Artemisinins ameliorate polycystic ovarian syndrome by mediating LONP1-CYP11A1 interaction.

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.

CHCHD10 Modulates Thermogenesis of Adipocytes by Regulating Lipolysis.

Brown and beige adipocytes dissipate energy in a nonshivering 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. We identify CHCHD10 as a novel regulator for adipocyte thermogenesis. CHCHD10 is dramatically upregulated during thermogenic adipocyte activation by PPARγ-PGC1α and positively correlated with UCP1 expression in adipose tissues from humans and mice. We generated adipocyte-specific Chchd10 knockout mice (Chchd10-AKO) and found that depleting CHCHD10 leads to impaired UCP1-dependent thermogenesis and energy expenditure in the fasting state, with no effect in the fed state. Lipolysis in adipocytes is disrupted by CHCHD10 deficiency, while augmented lipolysis through 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 an essential role of CHCHD10 in regulating lipolysis and the thermogenic program in adipocytes.

The protease SENP2 controls hepatic gluconeogenesis by regulating the SUMOylation of the fuel sensor AMPKα.

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 glucagon-induced 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, 5'-AMP-activated protein kinase alpha (AMPKα), which is a negative regulator of gluconeogenesis. SENP2 interacted with and deSUMOylated AMPKα, thereby promoting its ubiquitination and reducing its protein stability. Inhibition of AMPKα 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.

Hepatic Small Ubiquitin-Related Modifier (SUMO)-Specific Protease 2 Controls Systemic Metabolism Through SUMOylation-Dependent Regulation of Liver-Adipose Tissue Crosstalk.

BACKGROUND AND AIMS: NAFLD, characterized by aberrant triglyceride accumulation in liver, affects the metabolic remodeling of hepatic and nonhepatic tissues by secreting altered hepatokines. Small ubiquitin-related modifier (SUMO)-specific protease 2 (SENP2) is responsible for de-SUMOylation of target protein, with broad effects on cell growth, signal transduction, and developmental processes. However, the role of SENP2 in hepatic metabolism remains unclear. APPROACH AND RESULTS: We found that SENP2 was the most dramatically increased SENP in the fatty liver and that its level was modulated by fed/fasted conditions. To define the role of hepatic SENP2 in metabolic regulation, we generated liver-specific SENP2 knockout (Senp2-LKO) mice. Senp2-LKO mice exhibited resistance to high-fat diet-induced hepatic steatosis and obesity. RNA-sequencing analysis showed that Senp2 deficiency up-regulated genes involved in fatty acid oxidation and down-regulated genes in lipogenesis in the liver. Additionally, ablation of hepatic SENP2 activated thermogenesis of adipose tissues. Improved energy homeostasis of both the liver and adipose tissues by SENP2 disruption prompted us to detect the hepatokines, with FGF21 identified as a key factor markedly elevated in Senp2-LKO mice that maintained metabolic homeostasis. Loss of FGF21 obviously reversed the positive effects of SENP2 deficiency on metabolism. Mechanistically, by screening transcriptional factors of FGF21, peroxisome proliferator-activated receptor alpha (PPARα) was defined as the mediator for SENP2 and FGF21. SENP2 interacted with PPARα and deSUMOylated it, thereby promoting ubiquitylation and subsequent degradation of PPARα, which in turn inhibited FGF21 expression and fatty acid oxidation. Consistently, SENP2 overexpression in liver facilitated development of metabolic disorders. CONCLUSIONS: Our finding demonstrated a key role of hepatic SENP2 in governing metabolic balance by regulating liver-adipose tissue crosstalk, linking the SUMOylation process to metabolic regulation.

BMP4 facilitates beige fat biogenesis via regulating adipose tissue macrophages.

Thermogenic beige fat improves metabolism and prevents obesity. Emerging evidence shows that the activation of M2 macrophages stimulates beige adipogenesis, whereas the activation of M1 macrophages, which play a major role in inflammation, impedes beige adipogenesis. Thus, the identification of factors that regulate adipose tissue macrophages (ATMs) will help clarify the mechanism involved in beiging. Here, we found that one of the secreted proteins in adipose tissue, namely, BMP4, alters the ATM profile in subcutaneous adipose tissue by activating M2 and inhibiting M1 macrophages. Mechanistically, the BMP4-stimulated p38/MAPK/STAT6/PI3K-AKT signalling pathway is involved. Meanwhile, BMP4 improved the potency of M2 macrophages to induce beige fat biogenesis. Considering that the overexpression of BMP4 in adipose tissue promotes the beiging of subcutaneous adipose tissue and improves insulin sensitivity, these findings provide evidence that BMP4 acts as an activator of beige fat by targeting immuno-metabolic pathways.

