PPARγ in Atherosclerotic Endothelial Dysfunction: Regulatory Compounds and PTMs.

The formation of atherosclerotic plaques is one of the main sources of cardiovascular disease. In addition to known risk factors such as dyslipidemia, diabetes, obesity, and hypertension, endothelial dysfunction has been shown to play a key role in the formation and progression of atherosclerosis. Peroxisome proliferator-activated receptor-gamma (PPARγ), a transcription factor belonging to the steroid superfamily, is expressed in the aorta and plays a critical role in protecting endothelial function. It thereby serves as a target for treating both diabetes and atherosclerosis. Although many studies have examined endothelial cell disorders in atherosclerosis, the role of PPARγ in endothelial dysfunction is still not well understood. In this review, we summarize the possible mechanisms of action behind PPARγ regulatory compounds and post-translational modifications (PTMs) of PPARγ in the control of endothelial function. We also explore the potential use of endothelial PPARγ-targeted agents in the prevention and treatment of atherosclerosis.

PPARγ Acetylation Orchestrates Adipose Plasticity and Metabolic Rhythms.

Systemic glucose metabolism and insulin activity oscillate in response to diurnal rhythms and nutrient availability with the necessary involvement of adipose tissue to maintain metabolic homeostasis. However, the adipose-intrinsic regulatory mechanism remains elusive. Here, the dynamics of PPARγ acetylation in adipose tissue are shown to orchestrate metabolic oscillation in daily rhythms. Acetylation of PPARγ displays a diurnal rhythm in young healthy mice, with the peak at zeitgeber time 0 (ZT0) and the trough at ZT18. This rhythmic pattern is deranged in pathological conditions such as obesity, aging, and circadian disruption. The adipocyte-specific acetylation-mimetic mutation of PPARγ K293Q (aKQ) restrains adipose plasticity during calorie restriction and diet-induced obesity, associated with proteolysis of a core circadian component BMAL1. Consistently, the rhythmicity in glucose tolerance and insulin sensitivity is altered in aKQ and the complementary PPARγ deacetylation-mimetic K268R/K293R (2KR) mouse models. Furthermore, the PPARγ acetylation-sensitive downstream target adipsin is revealed as a novel diurnal factor that destabilizes BMAL1 and mediates metabolic rhythms. These findings collectively signify that PPARγ acetylation is a hinge connecting adipose plasticity and metabolic rhythms, the two determinants of metabolic health.

PPARγ (Peroxisome Proliferator-Activated Receptor γ) Deacetylation Suppresses Aging-Associated Atherosclerosis and Hypercholesterolemia.

BACKGROUND: Atherosclerosis is a medical urgency manifesting at the onset of hypercholesterolemia and is associated with aging. Activation of PPARγ (peroxisome proliferator-activated receptor γ) counteracts metabolic dysfunction influenced by aging, and its deacetylation displays an atheroprotective property. Despite the marked increase of PPARγ acetylation during aging, it is unknown whether PPARγ acetylation is a pathogenic contributor to aging-associated atherosclerosis. METHODS: Mice with constitutive deacetylation-mimetic PPARγ mutations on lysine residues K268 and K293 (2KR) in an LDL (low-density lipoprotein)-receptor knockout (Ldlr-/-) background (2KR:Ldlr-/-) were aged for 18 months on a standard laboratory diet to examine the cardiometabolic phenotype, which was confirmed in Western-type diet-fed 2KR:Ldlr+/- mice. Whole-liver RNA-sequencing and in vitro studies in bone marrow-derived macrophages were conducted to decipher the mechanism. RESULTS: In contrast to severe atherosclerosis in WT:Ldlr-/- mice, aged 2KR:Ldlr-/- mice developed little to no plaque, which was underlain by a significantly improved plasma lipid profile, with particular reductions in circulating LDL. The protection from hypercholesterolemia was recapitulated in Western-type diet-fed 2KR:Ldlr+/- mice. Liver RNA-sequencing analysis revealed suppression of liver inflammation rather than changes in cholesterol metabolism. This anti-inflammatory effect of 2KR was attributed to polarized M2 activation of macrophages. Additionally, the upregulation of core circadian component Bmal1 (brain and muscle ARNT-like 1), perceived to be involved in anti-inflammatory immunity, was observed in the liver and bone marrow-derived macrophages. CONCLUSIONS: PPARγ deacetylation in mice prevents the development of aging-associated atherosclerosis and hypercholesterolemia, in association with the anti-inflammatory phenotype of 2KR macrophages.

