Circular RNA circMYLK4 shifts energy metabolism from glycolysis to OXPHOS by binding to the calcium channel auxiliary subunit CACNA2D2.

Skeletal muscle is heterogeneous tissue, composed of fast-twitch fibers primarily relying on glycolysis and slow-twitch fibers primarily relying on oxidative phosphorylation. The relative expression and balance of glycolysis and oxidative phosphorylation in skeletal muscle are crucial for muscle growth and skeletal muscle metabolism. Here, we employed multi-omics approaches including transcriptomics, proteomics, phosphoproteomics, and metabolomics to unravel the role of circMYLK4, a differentially expressed circRNA in fast and slow-twitch muscle fibers, in muscle fiber metabolism. We discovered that circMYLK4 inhibits glycolysis and promotes mitochondrial oxidative phosphorylation. Mechanistically, circMYLK4 interacts with the voltage-gated calcium channel auxiliary subunit CACNA2D2, leading to the inhibition of Ca2+ release from the sarcoplasmic reticulum. The decrease in cytoplasmic Ca2+ concentration inhibits the expression of key enzymes, PHKB and PHKG1, involved in glycogen breakdown, thereby suppressing glycolysis. On the other hand, the increased fatty acid ß-oxidation enhances the tricarboxylic acid cycle and mitochondrial oxidative phosphorylation. In general, circMYLK4 plays an indispensable role in maintaining the metabolic homeostasis of skeletal muscle.

Identification of different myofiber types in pigs muscles and construction of regulatory networks.

BACKGROUND: Skeletal muscle is composed of muscle fibers with different physiological characteristics, which plays an important role in regulating skeletal muscle metabolism, movement and body homeostasis. The type of skeletal muscle fiber directly affects meat quality. However, the transcriptome and gene interactions between different types of muscle fibers are not well understood. RESULTS: In this paper, we selected 180-days-old Large White pigs and found that longissimus dorsi (LD) muscle was dominated by fast-fermenting myofibrils and soleus (SOL) muscle was dominated by slow-oxidizing myofibrils by frozen sections and related mRNA and protein assays. Here, we selected LD muscle and SOL muscle for transcriptomic sequencing, and identified 312 differentially expressed mRNA (DEmRs), 30 differentially expressed miRNA (DEmiRs), 183 differentially expressed lncRNA (DElRs), and 3417 differentially expressed circRNA (DEcRs). The ceRNA network included ssc-miR-378, ssc-miR-378b-3p, ssc-miR-24-3p, XR_308817, XR_308823, SMIM8, MAVS and FOS as multiple core nodes that play important roles in muscle development. Moreover, we found that different members of the miR-10 family expressed differently in oxidized and glycolytic muscle fibers, among which miR-10a-5p was highly expressed in glycolytic muscle fibers (LD) and could target MYBPH gene mRNA. Therefore, we speculate that miR-10a-5p may be involved in the transformation of muscle fiber types by targeting the MYHBP gene. In addition, PPI analysis of differentially expressed mRNA genes showed that ACTC1, ACTG2 and ACTN2 gene had the highest node degree, suggesting that this gene may play a key role in the regulatory network of muscle fiber type determination. CONCLUSIONS: We can conclude that these genes play a key role in regulating muscle fiber type transformation. Our study provides transcriptomic profiles and ceRNA interaction networks for different muscle fiber types in pigs, providing reference for the transformation of pig muscle fiber types and the improvement of meat quality.

Proto-oncogene FAM83A contributes to casein kinase 1-mediated mitochondrial maintenance and white adipocyte differentiation.