The BMP4-Smad signaling pathway regulates hyperandrogenism development in a female mouse model.

Polycystic ovary syndrome is a common endocrine disorder and a major cause of anovulatory sterility in women at reproductive age. Most patients with polycystic ovary syndrome have hyperandrogenism, caused by excess androgen synthesis. Bone morphogenetic protein 4 (BMP4) is an essential regulator of embryonic development and organ formation, and recent studies have also shown that BMP4 may be involved in female steroidogenesis process. However, the effect of BMP4 on hyperandrogenism remains unknown. Here, using a female mouse model of hyperandrogenism, we found that ovarian BMP4 levels were significantly decreased in hyperandrogenism. Elevated androgens inhibited BMP4 expression via activation of androgen receptors. Moreover, BMP4 treatment suppressed androgen synthesis in theca cells and promoted estrogen production in granulosa cells by regulating the expression of steroidogenic enzymes, including CYP11A, HSD3B2, CYP17A1, and CYP19A1 Consistently, knockdown of BMP4 augmented androgen levels and inhibited estrogen levels. Mechanistically, Smad signaling rather than the p38 MAPK pathway regulated androgen and estrogen formation, thereby mediating the effect of BMP4. Of note, BMP4-transgenic mice were protected against hyperandrogenism. Our observations clarify a vital role of BMP4 in controlling sex hormone levels and offer new insights into intervention for managing hyperandrogenism by targeting the BMP4-Smad signaling pathway.

BMP4 Cross-talks With Estrogen/ERα Signaling to Regulate Adiposity and Glucose Metabolism in Females.

Similar to estrogens, bone morphogenetic protein 4 (BMP4) promotes the accumulation of more metabolically active subcutaneous fat and reduction of visceral fat. However, whether there is a cross-talk between BMP4 and estrogen signaling remained unknown. Herein, we found that BMP4 deficiency in white adipose tissue (WAT) increased the estrogen receptor α (ERα) level and its signaling, which prevented adult female mice from developing high fat diet (HFD)-induced obesity and insulin resistance; estrogens depletion up regulated BMP4 expression to overcome overt adiposity and impaired insulin sensitivity with aging, and failure of BMP4 regulation due to genetic knockout led to more fat gain in aged female mice. This mutual regulation between BMP4 and estrogen/ERα signaling may also happen in adipose tissue of women, since the BMP4 level significantly increased after menopause, and was inversely correlated with body mass index (BMI). These findings suggest a counterbalance between BMP4 and estrogen/ERα signaling in the regulation of adiposity and relative metabolism in females.

Acetylation of Mitochondrial Trifunctional Protein α-Subunit Enhances Its Stability To Promote Fatty Acid Oxidation and Is Decreased in Nonalcoholic Fatty Liver Disease.

Nonalcoholic fatty liver disease (NAFLD) has become the most common liver disease, and decreased fatty acid oxidation is one of the important contributors to NAFLD. Mitochondrial trifunctional protein α-subunit (MTPα) functions as a critical enzyme for fatty acid ß-oxidation, but whether dysregulation of MTPα is pathogenically connected to NAFLD is poorly understood. We show that MTPα is acetylated at lysine residues 350, 383, and 406 (MTPα-3K), which promotes its protein stability by antagonizing its ubiquitylation on the same three lysines (MTPα-3K) and blocking its subsequent degradation. Sirtuin 4 (SIRT4) has been identified as the deacetylase, deacetylating and destabilizing MTPα. Replacement of MTPα-3K with either MTPα-3KR or MTPα-3KQ inhibits cellular lipid accumulation both in free fatty acid (FFA)-treated alpha mouse liver 12 (AML12) cells and primary hepatocytes and in the livers of high-fat/high-sucrose (HF/HS) diet-fed mice. Moreover, knockdown of SIRT4 could phenocopy the effects of MTPα-3K mutant expression in mouse livers, and MTPα-3K mutants more efficiently attenuate SIRT4-mediated hepatic steatosis in HF/HS diet-fed mice. Importantly, acetylation of both MTPα and MTPα-3K is decreased while SIRT4 is increased in the livers of mice and humans with NAFLD. Our study reveals a novel mechanism of MTPα regulation by acetylation and ubiquitylation and a direct functional link of this regulation to NAFLD.

BMP4 mediates the interplay between adipogenesis and angiogenesis during expansion of subcutaneous white adipose tissue.