The Potential Roles of Post-Translational Modifications of PPARγ in Treating Diabetes.

The number of patients with type 2 diabetes mellitus (T2DM), which is mainly characterized by insulin resistance and insulin secretion deficiency, has been soaring in recent years. Accompanied by many other metabolic syndromes, such as cardiovascular diseases, T2DM represents a big challenge to public health and economic development. Peroxisome proliferator-activated receptor γ (PPARγ), a ligand-activated nuclear receptor that is critical in regulating glucose and lipid metabolism, has been developed as a powerful drug target for T2DM, such as thiazolidinediones (TZDs). Despite thiazolidinediones (TZDs), a class of PPARγ agonists, having been proven to be potent insulin sensitizers, their use is restricted in the treatment of diabetes for their adverse effects. Post-translational modifications (PTMs) have shed light on the selective activation of PPARγ, which shows great potential to circumvent TZDs' side effects while maintaining insulin sensitization. In this review, we will focus on the potential effects of PTMs of PPARγ on treating T2DM in terms of phosphorylation, acetylation, ubiquitination, SUMOylation, O-GlcNAcylation, and S-nitrosylation. A better understanding of PTMs of PPARγ will help to design a new generation of safer compounds targeting PPARγ to treat type 2 diabetes.

Adipokines, adiposity, and atherosclerosis.

Characterized by a surplus of whole-body adiposity, obesity is strongly associated with the prognosis of atherosclerosis, a hallmark of coronary artery disease (CAD) and the major contributor to cardiovascular disease (CVD) mortality. Adipose tissue serves a primary role as a lipid-storage organ, secreting cytokines known as adipokines that affect whole-body metabolism, inflammation, and endocrine functions. Emerging evidence suggests that adipokines can play important roles in atherosclerosis development, progression, as well as regression. Here, we review the versatile functions of various adipokines in atherosclerosis and divide these respective functions into three major groups: protective, deteriorative, and undefined. The protective adipokines represented here are adiponectin, fibroblast growth factor 21 (FGF-21), C1q tumor necrosis factor-related protein 9 (CTRP9), and progranulin, while the deteriorative adipokines listed include leptin, chemerin, resistin, Interleukin- 6 (IL-6), and more, with additional adipokines that have unclear roles denoted as undefined adipokines. Comprehensively categorizing adipokines in the context of atherosclerosis can help elucidate the various pathways involved and potentially pave novel therapeutic approaches to treat CVDs.

The Role of Peroxisome Proliferator-Activated Receptor Gamma and Atherosclerosis: Post-translational Modification and Selective Modulators.

Atherosclerosis is the hallmark of cardiovascular disease (CVD) which is a leading cause of death in type 2 diabetes patients, and glycemic control is not beneficial in reducing the potential risk of CVD. Clinically, it was shown that Thiazolidinediones (TZDs), a class of peroxisome proliferator-activated receptor gamma (PPARγ) agonists, are insulin sensitizers with reducing risk of CVD, while the potential adverse effects, such as weight gain, fluid retention, bone loss, and cardiovascular risk, restricts its use in diabetic treatment. PPARγ, a ligand-activated nuclear receptor, has shown to play a crucial role in anti-atherosclerosis by promoting cholesterol efflux, repressing monocytes infiltrating into the vascular intima under endothelial layer, their transformation into macrophages, and inhibiting vascular smooth muscle cells proliferation as well as migration. The selective activation of subsets of PPARγ targets, such as through PPARγ post-translational modification, is thought to improve the safety profile of PPARγ agonists. Here, this review focuses on the significance of PPARγ activity regulation (selective activation and post-translational modification) in the occurrence, development and treatment of atherosclerosis, and further clarifies the value of PPARγ as a safe therapeutic target for anti-atherosclerosis especially in diabetic treatment.

