Effects of dietary multienzymes on the growth performance, digestive enzyme activity, nutrient digestibility, excreta noxious gas emission, and nutrient transporter gene expression in white feather broilers.

Adding multienzymes to poultry feed rations is recognized as a nutritional strategy aimed at improving poultry performance and health status. Nonetheless, some literatures present an ongoing debate about the extent of multienzymes beneficial impact on poultry growth performance. This study aimed to explore the impacts of dietary multienzyme supplementation on broilers, focusing specifically on growth performance, carcass characteristics, apparent nutrient digestibility, excreta noxious gas emission, and intestinal nutrient transporter gene expression. A total of 3,200 broilers were randomly assigned to five groups (eight replicates per treatment group) and treated with the following: normal control (CON), CON + 100 g/t multienzyme (ME100), CON + 150 g/t multienzyme (ME150), CON + 200 g/t multienzyme (ME200), and CON + 250 g/t multienzyme (ME250). Supplementing with multienzymes significantly influenced the feed conversion rate (linear, P = 0.007; quadratic, P = 0.024) and the European broiler index (linear, P = 0.004; quadratic, P = 0.016) in broilers. Dietary multienzymes significantly influenced apparent metabolizable energy (quadratic, P = 0.015) and neutral detergent fiber (quadratic, P < 0.001). Moreover, multienzyme supplementation in the diet also decreased the emission of ammonia (linear, P = 0.001; quadratic, P = 0.006) and hydrogen sulfide (quadratic, P = 0.006) in the excreta. In addition, dietary multi-enzyme notably elevated (P < 0.05) the mRNA expression of nutrient transporter genes, including peptide transporter 1 (PePT1), Na-dependent neutral amino acid transporter (B0AT), glucose transporter 2 (GLUT2), and fatty acid binding protein1 (FABP1). These findings suggest that dietary supplementation with multienzymes can improve the efficiency of feed utilization, and the digestion and absorption of nutrients and reduce excreta gas emission. Furthermore, this study provides a theoretical basis for advancing the use of multienzymes in broiler production.

Multienzyme additives are increasingly used in animal feed, primarily to enhance growth performance and nutrient digestibility. This study focused on the effects of multienzyme additives (xylanase, mannanase, cellulase, arabinofuranosidase, ferulic acid esterase, amylase, and protease) on various aspects of broilers, including growth performance, carcass characteristics, digestive enzyme activities, apparent nutrient digestibility, excreta noxious gas emission, and intestinal nutrient transporter gene expression. The inclusion of multienzymes in the diet was found to significantly increase the weight of breast muscle in broilers. Additionally, it led to a notable decrease in the viscosity of the fecal and jejunal digesta. Furthermore, the present study revealed an increase in the mRNA expression of key nutrient transporters­peptide transporter 1 (PePT1), Na-dependent neutral amino acid transporter (B0AT), and fatty acid binding protein 1 (FABP1), in the intestine of broilers. These findings indicate that dietary multienzymes enhance the efficiency of feed nutrient digestion and absorption in broilers.

Effect of a novel alkaline protease from Bacillus licheniformis on growth performance, carcass characteristics, meat quality, antioxidant capacity, and intestinal morphology of white feather broilers.

BACKGROUND: The present study was conducted to investigate the effects of dietary novel alkaline protease from Bacillus licheniformis on the growth performance, meat quality, antioxidant status and intestinal morphology of broilers. In total, 4000 broilers were randomly assigned into five groups and treated with normal control, normal control + 100 mg kg-1 protease, normal control + 200 mg kg-1 protease, normal control + 300 mg kg-1 protease and normal control + 400 mg kg-1 protease. RESULTS: Supplementing protease impacted final body weight (linear, P = 0.003; quadratic, P = 0.006) and decreased feed conversion rate (linear, P = 0.036) in broilers. Moreover, dietary protease significantly increased breast muscle rate (linear, P = 0.005; quadratic, P = 0.021) and decreased drip loss (linear, P < 0.001; quadratic, P < 0.001). In addition, dietary protease notably increased protein digestibility (linear, P = 0.001; quadratic, P = 0.006) and trypsin activity (linear, P = 0.002; quadratic, P = 0.009) in jejunum. Light microscopy revealed that the jejunum villi in the 300 mg kg-1 and 400 mg kg-1 groups exhibited greater height and a denser arrangement compared to those in the control group. The addition of protease decreased malondialdehyde content (linear, P < 0.001; quadratic, P < 0.001) and increased total antioxidant capacity (linear, P = 0.001; quadratic, P < 0.001) in pectoral muscles. CONCLUSION: The results of the present study suggest that dietary novel alkaline protease from B. licheniformis improved growth performance by affecting trypsin activity, protein digestibility, antioxidant capacity and intestinal health. © 2024 Society of Chemical Industry.