Family with sequence similarity 83 A (FAM83A) is a newly discovered proto-oncogene that has been shown to play key roles in various cancers. However, the function of FAM83A in other physiological processes is not well known. Here, we report a novel function of FAM83A in adipocyte differentiation. We used an adipocyte-targeting fusion oligopeptide (FITC-ATS-9R) to deliver a FAM83A-sgRNA/Cas9 plasmid to knockdown Fam83a (ATS/sg-FAM83A) in white adipose tissue in mice, which resulted in reduced white adipose tissue mass, smaller adipocytes, and mitochondrial damage that was aggravated by a high-fat diet. In cultured 3T3-L1 adipocytes, we found loss or knockdown of Fam83a significantly repressed lipid droplet formation and downregulated the expression of lipogenic genes and proteins. Furthermore, inhibition of Fam83a decreased mitochondrial ATP production through blockage of the electron transport chain, associated with enhanced apoptosis. Mechanistically, we demonstrate FAM83A interacts with casein kinase 1 (CK1) and promotes the permeability of the mitochondrial outer membrane. Furthermore, loss of Fam83a in adipocytes hampered the formation of the TOM40 complex and impeded CK1-driven lipogenesis. Taken together, these results establish FAM83A as a critical regulator of mitochondria maintenance during adipogenesis.

MicroRNA-668-3p inhibits myoblast proliferation and differentiation by targeting Appl1.

BACKGROUND: Skeletal muscle is the largest tissue in the body, and it affects motion, metabolism and homeostasis. Skeletal muscle development comprises myoblast proliferation, fusion and differentiation to form myotubes, which subsequently form mature muscle fibres. This process is strictly regulated by a series of molecular networks. Increasing evidence has shown that noncoding RNAs, especially microRNAs (miRNAs), play vital roles in regulating skeletal muscle growth. Here, we showed that miR-668-3p is highly expressed in skeletal muscle. METHODS: Proliferating and differentiated C2C12 cells were transfected with miR-668-3p mimics and/or inhibitor, and the mRNA and protein levels of its target gene were evaluated by RT‒qPCR and Western blotting analysis. The targeting of Appl1 by miR-668-3p was confirmed by dual luciferase assay. The interdependence of miR-668-3p and Appl1 was verified by cotransfection of C2C12 cells. RESULTS: Our data reveal that miR-668-3p can inhibit myoblast proliferation and myogenic differentiation. Phosphotyrosine interacting with PH domain and leucine zipper 1 (Appl1) is a target gene of miR-668-3p, and it can promote myoblast proliferation and differentiation by activating the p38 MAPK pathway. Furthermore, the inhibitory effect of miR-668-3p on myoblast cell proliferation and myogenic differentiation could be rescued by Appl1. CONCLUSION: Our results indicate a new mechanism by which the miR-668-3p/Appl1/p38 MAPK pathway regulates skeletal muscle development.

Rosiglitazone-induced PPARγ activation promotes intramuscular adipocyte adipogenesis of pig.

Intramuscular fat (IMF) positively influences various aspects of meat quality, while the subcutaneous fat (SF) has negative effect on carcass characteristics and fattening efficiency. Peroxisome proliferator-activated receptor gamma (PPARγ) is a key regulator of adipocyte differentiation, herein, through bioinformatic screen for the potential regulators of adipogenesis from two independent microarray datasets, we identified that PPARγ is a potentially regulator between porcine IMF and SF adipogenesis. Then we treated subcutaneous preadipocytes (SA) and intramuscular preadipocytes (IMA) of pig with RSG (1 µmol/L), and we found that RSG treatment promoted the differentiation of IMA via differentially activating PPARγ transcriptional activity. Besides, RSG treatment promoted apoptosis and lipolysis of SA. Meanwhile, by the treatment of conditioned medium, we excluded the possibility of indirect regulation of RSG from myocyte to adipocyte and proposed that AMPK may mediate the RSG-induced differential activation of PPARγ. Collectively, the RSG treatment promotes IMA adipogenesis, and advances SA lipolysis, this effect may be associated with AMPK-mediated PPARγ differential activation. Our data indicates that targeting PPARγ might be an effective strategy to promote intramuscular fat deposition while reduce subcutaneous fat mass of pig.

CRISPR/dCas9 Tools: Epigenetic Mechanism and Application in Gene Transcriptional Regulation.

