Dynamic fecal microenvironment properties enable predictions and understanding of peripartum blood oxidative status and nonesterified fatty acids in dairy cows.

The transition period in dairy cows is a critical stage and peripartum oxidative status, negative energy balance (NEB), and inflammation are highly prevalent. Fecal microbial metabolism is closely associated with blood oxidative status and nonesterified fatty acids (NEFA) levels. Here, we investigated dynamic changes in total oxidative status markers and NEFA in blood, fecal microbiome, and metabolome of 30 dairy cows during transition (-21, -7, +7, +21 d relative to calving). Then the Bayesian network and 9 machine-learning algorithms were applied to dismantle their relationship. Our results show that the oxidative status indicator (OSI) of -21, -7, +7 d was higher than +21 d. The plasma concentration of NEFA peaked on +7 d. For fecal microenvironment, a decline in bacterial α diversity was observed at postpartum and in bacterial interactions at +7 d. Conversely, microbial metabolites involved in carbohydrate, lipid, and energy metabolism increased on +7 d. A correlation analysis revealed that 11 and 10 microbial metabolites contributed to OSI and NEFA variations, respectively (arc strength >0.5). The support vector machine (SVM) radial model showed the highest average predictive accuracy (100% and 88.9% in the test and external data sets) for OSI using 1 metabolite and 3 microbiota. The SVM radial model also showed the highest average diagnostic accuracy (100% and 91% in the test and external data sets) for NEFA with 2 metabolites and 3 microbiota. Our results reveal a relationship between variation in the fecal microenvironment and indicators of oxidative status, NEB, and inflammation, which provide a theoretical basis for the prevention and precise regulation of peripartum oxidative status and NEB.

mRNA m5C inhibits adipogenesis and promotes myogenesis by respectively facilitating YBX2 and SMO mRNA export in ALYREF-m5C manner.

Although 5-methylcytosine (m5C) has been identified as a novel and abundant mRNA modification and associated with energy metabolism, its regulation function in adipose tissue and skeletal muscle is still limited. This study aimed at investigating the effect of mRNA m5C on adipogenesis and myogenesis using Jinhua pigs (J), Yorkshire pigs (Y) and their hybrids Yorkshire-Jinhua pigs (YJ). We found that Y grow faster than J and YJ, while fatness-related characteristics observed in Y were lower than those of J and YJ. Besides, total mRNA m5C levels and expression rates of NSUN2 were higher both in backfat layer (BL) and longissimus dorsi muscle (LDM) of Y compared to J and YJ, suggesting that higher mRNA m5C levels positively correlate with lower fat and higher muscle mass. RNA bisulfite sequencing profiling of m5C revealed tissue-specific and dynamic features in pigs. Functionally, hyper-methylated m5C-containing genes were enriched in pathways linked to impaired adipogenesis and enhanced myogenesis. In in vitro, m5C inhibited lipid accumulation and promoted myogenic differentiation. Furthermore, YBX2 and SMO were identified as m5C targets. Mechanistically, YBX2 and SMO mRNAs with m5C modification were recognized and exported into the cytoplasm from the nucleus by ALYREF, thus leading to increased YBX2 and SMO protein expression and thereby inhibiting adipogenesis and promoting myogenesis, respectively. Our work uncovered the critical role of mRNA m5C in regulating adipogenesis and myogenesis via ALYREF-m5C-YBX2 and ALYREF-m5C-SMO manners, providing a potential therapeutic target in the prevention and treatment of obesity, skeletal muscle dysfunction and metabolic disorder diseases.

Ileum tissue single-cell mRNA sequencing elucidates the cellular architecture of pathophysiological changes associated with weaning in piglets.