Exercise Promotes Bone Marrow Microenvironment by Inhibiting Adipsin in Diet-Induced Male Obese Mice.

Obesity is a growing global epidemic linked to many diseases, including diabetes, cardiovascular diseases, and musculoskeletal disorders. Exercise can improve bone density and decrease excess bone marrow adipose tissue (BMAT) in obese individuals. However, the mechanism of exercise regulating bone marrow microenvironment remains unclear. This study examines how exercise induces bone marrow remodeling in diet-induced obesity. We employed unbiased RNA-Seq to investigate the effect of exercise on the bone marrow of diet-induced obese male mice. Bone mesenchymal stem cells (BMSCs) were isolated to explore the regulatory effects of exercise in vitro. Our data demonstrated that exercise could slow down the progression of obesity and improve trabecular bone density. RNA-seq data revealed that exercise inhibited secreted phosphoprotein 1 (Spp1), which was shown to mediate bone resorption through mechanosensing mechanisms. Interactome analysis of Spp1 using the HINT database showed that Spp1 interacted with the adipokine adipsin. Moreover, exercise decreased BMAT, which induced osteoclast differentiation and promoted bone loss. Our study reveals that exercise improves the bone marrow microenvironment by at least partially inhibiting the adipsin-Spp1 signaling pathway so as to inhibit the alternative complement system from activating osteoclasts in diet-induced obese mice.

Adipsin promotes bone marrow adiposity by priming mesenchymal stem cells.

Background: Marrow adipose tissue (MAT) has been shown to be vital for regulating metabolism and maintaining skeletal homeostasis in the bone marrow (BM) niche. As a reflection of BM remodeling, MAT is highly responsive to nutrient fluctuations, hormonal changes, and metabolic disturbances such as obesity and diabetes mellitus. Expansion of MAT has also been strongly associated with bone loss in mice and humans. However, the regulation of BM plasticity remains poorly understood, as does the mechanism that links changes in marrow adiposity with bone remodeling. Methods: We studied deletion of Adipsin, and its downstream effector, C3, in C57BL/6 mice as well as the bone-protected PPARγ constitutive deacetylation 2KR mice to assess BM plasticity. The mice were challenged with thiazolidinedione treatment, calorie restriction, or aging to induce bone loss and MAT expansion. Analysis of bone mineral density and marrow adiposity was performed using a µCT scanner and by RNA analysis to assess adipocyte and osteoblast markers. For in vitro studies, primary bone marrow stromal cells were isolated and subjected to osteoblastogenic or adipogenic differentiation or chemical treatment followed by morphological and molecular analyses. Clinical data was obtained from samples of a previous clinical trial of fasting and high-calorie diet in healthy human volunteers. Results: We show that Adipsin is the most upregulated adipokine during MAT expansion in mice and humans in a PPARγ acetylation-dependent manner. Genetic ablation of Adipsin in mice specifically inhibited MAT expansion but not peripheral adipose depots, and improved bone mass during calorie restriction, thiazolidinedione treatment, and aging. These effects were mediated through its downstream effector, complement component C3, to prime common progenitor cells toward adipogenesis rather than osteoblastogenesis through inhibiting Wnt/ß-catenin signaling. Conclusions: Adipsin promotes new adipocyte formation and affects skeletal remodeling in the BM niche. Our study reveals a novel mechanism whereby the BM sustains its own plasticity through paracrine and endocrine actions of a unique adipokine. Funding: This work was supported by the National Institutes of Health T32DK007328 (NA), F31DK124926 (NA), R01DK121140 (JCL), R01AR068970 (BZ), R01AR071463 (BZ), R01DK112943 (LQ), R24DK092759 (CJR), and P01HL087123 (LQ).