CRISPR/Cas9-mediated cleavage of DNA, which depends on the endonuclease activity of Cas9, has been widely used for gene editing due to its excellent programmability and specificity. However, the changes to the DNA sequence that are mediated by CRISPR/Cas9 affect the structures and stability of the genome, which may affect the accuracy of results. Mutations in the RuvC and HNH regions of the Cas9 protein lead to the inactivation of Cas9 into dCas9 with no endonuclease activity. Despite the loss of endonuclease activity, dCas9 can still bind the DNA strand using guide RNA. Recently, proteins with active/inhibitory effects have been linked to the end of the dCas9 protein to form fusion proteins with transcriptional active/inhibitory effects, named CRISPRa and CRISPRi, respectively. These CRISPR tools mediate the transcription activity of protein-coding and non-coding genes by regulating the chromosomal modification states of target gene promoters, enhancers, and other functional elements. Here, we highlight the epigenetic mechanisms and applications of the common CRISPR/dCas9 tools, by which we hope to provide a reference for future related gene regulation, gene function, high-throughput target gene screening, and disease treatment.

miR-196b-5p promotes myoblast proliferation and differentiation.

MicroRNAs (miRNAs) are a class of non-coding single-stranded RNA molecules about 22 nucleotides in length and are encoded by endogenous genes, and are involved in the regulation of post-transcriptional gene expression in animals and plants. Many studies have shown that microRNAs regulate the development of skeletal muscle, mainly manifested in the activation of muscle satellite cells and biological processes such as proliferation, differentiation, and formation of muscle tubes. In this study, miRNA sequencing screening of longissimus dorsi (LD, mainly fast-twitch fibers) and soleus muscle (Sol, dominated by slow-twitch fibers) identified the miR-196b-5p as a differentially expressed and highly conserved sequence in different skeletal muscles. Studies of miR-196b-5p in skeletal muscle have not been reported. In this study, miR-196b-5p mimics and inhibitor were used in miR-196b-5p overexpression and interference experiments in C2C12 cells. The effect of miR-196b-5p on myoblast proliferation and differentiation was analyzed by western blotting, real-time quantitative RT-PCR, flow cytometry, immunofluorescence staining, and the target gene of miR-196b-5p was identified by bioinformatics prediction and analyzed by dual luciferase reporter assays. The results showed that overexpression of miR-196b-5p could significantly increase the mRNA and protein expression of Cyclin B, Cyclin D and Cyclin E (P<0.05); Cell cycle analysis showed that overexpression of miR-196b-5p significantly increased the proportion of cells in the S phase (P<0.05), indicating that miR-196b-5p could accelerate cell cycle progress. Results of EdU staining showed that overexpression of miR-196b-5p significantly promoted cell proliferation. Conversely, inhibition of miR-196b-5p expression could significantly reduce the proliferation capacity of myoblasts. Further, overexpression of miR-196b-5p could significantly increase the expression levels of myogenic marker genes MyoD, MoyG and MyHC (P<0.05), thereby promoting myoblast fusion and accelerating C2C12 cell differentiation. Bioinformatics predictions and dual luciferase experiments demonstrated that miR-196b-5p could target and inhibit the expression of the Sirt1 gene. Altering the Sirt1 expression could not rescue the effects of miR-196b-5p on the cell cycle, but could weaken the promoting effects of miR-196b-5p on myoblast differentiation, suggesting that miR-196b-5p promoted myoblast differentiation by targeting Sirt1.

Hoxa5 inhibits adipocyte proliferation through transcriptional regulation of Ccne1 and blocking JAK2/STAT3 signaling pathway in mice.