BACKGROUND: In mammals, transitioning from sole milk uptake to the intake of solid feed results in dramatic developmental changes in intestinal function and immunological status. In fact, weaning stress is often accompanied by intestinal inflammatory processes. To develop effective intervention strategies, it is necessary to characterize the developmental pattern and immune response that occurs on weaning, as we have done in this study for piglets. RESULTS: To comprehensively delineate cell heterogeneity in ileum tissues and the underlying mechanisms in weaning-induced intestinal inflammation of piglets, we have analyzed the transcriptomes of 42,149 cells from ileum mucosa of normally suckling and post-weaned piglets. There were 31 cell subtypes including epithelial, stromal, and immune cells. A bifurcating trajectory was inferred to separate secretory and absorptive lineages. Integrated cross-species datasets showed well-conserved cellular architectures and transcription signatures between human and pig. Comparative analyses of cellular components, cell-cell communications, and molecular states revealed that T cell subpopulations were significantly altered in weaned piglets. We found that T helper (Th) 17 functional plasticity across changes in the cytokine milieu and the enrichment of granzyme B (GZMB)-expressing cytotoxic T cells potentially exacerbate mucosal inflammation via mitochondrial dysfunction in epithelial cells. CONCLUSIONS: Our work has elucidated the single-cell molecular characteristics of the piglet ileum before and after weaning. We have provided an atlas that portrays the landscape of the intestinal pathophysiological inflammatory process associated with weaning, finding a level of conservation between human and pig that support the use of piglets as a model for human infants.

Potential key factors involved in regulating adipocyte dedifferentiation.

Adipocytes are the key constituents of adipose tissue, and their de-differentiation process has been widely observed in physiological and pathological conditions. For obese people, the promotion of adipocyte de-differentiation or maintenance of an undifferentiated state of adipocytes may help to improve their metabolic condition. Thus, understanding the regulatory mechanisms of adipocyte de-differentiation is necessary for treating metabolic diseases. Attractively, in addition to intracellular signals regulating adipocyte de-differentiation, external factors such as temperature and pressure also affect adipocyte de-differentiation. In this review, we summarize the recent progress in the field and discuss the regulatory roles and mechanisms of involved endogenous and exogenous factors during the process of de-differentiation.

Limits to sustained energy intake. XXIX. The case of the golden hamster (Mesocricetus auratus).

Golden hamster females have the shortest known gestation period among placental mammals, and at the same time raise very large litters of up to 16 offspring, which are born in a naked and blind state and are only able to pick up food from days 12 to 14 onwards. We quantified energy metabolism and milk production in female golden hamsters raising offspring under cold (8°C), normal (22°C) and hot (30°C) ambient temperature conditions. We monitored energy intake, subcutaneous body temperature, daily energy expenditure, litter size and pup masses over the course of lactation. Our results show that, in line with the concept of heat dissipation limitation, female golden hamsters had the largest energy intake under the coldest conditions and a significantly lower intake at 30°C (partial for influence of ambient temperature: F2,403=5.6; P=0.004). Metabolisable energy intake as well as milk energy output showed the same pattern and were significantly different between the temperatures (partial for milk energy production: F1,40=86.4; P<0.0001), with consistently higher subcutaneous temperatures in the reproductive females (F1,813=36.77; P<0.0001) compared with baseline females. These data suggest that raising offspring in golden hamsters comes at the cost of producing large amounts of body heat up to a level constraining energy intake, similar to that observed in some laboratory mice. Notably, we observed that females seemed to adjust litter size according to their milk production, with the smallest litters (3.4±0.7 pups) being raised by hot-exposed mothers. Future research is needed to unravel the mechanism by which females assess their own milk production capabilities and how this may be linked to litter size at different ambient temperatures. Golden hamsters reach 8-10 times resting metabolic rate when raising offspring under cold conditions, which is compatible with the findings from laboratory mice and other rodents.

Dorsal shaving affects concentrations of faecal cortisol metabolites in lactating golden hamsters.

Breeding of golden hamsters is classically performed at thermal conditions ranging from 20 to 24 °C. However, growing evidence suggests that lactating females suffer from heat stress. We hypothesised that shaving females dorsally to maximise heat dissipation may reduce stress during reproduction. We thus compared faecal cortisol metabolites (FCM) from shaved golden hamster mothers with those from unshaved controls. We observed significantly lower FCM levels in the shaved mothers (F1,22 = 8.69, p = 0.0075) pointing to lower stress due to ameliorated heat dissipation over the body surface. In addition, we observed 0.4 °C lower mean subcutaneous body temperatures in the shaved females, although this effect did not reach significance (F1,22 = 1.86, p = 0.18). Our results suggest that golden hamsters having body masses being more than four times that of laboratory mice provide a very interesting model to study aspects of lactation and heat production at the same time.