The expansion of subcutaneous (SC) white adipose tissue (WAT) has beneficial effects on metabolic health. Our previous work showed an increased number of bone morphogenetic protein 4 (BMP4)-activated beige adipocytes in SC WAT, indicating a potential role of BMP4 in adipocyte recruitment. It was also demonstrated that BMP4 committed multipotent mesodermal C3H10T1/2 stem cells to the adipocyte lineage ex vivo However, the mechanism by which BMP4 regulates adipogenesis in vivo has not been clarified. In this study, we found that BMP4 stimulated de novo adipogenesis in SC WAT concomitant with enhanced blood vessel formation, thus promoting adipose tissue angiogenesis. Platelet-derived growth factor receptor-ß-positive (PDGFRß(+)) multipotent stem cells within the neoangiogenic vessels were found to be adipocyte progenitors. Moreover, BMP4 downregulated PDGFRß by stimulating the lysosome-dependent degradation, which efficiently initiated adipogenic differentiation. These results suggest how BMP4 regulates adipocyte recruitment in SC WAT, and thus promote its beneficial metabolic effects.

Protein Inhibitor of Activated STAT 1 (PIAS1) Protects Against Obesity-Induced Insulin Resistance by Inhibiting Inflammation Cascade in Adipose Tissue.

Obesity is associated with chronic low-level inflammation, especially in fat tissues, which contributes to insulin resistance and type 2 diabetes mellitus (T2DM). Protein inhibitor of activated STAT 1 (PIAS1) modulates a variety of cellular processes such as cell proliferation and DNA damage responses. Particularly, PIAS1 functions in the innate immune system and is a key regulator of the inflammation cascade. However, whether PIAS1 is involved in the regulation of insulin sensitivity remains unknown. Here, we demonstrated that PIAS1 expression in white adipose tissue (WAT) was downregulated by c-Jun N-terminal kinase in prediabetic mice models. Overexpression of PIAS1 in inguinal WAT of prediabetic mice significantly improved systemic insulin sensitivity, whereas knockdown of PIAS1 in wild-type mice led to insulin resistance. Mechanistically, PIAS1 inhibited the activation of stress-induced kinases and the expression of nuclear factor-κB target genes in adipocytes, mainly including proinflammatory and chemotactic factors. In doing so, PIAS1 inhibited macrophage infiltration in adipose tissue, thus suppressing amplification of the inflammation cascade, which in turn improved insulin sensitivity. These results were further verified in a fat transplantation model. Our findings shed light on the critical role of PIAS1 in controlling insulin sensitivity and suggest a therapeutic potential of PIAS1 in T2DM.

Upregulation of miR-125b by estrogen protects against non-alcoholic fatty liver in female mice.

BACKGROUND & AIMS: Due to the protective effect of estrogen against hepatic fat accumulation, the prevalence of non-alcoholic fatty liver disease (NAFLD) in premenopausal women is lower than that in men at the same age and in postmenopausal women. Our study was to further elucidate an underlying mechanism by which estrogen prevents NAFLD from miRNA perspective in female mice. METHODS: miRNA expression was evaluated by TaqMan miRNA assay. Luciferase and ChIP assay were done to validate regulation of miR-125b by estrogen via estrogen receptor alpha (ERα). Nile red and Oil red O staining were used to check lipid content. Overexpressing or inhibiting the physiological role of miR-125b in the liver of mice through injecting adenovirus were used to identify the function of miR-125b in vivo. RESULTS: miR-125b expression was activated by estrogen via ERα in vitro and in vivo. miR-125b inhibited lipid accumulation both in HepG2 cells and primary mouse hepatocytes. Consistently, ovariectomized or liver-specific ERα knockdown mice treated with miR-125b overexpressing adenoviruses were resistant to hepatic steatosis induced by high-fat diet, due to decreased fatty acid uptake and synthesis and decreased triglyceride synthesis. Conversely, inhibiting the physiological role of miR-125b with a sponge decoy slightly promoted liver steatosis with a high-fat diet. Notably, we provided evidence showing that fatty acid synthase was a functional target of miR-125b. CONCLUSION: Our findings identify a novel mechanism by which estrogen protects against hepatic steatosis in female mice via upregulating miR-125b expression.

Transactivation of Atg4b by C/EBPß promotes autophagy to facilitate adipogenesis.

Autophagy is a highly conserved self-digestion pathway involved in various physiological and pathophysiological processes. Recent studies have implicated a pivotal role of autophagy in adipocyte differentiation, but the molecular mechanism for its role and how it is regulated during this process are not clear. Here, we show that CCAAT /enhancer-binding protein ß (C/EBPß), an important adipogenic factor, is required for the activation of autophagy during 3T3-L1 adipocyte differentiation. An autophagy-related gene, Atg4b, is identified as a de novo target gene of C/EBPß and is shown to play an important role in 3T3-L1 adipocyte differentiation. Furthermore, autophagy is required for the degradation of Klf2 and Klf3, two negative regulators of adipocyte differentiation, which is mediated by the adaptor protein p62/SQSTM1. Importantly, the regulation of autophagy by C/EBPß and the role of autophagy in Klf2/3 degradation and in adipogenesis are further confirmed in mouse models. Our data describe a novel function of C/EBPß in regulating autophagy and reveal the mechanism of autophagy during adipocyte differentiation. These new insights into the molecular mechanism of adipose tissue development provide a functional pathway with therapeutic potential against obesity and its related metabolic disorders.

