05) which is consistent with the change of protein turnover signal pathway in the cell. Mechanistically, there is an interaction between PHD3, NF-κB, and IKBα which was detected by immunoprecipitation. With the interfering of PHD3, the expression of the inflammatory signal pathway also significantly decreased (P < .05). These results suggest that PHD3 may affect protein turnover in muscle tissue by mediating inflammatory signal pathway. Finally, we knocked out PHD3 in denervated muscle atrophy mice and LPS-induced myotubes atrophy model. Then, we found that the decrease of PHD3 protein level could alleviate the muscle weight and muscle fiber reduction induced by denervation in mice. Meanwhile, the protein level of the inflammatory signal pathway and the content of 3-methylhistidine in denervated atrophic muscle were also significantly reduced (P < .05). In vitro, PHD3 knock-out could alleviate the decrease of myotube diameter induced by LPS, and the expression of protein synthesis pathway was also significantly increased (P < .05). On the contrary, the expression level of protein degradation and inflammatory signal pathway was significantly decreased (P < .05). Through these series of studies, we found that the increased expression of PHD3 in denervated muscle might be an important regulator in inducing muscle atrophy, and this process is likely to be mediated by the inflammatory NF-κB signal pathway.

Hypoxanthine (Hx), an intermediate metabolite of the purine metabolism pathway which is dramatically increased in blood and skeletal muscle during muscle contraction and metabolism, is characterized as a marker of exercise exhaustion. However, the physiological effects of Hx on skeletal muscle remain unknown. Herein, we demonstrate that chronic treatment with Hx through dietary supplementation resulted in skeletal muscle fatigue and impaired the exercise performance of mice without affecting their growth and skeletal muscle development. Hx increased the uncoupling protein 2 (UCP2) expression in the skeletal muscle, which led to decreased energy substrate storage and enhanced glycolysis. These effects could also be verified in acute treatment with Hx through intraperitoneal injection. In addition, muscular specifically knockout of UCP2 through intra-muscle tissue injection of adenovirus-associated virus reversed the effects of Hx. In conclusion, we identified a novel role of Hx in the skeletal muscular fatigue mediated by UCP2-dependent mitochondrial uncoupling. This finding may shed light on the pathological mechanism of clinical muscle dysfunctions due to abnormal metabolism, such as muscle fatigue and weakness.

While resistance exercise effectively improves overall health in diabetic patients, the underlying biological mechanism by which resistance exercise improves metabolic function and glucose homeostasis remain mostly unknown. Previously, we identified a myometabolite-mediated metabolic pathway that is essential for the beneficial effects of resistance exercise on metabolic function. We found that resistance exercise-induced α-ketoglutaric acid (AKG) stimulates muscle hypertrophy and fat loss through 2-oxoglutarate receptor 1 (OXGR1)-dependent adrenal activation. Here, we provided evidence for the beneficial effects of AKG on glucose homeostasis in a diet-induced obesity (DIO) mouse model, which are independent of OXGR1. We showed that circulating AKG levels are negatively correlated with the fraction of blood glycated hemoglobin (HbA1c) in both humans and mice and significantly decreased in DIO mice. Consistently, pharmacological elevation of AKG effectively decreased body weight, blood glucose, and hepatic gluconeogenesis without changing insulin sensitivity and glucose tolerance in DIO mice. Notably, OXGR1KO blocked the inhibitory effects of AKG on body weight but failed to affect AKG’s suppression on blood glucose and hepatic gluconeogenesis, indicating distinct mechanisms for AKG’s regulation on energy balance and glucose homeostasis. In supporting this view, we showed that serpina1e, a member of protease inhibitor serpins superfamily, mediates the direct inhibitory effects of AKG on gluconeogenesis in both in vitro hepatocytes and liver slice. By using a liver-specific serpina1e deletion mouse model, we further demonstrated that liver serpina1e is required for the inhibitory effects of AKG on hepatic gluconeogenesis and hyperglycemia in DIO mice. Finally, we provided in vitro evidence to support a model in which AKG decreases hepatic gluconeogenesis by targeting trimethylation of lysine 27 on histone 3 (H3K27me3) in seprina1e promoter region. Our studies established an important role of AKG in glucose homeostasis, and identified the AKG-serpina1e pathway as potential therapeutic targets to attenuate hyperglycemia.

Lauric acid (LA) has been implicated in the prevention/treatment of obesity. However, the role of LA in modulating an obesity-related female reproductive disorder remains largely unknown. Here, female mice were fed a control diet, high-fat diet (HFD), or HFD supplemented with 1% LA. The results demonstrated that the HFD-induced estrous cycle irregularity and the reduction of serum follicle-stimulating hormone (FSH) were alleviated by LA supplementation. In possible mechanisms, LA supplementation led to significant increase in serum lipid metabolites such as sphingomyelin and lysophosphatidylcholine containing LA (C12:0) and the improvement of glucose metabolism in mice fed HFD. Moreover, impaired body energy metabolism and weakened brown adipose tissue (BAT) thermogenesis of HFD-fed mice were improved by LA supplementation. Together, these findings showed that LA supplementation alleviated HFD-induced estrous cycle irregularity, possibly associated with altered serum lipid metabolites, improved glucose metabolism, body energy metabolism, and BAT thermogenesis. These findings suggested the potential application of LA in alleviating obesity and its related reproductive disorders.

Recently, embryo muscle development, which is crucial for postnatal skeletal muscle growth, has been investigated widely. Nutrients in ovo were suggested to be critical in embryo muscle development since the chick growth mostly relies on nutrients in eggs at the early developmental stage. Phytosterol esters (PE), which are derived from the reactions between phytosterols and fatty acids, were demonstrated to have important effects on lipid and cholesterol metabolism regulation. In order to reveal the effect of maternal lipid metabolism on the deposition of nutrients in eggs and the development of embryonic muscles, broiler hens were fed with a diet supplemented with 5% PE or control diet. Lipid deposition in eggs and growth of the hatched chicks were studied. We found that PE increased bile acid (BA) deposition in the eggs and serum of hens (p=0.02 and p

Beneficial effects of resistance exercise on metabolic health and particularly muscle hypertrophy and fat loss are well established, but the underlying chemical and physiological mechanisms are not fully understood. Here, we identified a myometabolite‐mediated metabolic pathway that is essential for the beneficial metabolic effects of resistance exercise in mice. We showed that substantial accumulation of the tricarboxylic acid cycle intermediate α‐ketoglutaric acid (AKG) is a metabolic signature of resistance exercise performance. Interestingly, human plasma AKG level is also negatively correlated with BMI. Pharmacological elevation of circulating AKG induces muscle hypertrophy, brown adipose tissue (BAT) thermogenesis, and white adipose tissue (WAT) lipolysis in vivo. We further found that AKG stimulates the adrenal release of adrenaline through 2‐oxoglutarate receptor 1 (OXGR1) expressed in adrenal glands. Finally, by using both loss‐of‐function and gain‐of‐function mouse models, we showed that OXGR1 is essential for AKG‐mediated exercise‐induced beneficial metabolic effects. These findings reveal an unappreciated mechanism for the salutary effects of resistance exercise, using AKG as a systemically derived molecule for adrenal stimulation of muscle hypertrophy and fat loss., The Krebs cycle intermediate α‐ketoglutaric acid acts as a systemic myometabolite promoting energy expenditure and lipolysis in mice.