Energy Metabolism and Ageing in the Mouse: A Mini-Review.

The mouse has rapidly become the mammalian model organism of choice in ageing research due to its relatively short lifespan, the proximity of its genome and physiology to humans, and most importantly due to its genetic pliability and the availability of mutant strains. Mouse models have provided great insights into the ageing process, which in its broadest sense is the progressive decline of body functions over time. In this mini-review, we briefly cover the historical views on the link between ageing and metabolic rate, highlight genetically modified transgenic mouse models of extended lifespan, discuss endocrine pathways linked to senescence and ageing, and then examine pathways by which caloric restriction is postulated to result in longevity.

Ecophysiology of omega Fatty acids: a lid for every jar.

Omega fatty acids affect various physiological functions, such as locomotion, cardiac function, and thermogenesis. We highlight evidence from animal models that points to pathways by which specific omega fatty acids exert differential effects. We suggest that optimizing the omega fatty acid composition of tissues involves trade-offs between costs and benefits of specific fatty acids.

Ambient temperature affects postnatal litter size reduction in golden hamsters.

BACKGROUND: To better understand how different ambient temperatures during lactation affect survival of young, we studied patterns of losses of pups in golden hamsters (Mesocricetus auratus) at different ambient temperatures in the laboratory, mimicking temperature conditions in natural habitats. Golden hamsters produce large litters of more than 10 young but are also known to wean fewer pups at the end of lactation than they give birth to. We wanted to know whether temperature affects litter size reductions and whether the underlying causes of pup loss were related to maternal food (gross energy) intake and reproductive performance, such as litter growth. For that, we exposed lactating females to three different ambient temperatures and investigated associations with losses of offspring between birth and weaning. RESULTS: Overall, around one third of pups per litter disappeared, obviously consumed by the mother. Such litter size reductions were greatest at 30 °C, in particular during the intermediate postnatal period around peak lactation. Furthermore, litter size reductions were generally higher in larger litters. Maternal gross energy intake was highest at 5 °C suggesting that mothers were not limited by milk production and might have been able to raise a higher number of pups until weaning. This was further supported by the fact that the daily increases in litter mass as well as in the individual pup body masses, a proxy of mother's lactational performance, were lower at higher ambient temperatures. CONCLUSIONS: We suggest that ambient temperatures around the thermoneutral zone and beyond are preventing golden hamster females from producing milk at sufficient rates. Around two thirds of the pups per litter disappeared at high temperature conditions, and their early growth rates were significantly lower than at lower ambient temperatures. It is possible that these losses are due to an intrinsic physiological limitation (imposed by heat dissipation) compromising maternal energy intake and milk production.

Detection of rare species of volatile organic selenium metabolites in male golden hamster urine.

Selenium has been considered as an essential trace element in mammals and its intake comes mainly from food. Mammals can metabolize both inorganic and organic species, and urinary excretion is the primary elimination route of selenium. Selenosugars and trimethylselenonium ion have been identified as major urinary metabolites. Other metabolites have been reported, but they were detected in some studies and not in others. Still, a large portion of the ingested selenium eliminated from the body is unknown. Volatile selenium species may account for a certain portion of the unknown species since they can easily be lost during sample analyses. While we analyzed male golden hamster urine in search of potential volatile pheromone(s), four volatile selenium compounds were detected. They were dimethyl selenenylsulfide, dimethyl diselenide, dimethyl bis(thio)selenide, and dimethyl selenodisulfide. When the urine samples were aged and dried for 48 h, dimethyl selenodisulfide tended to increase, while others decreased. The increase might be due to the formation of dimethyl selenodisulfide via reaction of dimethyl diselenide and dimethyl trisulfide whose concentration increased as urine aged. To our knowledge, dimethyl bis(thio)selenide and dimethyl selenodisulfide have never been demonstrated in urine. It remains to be determined whether these species are common metabolites in other animals or hamster-specific.

