Cell surface proteins are responsible for many crucial physiological roles, and they are also the major category of drug targets as the majority of therapeutics target membrane proteins on the surface of cells to alter cellular signaling. Despite its great significance, ligand discovery against membrane proteins has posed a great challenge mainly due to the special property of their natural habitat. Here, we design a new chemical proteomic probe OPA-S-S-alkyne that can efficiently and selectively target the lysines exposed on the cell surface and develop a chemical proteomics strategy for global analysis of surface functionality (GASF) in living cells. In total, we quantified 2639 cell surface lysines in Hela cell and several hundred residues with high reactivity were discovered, which represents the largest dataset of surface functional lysine sites to date. We discovered and validated that hyper-reactive lysine residues K382 on tyrosine kinase-like orphan receptor 2 (ROR2) and K285 on Endoglin (ENG/CD105) are at the protein interaction interface in co-crystal structures of protein complexes, emphasizing the broad potential functional consequences of cell surface lysines and GASF strategy is highly desirable for discovering new active and ligandable sites that can be functionally interrogated for drug discovery. Ligand discovery against membrane proteins has been a major challenge, mainly due to the peculiar nature of their natural habitat. Here, the authors designed a new chemical proteomic probe that targets the lysines exposed on the cell surface and developed a chemical proteomic strategy for global analysis of surface functionality in living cells.

ObjectiveBrown adipose tissue (BAT) development and function are essential for maintaining energy balance. However, the key factors that specifically regulate brown adipogenesis require further identification. Here, we demonstrated that the nuclear receptor subfamily 2 group F member 6 (NR2F6) played a pivotal role in brown adipogenesis and energy homeostasis.MethodsWe examined the differentiation of immortalized brown adipocytes and primary brown adipocytes when NR2F6 were deleted, and explored the mechanism through which NR2F6 regulated adipogenesis using ChIP-qPCR in vitro. Male wild type (WT) and Pdgfra-Cre-mediated deletion of Nr2f6 in preadipocytes (NR2F6-PKO) mice were fed with high fat diet (HFD) for 12 weeks, and adiposity, glucose intolerance, insulin resistance and inflammation were assessed.ResultsNR2F6 exhibited abundant expression in BAT, while its expression was minimal in white adipose tissue (WAT). Within BAT, NR2F6 was highly expressed in preadipocytes, experienced a transient increase in the early stage of brown adipocyte differentiation, and significantly decreased in the mature adipocytes. Depletion of NR2F6 in preadipocytes inhibited brown adipogenesis, caused hypertrophy of brown adipocytes, and impaired thermogenic function of BAT, but without affecting WAT development. NR2F6 transcriptionally regulated PPARγ expression to promote adipogenic process in brown adipocytes. Loss of NR2F6 in preadipocytes led to increased susceptibility to diet-induced metabolic disorders.ConclusionsOur findings unveiled NR2F6 as a novel key regulator of brown adipogenesis, potentially opening up new avenues for maintaining metabolic homeostasis by targeting NR2F6.

Polycystic ovary syndrome (PCOS), a prevalent reproductive disorder in women of reproductive age, features androgen excess, ovulatory dysfunction, and polycystic ovaries. Despite its high prevalence, specific pharmacologic intervention for PCOS is challenging. In this study, we identified artemisinins as anti-PCOS agents. Our finding demonstrated the efficacy of artemisinin derivatives in alleviating PCOS symptoms in both rodent models and human patients, curbing hyperandrogenemia through suppression of ovarian androgen synthesis. Artemisinins promoted cytochrome P450 family 11 subfamily A member 1 (CYP11A1) protein degradation to block androgen overproduction. Mechanistically, artemisinins directly targeted lon peptidase 1 (LONP1), enhanced LONP1-CYP11A1 interaction, and facilitated LONP1-catalyzed CYP11A1 degradation. Overexpression of LONP1 replicated the androgen-lowering effect of artemisinins. Our data suggest that artemisinin application is a promising approach for treating PCOS and highlight the crucial role of the LONP1-CYP11A1 interaction in controlling hyperandrogenism and PCOS occurrence.