The highly regulated proliferation of adipocytes plays a momentous role in fat development and obesity. Hoxa5 is an important member of the Hox family, its encoded protein is an important transcription factor related to development, and its differential expression in different adipose tissues seems to indicate that Hoxa5 may be involved in the regulation of adipocyte proliferation. To evaluate the regulatory mechanism of Hoxa5 on adipocyte proliferation, we constructed a variety of Hoxa5 expression vectors in vivo and in vitro to explore its mechanism on adipocyte proliferation and its potential impact on obesity. We observed that the overexpression of Hoxa5 strongly reduces cell counts and Hoxa5 can inhibit cell proliferation and block cell cycle progression by regulating the expression of genes such as Cyclin E, Cyclin D1, and p53. Most importantly, we demonstrated that Hoxa5 exerts its effect by regulating the signaling pathway of Janus kinase 2 (JAK2) signal transduction and transcription 3 (STAT3) activator, as well as binding to the promoter region of Ccne1 and inhibiting the transcription of Ccne1. This study provides an in-depth understanding of the potential molecular mechanism of Hoxa5 inhibiting adipocyte proliferation. Our results suggest the importance of Hoxa5 in the treatment of obesity.

Circular RNA screening identifies circMYLK4 as a regulator of fast/slow myofibers in porcine skeletal muscles.

The type of myofiber is related to the quality of meat. The slow oxidized myofiber helps to increase the tenderness and juiciness of muscle. Numerous studies have shown that circRNA plays a key role in skeletal muscle development. However, the role of circRNA in porcine skeletal myofiber types is unclear. In this study, we performed high-throughput RNA sequencing to study the differential expression of circRNA in the longissimus dorsi and the soleus muscle. A total of 40,757 circRNAs were identified, of which 181 were significantly different. Interestingly, some circRNAs were involved in metabolism pathways, AMPK, FoxO, and PI3K-Akt signaling pathways. Besides, we focused on a novel circRNA-circMYLK4. By injecting circMYLK4-AAV into piglets, we found that circMYLK4 significantly increased the mRNA and protein levels of the slow muscle marker genes. In summary, our study laid an essential foundation for further research of circRNA in myofiber type conversion and higher meat quality.

MicroRNA-129-5p inhibits C2C12 myogenesis and represses slow fiber gene expression in vitro.

The miR-129 family is widely reported as tumor repressors, although their roles in skeletal muscle have not been fully investigated. Here, the function and mechanism of miR-129-5p in skeletal muscle, a member of the miR-129 family, were explored using C2C12 cell line. Our study showed that miR-129-5p was widely detected in mouse tissues, with the highest expression in skeletal muscle. Gain- and loss-of-function study showed that miR-129-5p could negatively regulate myogenic differentiation, indicated by reduced ratio of MyHC-positive myofibers and repressed expression of myogenic genes, such as MyoD, MyoG, and MyHC. Furthermore, miR-129-5p was more enriched in fast extensor digitorum longus (EDL) than in slow soleus (SOL). Enhanced miR-129-5p could significantly reduce the expression of mitochondrial cox family, together with that of MyHC I, and knockdown of miR-129-5p conversely increased the expression of cox genes and MyHC I. Mechanistically, miR-129-5p directly targeted the 3'-UTR of Mef2a, which was suppressed by miR-129-5p agomir at both mRNA and protein levels in C2C12 cells. Moreover, overexpression of Mef2a could rescue the inhibitory effects of miR-129-5p on the expression of myogenic factors and MyHC I. Collectively, our data revealed that miR-129-5p is a negative regulator of myogenic differentiation and slow fiber gene expression, thus affecting body metabolic homeostasis.

The long noncoding RNA MyHC IIA/X-AS contributes to skeletal muscle myogenesis and maintains the fast fiber phenotype.