Adipsin deficiency does not impact atherosclerosis development in Ldlr-/- mice.

Obesity is a potent risk factor for atherosclerotic morbidity and mortality. Cytokines secreted from adipose tissue, namely, adipokines, have been suggested to be actively involved in atherosclerosis. One of the most abundant adipokines, adipsin, is downregulated in obesity. It catalyzes the rate-limiting step of alternative complement activation, which is one of the three complement pathways potentially involved in inflammation in atherosclerosis. Interestingly, adipsin has been identified as a novel biomarker in human coronary artery disease. However, its role in the development of atherosclerosis remains unexplored. We crossed adipsin-/- mice onto an Ldlr-/- background [double-knockout (DKO) mice] and induced atherogenesis by high-fat and high-cholesterol feeding. Metabolic profiles were systemically characterized, and atherosclerotic plaques were measured at both aortic root and arch regions. Western blotting was conducted to assess adipsin level and complement activity. The DKO mice exhibited similar sizes of atherosclerotic lesions as Ldlr-/- control mice at both the aortic root and arch regions. Accordingly, they displayed comparable metabolic parameters, including body weight, insulin sensitivity, and lipid profiles, along with compensated complement activity. Adipsin deficiency does not impact the development of atherosclerosis in Ldlr-/- mice despite its crucial function in alternative complement activation. Therefore, it is unlikely to play an important role in mediating the risk of atherosclerotic complications in obesity.NEW & NOTEWORTHY Adipsin deficiency does not impact the development of atherosclerosis in Ldlr-/- mice despite its crucial function in alternative complement activation. Therefore, it is unlikely to play an important role in mediating the risk of atherosclerotic complications in obesity.

PPARγ Deacetylation Confers the Antiatherogenic Effect and Improves Endothelial Function in Diabetes Treatment.

Cardiovascular disease (CVD) is the leading cause of death in patients with diabetes, and tight glycemic control fails to reduce the risk of developing CVD. Thiazolidinediones (TZDs), a class of peroxisome proliferator-activated receptor γ (PPARγ) agonists, are potent insulin sensitizers with antiatherogenic properties, but their clinical use is limited by side effects. PPARγ deacetylation on two lysine residues (K268 and K293) induces brown remodeling of white adipose tissue and uncouples the adverse effects of TZDs from insulin sensitization. Here we show that PPARγ deacetylation confers antiatherogenic properties and retains the insulin-sensitizing effects of TZD while circumventing its detriments. We generated mice homozygous with mice with deacetylation-mimetic PPARγ mutations K268R/K293R (2KR) on an LDL-receptor knockout (Ldlr -/- ) background. 2KR:Ldlr -/- mice showed smaller atherosclerotic lesion areas than Ldlr -/- mice, particularly in aortic arches. With rosiglitazone treatment, 2KR:Ldlr -/- mice demonstrated a residual antiatherogenic response and substantial protection against bone loss and fluid retention. The antiatherosclerotic effect of 2KR was attributed to the protection of endothelium, indicated by improved endothelium-dependent vasorelaxation and repressed expression of proatherogenic factors including inducible nitric oxide synthase, interleukin-6, and NADPH oxidase 2. Therefore, manipulating PPARγ acetylation is a promising therapeutic strategy to control risk of CVD in diabetes treatment.

Identification of a natural beige adipose depot in mice.