Obesity and adverse lipid profile leads to female infertility. The cardiometabolic index (CMI) is a promising indicator for predicting obesity-related diseases. The correlation between CMI and female infertility merits further investigation. The data for this study were acquired from the 2013–2020 National Health and Nutrition Examination Survey (NHANES), with 2333 women enrolled. The cardiometabolic index (CMI) of each participant was calculated as the ratio of triglycerides and high-density lipoprotein cholesterol multiplied by waist-to-height ratio. Weighted multivariate logistic regression models were used to assess the independent correlation between the log-transformed CMI and infertility. Subgroup analyses were carried out to assess the reliability of the findings. Interaction tests were employed to determine whether variables affected infertility by interacting with log CMI. A total of 2333 participants aged 18–45 years were enrolled, 274 of whom were infertile. Log CMI of the infertility group was significantly higher than that of the non-infertility group (P < 0.001). After adjustment for potential confounders, women with higher CMI were at an increased risk of infertility (OR = 2.411, 95

Adipose tissue development begins in the fetal period, and continues to expand after birth. Dysregulation of adipose tissue during weaning may predispose individuals to lifelong metabolic disorders. However, the developmental remodeling of adipose tissue during weaning remains largely unexplored. Here we comprehensively compare the changes in mouse subcutaneous white adipose tissue from 7 days after birth to 7 days after weaning using single-cell RNA sequencing along with other molecular and histologic assays. We characterize the developmental trajectory of preadipocytes and indicate the commitment of preadipocytes with beige potential during weaning. Meanwhile, we find immune cells unique to weaning period, whose expression of extracellular matrix proteins implies potential regulation on preadipocyte. Finally, the strongest cell-cell interaction during weaning determined by the TGFβ ligand-receptor pairs is between preadipocytes and endotheliocytes. Our results provide a detailed and unbiased cellular landscape and offer insights into the potential regulation of adipose tissue remodeling during weaning.

S-acylation is a widespread lipidation form in eukaryotes in which various fatty acids can be covalently attached to specific cysteine residues. However, due to the low reactivity of the lipid moieties and lack of specific antibodies, purification of intact S-acylated peptides remains challenging. Here, we developed a pretreatment method for direct separation and global analysis of endogenously intact S-acylated peptides by nanographite fluoride-based solid-phase extraction (nGF-SPE), together with the investigation and optimization of the enrichment procedure as well as the LC-MS/MS analysis process. Consequently, we performed the first global profiling of endogenously intact S-acylated peptides, with 701 S-palmitoylated peptides from HeLa cell lysates in a restricted search. Furthermore, coupling the nGF-SPE method with open search mode, altogether 1119 intact S-acylated peptides were identified with the attached palmitate, palmitoleate, myristate, and octanoate chain, respectively, providing a global insight into the endogenously heterogeneous modification state. Notably, we found and validated that S-palmitoleoylation (C16:1) provided less affinity toward lipid rafts compared with S-palmitoylation (C16:0). This study developed the first straightforward way to characterize endogenously intact S-acylated peptides on a proteome-wide scale, providing the modified residues together with their attached lipid moieties simultaneously, which paves the way for further understanding of protein S-acylation.

Obesity, particularly increased visceral fat, positively correlates with various metabolic challenges, including atherosclerosis, but the mechanism is not fully understood. The aim of this study is to determine the role of visceral-fat-derived exosomes (Exo) in endothelial cells and atherosclerosis. We show that obesity changes the miRNA profile of visceral adipose exosomes in mice. Importantly, exosomal miR-27b-3p efficiently enters into the vascular endothelial cells and activates the NF-κB pathway by downregulating PPARα. Mechanistically, miR-27b-3p binds directly to the CDS region of PPARα mRNA, thereby promoting mRNA degradation and suppressing translation. In ApoE-deficient mice, administration of miR-27b-3p mimic increases inflammation and atherogenesis, while overexpression of PPARα protects against atherosclerosis. Thus, obesity-induced exosomal miR-27b-3p promotes endothelial inflammation and facilitates atherogenesis by PPARα suppression. We reveal an exosomal pathway by which obesity aggravates atherosclerosis and proposed therapeutic strategies for atherosclerosis in people with obesity.