// Yingying Meng 1, 2, * , Jing Zhang 1, 2, * , Cong Yuan 1, 2 , Fenglin Zhang 1, 2 , Qin Fu 1, 2 , Han Su 1, 2 , Xiaotong Zhu 1, 2 , Lina Wang 1, 2 , Ping Gao 1, 2 , Gang Shu 1, 2 , Qingyan Jiang 1, 2 and Songbo Wang 1, 2 1 Guangdong Provincial Key Laboratory of Animal Nutrition Control, College of Animal Science, South China Agricultural University, Guangzhou 510642, P. R. China 2 National Engineering Research Center for Breeding Swine Industry and UBT Lipid Suite Functional Fatty Acids Research Center, South China Agricultural University, Guangzhou 510642, P. R. China * These authors are co-first authors Correspondence to: Songbo Wang, email: songbowang@scau.edu.cn Keywords: oleic acid (OA); mammary gland development; CD36-[Ca 2+ ]i; PI3K/Akt; peripubertal mice Received: October 21, 2017 Accepted: December 05, 2017 Published: January 12, 2018 ABSTRACT This study aimed to investigate the effects of oleic acid (OA), a monounsaturated fatty acid, on HC11 mammary epithelial cells proliferation and peripubertal mammary gland development and explore the underlying mechanisms. HC11 cells and C57BL/6J mice were treated with OA. HC11 proliferation, peripubertal mammary gland development, and the involvement of CD36 and PI3K/Akt were assessed. In vitro , 100 μM OA significantly promoted HC11 proliferation by increasing Cyclin D1/3 and PCNA expression and decreasing p21 expression. Meanwhile, OA enhanced CD36 expression, elevated [Ca 2+ ] i and activated PI3K/Akt signaling pathway. However, knockdown of CD36, chelation of [Ca 2+ ] i or inhibition of PI3K eliminated the OA-induced promotion of HC11 proliferation and change in proliferative markers expression. In vivo , peripubertal exposure to diet containing 2% OA stimulated mammary duct development, with increased terminal duct end (TDE) and ductal branch. Moreover, dietary OA increased the serum levels of IGF-1 and E2, enhanced the expression of CD36 and Cyclin D1, and activated PI3K/Akt pathway in mammary glands. In conclusion, OA stimulated HC11 cells proliferation and mammary gland development in peripubertal mice, which was associated with activation of CD36-[Ca 2+ ] i and PI3K/Akt signaling pathway. These data provided new insights into the stimulation of mammary gland development by dietary oleic acid.

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.

This study mainly investigated the effects of different dietary levels of glutamate (Glu) and aspartate (Asp) on growth performance, blood amino acids, and amino acid transporters in piglets. Forty-two healthy piglets were randomly divided into six groups (n = 7): a control group in which piglets were fed 2.9% Glu and 1.5% Asp and other groups in which piglets received 1.3% or 1.7% Asp and 2.6%, 3.2%, or 3.5% Glu for 21 days. Growth performance, serum amino acid profiles from the mesenteric vein, portal vein, and anterior vena cava, and amino acid transporters in the liver were determined. The results showed that lower doses of Asp promoted growth and enhanced the amino acids, while high doses of Asp and Glu reduced growth and the amino acid pool in piglets (P

Capsaicin is a major pungent content in green and red peppers which are widely used as spice, and capsaicin may activate different receptors. To determine whether capsaicin has different effects on different types of skeletal muscle, we applied different concentrations (0, 0.01, and 0.02%) of capsaicin in the normal diet and conducted a four-week experiment on Sprague-Dawley rats. The fiber type composition, glucose metabolism enzyme activity, and different signaling molecules' expressions of receptors were detected. Our results suggested that capsaicin reduced the body fat deposition, while promoting the slow muscle-related gene expression and increasing the enzyme activity in the gastrocnemius and soleus muscles. However, fatty acid metabolism was significantly increased only in the soleus muscle. The study of intracellular signaling suggested that the transient receptor potential vanilloid 1 (TRPV1) and cannabinoid receptors in the soleus muscle were more sensitive to capsaicin. In conclusion, the distribution of TRPV1 and cannabinoid receptors differs in different types of muscle, and the different roles of capsaicin in different types of muscle may be related to the different degrees of activation of receptors.

The rising prevalence of obesity and being overweight is a worldwide health concern. Food reward dysregulation is the basic factor for the development of obesity. Dopamine (DA) neurons in the ventral tegmental area (VTA) play a vital role in food reward. Toll-like receptor 4 (TLR4) is a transmembrane pattern recognition receptor that can be activated by saturated fatty acids. Here, we show that the deletion of TLR4 specifically in DA neurons increases body weight, increases food intake, and decreases food reward. Conditional deletion of TLR4 also decreased the activity of DA neurons while suppressing the expression of tyrosine hydroxylase (TH) in the VTA, which regulates the concentration of DA in the nucleus accumbens (NAc) to affect food reward. Meanwhile, AAV-Cre-GFP mediated VTA-specific TLR4-deficient mice recapitulates food reward of DAT-TLR4-KO mice. Food reward could be rescued by re-expressing TLR4 in VTA DA neurons. Moreover, effects of intra-VTA infusion of lauric acid (a saturated fatty acid with 12 carbon) on food reward were abolished in mice lacking TLR4 in DA neurons. Our study demonstrates the critical role of TLR4 signaling in regulating the activity of VTA DA neurons and the normal function of the mesolimbic DA system that may contribute to food reward.

Skeletal muscle atrophy due to excessive protein degradation is the main cause for muscle dysfunction, fatigue, and weakening of athletic ability. Endurance exercise is effective to attenuate muscle atrophy, but the underlying mechanism has not been fully investigated. α-Ketoglutarate (AKG) is a key intermediate of tricarboxylic acid cycle, which is generated during endurance exercise. Here, we demonstrated that AKG effectively attenuated corticosterone-induced protein degradation and rescued the muscle atrophy and dysfunction in a Duchenne muscular dystrophy mouse model. Interestingly, AKG also inhibited the expression of proline hydroxylase 3 (PHD3), one of the important oxidoreductases expressed under hypoxic conditions. Subsequently, we identified the β2 adrenergic receptor (ADRB2) as a downstream target for PHD3. We found AKG inhibited PHD3/ADRB2 interaction and therefore increased the stability of ADRB2. In addition, combining pharmacologic and genetic approaches, we showed that AKG rescues skeletal muscle atrophy and protein degradation through a PHD3/ADRB2 mediated mechanism. Taken together, these data reveal a mechanism for inhibitory effects of AKG on muscle atrophy and protein degradation. These findings not only provide a molecular basis for the potential use of exercise-generated metabolite AKG in muscle atrophy treatment, but also identify PHD3 as a potential target for the development of therapies for muscle wasting.—Cai, X., Yuan, Y., Liao, Z., Xing, K., Zhu, C., Xu, Y., Yu, L., Wang, L., Wang, S., Zhu, X., Gao, P., Zhang, Y., Jiang, Q., Xu, P., Shu, G. α-Ketoglutarate prevents skeletal muscle protein degradation and muscle atrophy through PHD3/ADRB2 pathway.