Bacillus subtilis and Enterococcus faecium co-fermented feed alters antioxidant capacity, muscle fibre characteristics and lipid profiles of finishing pigs.

This study aimed to assess how Bacillus subtilis and Enterococcus faecium co-fermented feed (FF) affects the antioxidant capacity, muscle fibre types and muscle lipid profiles of finishing pigs. In this study, a total of 144 Duroc × Berkshire × Jiaxing Black finishing pigs were randomly assigned into three groups with four replicates (twelve pigs per replication). The three treatments were a basal diet (0 % FF), basal diet + 5 % FF and basal diet + 10 % FF, respectively. The experiment lasted 38 d after 4 d of acclimation. The study revealed that 10 % FF significantly increased the activity of superoxide dismutase (SOD) and catalase (CAT) compared with 0 % FF group, with mRNA levels of up-regulated antioxidant-related genes (GPX1, SOD1, SOD2 and CAT) in 10 % FF group. 10 % FF also significantly up-regulated the percentage of slow-twitch fibre and the mRNA expression of MyHC I, MyHC IIa and MyHC IIx, and slow MyHC protein expression while reducing MyHC IIb mRNA expression. Lipidomics analysis showed that 5 % FF and 10 % FF altered lipid profiles in longissimus thoracis. 10 % FF particularly led to an increase in the percentage of TAG. The Pearson correlation analysis indicated that certain molecular markers such as phosphatidic acid (PA) (49:4), Hex2Cer (d50:6), cardiolipin (CL) (72:8) and phosphatidylcholine (PC) (33:0e) could be used to indicate the characteristics of muscle fibres and were closely related to meat quality. Together, our findings suggest that 10 % FF improved antioxidant capacity, enhanced slow-twitch fibre percentage and altered muscle lipid profiles in finishing pigs.

Growth arrest and DNA damage-inducible alpha regulates muscle repair and fat infiltration through ATP synthase F1 subunit alpha.