Great progress has been made in identifying positive regulators that activate adipocyte thermogenesis, but negative regulatory signaling of thermogenesis remains poorly understood. Here, we found that cardiotrophin-like cytokine factor 1 (CLCF1) signaling led to loss of brown fat identity, which impaired thermogenic capacity. CLCF1 levels decreased during thermogenic stimulation but were considerably increased in obesity. Adipocyte-specific CLCF1 transgenic (CLCF1-ATG) mice showed impaired energy expenditure and severe cold intolerance. Elevated CLCF1 triggered whitening of brown adipose tissue by suppressing mitochondrial biogenesis. Mechanistically, CLCF1 bound and activated ciliary neurotrophic factor receptor (CNTFR) and augmented signal transducer and activator of transcription 3 (STAT3) signaling. STAT3 transcriptionally inhibited both peroxisome proliferator-activated receptor-γ coactivator (PGC) 1α and 1β, which thereafter restrained mitochondrial biogenesis in adipocytes. Inhibition of CNTFR or STAT3 could diminish the inhibitory effects of CLCF1 on mitochondrial biogenesis and thermogenesis. As a result, CLCF1-TG mice were predisposed to develop metabolic dysfunction even without external metabolic stress. Our findings revealed a brake signal on nonshivering thermogenesis and suggested that targeting this pathway could be used to restore brown fat activity and systemic metabolic homeostasis in obesity.

Aging is a pressing global health issue that is linked to various diseases, such as diabetes and Alzheimer's disease. It is well known that glycation plays a pathological role in the aging process and age-related diseases. Thus, it is of great significance to discover protein glycation at an early stage for monitoring and intervention in the aging process. However, the endogenous age-related early-stage glycated proteome remains insufficiently profiled. To address this research gap, our study focuses on assessing glycated proteomics profiles in the serum of mice. We employ a robust and quantitative strategy previously developed by our team, to analyze endogenous glycated proteome in serum samples of 4 age groups of mice (10 weeks, 16 weeks, 48 weeks and 80 weeks). In total, 2959 endogenous glycated peptides corresponding to 296 serum proteins are identified from 48 runs of serum samples from 16 mice across the four age groups. By comparing these glycated peptides between adjacent age groups, we discover 49 glycated peptides from 35 proteins that show significant upregulation between the 48-week and 80-week age groups. Furthermore, we identify 10 glycated proteins (or protein groups) that are significantly upregulated only between the 48-week and 80-week age groups, including lecithin-cholesterol acyltransferase (LCAT) and apolipoprotein A-II (Apo A-II). These novel findings provide unique signatures for understanding the aging process and age-related diseases. By shedding light on the early-stage glycated proteome, our study contributes valuable insights that may have implications for future interventions and therapeutic approaches.

Energy-dissipating adipocytes have the potential to improve metabolic health. Here, we identify hypoxia-induced gene domain protein-1a (HIGD1A), a mitochondrial inner membrane protein, as a positive regulator of adipose browning. HIGD1A is induced in thermogenic fats by cold exposure. Peroxisome proliferator-activated receptor gamma (PPARγ) transactivates HIGD1A expression synergistically with peroxisome proliferators-activated receptor γ coactivator α (PGC1α). HIGD1A knockdown inhibits adipocyte browning, whereas HIGD1A upregulation promotes the browning process. Mechanistically, HIGD1A deficiency impairs mitochondrial respiration to increase reactive oxygen species (ROS) level. This increases NAD+ consumption for DNA damage repair and curtails the NAD+/NADH ratio, which inhibits sirtuin1 (SIRT1) activity, thereby compromising adipocyte browning. Conversely, overexpression of HIGD1A blunts the above process to promote adaptive thermogenesis. Furthermore, mice with HIGD1A knockdown in inguinal and brown fat have impaired thermogenesis and are prone to diet-induced obesity (DIO). Overexpression of HIGD1A favors adipose tissue browning, ultimately preventing DIO and metabolic disorders. Thus, the mitochondrial protein HIGD1A links SIRT1 activity to adipocyte browning by inhibiting ROS levels.

