BACKGROUND: Muscle atrophy, fibrosis and fatty infiltration (FI) are commonly seen in rotator cuff tears (RCT), which are critical factors that directly determine the clinical outcomes for patients with this injury. Therefore, improving muscle quality after RCT is crucial in improving the clinical outcome of tendon repair. In recent years, it has been discovered that adults have functional beige/brown adipose tissue (BAT) which can secrete batokines to promote muscle growth. PRDM16, a PR-domain containing protein, was discovered with the ability to determine the brown fat cell fate and stimulate its development. Thus, the goal of this study is to discover the role of PRDM16 in improving muscle function after massive tendon tears using a transgenic mouse model with an elevated level of PRDM16 expression.
METHODS: Transgenic aP2 driven PRDM16 overexpression mice and C57BL/6J mice underwent unilateral supraspinatus (SS) tendon transection and suprascapular nerve transection (TTDN) as described previously (N=8 in each group). DigiGait was performed to evaluate forelimb function at 6 weeks post the TTDN injury. Bilateral SS muscles, interscapular brown fat, epididymal white fat, and inguinal beige fat were harvested for analysis. The expression of PRDM16 in adipose tissue was detected by Western Blot. Masson\'s trichome staining was conducted to evaluate the muscle fibrosis and Oil Red O staining was used to determine the fat infiltration. Muscle fiber type was determined by MHC expression via immunostaining. All data was presented in the form of mean±SD. T-test and two-way ANOVA analysis was performed to determine a statistically significant difference between groups. Significance was considered when P<0.05.
RESULTS: Western blot data showed an increased expression of PRDM16 protein in both white and brown fat in PRDM16-overexpression mice compared to wild-type (WT) mice. Even though PRDM16 overexpression had no effect on increasing muscle weight, it significantly improved the forelimbs function with longer brake, stance and stride time, larger stride length and paw area in mice after RCT. Additionally, PRDM16 overexpression mice showed no difference in amount of fibrosis when compared to WT mice, however, they had significantly reduced area of fatty infiltration. These mice also exhibited abundant MHC-IIx fiber percentage in supraspinatus muscle after TTDN.
CONCLUSIONS: Overexpression of PRDM16 significantly improved muscle function and reduced fatty infiltration after rotator cuff tears. Promoting BAT activity is beneficial in improving rotator cuff muscle quality and shoulder function after RCT.

Chronic rotator cuff (RC) injuries can lead to a degenerative microenvironment that favors chronic inflammation, fibrosis, and fatty infiltration. Recovery of muscle structure and function will ultimately require a complex network of muscle resident cells, including satellite cells, fibro-adipogenic progenitors (FAPs), and immune cells. Recent work suggests that signaling from adipose tissue and progenitors could modulate regeneration and recovery of function, particularly promyogenic signaling from brown or beige adipose (BAT). In this study, we sought to identify cellular targets of BAT signaling during muscle regeneration using a RC BAT transplantation mouse model. Cardiotoxin injured supraspinatus muscle had improved mass at 7 days postsurgery (dps) when transplanted with exogeneous BAT. Transcriptional analysis revealed transplanted BAT modulates FAP signaling early in regeneration likely via crosstalk with immune cells. However, this conferred no long-term benefit as muscle mass and function were not improved at 28 dps. To eliminate the confounding effects of endogenous BAT, we transplanted BAT in the \"BAT-free\" uncoupling protein-1 diphtheria toxin fragment A (UCP1-DTA) mouse and here found improved muscle contractile function, but not mass at 28 dps. Interestingly, the transplanted BAT increased fatty infiltration in all experimental groups, implying modulation of FAP adipogenesis during regeneration. Thus, we conclude that transplanted BAT modulates FAP signaling early in regeneration, but does not grant long-term benefits.

BACKGROUND: Lotus (Nelumbo nucifera) leaf has been described to have anti-obesity activity, but the role of white fat \'browning\' or \'beiging\' in its beneficial metabolic actions remains unclear. Here, 3T3-L1 cells and high-fat-diet (HFD)-fed mice were used to evaluate the effects of miquelianin-rich lotus leaf extract (LLE) on white-to-beige fat conversion and its regulatory mechanisms.
RESULTS: Treatment with LLE increased mitochondrial abundance, mitochondrial membrane potential and NAD+ /NADH ratio in 3T3-L1 cells, suggesting its potential in promoting mitochondrial activity. qPCR and/or western blotting analysis confirmed that LLE induced the expression of beige fat-enriched gene signatures (e.g. Sirt1, Cidea, Dio2, Prdm16, Ucp1, Cd40, Cd137, Cited1) and mitochondrial biogenesis-related markers (e.g. Nrf1, Cox2, Cox7a, Tfam) in 3T3-L1 cells and inguinal white adipose tissue of HFD-fed mice. Furthermore, we found that LLE treatment inhibited mitochondrial fission protein DRP1 and blocked mitophagy markers such as PINK1, PARKIN, BECLIN1 and LC-3B. Chemical inhibition experiments revealed that AMPK/DRP1 signaling was required for LLE-induced beige fat formation via suppressing PINK1/PARKIN/mitophagy.
CONCLUSIONS: Our data reveal a novel mechanism underlying the anti-obesity effect of LLE, namely the induction of white fat beiging via AMPK/DRP1/mitophagy signaling. © 2023 Society of Chemical Industry.