Fat infiltration in skeletal muscle: Influential triggers and regulatory mechanism.

Fat infiltration in skeletal muscle (also known as myosteatosis) is now recognized as a distinct disease from sarcopenia and is directly related to declining muscle capacity. Hence, understanding the origins and regulatory mechanisms of fat infiltration is vital for maintaining skeletal muscle development and improving human health. In this article, we summarized the triggering factors such as aging, metabolic diseases and metabolic syndromes, nonmetabolic diseases, and muscle injury that all induce fat infiltration in skeletal muscle. We discussed recent advances on the cellular origins of fat infiltration and found several cell types including myogenic cells and non-myogenic cells that contribute to myosteatosis. Furthermore, we reviewed the molecular regulatory mechanism, detection methods, and intervention strategies of fat infiltration in skeletal muscle. Based on the current findings, our review will provide new insight into regulating function and lipid metabolism of skeletal muscle and treating muscle-related diseases.

Rapid UV Photo-Cross-Linking of α-Lactalbumin Hydrogel Biomaterial To Enable Wound Healing.

Although both the function and biocompatibility of protein-based biomaterials are better than those of synthetic materials, their usage as medical material is currently limited by their high costs, low yield, and low batch-to-batch reproducibility. In this article, we show how α-lactalbumin (α-LA), rich in tryptophan, was used to produce a novel type of naturally occurring, protein-based biomaterial suitable for wound dressing. To create a photo-cross-linkable polymer, α-LA was methacrylated at a 100-g batch scale with >95% conversion and 90% yield. α-LAMA was further processed using photo-cross-linking-based advanced processing techniques such as microfluidics and 3D printing to create injectable hydrogels, monodispersed microspheres, and patterned scaffolds. The obtained α-LAMA hydrogels show promising biocompatibility and degradability during in vivo testing. Additionally, the α-LAMA hydrogel can accelerate post-traumatic wound healing and promote new tissue regeneration. In conclusion, cheap and safe α-LAMA-based biomaterials could be produced, and they have a beneficial effect on wound healing. As a result, there may arise a potential partnership between the dairy industry and the development of pharmaceuticals.

Extracellular vesicles of Bacteroides uniformis induce M1 macrophage polarization and aggravate gut inflammation during weaning.

Weaning process is commonly associated with gastrointestinal inflammation and dysbiosis of the intestinal microbes. In particular, the impact of gut bacteria and extracellular vesicles on the etiology of intestinal inflammation during weaning is not well understood. We have uncovered a potential link between gut inflammation and the corresponding variation of macrophage bacterial sensing and pro-inflammatory polarization during the weaning process of piglets through single-cell transcriptomic analyses. We conducted a comprehensive analysis of bacterial distribution across the gastrointestinal tract and pinpointed Bacteroides uniformis enriching in piglets undergoing weaning. Next, we found out that exposure to B. uniformis-derived extracellular vesicles (BEVs) exacerbated gut inflammation in a murine colitis model while recruiting and polarizing intestinal macrophages toward a pro-inflammatory phenotype. BEVs modulated the function of macrophages cultured in vitro by suppressing the granulocyte-macrophage colony-stimulating factor/signal transducer and activator of transcription 5/arginase 1 pathway, thereby affecting polarization toward an M1-like state. The effects of BEVs were verified both in the macrophage clearance murine model and by using an adoptive transfer assay. Our findings highlight the involvement of BEVs in facilitating the polarization of pro-inflammatory macrophages and promoting gut inflammation during weaning.

Sexually Dimorphic Response to Hepatic Injury in Newborn Suffering from Intrauterine Growth Restriction.

