Asprosin contributes to pathogenesis of obesity by adipocyte mitophagy induction to inhibit white adipose browning in mice.

BACKGROUND: Asprosin (ASP) is a newly discovered adipokine secreted by white adipose tissue (WAT), which can regulate the homeostasis of glucose and lipid metabolism. However, it is not clear whether it can regulate the browning of WAT and mitophagy during the browning process. Accordingly, this study aims to investigate the effects and possible mechanisms of ASP on the browning of WAT and mitophagy in vivo and in vitro. METHODS: In in vivo experiments, some mouse models were used including adipose tissue ASP-specific deficiency (ASP-/-), high fat diet (HFD)-induced obesity and white adipose browning; in in vitro experiments, some cell models were also established and used, including ASP-deficient 3T3-L1 preadipocyte (ASP-/-) and CL-316243 (CL, 1 µM)-induced browning. Based on these models, the browning of WAT and mitophagy were evaluated by morphology, functionality and molecular markers. RESULTS: Our in vivo data show that adipose tissue-specific deletion of ASP contributes to weight loss in mice; supplementation of ASP inhibits the expressions of browning-related proteins including UCP1, PRDM16 and PGC1ɑ during the cold exposure-induced browning, and promotes the expressions of mitophagy-related proteins including PINK1 and Parkin under the conditions of whether normal diet (ND) or HFD. Similarly, our in vitro data also show that the deletion of ASP in 3T3-L1 cells significantly increases the expressions of the browning-related proteins and decreases the expressions of the mitophagy-related proteins. CONCLUSIONS: These data demonstrate that ASP deletion can facilitate the browning and inhibit mitophagy in WAT. The findings will lay an experimental foundation for the development of new drugs targeting ASP and the clinical treatment of metabolic diseases related to obesity.

Acacetin alleviates energy metabolism disorder through promoting white fat browning mediated by AC-cAMP pathway

Acacetin (ACA), a flavone isolated from Chinese traditional medical herbs, has numerous pharmacological activities. However, little is known about the roles in white fat browning and energy metabolism. In the present study, we investigated whether and how ACA would improve energy metabolism in vivo and in vitro. ACA (20 mg/kg) was intraperitoneally injected to the mice with obesity induced by HFD for 14 consecutive days (in vivo); differentiated 3T3-L1 adipocytes were treated with ACA (20 µmol/L and 40 µmol/L) for 24 h (in vitro). The metabolic profile, lipid accumulation, fat-browning and mitochondrial contents, and so on were respectively detected. The results in vivo showed that ACA significantly reduced the body weight and visceral adipose tissue weight, alleviated the energy metabolism disorder, and enhanced the browning-related protein expressions in adipose tissue of rats. Besides, the data in vitro revealed that ACA significantly reduced the lipid accumulation, induced the expressions of the browning-related proteins and cAMP-dependent protein kinase A (PKA), and increased the mitochondrium contents, especially enhanced the energy metabolism of adipocytes; however, treatment with beta-adrenergic receptor blocker (propranolol, Pro) or adenyl cyclase (AC) inhibitor (SQ22536, SQ) abrogated the ACA-mediated effects. The data demonstrate that ACA alleviates the energy metabolism disorder through the pro-browning effects mediated by the AC-cAMP pathway. The findings would provide the experimental foundation for ACA to prevent and treat obesity and related metabolism disorders. (AU)

Acacetin alleviates energy metabolism disorder through promoting white fat browning mediated by AC-cAMP pathway.

Acacetin (ACA), a flavone isolated from Chinese traditional medical herbs, has numerous pharmacological activities. However, little is known about the roles in white fat browning and energy metabolism. In the present study, we investigated whether and how ACA would improve energy metabolism in vivo and in vitro. ACA (20 mg/kg) was intraperitoneally injected to the mice with obesity induced by HFD for 14 consecutive days (in vivo); differentiated 3T3-L1 adipocytes were treated with ACA (20 µmol/L and 40 µmol/L) for 24 h (in vitro). The metabolic profile, lipid accumulation, fat-browning and mitochondrial contents, and so on were respectively detected. The results in vivo showed that ACA significantly reduced the body weight and visceral adipose tissue weight, alleviated the energy metabolism disorder, and enhanced the browning-related protein expressions in adipose tissue of rats. Besides, the data in vitro revealed that ACA significantly reduced the lipid accumulation, induced the expressions of the browning-related proteins and cAMP-dependent protein kinase A (PKA), and increased the mitochondrium contents, especially enhanced the energy metabolism of adipocytes; however, treatment with beta-adrenergic receptor blocker (propranolol, Pro) or adenyl cyclase (AC) inhibitor (SQ22536, SQ) abrogated the ACA-mediated effects. The data demonstrate that ACA alleviates the energy metabolism disorder through the pro-browning effects mediated by the AC-cAMP pathway. The findings would provide the experimental foundation for ACA to prevent and treat obesity and related metabolism disorders.