BACKGROUND: Skeletal muscle fat infiltration is a common feature during ageing, obesity and several myopathies associated with muscular dysfunction and sarcopenia. However, the regulatory mechanisms of intramuscular adipogenesis and strategies to reduce fat infiltration in muscle remain unclear. Here, we identified the growth arrest and DNA damage-inducible alpha (GADD45A), a stress-inducible histone folding protein, as a critical regulator of intramuscular fat (IMAT) infiltration. METHODS: To explore the role of GADD45A on IMAT infiltration and muscle regeneration, the gain or loss function of GADD45A in intramuscular preadipocytes was performed. The adipocyte-specific GADD45A knock-in (KI) mice and high IMAT-infiltrated muscle model by glycerol injection (50 µL of 50% v/v GLY) were generated. RNA-sequencing, histological changes, gene expression, lipid metabolism, mitochondrial function and the effect of dietary factor epigallocatechin-3-gallate (EGCG) treatment (100 mg/kg) on IMAT infiltration were studied. RESULTS: The unbiased transcriptomics data analysis indicated that GADD45A expression positively correlates with IMAT infiltration and muscle metabolic disorders in humans (correlation: young vs. aged people, Gadd45a and Cebpa, r2  = 0.20, P < 0.05) and animals (correlation: wild-type [WT] vs. mdx mice, Gadd45a and Cebpa, r2  = 0.38, P < 0.05; NaCl vs. GLY mice, Gadd45a and Adipoq/Fabp4, r2  = 0.80/0.71, both P < 0.0001). In vitro, GADD45A overexpression promotes intramuscular preadipocyte adipogenesis, upregulating the expression of adipogenic genes (Ppara: +47%, Adipoq: +28%, P < 0.001; Cebpa: +135%, Fabp4: +16%, P < 0.01; Pparg: +66%, Leptin: +77%, P < 0.05). GADD45A knockdown robustly decreased lipid accumulation (Pparg: -57%, Adipoq: -35%, P < 0.001; Fabp4: -37%, P < 0.01; Leptin: -28%, P < 0.05). GADD45A KI mice exhibit inhibited skeletal muscle regeneration (myofibres: -40%, P < 0.01) and enhanced IMAT infiltration (adipocytes: +20%, P < 0.05). These KI mice have impaired exercise endurance and mitochondrial function. Mechanistically, GADD45A affects ATP synthase F1 subunit alpha (ATP5A1) ubiquitination degradation (ubiquitinated ATP5A1, P < 0.001) by recruiting the E3 ubiquitin ligase TRIM25, which decreases ATP synthesis (ATP production: -23%, P < 0.01) and inactivates the cAMP/PKA/LKB1 signalling pathway (cAMP: -36%, P < 0.01; decreased phospho-PKA and phospho-LKB1 protein content, P < 0.01). The dietary factor EGCG can protect against muscle fat infiltration (triglyceride: -64%, P < 0.05) via downregulating GADD45A (decreased GADD45A protein content, P < 0.001). CONCLUSIONS: Our findings reveal a crucial role of GADD45A in regulating muscle repair and fat infiltration and suggest that inhibition of GADD45A by EGCG might be a potential strategy to combat fat infiltration and its associated muscle dysfunction.

CRTC3 Regulates the Lipid Metabolism and Adipogenic Differentiation of Porcine Intramuscular and Subcutaneous Adipocytes by Activating the Calcium Pathway.

Fat deposition, especially the intramuscular (IM) fat deposition, is directly associated with meat quality. The cyclic adenosine monophosphate (cAMP)-responsive element binding-protein (CREB)-regulated transcription coactivator 3 (CRTC3) plays an important role in energy metabolism and various biological processes. The expression of porcine CRTC3 in skeletal muscle is positively associated with intramuscular fat deposition and possesses the capacity to control the intramuscular (IM) adipocyte morphology. However, the metabolic effects and transcriptional mechanism of CRTC3 in porcine intramuscular (IM) adipocytes as well as the regulatory mechanism of CRTC3 on porcine adipocyte differentiation have not been studied. Here, we utilized metabolomics and RNA sequencing (RNA-seq) to determine the metabolic and transcriptome profiles of CRTC3-overexpressing IM adipocytes. Moreover, the effect and regulation mechanism of CRTC3 on porcine IM and subcutaneous (SC) adipocyte differentiation were also studied. Our results showed that CRTC3 overexpression dramatically altered the metabolites in IM adipocytes. Glycerophospholipid (GP) metabolism and related genes were significantly changed in CRTC3-overexpressing IM adipocytes. Moreover, we demonstrated that CRTC3 overexpression promotes adipogenic differentiation by upregulating the Ca2+-cAMP signaling pathway in IM and SC adipocytes. We showed alterations in metabolites and in the expression of genes involved in lipid metabolism in CRTC3-overexpressing adipocytes and demonstrated the regulatory mechanism of CRTC3 on the adipogenic differentiation of porcine adipocytes. These results provide new insights into the regulatory roles of CRTC3 in porcine adipocytes, which could be an important target to regulate fat deposition in animals.

Comprehensive evaluation of the metabolic effects of porcine CRTC3 overexpression on subcutaneous adipocytes with metabolomic and transcriptomic analyses.