Beige fat is a potential therapeutic target for obesity and other metabolic diseases due to its inducible brown fat-like functions. Inguinal white adipose tissue (iWAT) can undergo robust brown remodeling with appropriate stimuli and is therefore widely considered as a representative beige fat depot. However, adipose tissues residing in different anatomic depots exhibit a broad range of plasticity, raising the possibility that better beige fat depots with greater plasticity may exist. Here we identified and characterized a novel, naturally-existing beige fat depot, thigh adipose tissue (tAT). Unlike classic WATs, tAT maintains beige fat morphology at room temperature, whereas high-fat diet (HFD) feeding or aging promotes the development of typical WAT features, namely unilocular adipocytes. The brown adipocyte gene expression in tAT is consistently higher than in iWAT under cold exposure, HFD feeding, and rosiglitazone treatment conditions. Our molecular profiling by RNA-Seq revealed up-regulation of energy expenditure pathways and repressed inflammation in tAT relative to eWAT and iWAT. Furthermore, we demonstrated that the master fatty acid oxidation regulator peroxisome proliferator-activated receptor α is dispensable for maintaining and activating the beige character of tAT. Therefore, we have identified tAT as a natural beige adipose depot in mice with a unique molecular profile that does not require peroxisome proliferator-activated receptor α.

Autophagy in Adipocyte Differentiation.

Adipose tissue regulates metabolic homeostasis by acting as an endocrine organ and energy reservoir. Adipose tissue development and functional maintenance are dependent on adipocyte differentiation, in which autophagy plays an important role. It has been shown that autophagy deficiency dampens adipocyte differentiation, compromises adipose tissue development, dysregulates adipocytokine secretion, and even causes sudden death in young animals. Therefore, accurate assessment of autophagy in adipocyte is critical for the study of adipose biology or pathology of metabolic diseases. In this chapter, we described the procedure of autophagy analysis during adipocyte differentiation, and discussed the power of steady-state autophagy protein (e.g., beclin 1, LC3, and p62) levels versus autophagy flux to reflect autophagy activity.

The depot-specific and essential roles of CBP/p300 in regulating adipose plasticity.

Fat remodeling has been extensively explored through protein deacetylation, but not yet acetylation, as a viable therapeutic approach in the management of obesity and related metabolic disorders. Here, we investigated the functions of key acetyltransferases CBP/p300 in adipose remodeling and their physiological effects by generating adipose-specific deletion of CBP (Cbp-AKO), p300 (p300-AKO) and double-knockout (Cbp/p300-AKO) models. We demonstrated that Cbp-AKO exhibited marked brown remodeling of inguinal WAT (iWAT) but not epididymal WAT (eWAT) after cold exposure and that this pattern was exaggerated in diet-induced obesity (DIO). Despite this striking browning phenotype, loss of Cbp was insufficient to impact body weight or glucose tolerance. In contrast, ablation of p300 in adipose tissues had minimal effects on fat remodeling and adiposity. Surprisingly, double-knockout mice (Cbp/p300-AKO) developed severe lipodystrophy along with marked hepatic steatosis, hyperglycemia and hyperlipidemia. Furthermore, we demonstrated that pharmacological inhibition of Cbp and p300 activity suppressed adipogenesis. Collectively, these data suggest that (i) CBP, but not p300, has distinct functions in regulating fat remodeling and that this occurs in a depot-selective manner; (ii) brown remodeling occurs independently of the improvements in glucose metabolism and obesity and (iii) the combined roles of CBP and p300 are indispensable for normal adipose development.

Isolation of Mouse Stromal Vascular Cells for Monolayer Culture.

Positive energy balance contributes to adipose tissue expansion and dysfunction, which accounts largely for obesity and related metabolic disorders. Thermogenic fat can dissipate energy, activation or induction of which may promote energy balance and address the pressing health issues. Recent studies have shown that stromal vascular fraction (SVF) from white adipose tissue (WAT) can develop both white and brown-like adipocyte phenotypes, thus serving as a unique model to study adipogenesis and thermogenesis. Here, we describe a protocol for effective isolation of mouse SVF from WAT, induction of differentiation, and detection of adipogenesis. Success tips for isolation and culture of SVF are also discussed.

Coordinated Upregulation of Mitochondrial Biogenesis and Autophagy in Breast Cancer Cells: The Role of Dynamin Related Protein-1 and Implication for Breast Cancer Treatment.