It has been demonstrated that dietary high fat diet negatively affects the pubertal mammary gland development. The aim of the present study was to investigate the effects of stearic acid (SA), an 18-carbon chain saturated fatty acid, on mammary gland development in pubertal mice and to explore the underlying mechanism. Our results demonstrated that dietary supplementation of 2% SA suppressed mammary duct development, with significant reduction of terminal end bud (TEB) number and ductal branch. In accord, the expression of proliferative marker Cyclin D1 was markedly decreased by dietary SA. Furthermore, dietary SA led to increase of G protein-coupled receptor 120 (GPR120) expression and inhibition of PI3K/Akt signaling pathway in mammary gland of pubertal mice. In good agreement with the in vivo findings, the in vitro results showed that 40 μM SA significantly suppressed proliferation of mouse mammary epithelial cell HC11 by regulating mRNA and/or protein expression of proliferative markers such as Cyclin D1/3, p21, and PCNA. Meanwhile, SA activated GPR120 and inhibited PI3K/Akt signaling pathway in a GPR120-dependent manner. In addition, SA-induced inhibition of PI3K/Akt signaling pathway, suppression of HC11 proliferation, and alteration of proliferative markers expression were abolished by knockdown of GPR120 with siRNA. Collectively, these findings showed that SA suppressed mammary gland development of pubertal mice, which was coincident with the SA-inhibited HC11 proliferation, and was associated with inhibition of PI3K/Akt signaling pathway through activation of GPR120. These data provided new insights into the regulation of mammary gland development by dietary fatty acids.

SUMMARY Although the widely used anticoagulant drug heparin has been shown to have many other biological functions independent of its anticoagulant role, its effects on energy homeostasis are unknown. Here, we demonstrate that heparin level is negatively associated with nutritional states and that heparin treatment increases food intake and body weight gain. By using electrophysiological, pharmacological, molecular biological, and chemogenetic approaches, we provide evidence that heparin increases food intake by stimulating AgRP neurons and increasing AgRP release. Our results support a model whereby heparin competes with insulin for insulin receptor binding on AgRP neurons, and by doing so it inhibits FoxO1 activity to promote AgRP release and feeding. Heparin may be a potential drug target for food intake regulation and body weight control., Graphical Abstract, In Brief Zhu et al. demonstrate that heparin competes with insulin for insulin receptor binding on AgRP neurons, and by doing so it inhibits FoxO1 activity to promote AgRP release and feeding. Heparin is identified as a potential drug target for food intake regulation and body weight control.

Laminarin, a type of β-glucan isolated from brown seaweeds, exhibits verity of physiological activities, which include immunology modulation and antitumor function. To investigate the effect of laminarin on energy homeostasis, mice were orally administrated with laminarin to test food intake, fat deposition, and glucose homeostasis. Chronically, laminarin treatment significantly decreases high-fat-diet-induced body weight gain and fat deposition and reduces blood glucose level and glucose tolerance. Acutely, laminarin enhances serum glucagon-like peptide-1 (GLP-1) content and the mRNA expression level of proglucagon and prohormone convertase 1 in ileum. Subsequently, laminarin suppresses the food intake of mice, the hypothalamic AgRP neuron activity, and AgRP expression but activates pancreatic function. Furthermore, laminarin-induced appetite reduction was totally blocked by Exendin (9-39), a specific competitive inhibitor of GLP-1 receptor. Then, STC-1 cells were adopted to address the underlying mechanism, by which laminarin promoted GLP-1 secretion in vitro. Results showed that laminarin dose-dependently promoted GLP-1 secretion and c-Fos protein expression in STC-1 cells, which were independent of Dectin-1 and CD18. Interestingly, BAPTA-AM, a calcium-chelating agent, potently attenuated laminarin-induced [Ca2+]i elevation, c-Fos expression, and GLP-1 secretion. In summary, our data support that laminarin counteracts diet-induced obesity and stimulates GLP-1 secretion via [Ca2+]i; this finding provides an experimental basis for laminarin application to treat obesity and maintain glucose homeostasis.

It has been shown that adipose tissue hyperplasia (increased adipocyte number or adipogenesis) has beneficial effects on metabolic health. The aim of the present study was to determine whether phytol could modulate hyperplasia/adipogenesis and glucose homeostasis, and to explore the underlying mechanisms in mice fed high-fat and high fructose diet (HFFD). Our results demonstrated that phytol administration decreased body weight gain and inguinal subcutaneous white adipose tissue (iWAT) weight. However, phytol significantly increased the adipocyte number in iWAT, with the smaller average adipocyte diameter. Meanwhile, OGTT result showed that phytol improved glucose tolerance. In accord, phytol administration markedly increased expression of marker genes associated with adipogenesis (PPARγ and C/EBPα) and glucose uptake (AS160 and GLUT4) and activated PI3K/Akt signaling pathway in mice iWAT. In agreement with the in vivo findings, the in vitro results indicated that 100 μM phytol significantly enhanced 3T3-L1 adipogenesis and glucose uptake, and activated PI3K/Akt signaling pathway. However, phytol-induced enhancement of 3T3-L1 adipognesis and glucose uptake, activation of PI3K/Akt signaling pathway, elevation of marker genes involved in adipogensis and glucose uptake, as well as translocation of GLUT4 from cytoplasm to membrane were abolished by Wortmannin, a specific PI3K/Akt inhibitor. Taken together, phytol increased adipocyte number in iWAT and improved glucose tolerance in mice fed HFFD, which was coincident with the enhanced adipogenesis and glucose uptake in 3T3-L1, and was associated with activation of PI3K/Akt signaling pathway. These data suggested the application of phytol as a potential nutritional agent to combat obesity and type 2 diabetes.

FSH plays an essential role in processes involved in human reproduction, including spermatogenesis and the ovarian cycle. While the transcriptional regulatory mechanisms underlying its synthesis and secretion have been extensively studied, little is known about its posttranscriptional regulation. A bioinformatics analysis from our group indicated that a microRNA (miRNA; miR-361-3p) could regulate FSH secretion by potentially targeting the FSHB subunit. Herein, we sought to confirm these findings by investigating the miR-361-3p-mediated regulation of FSH production in primary pig anterior pituitary cells. Gonadotropin-releasing hormone (GnRH) treatment resulted in an increase in FSHB synthesis at both the mRNA, protein/hormone level, along with a significant decrease in miR-361-3p and its precursor (pre-miR-361) levels in time- and dose-dependent manner. Using the Dual-Luciferase Assay, we confirmed that miR-361-3p directly targets FSHB. Additionally, overexpression of miR-361-3p using mimics significantly decreased the FSHB production at both the mRNA and protein levels, with a reduction in both protein synthesis and secretion. Conversely, both synthesis and secretion were significantly increased following miR-361-3p blockade. To confirm that miR-361-3p targets FSHB, we designed FSH-targeted siRNAs, and co-transfected anterior pituitary cells with both the siRNA and miR-361-3p inhibitors. Our results indicated that the siRNA blocked the miR-361-3p inhibitor-mediated upregulation of FSH, while no significant effect on non-target expression. Taken together, our results demonstrate that miR-361-3p negatively regulates FSH synthesis and secretion by targeting FSHB, which provides more functional evidence that a miRNA is involved in the direct regulation of FSH.