Mammalian skeletal muscles comprise different types of muscle fibers, and this muscle fiber heterogeneity is generally characterized by the expression of myosin heavy chain (MyHC) isoforms. A switch in MyHC expression leads to muscle fiber-type transition under various physiological and pathological conditions, but the underlying regulator coordinating the switch of MyHC expression remains largely unknown. Experiments reported in this study revealed the presence of a skeletal muscle-specific antisense transcript generated from the intergenic region between porcine MyHC IIa and IIx and is referred to here as MyHC IIA/X-AS. We found that MyHC IIA/X-AS is identified as a long noncoding RNA (lncRNA) that is strictly expressed in skeletal muscles and is predominantly distributed in the cytoplasm. Genetic analysis disclosed that MyHC IIA/X-AS stimulates cell cycle exit of skeletal satellite cells and their fusion into myotubes. Moreover, we observed that MyHC IIA/X-AS is more enriched in fast-twitch muscle and represses slow-type gene expression and thereby maintains the fast phenotype. Furthermore, we found that MyHC IIA/X-AS acts as a competing endogenous RNA that sponges microRNA-130b (miR-130b) and thereby maintains MyHC IIx expression and the fast fiber type. We also noted that miR-130b was proved to down-regulate MyHC IIx by directly targeting its 3'-UTR. Together, the results of our study uncovered a novel pathway, which revealed that lncRNA derived from the skeletal MyHC cluster could modulate local MyHC expression in trans, highlighting the role of lncRNAs in muscle fiber-type switching.

Neohesperidin attenuates obesity by altering the composition of the gut microbiota in high-fat diet-fed mice.

Obesity and related metabolic disorders are associated with intestinal microbiota dysbiosis, disrupted intestinal barrier, and chronic inflammation. Neohesperidin (Neo), a natural polyphenol abundant in citrus fruits, is known for its preventative and therapeutic effects on numerous diseases. Here, we report that Neo administration attenuates weight gain, low-grade inflammation, and insulin resistance in mice fed high-fat diet (HFD). Also, Neo administration substantially restores gut barrier damage, metabolic endotoxemia, and systemic inflammation. Sequencing of 16S rRNA genes in fecal samples revealed that Neo administration reverses HFD-induced intestinal microbiota dysbiosis: an increase in the diversity of gut microbiota and alteration in the composition of intestinal microbiota (particularly in the relative abundances of Bacteroidetes and Firmicutes). Furthermore, systemic antibiotic treatment abolishes the beneficial effects of Neo in body weight control, suggesting that the effect of Neo on obesity attenuation largely depends on the gut microbiota. More importantly, we demonstrate that the impact of Neo on the regulation of obesity could be transferred from Neo-treated mice to HFD-fed mice via fecal microbiota transplantation. Collectively, our data highlight the efficacy of Neo as a prebiotic agent for attenuating obesity, implying a potential mechanism for gut microbiota mediated the beneficial effect of Neo.

Effect of fermented corn-soybean meal on carcass and meat quality of grower-finisher pigs.

The fermented feed has been identified as a potential alternative to antibiotics in feeds that markedly affects gut health and growth performance of pigs. Two recent studies performed in our laboratory investigated that the fermented corn-soybean meal (fermented feed, FF) improved the gut health of pigs. This study was conducted to determine the effect of a FF on the carcass, meat quality, muscle fatty acids profile, muscle amino acid and antioxidant ability of grower-finisher pigs. In this study, a total of 48 crossbred barrows (Duroc × Landrace × Large White) were randomly divided into 2 treatments with unfermented corn-soybean diet (Ctrl) and FF diet. Compared with control pigs fed a standard diet, the results showed that FF significantly increased the muscle colour of redness and significantly reduced muscle moisture loss rate. Furthermore, FF significantly increased the content of aromatic amino acids such as aspartic acid, glutamic acid and alanine. More importantly, FF increased monounsaturated fatty acid and polyunsaturated fatty acid content. Collectively, FF could be a promising feed strategy in improving meat quality and nutritional value in grower-finisher pig.

miR-324-5p promotes adipocyte differentiation and lipid droplet accumulation by targeting Krueppel-like factor 3 (KLF3).

miRNAs, a kind of noncoding small RNA, play a significant role in adipose differentiation. In this study, we explored the effect of miR-324-5p in adipose differentiation, and found that miR-324-5p could promote adipocytes differentiation and increase body weight in mice. We overexpressed miR-324-5p during adipocytes differentiation, by oil red O and bodipy staining found that lipid accumulation was increased, and the expression level of adipogenic related genes were significantly increased. And the opposite experimental results were obtained after inhibiting miR-324-5p. In vivo, we injected miR-324-5p agomiR in obese mice and found that body weight, adipocyte area, and adipogenic-related gene expression level were significantly increased but lipolytic genes were decreased. To further explore the mechanism of miR-324-5p regulation in lipid accumulation, we constructed Krueppel-like factor 3 (KLF3) 3'-untranslated region luciferase reporter vector and KLF3 pcDNA 3.1 overexpression vector, and found that miR-324-5p was able to directly target KLF3. Overall, in this study we found that miR-324-5p could promote mice preadipoytes differentiation and increase mice fat accumulation by targeting KLF3.