BACKGROUND: Meat quality is largely driven by fat deposition, which is regulated by several genes and signaling pathways. The cyclic adenosine monophosphate (cAMP) -regulated transcriptional coactivator 3 (CRTC3) is a coactivator of cAMP response element binding protein (CREB) that mediates the function of protein kinase A (PKA) signaling pathway and is involved in various biological processes including lipid and energy metabolism. However, the effects of CRTC3 on the metabolome and transcriptome of porcine subcutaneous adipocytes have not been studied yet. Here, we tested whether porcine CRTC3 expression would be related to fat deposition in Heigai pigs (a local fatty breed in China) and Duroc×Landrace×Yorkshire (DLY, a lean breed) pigs in vivo. The effects of adenovirus-induced CRTC3 overexpression on the metabolomic and transcriptomic profiles of subcutaneous adipocytes were also determined in vitro by performing mass spectrometry-based metabolomics combined with RNA sequencing (RNA-seq). RESULTS: Porcine CRTC3 expression is associated with fat deposition in vivo. In addition, CRTC3 overexpression increased lipid accumulation and the expression of mature adipocyte-related genes in cultured porcine subcutaneous adipocytes. According to the metabolomic analysis, CRTC3 overexpression induced significant changes in adipocyte lipid, amino acid and nucleotide metabolites in vitro. The RNA-seq analysis suggested that CRTC3 overexpression alters the expression of genes and pathways involved in adipogenesis, fatty acid metabolism and glycerophospholipid metabolism in vitro. CONCLUSIONS: We identified significant alterations in the metabolite composition and the expression of genes and pathways involved in lipid metabolism in CRTC3-overexpressing adipocytes. Our results suggest that CRTC3 might play an important regulatory role in lipid metabolism and thus affects lipid accumulation in porcine subcutaneous adipocytes.

Single-cell RNA sequencing and lipidomics reveal cell and lipid dynamics of fat infiltration in skeletal muscle.

BACKGROUND: Ageing is accompanied by sarcopenia and intramuscular fat (IMAT) infiltration. In skeletal muscle, fat infiltration is a common feature in several myopathies and is associated with muscular dysfunction and insulin resistance. However, the cellular origin and lipidomic and transcriptomic changes during fat infiltration in skeletal muscle remain unclear. METHODS: In the current study, we generated a high IMAT-infiltrated skeletal muscle model by glycerol (GLY) injection. Single-cell RNA sequencing and lineage tracing were performed on GLY-injured skeletal muscle at 5 days post-injection (DPI) to identify the cell origins and dynamics. Lipidomics and RNA sequencing were performed on IMAT-infiltrated skeletal muscle at 14 DPI (or 17 DPI for the cold treatment) to analyse alterations of lipid compositions and gene expression levels. RESULTS: We identified nine distinct major clusters including myeloid-derived cells (52.13%), fibroblast/fibro/adipogenic progenitors (FAPs) (23.24%), and skeletal muscle stem cells (2.02%) in GLY-injured skeletal muscle. Clustering and pseudotemporal trajectories revealed six subpopulations in fibroblast/FAPs and 10 subclusters in myeloid-derived cells. A subpopulation of myeloid-derived cells expressing adipocyte-enriched genes and Pdgfra- /Cd68+ cells displayed lipid droplets upon adipogenic induction, indicating their adipogenic potential. Lipidomic analysis revealed the changes of overall lipid classes composition (e.g. triglycerides (TAGs) increased by 19.3 times, P = 0.0098; sulfoquinovosyl diacylglycerol decreased by 83%, P = 0.0056) and in the distribution of lipids [e.g. TAGs (18:2/18:2/22:6) increased by 181.6 times, P = 0.021] between GLY-group and saline control. RNA-seq revealed 1847 up-regulated genes and 321 down-regulated genes and significant changes in lipid metabolism-related pathways (e.g. glycerolipid pathway and glycerophospholipid pathway) in our model of GLY-injured skeletal muscle. Notably, short-term cold exposure altered fatty acid composition (e.g. saturated fatty acid decreased by 6.4%, P = 0.058) in fat-infiltrated muscles through directly affecting lipid metabolism pathways including PI3K-AKT and MAPK signalling pathway. CONCLUSIONS: Our results showed that a subpopulation of myeloid-derived cells may contribute to IMAT infiltration. GLY-induced IMAT infiltration changed the lipid composition and gene expression profiles. Short-term cold exposure might regulate lipid metabolism and its related signalling pathways in fat-infiltrated muscle. Our study provides a comprehensive resource describing the molecular signature of fat infiltration in skeletal muscle.