N-Acyl amino acids (NAAAs) are conjugate products of fatty acids and amino acids, which are available in animal-derived food. We compared the effects of N-arachidonoylglycine (NAGly), N-arachidonoylserine (NASer), and N-oleoylglycine (OLGly) on in vivo food intake and in vitro [Ca2+]i of Agouti-related protein (AgRP) neurons to identify the role of these compounds in energy homeostasis. Hypothalamic neuropeptide expression and anxiety behavior in response to OLGly were also tested. To further identify the underlying mechanism of OLGly on food intake, we first detected the expression level of potential OLGly receptors. The cannabinoid receptor type 1 (CB1R) antagonist was cotreated with OLGly to analyze the activation of AgRP neuron, including [Ca2+]i, expression levels of PKA, CREB, and c-Fos, and neuropeptide secretion. Results demonstrated that only OLGly (intrapertioneal injection of 6 mg/kg) can induce hyperphagia without changing the expression of hypothalamic neuropeptides and anxiety-like behavior....

Obesity is a serious health challenge worldwide and is associated with various comorbidities, including dyslipidemia, type 2 diabetes, and cardiovascular disease. Developing effective strategies to prevent obesity is therefore of paramount importance. One potential strategy to reduce obesity is to consume calcium, which has been implicated to be involved in reducing body weight/fat. In this review, we compile the evidence for the anti-obesity roles of calcium in cells, animals, and humans. In addition, we summarize the possible anti-obesity mechanisms of calcium, including regulation of (a) adipogenesis, (b) fat metabolism, (c) adipocyte (precursor) proliferation and apoptosis, (d) thermogenesis, (e) fat absorption and excretion, and (f) gut microbiota. Although the exact anti-obesity roles of calcium in different subjects and how calcium induces the proposed anti-obesity mechanisms need to be further investigated, the current evidence demonstrates the anti-obesity effects of calcium and suggests the potential application of dietary calcium for prevention of obesity.

Introduction Ginseng polysaccharide (GPS, same as Panax polysaccharide) is a kind of polysaccharide extracted from ginseng. It has been reported that GPS has the ability to activate innate immunity, regulates blood sugar balance, and improves antioxidant capacity, but the effect on feeding behavior and its mechanism remains unclear. Method To investigate the possible effect of GPS on feeding behavior of animals, mice were supplied with GPS in water, and food intake, hedonic feeding behavior, anxiety‐like behavior, expression of appetite‐regulation peptides in the central nervous system and glucose‐related hormone levels in the serum of mice were measured. Results Ginseng polysaccharide significantly increased the average daily food intake in mice and promoted hedonic eating behavior. Meanwhile, the levels of serum glucose and glucagon were significantly reduced by GPS, and GPS promoted hypothalamic neuropeptide Y expression, inhibited proopiomelanocortin (POMC) expression, and reduced dopamine D1 receptor (DRD1) levels in the midbrain. We also found that the anxiety level of mice was significantly lower after GPS intake. In conclusion, oral supplementation with GPS promoted food intake in mice, most likely through the regulation of circulating glucose levels.

Stimulating the browning of white adipocytes contributes to the restriction of obesity and related metabolic disorders. This study aimed to investigate the browning effects of phytol on mice inguinal subcutaneous white adipose tissue (iWAT) and explore the underlying mechanisms. Our results demonstrated that phytol administration decreased body weight gain and iWAT index, and stimulated the browning of mice iWAT, with the increased expression of brown adipocyte marker genes (UCP1, PRDM16, PGC1α, PDH, and Cyto C). In addition, phytol treatment activated the AMPKα signaling pathway in mice iWAT. In good agreement with the in vivo findings, the in vitro results showed that 100 μM phytol stimulated brown adipogenic differentiation and formation of brown-like adipocytes in the differentiated 3T3-L1 by increasing the mitochondria content and oxygen consumption, and promoting mRNA and/or protein expression of brown adipocyte markers (UCP1, PRDM16, PGC1α, PDH, Cyto C, Cidea and Elovl3) and beige adipocyte markers (CD137 and TMEM26). Meanwhile, phytol activated the AMPKα signaling pathway in the differentiated 3T3-L1. However, the inhibition of AMPKα with Compound C totally abolished phytol-stimulated brown adipogenic differentiation and formation of brown-like adipocytes. In conclusion, these results showed that phytol stimulated the browning of mice iWAT, which was coincident with the increased formation of brown-like adipocytes in the differentiated 3T3-L1, and appeared to be primarily mediated by the AMPKα signaling pathway. These data provided new insight into the role of phytol in regulating the browning of WAT and suggested the potential application of phytol as a nutritional intervention for the restriction of obesity and related metabolic disorders.

Objective Growth hormone releasing hormone (GHRH) is a major positive regulator of growth hormone (GH) in the anterior pituitary gland, while cortistatin's (CST) role is negative. miRNAs (microRNAs or miRs) are small RNA molecules modulating gene expression at the post-transcriptional level. However, little is known about the function of miRNAs in the regulation of GH synthesis and/or secretion. This study investigated potential functional miRNAs involved in GH secretion in the normal porcine pituitary. Design Primary porcine anterior pituitary cells were cultivated and then treated with 10 nmol/L GHRH and 100 nmol/L CST, respectively. The effects of GHRH and CST on GH secretion were determined using RIA. miRNA microarrays were employed to analyze miRNA expression after treatment and then differentially expressed miRNAs were screened. Bioinformatics analysis was used to analyze the potential targets in growth hormone regulation of altered miRNAs. Furthermore, functional experiments were conducted to study the function of ssc-let-7c. Results GHRH significantly promoted GH secretion, while CST suppressed GH secretion. 19 and 35 differentially expressed miRNAs were identified in response to GHRH and CST treatments respectively. Verification of 5 randomly selected miRNAs by quantitative real-time PCR (qRT-PCR) showed similar changes with microarray analysis. Target analysis showed that some miRNAs may be involved in GH secretion-related pathways. Importantly, ssc-let-7c was predicted to target GH1 and GHRHR mRNA 3′untranslated regions (3′UTRs), which was supported by luciferase reporter assay. Furthermore, functional experimental results showed that ssc-let-7c was involved in GH secretion regulation, and overexpression of ssc-let-7c inhibited GH secretion in porcine anterior pituitary cells. Conclusions GHRH and CST modulated porcine pituitary cell miRNA expression. Bioinformatics analysis revealed a complicated network among differentially expressed miRNAs, GH regulation-related genes and hormones. More interestingly, ssc-let-7c inhibited both GH1 and GHRHR mRNA 3′UTR reporter vectors' luciferase activity and overexpression of ssc-let-7c led to a decrease of GH secretion.