Asprosin aggravates vascular endothelial dysfunction via disturbing mitochondrial dynamics in obesity models.

OBJECTIVE: The aim of the study was to investigate the contribution of asprosin (ASP), a fasting-induced hormone involved in metabolic disorders, to vascular endothelial dysfunction in obesity models. METHODS: Primary rat thoracic aortic endothelial cells treated with palmitic acid and mice fed with a high-fat diet (HFD) were used as the obesity models. The role and mechanism of ASP in endothelial dysfunction were investigated by the means of morphologic, functional, and genetic analysis. RESULTS: ASP aggravated the endothelial dysfunction induced by either palmitic acid in vitro or an HFD in vivo, characterized as the impairment of endothelium-dependent vasodilation, reduction of nitric oxide levels, elevation of malondialdehyde levels, and inhibition of phosphoinositide 3-kinase-AKT-endothelial nitric oxide synthase signaling. However, adipose conditional knockout of ASP or ASP neutralization significantly alleviated the endothelial dysfunction induced by an HFD. Mechanistically, ASP enhanced mitochondrial fission, and inhibition of the fission through knockdown of dynamin-related protein 1 (a fission-hallmark factor) rescued the endothelial dysfunction and the disturbance to mitochondrial dynamics induced by ASP. CONCLUSIONS: The findings demonstrate that ASP causes and even exacerbates vascular endothelial dysfunction through promoting mitochondrial fission in obesity, suggesting that ASP can act as an early predictive marker of blood vessel dysfunction and become a novel potential therapeutic target for obesity-related cardiovascular diseases.

Angiogenesis-Browning Interplay Mediated by Asprosin-Knockout Contributes to Weight Loss in Mice with Obesity.

Asprosin (ASP) is a recently identified adipokine secreted by white adipose tissue (WAT). It plays important roles in the maintenance of glucose homeostasis in the fasting state and in the occurrence and development of obesity. However, there is no report on whether and how ASP would inhibit angiogenesis and fat browning in the mouse adipose microenvironment. Therefore, the study sought to investigate the effects of ASP-knockout on angiogenesis and fat browning, and to identify the interaction between them in the ASP-knockout mouse adipose microenvironment. In the experiments in vivo, the ASP-knockout alleviated the obesity induced by a high fat diet (HFD) and increased the expressions of the browning-related proteins including uncoupling protein 1 (UCP1), PRD1-BF-1-RIZ1 homologus domain-containing protein-16 (PRDM16) and PPAR gamma coactivator 1 (PGC1-α) and the endothelial cell marker (CD31). In the experiments in vitro, treatment with the conditional medium (CM) from ASP-knockout adipocytes (ASP-/--CM) significantly promoted the proliferation, migration and angiogenesis of vascular endothelial cells, and increased the expressions of vascular endothelial growth factor (VEGF)/vascular endothelial growth factor receptor 2 (VEGFR2) and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/endothelial nitric oxide synthase (eNOS) pathway proteins. In addition, the treatment with CM from endothelial cells (EC-CM) markedly reduced the accumulation of lipid droplets and increased the expressions of the browning-related proteins and the mitochondrial contents. Moreover, the treatment with EC-CM significantly improved the energy metabolism in 3T3-L1 adipocytes. These results highlight that ASP-knockout can promote the browning and angiogenesis of WAT, and the fat browning and angiogenesis can interact in the mouse adipose microenvironment, which contributes to weight loss in the mice with obesity.

Interaction of FOXO1 and SUMOylated PPARγ1 induced by hyperlipidemia and hyperglycemia favors vascular endothelial insulin resistance and dysfunction.