The regulatory role of melatonin in skeletal muscle.

Melatonin (N-acetyl-5-methoxy-tryptamine) is an effective antioxidant and free radical scavenger, that has important biological effects in multiple cell types and species. Melatonin research in muscle has recently gained attention, mainly focused on its role in cells or tissue repair and regeneration after injury, due to its powerful biological functions, including its antioxidant, anti-inflammation, anti-tumor and anti-cancer, circadian rhythm, and anti-apoptotic effects. However, the effect of melatonin in regulating muscle development has not been systematically summarized. In this review, we outline the latest research on the involvement of melatonin in the regulation of muscle development and regeneration in order to better understand its underlying molecular mechanisms and potential applications.

AMPK facilitates intestinal long-chain fatty acid uptake by manipulating CD36 expression and translocation.

Cellular long-chain fatty acids' (LCFAs) uptake is a crucial physiological process that regulates cellular energy homeostasis. AMPK has been shown to modulate LCFAs uptake in several kinds of cells, but whether it exerts an impact on intestinal LCFAs uptake is not quite clear. In the current study, we found that AMPK reinforced LCFAs uptake in intestinal epithelial cells (IECs). Moreover, intestinal epithelium-specific AMPK deletion impaired intestinal LCFAs absorption and protected mice from high-fat diet-induced obesity. Mechanistically, we discovered that AMPK deletion reduced the CD36 protein level by upregulating Parkin-mediated polyubiquitination of CD36 in IECs. Furthermore, our results revealed that AMPK affected PARK2 (gene name of Parkin) mRNA stability in a YTHDF2-dependent manner through FTO-dependent demethylation of N6 -methyladenosine (m6 A). Besides, AMPK promoted the translocation of CD36 to the plasma membrane in IECs, but the inhibition of AKT signaling suppressed this effect, which also halted the accelerated fatty acid uptake induced by AMPK. These results suggest that AMPK facilitates the intestinal LCFAs uptake by upregulating CD36 protein abundance and promoting its membrane translocation simultaneously. Such findings shed light on the role of AMPK in the regulation of intestinal LCFAs uptake.

The regulatory role of Myomaker and Myomixer-Myomerger-Minion in muscle development and regeneration.

Skeletal muscle plays essential roles in motor function, energy, and glucose metabolism. Skeletal muscle formation occurs through a process called myogenesis, in which a crucial step is the fusion of mononucleated myoblasts to form multinucleated myofibers. The myoblast/myocyte fusion is triggered and coordinated in a muscle-specific way that is essential for muscle development and post-natal muscle regeneration. Many molecules and proteins have been found and demonstrated to have the capacity to regulate the fusion of myoblast/myocytes. Interestingly, two newly discovered muscle-specific membrane proteins, Myomaker and Myomixer (also called Myomerger and Minion), have been identified as fusogenic regulators in vertebrates. Both Myomaker and Myomixer-Myomerger-Minion have the capacity to directly control the myogenic fusion process. Here, we review and discuss the latest studies related to these two proteins, including the discovery, structure, expression pattern, functions, and regulation of Myomaker and Myomixer-Myomerger-Minion. We also emphasize and discuss the interaction between Myomaker and Myomixer-Myomerger-Minion, as well as their cooperative regulatory roles in cell-cell fusion. Moreover, we highlight the areas for exploration of Myomaker and Myomixer-Myomerger-Minion in future studies and consider their potential application to control cell fusion for cell-therapy purposes.

Myomaker, and Myomixer-Myomerger-Minion modulate the efficiency of skeletal muscle development with melatonin supplementation through Wnt/ß-catenin pathway.