Adipocyte dysfunction is associated with many metabolic diseases such as obesity, insulin resistance and diabetes. Previous studies found that phloretin promotes 3T3-L1 cells differentiation, but the underlying mechanisms for phloretin's effects on adipogenesis remain unclear. In this study, we demonstrated that phloretin enhanced the lipid accumulation in porcine primary adipocytes in a time-dependent manner. Furthermore, phloretin increased the utilization of glucose and nonesterified fatty acid, while it decreased the lactate output. Microarray analysis revealed that genes associated with peroxisome proliferator-activated receptor-γ (PPARγ), mitogen-activated protein kinase and insulin signaling pathways were altered in response to phloretin. We further confirmed that phloretin enhanced expression of PPARγ, CAAT enhancer binding protein-α (C/EBPα) and adipose-related genes, such as fatty acids translocase and fatty acid synthase. In addition, phloretin activated the Akt (Thr308) and extracellular signal-regulated kinase, and therefore, inactivated Akt targets protein. Wortmannin effectively blocked the effect of phloretin on Akt activity and the protein levels of PPARγ, C/EBPα and fatty acid binding protein-4 (FABP4/aP2). Oral administration of 5 or 10 mg/kg phloretin to C57BL BKS-DB mice significantly decreased the serum glucose level and improved glucose tolerance. In conclusion, phloretin promotes the adipogenesis of porcine primary preadipocytes through Akt-associated signaling pathway. These findings suggested that phloretin might be able to increase insulin sensitivity and alleviate the metabolic diseases.

Intramuscular fat (IMF) content is considered to be a key factor that affects the marbling, tenderness, juiciness and flavor of pork. To investigate the effects of myristic acid (MA) on the differentiation of porcine intramuscular adipocytes, cells were isolated from longissimus dorsi muscle (LDM) and treated with 0, 10, 50 or 100 μmol L−1 MA. The results showed that MA significantly promotes the differentiation of intramuscular adipocytes in a dose-dependent manner. MA also led to a parallel increase in the expression of peroxisome proliferator activated receptor-γ (PPARγ) and adipose-related genes, such as glucose transporter 1 (GLUT1), lipoprotein lipase (LPL), adipocyte fatty acid binding protein 4 (FABP4/aP2), fatty acid translocase (FAT), acetyl-CoA carboxylase α (ACCα), adipose triglyceride lipase (ATGL) and fatty acid synthase (FASN). However, no significant effects of MA were observed on the expression of CAAT enhancer binding protein-α (C/EBPα) or hormone sensitive lipase (HSL). The expression of pyruvate dehydrogenase kinase 4 (PDK4) was increased by MA during the early stages of differentiation (day 1–3). In addition, MA also increased the absolute content of C14 (P

Bone marrow mesenchymal stem cells (BMSCs) could differentiate into various cell types including adipocytes and myocytes, which had important scientific significance not only in the field of tissue regeneration, but also in the field of agricultural science. In an attempt to exhibit the characterization and differentiation into adipocytes and myocytes of porcine BMSCs, we isolated and purified porcine BMSCs by red blood cell lysis method and percoll gradient centrifugation. The purified cells presented a stretched fibroblast-like phenotype when adhered to the culture plate. The results of flow cytometry analysis and immunofluorescence staining demonstrated that the isolated cells were positive for mesenchymal surface markers CD29, CD44 and negative for hematopoietic markers CD45 and the adhesion molecules CD31. Cells were induced to differentiate into adipocytes with adipogenic medium containing insulin, dexamethasone, oleate and octanoate. Oil Red O staining demonstrated that the porcine BMSCs successfully differentiated to adipocytes. Moreover, the findings of real-time PCR and Western blotting indicated that the induced cells expressed adipogenic marker genes (PPAR-γ, C/EBP-α, perilipin, aP2) mRNA or proteins (PPAR-γ, perilipin, aP2). On the other hand, porcine BMSCs were induced into myoctyes with myogenic medium supplemented with 5-azacytidine, basic fibroblast growth factor, chick embryo extract and horse serum. Morphological observation by hochest 33342 staining showed that the induced cells presented as multi-nucleus muscular tube structure. And myogenic marker genes (Myf5, desmin) mRNA or proteins (Myf5, MyoD, myogenin, desmin) were found in the induced cells. In addition, the results of immunofluorescence staining revealed that myogenic marker (Myf5, MyoD, myogenin, desmin, S-MyHC) proteins was positive in the induced cells. Above all, these results suggested that the isolated porcine BMSCs were not only consistent with the characterization of mesenchymal stem cells, but also exhibited the multipotential capacity to form adipocytes and myocytes, which provided the basis to investigate the regulation mechanism involved in the selective differentiation of porcine BMSCs.

Fatty acid translocase (FAT/CD36) is a membrane receptor that facilitates long-chain fatty acid uptake. To investigate its role in the regulation of long-chain fatty acid composition in muscle tissue, we studied and compared FAT/CD36 gene expression in muscle tissues of commercial broiler chickens and Chinese local Silky fowls. The results from gas chromatography–mass spectrometry analysis of muscle samples demonstrated that Chinese local Silky fowls had significantly higher (P < 0.05) proportions of linoleic acid (LA) and palmitic acid, lower proportions (P < 0.05) of arachidonic acid (AA) and oleic acid than the commercial broiler chickens. The mRNA expression levels of fatty acid (FA) transporters (FA transport protein-1, membrane FA-binding protein, FAT/CD36 and caveolin-1) in the m. ipsilateral pectoralis and biceps femoris were analyzed by Q-PCR, and FAT/CD36 expression levels showed significant differences between these types of chickens (P < 0.01). Interestingly, the levels of FAT/CD36 expression are positively correlated with LA content (r = 0.567, P < 0.01) but negatively correlated with palmitic acid content (r = −0.568, P < 0.01). Further experiments in the stably transfected Chinese hamster oocytes cells with chicken FAT/CD36 cDNA demonstrated that overexpression of FAT/CD36 improves total FA uptake with a significant increase in the proportion of LA and AA, and a decreased proportion of palmitic acid. These results suggest that chicken FAT/CD36 may selectively transport LA and AA, which may lead to the higher LA deposition in muscle tissue.

Background/Aims: We previously reported that porcine intramuscular (i.m.) preadipocytes were different from subcutaneous (s.c.) preadipocytes on cell differentiation and lipid accumulation, but the underlying mechanisms remained unknown. The paper aims to investigate the underlying mechanisms by comparing the differences between i.m. and s.c. preadipocytes in glucose utilization, lipid metabolism, and the role of BMP signaling pathway. Methods: Experiments were performed in porcine primary i.m. and s.c. preadipocytes in culture. The mRNA and protein expression patterns were determined respectively by Quantitative real-time PCR and Western blot. Cytosolic triglycerides were examined by triglyceride assay. Results: The i.m. preadipocytes consumed more glucose by expression of GLUT1 and s.c. preadipocytes mainly utilized exogenic fatty acids for lipid synthesis by expression of LPL and FAT. Meanwhile, the expression of genes related to lipogenesis and lipolysis in s.c. preadipocytes increased more quickly than those in i.m. preadipocytes. The expression patterns of the genes involved in BMP-Smad signaling pathway were consistent with those of the genes participated in adipocytes differentiation in both i.m. and s.c. preadipocytes. Exogenous BMP2 significantly increased, whereas Noggin and Compound C, remarkablely decreased the triglycerides content in i.m. preadipoytes, without affecting s.c. preadipocytes. BMP2 shRNA significantly reduced the mRNA levels of the downstream genes of BMP-Smad signaling pathway and PPARγ in both i.m. and s.c. preadipocytes. Conclusion: These findings suggested that the differentiation and lipid accumulation differences between i.m. and s.c. preadipocytes might be caused by the different manners of glucose utilization, lipid metabolism and the BMP-Smad signaling pathway. The special feature of i.m. adipocytes implied that these cells might be a potential target for treatment of diabetes.