PPARγ1 and FOXO1 are the key transcription factors that regulate insulin sensitivity. We previously found that a small ubiquitin-related modifier of PPARγ1 at K77 (SUMOylation) favored endothelial insulin resistance (IR) induced by high fat/high glucose (HF/HG) administration. However, whether and how the crosstalk between SUMOylated PPARγ1 and FOXO1 would mediate the development of the endothelial IR and dysfunction remains unclear. Here, we emphasize how PPARγ1-K77 SUMOylation would interact with FOXO1 and participate in the development of the endothelial IR and dysfunction. Our results show that the combination of HF/HG and PPARγ1-K77 SUMOylation exhibits a synergistic deteriorative effect on the endothelial IR and dysfunction, presenting decreased NO levels and elevated ET-1 levels, weakened PI3K/Akt/eNOS signaling, and impaired endothelium-dependent vasodilation function. The further researches reveal that PPARγ1-K77 SUMOylation readily interacts with FOXO1, and FOXO1 occupies the PPAR response element (PPRE) which is supposed to be occupied by PPARγ, thus resulting in the decrease of PPARγ1 transcription activity and the mitigation of the PI3K/Akt signaling. Moreover, the mitigation of the PI3K/Akt signaling promotes in turn the accumulation of FOXO1 in the nucleus where FOXO1 interacts with the SUMOylated PPARγ1, thus exerting a positive feedback effect on IR pathogenesis. The findings uncover a novel association between PPARγ1-K77 SUMOylation and FOXO1, which contributes to our understanding of the pathogenesis of endothelial IR and dysfunction and provides novel pharmacological targets for diabetic angiopathy.

Downregulation of osteopontin inhibits browning of white adipose tissues through PI3K-AKT pathway in C57BL / 6 mice.

Brown adipose tissue (BAT) plays important roles in regulating energy homeostasis and combating obesity. Accordingly, increasing the abundance and/or activating BAT would be effective and promising approaches to combat obesity and obesity-relative diseases. Our previous data in vitro have shown that osteopontin (OPN) induces the brown adipogenesis in 3T3-L1 cells via a phosphatidylinositol 3 kinase (PI3K)-AKT pathway. However, it is currently unknown whether OPN exerts such an effect on animals in vivo. Therefore, in the study we sought to investigate the pro-browning effects of OPN and to explore its underlying mechanisms by transfecting with Ad-GFP-aP2-OPN-shRNA to specifically down-regulate the OPN of white adipose tissue (WAT) in mice. Our present results show that downregulation of OPN in WAT exacerbates obesity and inhibits WAT-browning. Moreover, immunohistochemical results also exhibit that the downregulation of OPN significantly diminishes the expression and sub-cellular localization of UCP-1, PRDM16 and PGC-1α. Besides, the western blotting results reveal that the expression levels of PI3K, AKT-pS473 and PPARγ markedly reduce. Consequently, we conclude that the downregulation of OPN inhibits the browning of WAT through inhibiting the expression of PPARγ mediated by the PI3K-AKT pathway. The findings suggest that OPN is involved in regulation of WAT-browning and regulating its expression would become a potential strategy to combat obesity and obesity-relative metabolic diseases.

Sacubitril/Valsartan Reduces Fibrosis and Alleviates High-Salt Diet-Induced HFpEF in Rats.

Previous studies have confirmed the clinical efficacy of sacubitril/valsartan (Sac/Val) for the treatment of heart failure with reduced ejection fraction (HFrEF). However, the role of Sac/Val in heart failure with preserved ejection fraction (HFpEF) remains unclear. Sac/Val is a combination therapeutic medicine comprising sacubitril and valsartan that acts as a first angiotensin receptor blocker and neprilysin inhibitor (angiotensin-receptor neprilysin inhibitor (ARNI)). Here, we investigated the role of Sac/Val in high-salt diet-induced HFpEF coupled with vascular injury as well as the underlying mechanism. Rats were fed with high-salt feed, followed by intragastric administration of Sac/Val (68 mg/kg; i.g.). The results of functional tests revealed that a high-salt diet caused pathological injuries in the heart and vascular endothelium, which were significantly reversed by treatment with Sac/Val. Moreover, Sac/Val significantly decreased the levels of fibrotic factors, including type I collagen and type Ⅲ collagen, thus, reducing the ratio of MMP2/TIMP2 while increasing Smad7 levels. Further investigation suggested that Sac/Val probably reversed the effects of high-salt diet-induced HFpEF by inhibiting the activation of the TGF-ß1/Smad3 signaling pathway. Thus, treatment with Sac/Val effectively alleviated the symptoms of high-salt diet-induced HFpEF, probably by inhibiting fibrosis via the TGF-ß1/Smad3 signaling pathway, supporting the therapeutic potential of Sac/Val for the treatment of HFpEF.