Down-regulation of type I Runx2 mediated by dexamethasone is required for 3T3-L1 adipogenesis.

Runx2, a runt-related transcriptional factor family member, is involved in the regulation of osteoblast differentiation. Interestingly, it is abundant in growth-arrested 3T3-L1 preadipocytes and was dramatically down-regulated during adipocyte differentiation. Knockdown of Runx2 expression promoted 3T3-L1 adipocyte differentiation, whereas overexpression inhibited adipocyte differentiation and promoted the trans-differentiation of 3T3-L1 preadipocytes to bone cells. Runx2 was down-regulated specifically by dexamethasone (DEX). Only type I Runx2 was expressed in 3T3-L1 preadipocytes. Using luciferase assay and chromatin immunoprecipitation-quantitative PCR analysis, it was found that DEX repressed this type of Runx2 at the transcriptional level through direct binding of the glucocorticoid receptor (GR) to a GR-binding element in the Runx2 P2 promoter. Further studies indicated that GR recruited histone deacetylase 1 to the Runx2 P2 promoter which then mediated the deacetylation of histone H4 and down-regulated Runx2 expression. Runx2 might play its repressive role through the induction of p27 expression, which blocked 3T3-L1 adipocyte differentiation by inhibiting mitotic clonal expansion. Taken together, we identified Runx2 as a new downstream target of DEX and explored a new pathway between DEX, Runx2, and p27 which contributed to the mechanism of the 3T3-L1 adipocyte differentiation.

Phosphorylation prevents C/EBPß from the calpain-dependent degradation.

CCAAT/enhancer-binding protein (C/EBP) ß plays an important role in proliferation and differentiation of 3T3-L1 preadipocytes. C/EBPß is sequentially phosphorylated during the 3T3-L1 adipocyte differentiation program, first by MAPK/Cyclin A/cdk2 on Thr(188) and subsequently by GSK3ß on Ser(184) or Thr(179). Dual phosphorylation is critical for the gain of DNA binding activity of C/EBPß. In this manuscript, we found that phosphorylation also contributed to the stability of C/EBPß. Both ex vivo and in vitro experiments showed that phosphorylation by MAPK/Cyclin A/cdk2 and GSK3ß protected C/EBPß from µ-calpain-mediated proteolysis, while phosphorylation on Thr(188) by MAPK/Cyclin A/cdk2 contributed more to the stabilization of C/EBPß, Further studies indicated that phosphorylation mimic C/EBPß was insensitive to both calpain accelerator and calpain inhibitor. Thus, phosphorylation might contribute to the stability as well as the gain of DNA binding activity of C/EBPß.

Characterization of adipocyte differentiation from human mesenchymal stem cells in bone marrow.

BACKGROUND: Adipocyte hyperplasia is associated with obesity and arises due to adipogenic differentiation of resident multipotent stem cells in the vascular stroma of adipose tissue and remote stem cells of other organs. The mechanistic characterization of adipocyte differentiation has been researched in murine pre-adipocyte models (i.e. 3T3-L1 and 3T3-F442A), revealing that growth-arrest pre-adipocytes undergo mitotic clonal expansion and that regulation of the differentiation process relies on the sequential expression of three key transcription factors (C/EBPbeta, C/EBPalpha and PPARgamma). However, the mechanisms underlying adipocyte differentiation from multipotent stem cells, particularly human mesenchymal stem cells (hBMSCs), remain poorly understood. This study investigated cell cycle regulation and the roles of C/EBPbeta, C/EBPalpha and PPARgamma during adipocyte differentiation from hBMSCs. RESULTS: Utilising a BrdU incorporation assay and manual cell counting it was demonstrated that induction of adipocyte differentiation in culture resulted in 3T3-L1 pre-adipocytes but not hBMSCs undergoing mitotic clonal expansion. Knock-down and over-expression assays revealed that C/EBPbeta, C/EBPalpha and PPARgamma were required for adipocyte differentiation from hBMSCs. C/EBPbeta and C/EBPalpha individually induced adipocyte differentiation in the presence of inducers; PPARgamma alone initiated adipocyte differentiation but the cells failed to differentiate fully. Therefore, the roles of these transcription factors during human adipocyte differentiation are different from their respective roles in mouse. CONCLUSIONS: The characteristics of hBMSCs during adipogenic differentiation are different from those of murine cells. These findings could be important in elucidating the mechanisms underlying human obesity further.