Both the baseline amount of brown adipose tissue (BAT) and the capacity to stimulate browning of white adipose tissue (WAT) may provide a protective effect to the patient in a critical care setting. Critical illness is associated with reduced mitochondrial volume and function resulting in the increased production of reactive oxygen species, greater demand for adenosine triphosphate, a switch to uncoupled fat metabolism, and hibernation of the organelle, which all contribute to multiple organ failure. Increasing insulin resistance, decreasing fatty acid oxidation, and dependence on carbohydrate metabolism result. Browning of WAT may oppose many of these adverse effects. The presence of BAT and the changes associated with browning may help dissipate oxidative stress, increase consumption and utilization of metabolites, and reduce pro-inflammatory actions. The number of mitochondria increases, and there is greater infiltration of macrophages into adipose tissue. A shift occurs in macrophage expression from the M1 to M2 phenotype, an effect which further dampens inflammation, increases insulin sensitivity, and improves tissue healing and remodeling. Any benefit from these responses may be lost in the disease states of chronic hypermetabolism (such as burns or cancer cachexia) in which the persistence of these physiologic effects may become detrimental, contributing to excessive weight loss, adipose wasting, and loss of lean body mass. This paper discusses the plasticity of adipose tissue and whether shifts in its physiology provide clinical advantages in the intensive care unit.

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The main purpose of the present study was to investigate the effect of miquelianin (quercetin 3-O-glucuronide, Q3G), one of the main flavonoids in the Folium Nelumbinis extract (FNE), on beige adipocyte formation and its underlying mechanisms. In 3T3-L1 adipocytes Q3G (12.8%)-rich FNE treatment upregulated beige-related markers such as SIRT1, COX2, PGC-1α, TFAM, and UCP1. Furthermore, Q3G enhanced mitochondrial biosynthesis and inhibited mitophagy by downregulating the expression of PINK1, PARKIN, BECLIN1 and LC-3B in 3T3-L1 cells. Moreover, in high-fat-diet (HFD)-fed mice, Q3G markedly inhibited body weight gain, reduced blood glucose/lipid levels, reduced white adipose tissues (WAT) and mitigated hepatic steatosis. Meanwhile, the induced beiging accompanied by suppressed mitophagy was also demonstrated in inguinal WAT (iWAT). Chemical intervention of AMPK activity with Compound C (Com C) and Acadesine (AICAR) revealed that AMPK/DRP1 signaling was involved in Q3G-mediated mitophagy and the beiging process. Importantly, 16S rRNA sequencing analysis showed that Q3G beneficially reshaped gut microbiota structure, specifically inhibiting unclassified_Lachnospiraceae, Faecalibaculum, Roseburia and Colidextribacter while increasing Bacteroides, Akkermansia and Mucispirillum, which may potentially facilitate WAT beiging. Collectively, our findings provide a novel biological function for Folium Nelumbinis and Q3G in the fight against obesity through activating the energy-dissipating capacity of beige fat.

N6-methyladenosine (m6 A) modification has been implicated in the progression of obesity and metabolic diseases. However, its impact on beige fat biology is not well understood. Here, via m6 A-sequencing and RNA-sequencing, this work reports that upon beige adipocytes activation, glycolytic genes undergo major events of m6 A modification and transcriptional activation. Genetic ablation of m6 A writer Mettl3 in fat tissues reveals that Mettl3 deficiency in mature beige adipocytes leads to suppressed glycolytic capability and thermogenesis, as well as reduced preadipocytes proliferation via glycolytic product lactate. In addition, specific modulation of Mettl3 in beige fat via AAV delivery demonstrates consistently Mettl3\'s role in glucose metabolism, thermogenesis, and beige fat hyperplasia. Mechanistically, Mettl3 and m6 A reader Igf2bp2 control mRNA stability of key glycolytic genes in beige adipocytes. Overall, these findings highlight the significance of m6 A on fat biology and systemic energy homeostasis.