Negative regulation of eNOS-NO signaling by over-SUMOylation of PPARγ contributes to insulin resistance and dysfunction of vascular endothelium in rats.

SUMOylation of peroxisome proliferator-activated receptor gamma (PPAR γ) plays important regulatory role in its transcriptional activity. Our recent studies in vitro found that over-SUMOylation of PPARγ, like high glucose and high fat (HG/HF), induced endothelial insulin resistance (IR). However, whether such an event occurs in rats remains unclear. Therefore, our study aimed at investigating whether PPARγ over-SUMOylation could mimic high sucrose/fat diet (HFD) to induce endothelial IR and dysfunction and explored its underlying mechanisms. Normal chow-fed rats were intravenously infected with adenoviruses carrying the wild type cDNAs encoding PPARγ, SUMO1 and PIAS1 (protein inhibitor of activated STAT1). HFD-fed rats were regarded as a positive control. Body physical and biochemical parameters, glucose tolerance and vessel function were detected. The expression and SUMOylation levels of PPARγ were measured by western blotting and co-immunoprecipitation. Our results showed that like HFD, PPARγ over-SUMOylation induced endothelial IR and dysfunction via a negative regulation of eNOS-NO pathway. More importantly, we found that PPARγ over-SUMOylation induced endogenous SUMOylation cascade and exacerbated endothelial IR and dysfunction.The findings will deepen the understanding on PPARγ SUMOylation-regulating insulin signaling network and offer a potential target for prevention and cure of diabetic vascular complications.

Secreted-Osteopontin Contributes to Brown Adipogenesis In Vitro via a CD44-Dependent Pathway.

Osteopontin (OPN), a secreted glycoprotein, is involved in various pathophysiological processes including immune response, inflammation, tumor formation, and metabolism. OPN exists in 2 forms, secreted-OPN (sOPN) and intracellular-OPN (iOPN). While they might have different biological activities, it remains largely unknown whether sOPN and iOPN induce the differentiation of brown adipocytes. To test this possibility, 3T3-L1 cells were induced by DMI induction with or without recombinant human OPN (rhOPN, 10, 50, 100, 200 µM), respectively. Meanwhile, another batch of 3T3-L1 cells were infected with Ad-GFP-ap2-OPN and followed by DMI differentiation. Subsequently, the infected cells were treated with either anti-CD44 antibody or immunoglobulin G (Ig G). Accumulation of lipid droplets was visualized by Oil red O staining and protein levels were assayed by western blotting analysis. The results showed that sOPN and not rhOPN, notably increased the accumulation of lipid droplets and the expression of brown adipocyte-related genes. Moreover, neutralization of CD44 partially abrogated the effects induced by sOPN. These data demonstrate that sOPN and not rhOPN has the capacity to induce the differentiation of white preadipocytes into brown adipocytes through a CD44-dependent mechanism. The findings might provide a potential target for sOPN to combat obesity.

Sumoylation of PPARγ contributes to vascular endothelium insulin resistance through stabilizing the PPARγ-NcoR complex.

Sumoylation of peroxisome proliferator-activated receptor Î³ (PPARγ) affects its stabilization, sublocalization, and transcriptional activity. However, it remains largely unknown whether PPARγ sumoylation inhibits the transactivation effect, leading to endothelium insulin resistance (IR). To test this possibility, human umbilical vascular endothelial cells (HUVECs) with a 90% confluence were randomly allocated to two batches. One batch was first pretreated with or without vitamin E for 24 hr and the other infected with adenoviruses containing either PIAS1-shRNA (protein inhibitor of activated STAT1-short hairpin RNA) or scramble shRNA. Cells were suffered from high glucose and palmitic acid (PA) exposure for further 48 hr. The levels of PPARγ, p-IKK, IKK, and NcoR (nuclear corepressors) were measured by western blot analysis. The interaction of IKK and PIAS1, as well as the PPARγ sumoylation, were examined by coimmunoprecipitation. The results showed that the exposure of high glucose and PA induced reactive oxygen species (ROS) production and IKK activation in HUVECs, promoting the interaction of IKK and PIAS1 and the sumoylation of PPARγ. However, vitamin E and PIAS1-shRNA partially decreased ROS production and IKK activation induced by high glucose and PA exposure. These data indicate that ROS-IKK-PIAS1 pathway mediates PPARγ sumoylation, leading to endothelium IR via stabilizing PPARγ-NcoR complex. These findings benefit understanding of regulatory networks of insulin signaling, which might provide a potential target to prevent and cure IR-related diseases.