Intrauterine growth restriction (IUGR), when a fetus does not grow as expected, is associated with a reduction in hepatic functionality and a higher risk for chronic liver disease in adulthood. Utilizing early developmental plasticity to reverse the outcome of poor fetal programming remains an unexplored area. Focusing on the biochemical profiles of neonates and previous transcriptome findings, piglets from the same fetus are selected as models for studying IUGR. The cellular landscape of the liver is created by scRNA-seq to reveal sex-dependent patterns in IUGR-induced hepatic injury. One week after birth, IUGR piglets experience hypoxic stress. IUGR females exhibit fibroblast-driven T cell conversion into an immune-adapted phenotype, which effectively alleviates inflammation and fosters hepatic regeneration. In contrast, males experience even more severe hepatic injury. Prolonged inflammation due to disrupted lipid metabolism hinders intercellular communication among non-immune cells, which ultimately impairs liver regeneration even into adulthood. Additionally, Apolipoprotein A4 (APOA4) is explored as a novel biomarker by reducing hepatic triglyceride deposition as a protective response against hypoxia in IUGR males. PPARα activation can mitigate hepatic damage and meanwhile restore over-expressed APOA4 to normal in IUGR males. The pioneering study offers valuable insights into the sexually dimorphic responses to hepatic injury during IUGR.

Limits to sustained energy intake. XXI. Effect of exposing the mother, but not her pups, to a cold environment during lactation in mice.

The capacity of females to dissipate heat may constrain sustained energy intake during lactation. However, some previous experiments supporting this concept have confounded the impact of temperature on the mothers with the impact on the pups. We aimed to separate these effects in lactating laboratory mice (MF1 strain) by giving the mothers access to cages at two ambient temperatures (10 and 21°C) joined by a tube. Food was available only in the cold cage, but females could also choose go to this cage to cool down while their pups were housed in the warmer cage. Control animals had access to the same configuration of cages but with both maintained at 21°C. We hypothesised that if females were limited by heat dissipation, alleviating the heat load by providing a cool environment would allow them to dissipate more heat, take in more food, generate more milk and hence wean heavier litters. We measured maternal energy budgets and monitored time courses of core body temperature and physical activity. To minimise the variance in energy budgets, all litters were adjusted to 12 (±1) pups. Females in the experimental group had higher energy intake (F1,14=15.8, P=0.0014) and higher assimilated energy (F1,13=10.7, P=0.006), and provided their pups with more milk (F1,13=6.65, P=0.03), consistent with the heat dissipation limit theory. Yet, despite keeping demand constant, mean pup growth rates were similar (F1,13=0.06, P=0.8); thus, our data emphasise the difficulties of inferring milk production indirectly from pup growth.

Season-dependent impact of forage quality on stress in alpine chamois.

Chronically heightened stress levels in wildlife species may have detrimental effects on individual life history traits, for example, through the increased likelihood of disease, parasitic infections, and overall reduced fitness. Understanding the drivers of stress may thus have great potential for informing wildlife conservation. Although the role of climate and individual status is well studied in stress ecology, the impact of related stressors such as dietary quality is of increasing interest to wildlife research and conservation. In this study, fecal cortisol metabolites (FCMs) in Alpine chamois Rupicapra r. rupicapra used as bioindicators of stress, and their relationship with forage quality-measured as the percentage of fecal crude protein (CP)-were investigated. Data collection took place in 2011 and 2012 in the Gran Paradiso National Park (Western Italian Alps), on 22 individually marked adult males. The relationship between FCMs and CPs was analyzed through linear models and separated between winter and summer months, accounting for the effect of potentially confounding exogenous and endogenous variables. After AICc-based model selection, we found that forage quality was negatively related to FCM levels in Alpine chamois during the summer months, meaning that higher quality forage was associated with the decreased expression of stress hormones. However, during the winter months, we did not find a significant relationship, potentially as a result of forage quality being ubiquitously poor. Although the mechanisms through which dietary variations impact FCM concentrations in wildlife populations are largely unknown, the occurrence of significant relationships between forage quality and stress levels supports potentially important implications for the long-term effect of climatic changes on the fitness of wildlife populations.