Infection occurs mainly in Asia; occupational and food exposures are the primary risk factors., Streptococcus suis, a bacterium that affects pigs, is a neglected pathogen that causes systemic disease in humans. We conducted a systematic review and meta-analysis to summarize global estimates of the epidemiology, clinical characteristics, and outcomes of this zoonosis. We searched main literature databases for all studies through December 2012 using the search term “streptococcus suis.” The prevalence of S. suis infection is highest in Asia; the primary risk factors are occupational exposure and eating of contaminated food. The pooled proportions of case-patients with pig-related occupations and history of eating high-risk food were 38.1% and 37.3%, respectively. The main clinical syndrome was meningitis (pooled rate 68.0%), followed by sepsis, arthritis, endocarditis, and endophthalmitis. The pooled case-fatality rate was 12.8%. Sequelae included hearing loss (39.1%) and vestibular dysfunction (22.7%). Our analysis identified gaps in the literature, particularly in assessing risk factors and sequelae of this infection.

Skeletal muscle weight loss is accompanied by small fiber size and low protein content. Alpha-ketoglutarate (AKG) participates in protein and nitrogen metabolism. The effect of AKG on skeletal muscle hypertrophy has not yet been tested, and its underlying mechanism is yet to be determined. In this study, we demonstrated that AKG (2%) increased the gastrocnemius muscle weight and fiber diameter in mice. Our in vitro study also confirmed that AKG dose increased protein synthesis in C2C12 myotubes, which could be effectively blocked by the antagonists of Akt and mTOR. The effects of AKG on skeletal muscle protein synthesis were independent of glutamate, its metabolite. We tested the expression of GPR91 and GPR99. The result demonstrated that C2C12 cells expressed GPR91, which could be upregulated by AKG. GPR91 knockdown abolished the effect of AKG on protein synthesis but failed to inhibit protein degradation. These findings demonstrated that AKG promoted skeletal muscle hypertrophy via Akt/mTOR signaling pathway. In addition, GPR91 might be partially attributed to AKG-induced skeletal muscle protein synthesis.

The anterior pituitary harbours five distinct hormone-producing cell types, and their cellular differentiation is a highly regulated and coordinated process. Here we show that ZBTB20 is essential for anterior pituitary development and lactotrope specification in mice. In anterior pituitary, ZBTB20 is highly expressed by all the mature endocrine cell types, and to some less extent by somatolactotropes, the precursors of prolactin (PRL)-producing lactotropes. Disruption of Zbtb20 leads to anterior pituitary hypoplasia, hypopituitary dwarfism and a complete loss of mature lactotropes. In ZBTB20-null mice, although lactotrope lineage commitment is normally initiated, somatolactotropes exhibit profound defects in lineage specification and expansion. Furthermore, endogenous ZBTB20 protein binds to Prl promoter, and its knockdown decreases PRL expression and secretion in a lactotrope cell line MMQ. In addition, ZBTB20 overexpression enhances the transcriptional activity of Prl promoter in vitro. In conclusion, our findings point to ZBTB20 as a critical regulator of anterior pituitary development and lactotrope specification., The pituitary is a complex structure with the anterior lobe containing five specialized cell types secreting different hormones. Here Cao et al. unravel a role for ZBTB20 in pituitary development and specifically in lactrotrope specification.

The regulation of food intake is a promising way to combat obesity. It has been implicated that various fatty acids exert different effects on food intake and body weight. However, the underlying mechanism remains poorly understood. The aim of the present study was to investigate the effects of linoleic acid (LA) and stearic acid (SA) on agouti-related protein (AgRP) expression and secretion in immortalized mouse hypothalamic N38 cells and to explore the likely underlying mechanisms. Our results demonstrated that LA inhibited, while SA stimulated AgRP expression and secretion of N38 cells in a dose-dependent manner. In addition, LA suppressed the protein expression of toll-like receptor 4 (TLR4), phosphorylation levels of JNK and IKKα/β, suggesting the inhibition of TLR4-dependent inflammation pathway. However, the above mentioned inhibitory effects of LA were eliminated by TLR4 agonist lipopolysaccharide (LPS). In contrast, SA promoted TLR4 protein expression and activated TLR4-dependent inflammation pathway, with elevated ratio of p-JNK/JNK. While TLR4 siRNA reversed the stimulatory effects of SA on AgRP expression and TLR4-dependent inflammation. Moreover, we found that TLR4 was also involved in LA-enhanced and SA-impaired leptin/insulin signal pathways in N38 cells. In conclusion, our findings indicated that LA elicited inhibitory while SA exerted stimulatory effects on AgRP expression and secretion via TLR4-dependent inflammation and leptin/insulin pathways in N38 cells. These data provided a better understanding of the mechanism underlying fatty acids-regulated food intake and suggested the potential role of long-chain unsaturated fatty acids such as LA in reducing food intake and treating obesity.

The main purposes of this study were to investigate the effects of α-linolenic acid (ALA) on the insulin-like growth factor (IGF) system of porcine primary hepatocytes with or without growth hormone (GH) or insulin and the potential role of peroxisome proliferator-activated receptor α and -γ (PPARα/γ) pathway. We found that 1 μM ALA increased IGF-I secretion from hepatocytes at 48 and 72 h. Expression of hepatocytes IGF-I, IGF-II, GH receptor (GHR), insulin receptor (IR), IGF-binding protein 3 (IGFBP3), and IGFBP4 mRNAs was up-regulated by ALA treatment. GH (15 nM) alone or co-treated with ALA increased hepatocytes IGF-I secretion and the expression of GHR and IGFBP1 mRNAs, but down-regulated IGFBP5 mRNA compared with appropriate control across ALA. GH also enhanced the ALA-induced increase in the transcript levels of IGF-II and GHR, but tended to attenuate that of IGFBP4. Insulin (1 μM) alone or co-treated with ALA improved IGF-I secretion and the expression of IGFBP3 mRNA, but decreased IGFBP1 mRNA versus appropriate control across ALA. Insulin also up-regulated the expression of GHR, IR, IGFBP3, and IGFBP4 mRNAs, and tended to prevent the transcript levels of IGF-I and IGFBP4 improved by ALA. Both PPARγ agonist rosiglitazone and its antagonist GW9662 could elevated the IGF-I secretion in dose-dependent manner but they had no interaction with ALA. However, GW7647, a PPARα agonist, increased IGF-I secretion dose-dependently, but the antagonist GW6471 was without effect. Moreover, GW6471 prevented the IGF-I promoting effect of ALA. This suggests that the IGF-I promoting effect of ALA may be mediated by the PPARα pathway.

Different amino acids have been shown to affect feed intake when injected directly into the central nervous system of birds. In the present study, we investigated the effects of L-glutamine and L-alanine on feed intake and the mRNA expression levels of hypothalamic neuropeptides involved in feed intake regulation in broiler chicks. L-Glutamine or L-alanine was intra-cerebroventricularly (ICV) administered to 4-d-old broiler chicks and the feed intake were recorded at various time points. Quantitative PCR was performed to determine the hypothalamic mRNA expression levels of neuropeptide Y (NPY), agouti related protein (AgRP), pro-opiomelanocortin (POMC), melanocortin receptor 4 (MC4R) and corticotropin releasing factor (CRF). Our results showed that ICV administration of L-glutamine (0.55 or 5.5 μmol) significantly increased feed intake up to 2 h post-administration period and the hypothalamic NPY mRNA expression levels, while it markedly decreased hypothalamic POMC and CRF mRNA expression levels. In contrast, ICV administration of L-alanine (4 μmol) significantly decreased feed intake for the first 0.5 h post-administration period, and reduced the hypothalamic AgRP mRNA expression levels, while it remarkablely enhanced the mRNA expression levels of MC4R and CRF. These findings suggested that L-glutamine and L-alanine could act within the hypothalamus to influence feed intake in broiler chicks, and that both orexigenic and anorexigenic neuropeptide genes might contribute directly to these effects.