Overactive mitochondrial fission was shown to promote cell transformation and tumor growth. It remains elusive how mitochondrial quality is regulated in such conditions. Here, we show that upregulation of mitochondrial fission protein, dynamin related protein-1 (Drp1), was accompanied with increased mitochondrial biogenesis markers (PGC1α, NRF1, and Tfam) in breast cancer cells. However, mitochondrial number was reduced, which was associated with lower mitochondrial oxidative capacity in breast cancer cells. This contrast might be owing to enhanced mitochondrial turnover through autophagy, because an increased population of autophagic vacuoles engulfing mitochondria was observed in the cancer cells. Consistently, BNIP3 (a mitochondrial autophagy marker) and autophagic flux were significantly upregulated, indicative of augmented mitochondrial autophagy (mitophagy). The upregulation of Drp1 and BNIP3 was also observed in vivo (human breast carcinomas). Importantly, inhibition of Drp1 significantly suppressed mitochondrial autophagy, metabolic reprogramming, and cancer cell viability. Together, this study reveals coordinated increase of mitochondrial biogenesis and mitophagy in which Drp1 plays a central role regulating breast cancer cell metabolism and survival. Given the emerging evidence of PGC1α contributing to tumor growth, it will be of critical importance to target both mitochondrial biogenesis and mitophagy for effective cancer therapeutics.

FoxO1 interacts with transcription factor EB and differentially regulates mitochondrial uncoupling proteins via autophagy in adipocytes.

Mitochondrial uncoupling proteins (UCPs) are inducible and play an important role in metabolic and redox homeostasis. Recent studies have suggested that FoxO1 controls mitochondrial biogenesis and morphology, but it remains largely unknown how FoxO1 may regulate mitochondrial UCPs. Here we show that FoxO1 interacted with transcription factor EB (Tfeb), a key regulator of autophagosome and lysosome, and mediated the expression of UCP1, UCP2 and UCP3 differentially via autophagy in adipocytes. UCP1 was down-regulated but UCP2 and UCP3 were upregulated during adipocyte differentiation, which was associated with increased Tfeb and autophagy activity. However, inhibition of FoxO1 suppressed Tfeb and autophagy, attenuating UCP2 and UCP3 but increasing UCP1 expression. Pharmacological blockade of autophagy recapitulated the effects of FoxO1 inhibition on UCPs. Chromatin immunoprecipitation assay demonstrated that FoxO1 interacted with Tfeb by directly binding to its promoter, and silencing FoxO1 led to drastic decrease in Tfeb transcript and protein levels. These data provide the first line of evidence that FoxO1 interacts with Tfeb to regulate autophagy and UCP expression in adipocytes. Dysregulation of FoxO1→autophagy→UCP pathway may account for metabolic changes in obesity.

FoxO1 antagonist suppresses autophagy and lipid droplet growth in adipocytes.

Obesity and related metabolic disorders constitute one of the most pressing heath concerns worldwide. Increased adiposity is linked to autophagy upregulation in adipose tissues. However, it is unknown how autophagy is upregulated and contributes to aberrant adiposity. Here we show a FoxO1-autophagy-FSP27 axis that regulates adipogenesis and lipid droplet (LD) growth in adipocytes. Adipocyte differentiation was associated with upregulation of autophagy and fat specific protein 27 (FSP27), a key regulator of adipocyte maturation and expansion by promoting LD formation and growth. However, FoxO1 specific inhibitor AS1842856 potently suppressed autophagy, FSP27 expression, and adipocyte differentiation. In terminally differentiated adipocytes, AS1842856 significantly reduced FSP27 level and LD size, which was recapitulated by autophagy inhibitors (bafilomycin-A1 and leupeptin, BL). Similarly, AS1842856 and BL dampened autophagy activity and FSP27 expression in explant cultures of white adipose tissue. To our knowledge, this is the first study addressing FoxO1 in the regulation of adipose autophagy, shedding light on the mechanism of increased autophagy and adiposity in obese individuals. Given that adipogenesis and adipocyte expansion contribute to aberrant adiposity, targeting the FoxO1-autophagy-FSP27 axis may lead to new anti-obesity options.