Lactating SKH-1 furless mice prioritize own comfort over growth of their pups.

Lactation is the most energetically demanding physiological process that occurs in mammalian females, and as a consequence of this energy expenditure, lactating females produce an enormous amount of excess heat. This heat is thought to limit the amount of milk a mother produces, and by improving heat dissipation, females may improve their milk production and offspring quality. Here we used SKH-1 hairless mice as a natural model of improved heat dissipation. Lactating mothers were given access to a secondary cage to rest away from their pups, and this secondary cage was kept either at room temperature (22 °C) in the control rounds or cooled to 8 °C in the experimental groups. We hypothesized that the cold exposure would maximize the heat dissipation potential, leading to increased milk production and healthier pups even in the hairless mouse model. However, we found the opposite, where cold exposure allowed mothers to eat more food, but they produced smaller weight pups at the end of lactation. Our results suggest that mothers prioritize their own fitness, even if it lowers the fitness of their offspring in this particular mouse strain. This maternal-offspring trade-off is interesting and requires future studies to understand the full interaction of maternal effects and offspring fitness in the light of the heat dissipation limitation.

Biodegradation of conventional plastics: Candidate organisms and potential mechanisms.

With the benefits of coming at low-cost, being light-weight and having a high formability and durability, conventional plastics are widely used in both industry and daily life. However, because of their durability and extensive half-life with poor degradability and the low recycling rate, large amounts of plastic waste are accumulated in various environments, posing a significant threat to organisms and ecosystems. Compared to conventional physical and chemical degradation, biodegradation of plastic might become a promising and environmentally friendly solution for this problem. One of the aims of this review is to briefly describe the impact of plastics (especially microplastics). To facilitate rapid advancements in the area of plastic biodegradation, this paper provides a comprehensive review of the candidate organisms capable of biodegrading plastics and originating from four categories including natural microorganisms, artificially derived microorganisms, algae and animal organisms. In addition, the potential mechanism during plastic biodegradation and associated driving factors are summarized and discussed. Furthermore, the recent biotechnological progress (e.g. synthetic biology, systems biology, etc.) is highlighted as being key for future research. Finally, innovative research avenues for future studies are proposed. Concluding, our review is addressing the practical application of plastic biodegradation and the plastic pollution, thus necessitating more sustainable developments.

Gut microbiome is linked to functions of peripheral immune cells in transition cows during excessive lipolysis.

BACKGROUND: Postpartum dairy cows experiencing excessive lipolysis are prone to severe immunosuppression. Despite the extensive understanding of the gut microbial regulation of host immunity and metabolism, its role during excessive lipolysis in cows is largely unknown. Herein, we investigated the potential links between the gut microbiome and postpartum immunosuppression in periparturient dairy cows with excessive lipolysis using single immune cell transcriptome, 16S amplicon sequencing, metagenomics, and targeted metabolomics. RESULTS: The use of single-cell RNA sequencing identified 26 clusters that were annotated to 10 different immune cell types. Enrichment of functions of these clusters revealed a downregulation of functions in immune cells isolated from a cow with excessive lipolysis compared to a cow with low/normal lipolysis. The results of metagenomic sequencing and targeted metabolome analysis together revealed that secondary bile acid (SBA) biosynthesis was significantly activated in the cows with excessive lipolysis. Moreover, the relative abundance of gut Bacteroides sp. OF04 - 15BH, Paraprevotella clara, Paraprevotella xylaniphila, and Treponema sp. JC4 was mainly associated with SBA synthesis. The use of an integrated analysis showed that the reduction of plasma glycolithocholic acid and taurolithocholic acid could contribute to the immunosuppression of monocytes (CD14+MON) during excessive lipolysis by decreasing the expression of GPBAR1. CONCLUSIONS: Our results suggest that alterations in the gut microbiota and their functions related to SBA synthesis suppressed the functions of monocytes during excessive lipolysis in transition dairy cows. Therefore, we concluded that altered microbial SBA synthesis during excessive lipolysis could lead to postpartum immunosuppression in transition cows. Video Abstract.

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.