MicroRNAs (miRNAs) are an abundant class of small regulatory RNAs that negatively regulate gene expression at the post-transcriptional level. Although an increasing number of porcine miRNAs recently have been identified, research has yet to identify the full repertoire of miRNAs in pig skeletal and adipose tissues and their differences between breeds. We extracted small RNA from skeletal muscle and adipose tissues of Landrace and Lantang pigs, and the expression of a total of 184 known porcine miRNAs (113 from Solexa sequencing and 171 from miRNA chip hybridization) as well as 521 novel miRNA candidates was detected. Moreover, 20 miRNAs were selected randomly from the 184 miRNAs and analysed by quantitative real-time PCR to confirm the aforementioned results. In the skeletal muscle tissues, 21 miRNAs were up-regulated in Lantang and another 33 were highly expressed in Landrace pigs. In the adipose tissues, 25 miRNAs were down-regulated in Lantang and another 23 were lowly expressed in Landrace pigs. miRNA divergence between tissues was also detected in this study. Ten miRNAs were highly expressed in the skeletal muscle tissue in comparison with adipose tissue, and another 10 miRNAs exhibited the opposite expression profile. To investigate the regulatory mechanism of the miRNAs in muscle and adipose tissues, the 10 miRNAs with the most divergent expression profiles were functionally categorized using the Kyoto Encyclopedia of Genes and Genomes database. Most of the miRNAs strongly corresponded to myogenesis and adipogenesis processes. In addition, 84 of the 521 miRNA candidates were potentially porcine-specific miRNAs. This study adds new valuable information to comparative miRNA profiles of skeletal muscle and adipose tissues in porcine species. The great diversity of miRNA composition and expression levels both between breeds and between tissues suggests that a complex regulatory network exists in porcine subcutaneous fat development.

Sorbic acid (SA) is a PUFA with a conjugated double bond. The conjugated fatty acids, including CLA, are multifunctional bioactive fatty acids with the ability to improve growth performance. The effect of SA on pig growth performance was examined to determine its mechanism of action. The ADG, ADFI, and serum IGF-I concentration were examined, as were IGF-I secretion and IGF system gene expression in hepatocytes. Two hundred forty 21-d-old Duroc × Landrace × Yorkshire weaned piglets (6.86 ± 0.02 kg) were randomly divided into 4 groups, each consisting of 3 pens of 20 piglets (10 female and 10 male). The 4 groups of piglets were kept in a temperature-controlled room (26 to 28 °C), and feed and water were provided to the pigs ad libitum. Weanling piglets were fed diets that included 0, 0.5, 2, or 4 g of SA/kg for 42 d. The diet supplemented with 0.5 g/kg of SA improved (P 0.05; 1 g of SA/kg, P 0.05) IGFBP or PPARγ mRNA expression, in pig primary hepatocytes. These results indicate that SA improves growth performance by regulating IGF system gene expression and hormone secretion.

Fatty acids translocase (FAT) is a transporter that facilitate long-chain fatty acids uptake as well as lipid accretion. To investigate the potential role of FAT in different adipose tissues, we investigated the cDNA structure of porcine FAT (pFAT) and analysed the tissue distribution of pFAT mRNA. The FAT mRNA expression profiles in the pre-adipocytes isolated from subcutaneous and intramuscular fat were also compared during cell differentiation. The results showed that 2389 bp porcine cDNA (DQ192230) had 87% homology with human FAT, 83% with mouse FAT, 82% with rat FAT and 67.5% with chicken FAT. Alignment of deduced amino acids sequence showed 82.4% homology with human FAT, 83.3% with mouse FAT and 85% with rat FAT. RT-PCR analysis revealed that the pFAT mRNA had a wide-spread expression in most tissues except for the brain. The higher level transcript was detected in visceral fat tissue by real-time quantitative RT-PCR. Interestingly, the pFAT mRNA expression level was dramatically increased in the primary culture pre-adipocytes derived from intramuscular fat and this consistent with the cellular lipid accretion. However, a sustained lower-level transcript was also found in the adipocytes from subcutaneous fat. The present study indicated that pFAT mRNA had a differential expression in subcutaneous, visceral and intramuscular fat depots. The data presented here provide further proof that pFAT might be involved in the modulation of the temporal and spatial fat depots.

We report here the ontogenic changes in mRNA expression of chicken ghrelin (cGhrelin) and its receptor (cGHS-R1a) and the effects of fasting and refeeding on cGhrelin and cGHS-R1a mRNAs expression in 30-day-old broiler chickens. The level of cGhrelin mRNA in the proventriculus was low from embryo—day 15 (E15) to E19, but dramatically increased at post-hatching-day 2 (P2), then remained constant until P30 and followed by a significant decrease at P44 when there was a diet transition at P31 and thereafter. The decreased level was reversed at P58. Hypothalamic cGhrelin mRNA and proventriculus and hepatic cGHS-R1a mRNA were significantly increased at P30. The cGhrelin mRNA level in the proventriculus significantly increased in response to either 12-h or 36-h fasting but did not decrease after subsequent 12-h refeeding. The level of cGHS-R1a mRNA in the proventriculus was significantly upregulated in response to a 12-h fast but not to a 36-h fast and returned to the control level upon 12-h refeeding. Interestingly, it was apparent that the mRNA levels of both cGhrelin and cGHS-R1a in the liver were upregulated in response to fasting in a time-dependent manner and returned to the control level with subsequent refeeding. These results suggest that the expression pattern of ghrelin and its receptor mRNAs distinctly change in tissues depending on ontogenic stages and feeding states in poultry.

TNF-α is a multifunctional cytokine participating in immune disorders, inflammation, and tumor development with regulatory effects on energy metabolism. Our work focused on the function of TNF-α in adipogenesis of primary porcine adipocytes. TNF-α could suppress the insulin receptor (IR) at the mRNA and protein levels. Microarray analysis of TNF-α-treated porcine adipocytes was used to screen out 29 differentially expressed microRNAs (miRNAs), 13 of which were remarkably upregulated and 16 were intensely downregulated. These 29 differentially expressed miRNAs were predicted to mainly participate in the insulin signaling pathway, adipocytokine signaling pathway, and type 2 diabetes mellitus pathway by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses. miR-146a-5p, reportedly involved in immunity and cancer relevant processes, was one of the most highly differentially expressed miRNAs after TNF-α treatment. Red Oil O staining and TG assay revealed that miR-146a-5p suppressed adipogenesis. A dual-luciferase reporter and siRNA assay verified that miR-146a-5p targeted IR and could inhibit its protein expression. miR-146a-5p was also validated to be involved in the insulin signaling pathway by reducing tyrosine phosphorylation of insulin receptor substrate-1. Our study provides the first evidence of miR-146a-5p targeting IR, which facilitates future studies related to obesity and diabetes using pig models.