Melatonin, a pleiotropic hormone secreted from the pineal gland, has been shown to exert beneficial effects in muscle regeneration and repair due to its functional diversity, including anti-inflammation, anti-apoptosis, and anti-oxidative activity. However, little is known about the negative role of melatonin in myogenesis. Here, using skeletal muscle cells, we found that melatonin promoted C2C12 cells proliferation and inhibits differentiation both in C2C12 cells and primary myoblasts in mice. Melatonin administration significantly down-regulated differentiation and fusion related genes and inhibited myotube formation both in C2C12 cells and primary myoblasts in mice. RNA-seq showed that melatonin down-regulated essential fusion pore components Myomaker and Myomixer-Myomerger-Minion. Moreover, melatonin suppressed Wnt/ß-catenin signaling. Inhibition of GSK3ß by LiCl rescued the influence of melatonin on differentiation efficiency, Myomaker, but not Myomxier in C2C12 cells. In conclusion, melatonin inhibits myogenic differentiation, Myomaker, and Myomixer through reducing Wnt/ß-catenin signaling. These data establish a link between melatonin and fusogenic membrane proteins Myomaker and Myomixer, and suggest the new perspective of melatonin in treatment or preventment of muscular diseases.

Roles of phosphatase and tensin homolog in skeletal muscle.

The phosphatase and tensin homolog (PTEN), originally identified as a tumor suppressor, is an important regulator of the PI3K-Akt pathway. PTEN plays crucial roles in various cellular processes, including cell survival, cell growth, cell proliferation, cell differentiation, and cell metabolism. In metabolic tissues, PTEN expression affects insulin sensitivity and glucose homeostasis. In skeletal muscle, the deletion of PTEN regulates muscle development and protects the mutant mice from insulin resistance and diabetes. Notably, the regulatory role of PTEN in skeletal muscle stem cells has been recently reported. In this review, we mainly discuss the role of PTEN in regulating the development, glucose metabolism, stem cell fate decision, and regeneration of skeletal muscle.

Cathelicidin-WA attenuates LPS-induced inflammation and redox imbalance through activation of AMPK signaling.

Dysregulated activation of inflammation is associated with the development and progression of many diseases. Generation of reactive oxygen species (ROS) has been shown to promote an inflammatory response. Cathelicidin peptides not only defend against the invasion of various microbes but also play an important role in regulating immune responses. The objective of this study was to investigate the effects and mechanisms of Cathelicidin-WA (CWA) on the inflammatory response and oxidative stress in macrophages. Our results showed that CWA efficiently attenuated lipopolysaccharide (LPS)-stimulated inflammation and oxidative stress both in vivo and in vitro. Mechanistically, we found that CWA significantly reduced the LPS-induced nuclear translocation of NF-κB, thus decreasing the production of the pro-inflammatory cytokines TNF-α and IL-6 in macrophages. On the other hand, CWA markedly promoted the nuclear translocation of Nrf2 via the AKT pathway and p38 signaling. This resulted in increased expression of the anti-oxidative genes NQO-1 and HO-1 and alleviated oxidative stress in LPS-stimulated macrophages. Interestingly, the effects of CWA were diminished when AMPK was knocked down. Consistently, we noticed that CWA failed to ameliorate the LPS-induced inflammatory response and oxidative stress in AMPK knockout mice. Furthermore, we discovered that LKB1 was essential for AMPK activation by CWA. These data demonstrated for the first time that CWA attenuated LPS-stimulated inflammation and redox imbalance through regulating LKB1-AMPK signaling. Such knowledge provides new insights into the mechanisms through which Cathelicidin peptides modulate immune responses.

Rapid Communication: Porcine CRTC3 gene clone, expression pattern, and its regulatory role in intestinal epithelial cells.

In the current study, we aimed to clone the full-length cDNA of porcine CRTC3 (pCRTC3) gene and examine its expression pattern and function in intestinal epithelial cells. The full-length cDNA sequence of pCRTC3 was 2,173 bp (GenBank accession no. MF964215), with a 1,860-bp open reading frame encoding a 620-AA protein. Comparison of the deduced AA sequence with different species including human, mouse, rat, Papio, cattle, and rabbit showed 89% to 91.9% similarity. The pCRTC3 was highly expressed in small intestine and spleen, to a lesser degree in lung, liver, and adipose tissue, and was expressed at a low but detectable level in skeletal muscle, kidney, and heart. In addition, high protein levels of pCRTC3 were found in IPEC-J2 cells, in which pCRTC3 was mainly localized in cytoplasm. Furthermore, we demonstrated that knockdown of pCRTC3 significantly decreased the expression of the porcine tight junction-related genes including zonula occludens-1 (ZO-1), ZO-2, occludin, and claudin-1 by 57.88% (P < 0.01), 40.19% (P < 0.01), 51.59% (P < 0.01), and 35.70% (P < 0.05), respectively. Taken together, we first cloned the full-length sequence of pCRTC3 and revealed the tissue-specific expression pattern, localization, and function of pCRTC3 in regulating the expression of intestinal tight junction-related genes. This study could provide some useful information for understanding the function of CRTC3 in pigs.