Uncontrolled gluconeogenesis results in elevated hepatic glucose production in type 2 diabetes (T2D). The small ubiquitin-related modifier (SUMO)-specific protease 2 (SENP2) is known to catalyze deSUMOylation of target proteins, with broad effects on cell growth, signal transduction, and developmental processes. However, the role of SENP2 in hepatic gluconeogenesis and the occurrence of T2D remain unknown. Herein, we established SENP2 hepatic knockout mice and found that SENP2 deficiency could protect against high-fat diet-induced hyperglycemia. Pyruvate-or glucagoninduced elevation in blood glucose was attenuated by disruption of SENP2 expression, whereas overexpression of SENP2 in the liver facilitated high-fat diet-induced hyperglycemia. Using an in vitro assay, we showed that SENP2 regulated hepatic glucose production. Mechanistically, the effects of SENP2 on gluconeogenesis were found to be mediated by the cellular fuel sensor kinase, 50-AMP-activated protein kinase alpha (AMPK alpha), which is a negative regulator of gluconeogenesis. SENP2 interacted with and deSUMOylated AMPK alpha, thereby promoting its ubiquitination and reducing its protein stability. Inhibition of AMPK alpha kinase activity dramatically reversed impaired hepatic gluconeogenesis and reduced blood glucose levels in SENP2-deficient mice. Our study highlights the novel role of hepatic SENP2 in regulating gluconeogenesis and furthers our understanding of the pathogenesis of T2D.

Cysteine dioxygenase 1 (Cdo1) is a key enzyme in taurine synthesis. Here we show that Cdo1 promotes lipolysis in adipose tissue. Adipose-specific knockout of Cdo1 in mice impairs energy expenditure, cold tolerance and lipolysis, exacerbates diet-induced obesity (DIO) and decreases adipose expression of the key lipolytic genes encoding ATGL and HSL, with little effect on adipose taurine levels. White-adipose-specific overexpression of ATGL and HSL blunts the role of adipose Cdo1 deficiency in promoting DIO. Mechanistically, Cdo1 interacts with PPARγ and facilitates the recruitment of Med24, the core subunit of mediator complex, to ATGL and HSL gene promoters, thereby transactivating their expression. Further, mice with transgenic overexpression of Cdo1 show better cold tolerance, ameliorated DIO and higher lipolysis capacity. Thus, we uncover an unexpected and important role of Cdo1 in regulating adipose lipolysis.

脂肪组织可将多余能量以甘油三酯(triglycerides,TG)形式储存,在饥饿状态下可分解TG产生游离脂肪酸(free fatty acids,FFAs)为机体供能.此外,脂肪组织还具有体温调节和器官保护功能,并且越来越多的证据表明,脂肪组织也是一种重要的内分泌组织.脂肪组织分泌的蛋白质物质被称为脂肪细胞因子(adipokine),可通过自分泌、旁分泌和内分泌方式发挥多种生物学功能,例如调节能量摄入和能量消耗,调节糖脂代谢,抗炎和促炎反应.对整体而言,脂肪细胞因子可调节大脑、肝、肌肉、血管系统、心、胰腺和免疫系统等不同靶器官的生物反应.其中,脂肪细胞因子在糖脂代谢中发挥特定的作用,包括:葡萄糖代谢[瘦素(leptin)、脂联素(adiponectin)、抵抗素(resistin)];胰岛素敏感性[瘦素、脂联素、锌-α2-糖蛋白(zinc-α2-glycoprotein,ZAG)];脂肪形成[骨形成蛋白4(bone morphogenetic protein 4,BMP4)]等生物反应过程.但目前对脂肪组织功能障碍与代谢之间机制的理解尚不完善.脂肪组织功能发生紊乱时,脂肪细胞因子的分泌会发生改变,并可能导致一系列与肥胖相关的代谢性疾病的发生.临床前和临床研究表明,激活或抑制特定脂肪细胞因子的信号转导可能是一种适合干预代谢疾病的方法.本文就部分脂肪细胞因子对代谢的调控作用做出综述,以增强对脂肪细胞因子功能的理解.