Insulin resistance is associated with epigenetic and genetic regulation of mitochondrial DNA in obese humans.

BACKGROUND: Mitochondrial alterations have been observed in subjects with metabolic disorders such as obesity and diabetes. Studies on animal models and cell cultures suggest aberrant glucose and lipid levels, and impaired insulin signaling might lead to mitochondrial changes. However, the molecular mechanism underlying mitochondrial aberrance remains largely unexplored in human subjects. RESULTS: Here we show that the mitochondrial DNA copy number (mtDNAn) was significantly reduced (6.9-fold lower, p < 0.001) in the leukocytes from obese humans (BMI >30). The reduction of mtDNAn was strongly associated with insulin resistance (HOMA-IR: -0.703, p < 0.05; fasting insulin level: -0.015, p < 0.05); by contrast, the correlation between fasting glucose or lipid levels and mtDNAn was not significant. Epigenetic study of the displacement loop (D-loop) region of mitochondrial genome, which controls the replication and transcription of the mitochondrial DNA as well as organization of the mitochondrial nucleoid, revealed a dramatic increase of DNA methylation in obese (5.2-fold higher vs. lean subjects, p < 0.05) and insulin-resistant (4.6-fold higher vs. insulin-sensitive subjects, p < 0.05) individuals. CONCLUSIONS: The reduction of mtDNAn in obese human subjects is associated with insulin resistance and may arise from increased D-loop methylation, suggesting an insulin signaling-epigenetic-genetic axis in mitochondrial regulation.

Targeting FoxO1 with AS1842856 suppresses adipogenesis.

Hyperplasia (i.e., increased adipogenesis) contributes to excess adiposity, the hallmark of obesity that can trigger metabolic complications. As FoxO1 has been implicated in adipogenic regulation, we investigated the kinetics of FoxO1 activation during adipocyte differentiation, and tested the effects of FoxO1 antagonist (AS1842856) on adipogenesis. We found for the first time that the kinetics of FoxO1 activation follows a series of sigmoid curves, and reveals the phases relevant to clonal expansion, cell cycle arrest, and the regulation of PPARγ, adiponectin, and mitochondrial proteins (complexes I and III). In addition, multiple activation-inactivation transitions exist in the stage of terminal differentiation. Importantly, persistent inhibition of FoxO1 with AS1842856 almost completely suppressed adipocyte differentiation, while selective inhibition in specific stages had differential effects on adipogenesis. Our data present a new view of FoxO1 in adipogenic regulation, and suggest AS1842856 can be an anti-obesity agent that warrants further investigation.

Evodiamine activates AMPK and promotes adiponectin multimerization in 3T3-L1 adipocytes.

Adiponectin, an adipokine with insulin-sensitizing effect, is secreted from adipocytes into circulation as high, medium, and low molecular weight (HMW, MMW, and LMW) forms. The HMW adiponectin is more metabolically active and the ratio of HMW adiponectin to total adiponectin directly correlates with insulin sensitivity. Evodiamine is an indole alkaloid found in the traditional Chinese medicinal plant Evodia rutaecarpa. In this study, evodiamine was found to activate AMP-activated protein kinase (AMPK) in both 3T3-L1 adipocytes and 293T cells. Activation of AMPK by evodiamine promoted the assembly of HMW adiponectin and increased the HMW/total ratio of adiponectin in 3T3-L1 adipocytes. The Ca(2+)-dependent PI3K/Akt/CaMKII-signaling pathway was demonstrated to be involved in evodiamine-induced AMPK activation. This study revealed a novel role of this Ca(2+)-mediated signaling pathway in promoting the multimerization of adiponectin.