Betaine promotes lipid accumulation in adipogenic-differentiated skeletal muscle cells through ERK/PPARγ signalling pathway.

Betaine, a neutral zwitterionic compound, could regulate intramuscular fat (IMF) deposition and meat quality. However, the efficacy is controversial. Moreover, the regulatory mechanism of betaine on lipid metabolism in skeletal muscle cells remains unclear. Therefore, in this study, we examined the effects and regulatory mechanism of betaine on lipid accumulation in adipogenic-differentiated C2C12 cells. We found that adipogenic-induced C2C12 cells treated with 10 mM betaine for 24 and 48 h had more lipid accumulation than the control group. Real-time PCR and Western blot results revealed that betaine treatment did not alter the expression of lipolysis and lipid oxidation-related genes, but dramatically increased the expression of peroxisome proliferator-activated receptor γ (PPARγ) and its target genes such as fatty acid binding protein 4 (aP2), fatty acid synthase (FAS) and lipoprteinlipase (LPL). Furthermore, betaine combined with PPARγ inhibitor GW9662 treatment showed that betaine elevated C2C12 lipid accumulation through upregulation of PPARγ. Mechanistically, we found that betaine promoted PPARγ expression and lipid accumulation through inhibition of extracellular regulated protein kinases1/2 (ERK1/2) signalling pathway. These results demonstrate that betaine acts through ERK1/2-PPARγ signalling pathway to regulate lipid metabolism in adipogenic-differentiated skeletal muscle cells, which could provide some useful information for controlling muscle lipid accumulation by manipulating ERK1/2 and PPARγ signalling pathway.

Regulation role of CRTC3 in skeletal muscle and adipose tissue.

The cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling pathway plays important role in regulating energy homeostasis. Many of the effects of the cAMP-PKA signaling is mediated through the cAMP responsive element binding protein (CREB) and its coactivator CREB-regulated transcription coactivators (CRTCs). CRTC3 is a member of CRTCs family proteins and plays important roles in glucose and energy metabolism. Previous studies show that global knockout of CRTC3 enhances oxygen consumption and energy expenditure and subsequently protects the knockout animal against obesity. In skeletal muscle, CRTC3 affects lipid and glycogen metabolism and mitochondrial biogenesis. In white adipocytes, CRTC3 regulates GLUT4 expression and glucose uptake. More recently, the localization and function of CRTC3 in brown fat have been reported. In this review, we mainly discuss the regulatory role of CRTC3 in skeletal muscle and adipose tissues.

Peripheral Neuropathy and Hindlimb Paralysis in a Mouse Model of Adipocyte-Specific Knockout of Lkb1.

Brown adipose tissues (BAT) burn lipids to generate heat through uncoupled respiration, thus representing a powerful target to counteract lipid accumulation and obesity. The tumor suppressor liver kinase b1 (Lkb1) is a key regulator of cellular energy metabolism; and adipocyte-specific knockout of Lkb1 (Ad-Lkb1 KO) leads to the expansion of BAT, improvements in systemic metabolism and resistance to obesity in young mice. Here we report the unexpected finding that the Ad-Lkb1 KO mice develop hindlimb paralysis at mid-age. Gene expression analyses indicate that Lkb1 KO upregulates the expression of inflammatory cytokines in interscapular BAT and epineurial brown adipocytes surrounding the sciatic nerve. This is followed by peripheral neuropathy characterized by infiltration of macrophages into the sciatic nerve, axon degeneration, reduced nerve conductance, and hindlimb paralysis. Mechanistically, Lkb1 KO reduces AMPK phosphorylation and amplifies mammalian target-of-rapamycin (mTOR)-dependent inflammatory signaling specifically in BAT but not WAT. Importantly, pharmacological or genetic inhibition of mTOR ameliorates inflammation and prevents paralysis. These results demonstrate that BAT inflammation is linked to peripheral neuropathy.