Brown and beige adipocytes dissipate energy in a non-shivering thermogenesis manner, exerting beneficial effects on metabolic homeostasis. CHCHD10 is a nuclear-encoded mitochondrial protein involved in cristae organization; however, its role in thermogenic adipocytes remains unknown. Herein, we identify CHCHD10 as a novel regulator for adipocyte thermogenesis. CHCHD10 is dramatically upregulated during thermogenic adipocytes activation by PPARγ-PGC1α, and positively correlated with UCP1 expression in the adipose tissues from human and mice. We generate adipocyte-specific Chchd10 knockout mice (Chchd10-AKO) and find that depleting CHCHD10 leads to impaired UCP1-dependent thermogenesis and energy expenditure in the fasting state, with no effect in fed state. Lipolysis in adipocytes is disrupted by CHCHD10 deficiency, while augmented lipolysis via ATGL overexpression recovers adipocyte thermogenesis in Chchd10-AKO mice. Consistently, overexpression of Chchd10 activates thermogenic adipocytes. Mechanistically, CHCHD10 deficiency results in the disorganization of mitochondrial cristae, leading to impairment of oxidative phosphorylation complex assembly in mitochondria, which in turn inhibits ATP generation. Decreased ATP results in downregulation of lipolysis by reducing nascent protein synthesis of ATGL, thereby suppressing adipocyte thermogenesis. As a result, Chchd10-AKO mice are prone to develop high-fat diet-induced metabolic disorders. Together, our findings reveal essential role of CHCHD10 in regulating lipolysis and thermogenic program in adipocytes.

Accumulating evidence from both animal and human studies suggests that activation of beige fat increases cellular energy expenditure, ultimately reducing adiposity. Here, we report the central role of adipocyte-derived lysyl oxidase (Lox) in the formation of thermogenic beige fat. Mice exposed to cold or a β3 agonist showed drastically lower Lox expression in thermogenically activated beige fat. Importantly, inhibition of Lox activity with BAPN stimulated biogenesis of beige fat in inguinal white adipose tissue (iWAT) under housing conditions and potentiated cold-induced adaptive thermogenesis and beiging in both iWAT and epididymal white adipose tissue (eWAT). Notably, white adipocytes with Lox repression undergo transdifferentiation into beige adipocytes which can be suppressed by tumor necrosis factor-α (TNFα) via ERK activation. This work provides new insight into the molecular control to expand beige fat by Lox inhibition and suggest the potential for utilizing inhibitor of Lox to treat the emerging epidemics of obesity and diabetes.

Pluripotent stem cells (PSCs) are promising in regenerative medicine. A major challenge of PSC therapy is the risk of teratoma formation because of the contamination of undifferentiated stem cells. Constitutive promoters or endogenous SOX2 promoters have been used to drive inducible caspase-9 (iCasp9) gene expression but cannot specifically eradicate undifferentiated PSCs. Here, we inserted iCasp9 gene into the endogenous OCT4 locus of human and mouse PSCs without affecting their pluripotency. A chemical inducer of dimerization (CID), AP1903, induced iCasp9 activation, which led to the apoptosis of specific undifferentiated PSCs in vitro and in vivo. Differentiated cell lineages survived because of the silence of the endogenous OCT4 gene. Human and mouse PSCs were controllable when CID was administrated within 2 weeks after PSC injection in immunodeficient mice. However, an interval longer than 2 weeks caused teratoma formation and mouse death because a mass of somatic cells already differentiated from the PSCs. In conclusion, we have developed a specific and efficient PSC suicide system that will be of value in the clinical applications of PSC-based therapy.

Beiging of white adipose tissue (WAT) is capable of adaptive thermogenesis and dissipating energy. The beiging processes have been associated with the increase of anti-inflammatory M2 macrophages, however the function of M2 macrophage on beiging and the underlying mechanism are not fully understood. Here we identified a macrophage cytokine Slit3 by analyzing the transcriptome of M2 macrophages collected with FACS in inguinal WAT (iWAT) of mice after cold exposure. Once released from macrophages, Slit3 bound to the ROBO1 receptor on sympathetic neuron and activated tyrosine hydrolase (TH) through PKA and CaMKⅡ signaling, and thus stimulated norepinephrine (NE) synthesis and release. NE acts on adipocytes and stimulate thermogenesis. Adoptive transfer of Slit3-overexpressing M2 macrophages to iWAT depot acquired local adipocytes with beiging phenotype and enhanced thermogenesis. In addition, mice bearing the myeloid inactivation of Slit3 were cold intolerant and gained more weight due to the lowered metabolic rate. Collectively, we demonstrate Slit3 is a macrophage cytokine and promotes beiging and thermogenesis through intensifying the sympathetic nerve function. As the expanded M2 macrophages are integral cell population in adipose tissue, the macrophage-Slit3-sympathetic neuron-adipocyte axis assures the long-term cold adaption.