Cold stimuli trigger the conversion of white adipose tissue into beige adipose tissue, which is capable of non-shivering thermogenesis. However, what process drives this activation of thermogenesis in beige fat is not well understood. Here, we examine the ER protein NNAT as a regulator of thermogenesis in adipose tissue.
We investigated the regulation of adipose tissue NNAT expression in response to changes in ambient temperature. We also evaluated the functional role of NNAT in thermogenic regulation using Nnat null mice and primary adipocytes that lack or overexpress NNAT.
Cold exposure or treatment with a β3-adrenergic agonist reduces the expression of adipose tissue NNAT in mice. Genetic disruption of Nnat in mice enhances inguinal adipose tissue thermogenesis. Nnat null mice exhibit improved cold tolerance both in the presence and absence of UCP1. Gain-of-function studies indicate that ectopic expression of Nnat abolishes adrenergic receptor-mediated respiration in beige adipocytes. NNAT physically interacts with the ER Ca2+-ATPase (SERCA) in adipocytes and inhibits its activity, impairing Ca2+ transport and heat dissipation. We further demonstrate that NHLRC1, an E3 ubiquitin protein ligase implicated in proteasomal degradation of NNAT, is induced by cold exposure or β3-adrenergic stimulation, thus providing regulatory control at the protein level. This serves to link cold stimuli to NNAT degradation in adipose tissue, which in turn leads to enhanced SERCA activity.
Our study implicates NNAT in the regulation of adipocyte thermogenesis.

Brown and beige fat protect against cold environments and obesity by catabolizing stored energy to generate heat. This process is achieved by controlling thermogenesis-related gene expression and the development of brown/beige fat through the induction of transcription factors, most notably PPARγ. However, the cofactors that induce the expression of thermogenic genes with PPARγ are still not well understood. In this study, we explored the role of SOX4 in adaptive thermogenesis and its relationship with PPARγ. Methods: Whole transcriptome deep sequencing (RNA-seq) analysis of inguinal subcutaneous white adipose tissue (iWAT) after cold stimulation was performed to identify genes with differential expression in mice. Indirect calorimetry detected oxygen consumption rate and heat generation. mRNA levels were analyzed by qPCR assays. Proteins were detected by immunoblotting and immunofluorescence. Interaction of proteins was detected by endogenous and exogenous Co-IP. ChIP-qPCR, FAIRE assay and luciferase reporter assays were used to investigate transcriptional regulation. Results: SOX4 was identified as the main transcriptional effector of thermogenesis. Mice with either adipocyte-specific or UCP1+ cells deletion of SOX4 exhibited significant cold intolerance, decreased energy expenditure, and beige adipocyte formation, which was attributed to decreased thermogenic gene expression. In addition, these mice developed obesity on a high-fat diet, with severe hepatic steatosis, insulin resistance, and inflammation. At the cell level, loss of SOX4 from preadipocytes inhibited the development of beige adipocytes, and loss of SOX4 from mature beige adipocytes reduced the expression of thermogenesis-related genes and energy metabolism. Mechanistically, SOX4 stimulated the transcriptional activity of Ucp1 by binding to PPARγ and activating its transcriptional function. These actions of SOX4 were, at least partly, mediated by recruiting PRDM16 to PPARγ, thus forming a transcriptional complex to elevate the expression of thermogenic genes. Conclusion: SOX4, as a coactivator of PPARγ, drives the thermogenic gene expression program and thermogenesis of beige fat, promoting energy expenditure. It has important physiological significance in resisting cold and obesity.

Irisin, out-membrane part of fibronectin type III domain-containing 5 protein (FNDC5), was activated by Peroxisome proliferator-activated receptor γ (PPARγ) coactivator-1α (PGC-1α) during physical exercise in skeletal muscle tissues. Most studies have reported that the concentration of irisin is highly associated with health status. For instance, the level of irisin is significantly lower in patients with obesity, osteoporosis/fractures, muscle atrophy, Alzheimer\'s disease, and cardiovascular diseases (CVDs) but higher in patients with cancer. Irisin can bind to its receptor integrin αV/β5 to induce browning of white fat, maintain glucose stability, keep bone homeostasis, and alleviate cardiac injury. However, it is unclear whether it works by directly binding to its receptors to regulate muscle regeneration, promote neurogenesis, keep liver glucose homeostasis, and inhibit cancer development. Supplementation of recombinant irisin or exercise-activated irisin might be a successful strategy to fight obesity, osteoporosis, muscle atrophy, liver injury, and CVDs in one go. Here, we summarize the publications of FNDC5/irisin from PubMed/Medline, Scopus, and Web of Science until March 2022, and we review the role of FNDC5/irisin in physiology and pathology.