AMPK regulates lipid accumulation in skeletal muscle cells through FTO-dependent demethylation of N6-methyladenosine.

Skeletal muscle plays important roles in whole-body energy homeostasis. Excessive skeletal muscle lipid accumulation is associated with some metabolic diseases such as obesity and Type 2 Diabetes. The energy sensor AMPK (AMP-activated protein kinase) is a key regulator of skeletal muscle lipid metabolism, but the precise regulatory mechanism remains to be elucidated. Here, we provide a novel mechanism by which AMPK regulates skeletal muscle lipid accumulation through fat mass and obesity-associated protein (FTO)-dependent demethylation of N6-methyladenosine (m6A). We confirmed an inverse correlation between AMPK and skeletal muscle lipid content. Moreover, inhibition of AMPK enhanced lipid accumulation, while activation of AMPK reduced lipid accumulation in skeletal muscle cells. Notably, we found that mRNA m6A methylation levels were inversely correlated with lipid content in skeletal muscle. Furthermore, AMPK positively regulated the m6A methylation levels of mRNA, which could negatively regulate lipid accumulation in C2C12. At the molecular level, we demonstrated that AMPK regulated lipid accumulation in skeletal muscle cells by regulating FTO expression and FTO-dependent demethylation of m6A. Together, these results provide a novel regulatory mechanism of AMPK on lipid metabolism in skeletal muscle cells and suggest the possibility of controlling skeletal muscle lipid deposition by targeting AMPK or using m6A related drugs.

Pten is necessary for the quiescence and maintenance of adult muscle stem cells.

Satellite cells (SCs) are myogenic stem cells required for regeneration of adult skeletal muscles. A proper balance among quiescence, activation and differentiation is essential for long-term maintenance of SCs and their regenerative function. Here we show a function of Pten (phosphatase and tensin homologue) in quiescent SCs. Deletion of Pten in quiescent SCs leads to their spontaneous activation and premature differentiation without proliferation, resulting in depletion of SC pool and regenerative failure. However, prior to depletion, Pten-null activated SCs can transiently proliferate upon injury and regenerate injured muscles, but continually decline during regeneration, suggesting an inability to return to quiescence. Mechanistically, Pten deletion increases Akt phosphorylation, which induces cytoplasmic translocation of FoxO1 and suppression of Notch signalling. Accordingly, constitutive activation of Notch1 prevents SC depletion despite Pten deletion. Our findings delineate a critical function of Pten in maintaining SC quiescence and reveal an interaction between Pten and Notch signalling.

Mammalian target of rapamycin is essential for cardiomyocyte survival and heart development in mice.

Mammalian target of rapamycin (mTOR) is a critical regulator of protein synthesis, cell proliferation and energy metabolism. As constitutive knockout of Mtor leads to embryonic lethality, the in vivo function of mTOR in perinatal development and postnatal growth of heart is not well defined. In this study, we established a muscle-specific mTOR conditional knockout mouse model (mTOR-mKO) by crossing MCK-Cre and Mtor(flox/flox) mice. Although the mTOR-mKO mice survived embryonic and perinatal development, they exhibited severe postnatal growth retardation, cardiac muscle pathology and premature death. At the cellular level, the cardiac muscle of mTOR-mKO mice had fewer cardiomyocytes due to apoptosis and necrosis, leading to dilated cardiomyopathy. At the molecular level, the cardiac muscle of mTOR-mKO mice expressed lower levels of fatty acid oxidation and glycolysis related genes compared to the WT littermates. In addition, the mTOR-mKO cardiac muscle had reduced Myh6 but elevated Myh7 expression, indicating cardiac muscle degeneration. Furthermore, deletion of Mtor dramatically decreased the phosphorylation of S6 and AKT, two key targets downstream of mTORC1 and mTORC2 mediating the normal function of mTOR. These results demonstrate that mTOR is essential for cardiomyocyte survival and cardiac muscle function.