Knock-down Sox5 suppresses porcine adipogenesis through BMP R-Smads signal pathway.

Adipogenesis, a differentiation process that transitions preadipocytes to adipocytes, is key to understanding the biology of fat accumulation and obesity. During this process, there many crucial transcription factors, such as PPARγ and the C/EBP family. Here we show a transcription factor in preadipocytes --- Sox5, that has a function in porcine adipogenesis. In our porcine subcutaneous-derived preadipocyte differentiation model, we found Sox5 expression displayed a significant upregulation after initial induction and decreased afterwards, which resembles the PPARγ expression pattern. siRNA knockdown of Sox5 in porcine preadipocytes significantly promoted cell growth and accelerated cell cycle progression. After inducing differentiation, knockdown of Sox5 notably down-regulated the expression of adipogenic marker genes: PPARγ, aP2, FAS and impaired lipid accumulation. Mechanistically, the deletion of Sox5 down-regulated the BMP R-Smads signal pathway, a crucial signal pathway for controlling preadipocyte fate commitment and adipogenesis. After using BMP4 recombinant protein to activate the BMP R-Smads signal, Sox5 function was partially rescued. In conclusion, our findings uncovered a function of Sox5 in porcine adipogenesis and reveal an interaction between Sox5 and BMP signaling.

Elevated miR-10a-5p facilitates cell cycle and restrains adipogenic differentiation via targeting Map2k6 and Fasn, respectively.

miRNAs are a small class of noncoding RNAs that perform biological functions by regulating the stability or translation of target genes in various biological processes. This study illustrated the role of miR-10a-5p, which is relatively enriched in adipose tissues, using primary mouse preadipocytes as model. With elevated miR-10a-5p expression, the proliferative ability of mouse preadipocytes was significantly enhanced, indicated by increased EdU+ cells and G1/S transition, accompanied by upregulated Cyclin B, Cyclin D and PCNA and downregulated p21 and p27. Meanwhile, the adipogenic differentiation was significantly attenuated by elevated miR-10a-5p, supported by Oil Red O staining and suppressed PPARγ and aP2 expression. Furthermore, Map2k6 and Fasn were predicted to be the target genes of miR-10a-5p in silico, and dual luciferase reporter assay confirmed the direct targeting effects. Western blot analysis results showed that miR-10a-5p specially reduced Map2k6 expression at the proliferative stage without affecting Fasn expression, while significantly restrained Fasn expression with unchanged Map2k6 expression during adipogenic differentiation. Taken together, these results revealed a potential role of miR-10a-5p in adipogenesis and in the treatment of obesity.

Triptolide enhances lipolysis of adipocytes by enhancing ATGL transcription via upregulation of p53.

Lipolysis is an essential physiological activity of adipocytes. The Patatin Like Phospholipase Domain Containing 2 (PNPLA2) gene encodes the enzyme adipose triglyceride lipase (ATGL) responsible for triglyceride hydrolysis, the first step in lipolysis. In this study, we investigated the potential of triptolide (TP), a natural plant extract, to induce weight loss by examining its effect on ATGL expression. We found that long- and short-term TP administration reduced body weight and fat weight and increased heat production in brown adipose tissue in wild-type C57BL/6 mice. In 3T3-L1 fibroblasts and porcine adipocytes, TP treatment reduced the number of lipid droplets as determined by Oil Red O and BODIPY staining, with concomitant increases in free fatty acid and triglyceride levels in the culture medium. Combined treatment with TP and p53 inhibitor reversed these lipolytic effects. We next amplified the ATGL promoter region and identified conserved p53 binding sites in the sequence by in silico analysis. The results of the dual-luciferase reporter assay using a construct containing the ATGL promoter harboring the p53 binding site showed that p53 induces ATGL promoter activity and consequently, ATGL transcription. These results demonstrate that TP has therapeutic value as an anti-obesity agent and acts by promoting lipolysis via upregulation of p53 and ATGL transcription.