Loss of perivascular adipose tissue (PVAT) impairs endothelial function and enhances atherosclerosis. However, the roles of PVAT thermoregulation in vascular inflammation and the development of atherosclerosis remains unclear. Bone morphogenetic protein 4 (BMP4) transforms white adipocyte to beige adipocyte, while promotes a brown-to-white shift in inter-scapular brown adipose tissue (BAT). Here, we found that knockdown of BMP4 in PVAT reduced expression of brown adipocyte-characteristic genes and increased endothelial inflammation in vitro co-culture system. Ablating BMP4 expression either in adipose tissues or specifically in BAT in ApoE−/− mice demonstrated a marked exacerbation of atherosclerotic plaque formation in vivo. We further demonstrated that proinflammatory factors (especially IL-1β) increased in the supernatant of BMP4 knockdown adipocytes. Overexpression of BMP4 in adipose tissues promotes browning of PVAT and protects against atherosclerosis in ApoE−/− mice. These findings uncover an organ crosstalk between PVAT and blood endothelial cells that is engaged in atherosclerosis.

Obesity is one of the most important risk factors for type 2 diabetes (T2DM). The CREBRF missense allele of rs373863828 (p.Arg457Gln) is associated with increased body mass index (BMI), yet reduced risk of T2DM in people with Pacific ancestry. To investigate the functional consequences of the CREBRF variant, we introduced the corresponding human mutation p.Arg457Gln into porcine genome by using a CRISPR/Cas9-mediated homologous recombination (HR)-dependent approach. The CREBRF p.Arg457Gln pig models displayed dramatically increased fat deposition, yet improved sensitivity to insulin. Transcriptome and metabolome analyses of subcutaneous white adipose tissues showed that the CREBRF p.Arg457Gln mutation promoted preadipocyte differentiation, which indicated that obesity was caused by increased number (hyperplasia) rather than size (hypertrophy) of adipocytes. In addition, the oxidative capacity decreased in the adipose tissue of pigs with CREBRF p.Arg457Gln variant. The pre-oxidative metabolite content (4-HNE and MDA) significantly decreased, while activity of antioxidant enzymes (GPX, SOD, and CAT) increased, thereby repressing oxidative metabolism of adipose tissue and reducing level of reactive oxygen species (ROS). The low reactive oxygen species could prevent insulin resistance and reduce risk of obesity-induced type 2 diabetes. This study provides further mechanistic insights into favourable adiposity resulting from CREBRF p.Arg457Gln.

Beiging of white adipose tissue (WAT) is associated with an increase of anti-inflammatory M2-like macrophages in WAT. However, mechanisms through which M2-like macrophages affect beiging are incompletely understood. Here, we show that the macrophage cytokine Slit3 is secreted by adipose tissue macrophages and promotes cold adaptation by stimulating sympathetic innervation and thermogenesis in mice. Analysing the transcriptome of M2-like macrophages in murine inguinal WAT (iWAT) after cold exposure, we identify Slit3 as a secreted cytokine. Slit3 binds to the ROBO1 receptor on sympathetic neurons to stimulate Ca2+/calmodulin-dependent protein kinase II signalling and norepinephrine release, which enhances adipocyte thermogenesis. Adoptive transfer of Slit3-overexpressing M2 macrophages to iWAT promotes beiging and thermogenesis, whereas mice that lack Slit3 in myeloid cells are cold-intolerant and gain more weight. Our findings shed new light on the integral role of M2-like macrophages for adipose tissue homeostasis and uncover the macrophage–Slit3–sympathetic neuron–adipocyte signalling axis as a regulator of long-term cold adaptation. Slit3 is shown to be secreted from M2-like macrophages resident in adipose tissue, where it enables cold adaptation by stimulating norepinephrine release from sympathetic neurons.