Adipose tissue plays a vital role in the development of obesity and related diseases. The aim of the present study was to investigate the effects of N-Oleoyl glycine (OLGly), a lipoamino acid, on 3T3-L1 adipogenesis and to explore the likely mechanisms underlying this process. Lipid accumulation were evaluated using Oil Red O staining and triglyceride content assay. The mRNA expressions of cannabinoid receptors and the protein expressions of adipogenic genes and intracellular signaling pathway were determined by real-time quantitative PCR and western blot, respectively. The results indicated that OLGly itself, but not its degradation products, stimulated lipid accumulation and significantly increased adipogenic genes (PPARγ and aP2), in a dose- and time-dependent manner. Additionally, OLGly markedly increased the mRNA expression of CB1 receptor (CB1R) and the inhibition of CB1R by its antagonist SR141716 abolished the promotive effects of OLGly on lipid accumulation and the protein expression of PPARγ and aP2. Furthermore, OLGly increased the ratio of p-Akt/Akt and p-FoxO1/FoxO1, which could be reversed by SR141716. Moreover, OLGly-induced enhancement of adipogenesis, activation of insulin-mediated Akt signaling pathway and inactivation of FoxO1 were effectively blocked by Wortmannin, a specific PI3K/Akt inhibitor, indicating the essential role of Akt signaling pathway in the process of OLGly-stimulated 3T3-L1 adipogenesis. In conclusion, OLGly, a lipoamino acid, was able to promote 3T3-L1 adipogenesis through the activation of CB1 receptor and the enhancement of insulin-mediated Akt signaling pathway. These findings suggested the potential role of OLGly in increasing insulin sensitivity and suppressing obesity and diabetes.

Dietary protein or amino acid (AA) is a crucial nutritional factor to regulate hepatic insulin-like growth factor-1 (IGF-1) expression and secretion. However, the underlying intracellular mechanism by which dietary protein or AA induces IGF-1 expression remains unknown. We compared the IGF-1 gene expression and plasma IGF-1 level of pigs fed with normal crude protein (CP, 20%) and low-protein levels (LP, 14%). RNA sequencing (RNA-seq) was performed to detect transcript expression in the liver in response to dietary protein. The results showed that serum concentrations and mRNA levels of IGF-1 in the liver were higher in the CP group than in the LP group. RNA-seq analysis identified a total of 1319 differentially expressed transcripts (667 upregulated and 652 downregulated), among which the terms "oxidative phosphorylation", "ribosome", "gap junction", "PPAR signaling pathway", and "focal adhesion" were enriched. In addition, the porcine primary hepatocyte and HepG2 cell models also demonstrated that the mRNA and protein levels of IGF-1 and PPARγ increased with the increasing AA concentration in the culture. The PPARγ activator troglitazone increased IGF-1 gene expression and secretion in a dose dependent manner. Furthermore, inhibition of PPARγ effectively reversed the effects of the high AA concentration on the mRNA expression of IGF-1 and IGFBP-1 in HepG2 cells. Moreover, the protein levels of IGF-1 and PPARγ, as well as the phosphorylation of mTOR, significantly increased in HepG2 cells under high AA concentrations. mTOR phosphorylation can be decreased by the mTOR antagonist, rapamycin. The immunoprecipitation results also showed that high AA concentrations significantly increased the interaction of mTOR and PPARγ. In summary, PPARγ plays an important role in the regulation of IGF-1 secretion and gene expression in response to dietary protein.

Fasting-induced hypothalamic metabolic reprogramming is involved in regulating energy homeostasis and appetite in mammals, but this phenomenon remains unclear in poultry. In this study, the expression patterns of a panel of genes related to neuropeptides, glucose, and lipid metabolism enzymes in the hypothalamus of chickens during fasting and refeeding were characterized by microarray analysis and quantitative PCR. Results showed that 48 h of fasting upregulated (P < 0.05) the mRNA expressions of orexigenic neuropeptide Y and agouti-related protein but downregulated (P < 0.05) that of anorexigenic neuropeptide pro-opiomelanocortin; growth hormone-releasing hormone; islet amyloid polypeptide; thyroid-stimulating hormone, β; and glycoprotein hormones, α polypeptide. After 48 h of fasting, the mRNA expression of fatty acid β-oxidation [peroxisome proliferator-activated receptor α (PPARα), carnitine palmitoyltransferase 1A, and forkhead box O1], energy sensor protein [sirtuin 1 (SIRT1) and forkhead box O1], and glycolysis inhibitor (pyruvate dehydrogenase kinase, isozyme 4) were enhanced, but that of fatty acid synthesis and transport associated genes (acetyl-CoA carboxylase α, fatty acid synthase, apolipoprotein A-I, endothelial lipase, and fatty acid binding protein 7) were suppressed. Liver and muscle also demonstrated similar expression patterns of genes related to glucose and lipid metabolism with hypothalamus, except for that of acetyl-CoA carboxylase α, acyl-CoA synthetase long-chain family member 4, and apolipoprotein A-I. The results of intracerebroventricular (ICV) injection experiments confirmed that α-lipoic acid (ALA, pyruvate dehydrogenase kinase, isozyme 4 inhibitor, 0.10 μmol) and NADH (SIRT1 inhibitor, 0.80 μmol) significantly suppressed the appetite of chickens, whereas 2-deoxy-d-glucose (glycolytic inhibitor, 0.12 to 1.20 μmol) and NAD(+) (SIRT1 activator, 0.08 to 0.80 μmol) increased feed intake in chickens. The orexigenic effect of NAD(+) was also blocked by cotreatment with NADH. However, ICV injection of either GW7647 (PPARα agonist) or GW6471 (PPARα antagonist) showed no effects on feed intake. Results suggested that hypothalamic glycolysis (inhibited by ALA and promoted by 2-deoxy-d-glucose) and SIRT1 (inhibited by NADH and promoted by NAD(+)), not PPARα, were probably involved in feed intake regulation in chickens.

Serotonin (5-HT) is a central inhibitor of food intake in mammals. Thus far, the intracellular mechanisms for the effect of serotonin on appetite regulation remain unclear. It has been recently demonstrated that reactive oxygen species (ROS) in the hypothalamus are a crucial integrative target for the regulation of food intake. To investigate the role of ROS in the serotonin-induced anorexigenic effects, conscious mice were treated with 5-HT alone or combination with Trolox (a ROS scavenger) or Apocynin (an NADPH oxidase inhibitor) by acute intracerebroventricular injection. Both Trolox and Apocynin reversed the anorexigenic action of 5-HT and the 5-HT-induced hypothalamic ROS elevation. The mRNA and protein expression levels of pro-opiomelanocortin (POMC) were dramatically increased after ICV injection with 5-HT. The anorexigenic action of 5-HT was accompanied by markedly elevated hypothalamic MDA levels and GSH-Px activity, while the SOD activity was decreased. Moreover, 5-HT significantly increased the mRNA expression of UCP-2 but reduced the levels of UCP-3. Both Trolox and Apocynin could block the 5-HT-induced changes in UCP-2 and UCP-3 gene expression. Our study demonstrates for the first time that the anorexigenic effect of 5-HT is mediated by the generation of ROS in the hypothalamus through an NADPH oxidase-dependent pathway.