Ameliorative effects of indomethacin at different concentrations on endothelial insulin resistance through two distinct pathways.

Indomethacin (IDMT), a non-selective inhibitor of cycloxygenase-2 (COX-2), plays important roles in anti-inflammation and analgesia and it is commonly used to treat the patients with rheumatic and rheumatoid arthritis. Besides, various literatures reported that IDMT is a synthetic ligand of peroxisome proliferator activated receptor gamma (PPARγ). Rosiglitazone (RSG), an insulin-sensitizer, is also a synthetic ligand and applied clinically to cure the patients with type 2 diabetes mellitus. However, up to date little is known about whether IDMT ameliorates endothelial insulin resistance (IR). Accordingly, the purpose of this study is to investigate the effects of IDMT on endothelial IR and its underlying mechanism. Our present results showed that IDMT improved the endothelial IR induced by high glucose and fat concentration (HG/HF) in a concentration and time-dependent manner. Intriguingly, we further identified that 0.25 mM of IDMT noticeably induced the expression levels of PPARγ, AKT and endothelial nitric oxide synthase (eNOS) but failed to notably reverse the increases in expression levels of COX-2, inhibitory κB kinase (IKK) and tumor necrosis factor alpha (TNFα) induced by HG/HF; whereas 1.0 mM of IDMT exerted opposite effects compared with 0.25 mM of IDMT. Therefore, we conclude that IDMT ameliorates the endothelial IR induced by HG/HF through two distinct pathways, i.e., a lower concentration of IDMT through a PPARγ-AKT-eNOS pathway while a higher concentration mainly via an IKK-COX-2/TNFα pathway. The findings might provide a novel clinical use for IDMT to cure IR-related disorders.

Epigallocatechin-3-gallate suppresses differentiation of adipocytes via regulating the phosphorylation of FOXO1 mediated by PI3K-AKT signaling in 3T3-L1 cells.

Epigallocatechin-3-gallate (EGCG) is a pivotal effective component of green tea. It is known that EGCG has antioxidant activity, anti-angiogenesis, anti-tumor, cardiovascular protection and blood lipid regulation functions. Forkhead box-O1 (FOXO1) is one of the downstream signals of protein kinase B (AKT) and takes part in adipogenesis. The purpose of this study is to investigate the effects of EGCG on adipose differentiation and the likely mechanisms. 3T3-L1 cells were induced by DMI for 2, 4, 6 and 8 days, respectively. During induction, the cells were treated with EGCG (5 µM, 10 µM, 50 µM and 100 µM) or DMSO for the first 2 days. In addition, another batch of 3T3-L1cells were treated with SC-3036 (PI3K activator, 10 µM), or LY294002 (PI3K inhibitor, 10 µM) alone or combined with EGCG (100 µM) for the indicated times. Medium glucose concentration, lipid accumulation, the levels of TNF-α, resistin, adiponectin and leptin and the expression of FOXO1, phosphorylated-FOXO1 (P-FOXO1), PPARγ, fatty acid synthase (FAS) were detected, respectively. The present study demonstrated that EGCG inhibited glucose uptake, lipid accumulation and adipokine secretion in a concentration-dependent manner during adipogenesis, which suggests that EGCG inhibits adipocyte's differentiation, maturation and functions. Moreover, EGCG also down-regulated the expression levels of PPARγ and P-FOXO1. Conversely, the PI3K activator reversed these changes caused by EGCG, suggesting that the inhibitory effects of EGCG may be mediated by PI3K-AKT-FOXO1 pathway to negatively regulate the expression of PPARγ. The findings will provide a solid foundation for EGCG to prevent and cure the obesity-associated diseases.