Morus nigra L. leaves improve the meat quality in finishing pigs.

This study was conducted to evaluate effects of dietary Mulberry leaves on growth performance, carcass traits and meat quality in finishing pigs. Here, a total of 72 crossbred [(Landrace × Yorkshire) × Duroc] pigs with an average initial body weight of 70.03 ± 0.48 kg were used in this 45-day feeding trial. The pigs were randomly divided into three groups (6 pigs/pen and 4 replicates/group). Dietary treatments included a control diet (without any Mulberry leaves) and diets supplemented with 5% non- or fermented Mulberry leaf powder (MF or FMF respectively). The present findings indicated that compared with the control group, administration of MF or FMF significantly improved gain: feed ratio (p < .05) and reduced the backfat thickness (p < .05). Meanwhile, dietary MF and FMF significantly enhanced triglyceride deposition in Longissimus dorsi muscles (p < .05). Besides, both of MF and FMF could effectively improve the antioxidant capacity by increasing the content of T-AOC and SOD in serum and reduce the rancidity of pork. In conclusion, supplementary MF and FMF could promote gain: feed ratio, reduce backfat thickness, increase fat deposition in muscle and reduce the rancidity of pork.

Wnt3a regulates mitochondrial biogenesis through p38/CREB pathway.

Wnt3a is established as an important regulator of various developmental processes, especially in osteogenesis, adipogenesis and mitochondrial biogenesis. Numerous studies reported Wnt3a regulates osteogenesis and adipogenesis, but the mechanisms by which Wnt3a regulates mitochondrial biogenesis are not well understood. In this study, results suggested that Wnt3a stimulates mitochondrial biogenesis by increasing the expression of mitochondrial biogenesis genes and regulators, as well as mitochondrial copy number in adipocytes. As a key mediator of canonical Wnt/ß-catenin pathway, ß-catenin knockdown had no effect on basal or Wnt3a-mediated mitochondrial biogenesis in adipocytes, which suggested that Wnt3a-mediated mitochondrial biogenesis was independent of ß-catenin-dependent canonical Wnt/ß-catenin pathway. However, Wnt3a inhibited p38/CREB (p38 mitogen-activated protein kinase/cAMP response element-binding protein) signaling activation and p38 inhibitor impaired Wnt3a-stimulated mitochondrial biogenesis, indicating p38/CREB pathway could be involved in the regulation of Wnt3a-mediated mitochondrial biogenesis in adipocytes. In conclusion, our data showed that Wnt3a stimulates mitochondrial biogenesis in adipocytes, which is at least partially through activation of p38/CREB pathway.

Hhip inhibits proliferation and promotes differentiation of adipocytes through suppressing hedgehog signaling pathway.

Adipogenesis, which directly control body fat mass, plays a crucial role in lipid metabolism and obesity-related diseases. Hedgehog interacting protein (Hhip) belongs to Hedgehog (Hh) signaling pathway. The Hh signaling pathway was already linked with adipogenesis in previous reports, however, the physiological functions of Hhip on lipid deposition are still poorly understood. In this study, the level of Hhip was down-regulated during the development of porcine adipose tissues. Recombinant Hedgehog interacting protein (rHhip) could down-regulate cell cycle related genes and cell numbers in S phage to inhibit cell proliferation. Moreover, rHhip could increase adipocytes differentiation by targeting canonical Hh signaling, indicated by the increase of lipid accumulation and up-regulation of Glut4 and PPARγ expression. Collectively, these findings illustrated the essential role of Hhip in the proliferation and differentiation of adipocytes, and provided a potential novel target for preventing obesity.