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Aim: Increasing brown adipocytes activity and inducting browning of white adipocytes are potential therapeutic targets for the treatment of obesity. In the present study, we investigated the effects of Tanshinone 1 (Tan 1), a major compound from Salvia miltiorrhiza Bunge, on the activation of brown adipocytes and browning of white adipocytes in vivo and in vitro., Materials and Methods: Expression of genes associated with brown adipocyte function including thermogenesis, mitochondria biogenesis and fatty acid oxidation was examined in brown adipose tissue (BAT) and white adipose tissue (WAT) of high fat diet (HFD)-fed obese mice administrated with Tan 1 or in immortalized brown adipocytes (iBAs) and 3T3-L1 adipocytes treated with Tan 1. Mitochondria DNA (mtDNA) content, lipolysis and phosphorylated AMP-activated protein kinase (AMPK) were further assessed in Tan 1 treated-iBAs and 3T3-L1 adipocytes., Key Findings: The administration of Tan 1 protected against HFD-induced obesity in mice, which was associated with enhanced expression of brown adipocyte function-related genes in BAT and WAT. Tan 1 treatment also upregulated brown adipocyte function-related genes in iBA and induced beige adipocytes genes in 3T3-L1 adipocytes, resulting in increased mtDNA content and lipolysis. Tan 1 activated AMPK in BAT and WAT of HFD-fed obese mice as well as in iBAs and 3T3-L1 adipocytes. Inhibition of AMPK by compound C prevented Tan 1-induced expression of beige adipocytes genes., Significance: These results indicate that Tan 1 activates brown adipocytes and induces browning of white adipocytes, which may contribute to anti-obesity activity of Tan 1., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Gomisin N (GN) is a lignan derived from Schisandra chinensis. AMP-activated kinase (AMPK) has gained attention as a therapeutic target for the treatment of metabolic syndrome. Previously, we reported that GN activated the AMPK pathway and ameliorated high-fat diet (HFD)-induced hepatic steatosis. In this study, we investigated the anti-diabetic effects of GN in C2C12 myotubes and HFD obese mice. GN enhanced the phosphorylation of AMPK/acetyl-CoA carboxylase (ACC) and Akt. In addition, GN promoted glucose uptake in C2C12 myotubes, which was accompanied by the translocation of glucose transporter 4 (GLUT4) to the plasma membrane. Treatment with compound C, an AMPK inhibitor, suppressed GN-mediated stimulation of glucose uptake. Furthermore, GN increased the expression of mitochondria biogenesis and fatty acid oxidation genes in C2C12 myotubes. In the in vivo study, administration of GN to HFD mice decreased the levels of fasting blood glucose and insulin, and improved glucose tolerance in HFD obese mice. GN administration rescued the decreased phosphorylation of AMPK and Akt and stimulated the expression of mitochondria biogenesis genes in the skeletal muscle of HFD mice. These findings suggested that GN exerted anti-hyperglycemic effects through AMPK activation., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Gomisin N (GN) is a phytochemical derived from Schisandra chinensis. It has been reported to exert a protective effect against hepatic steatosis by attenuating endoplasmic reticulum (ER) stress. However, the detailed mechanism by which GN inhibits hepatic steatosis remains to be elucidated. In this study, we examined whether GN activates AMP-activated protein kinase (AMPK) and exerts therapeutic effects on liver X receptor (LXR)- or palmitic acid (PA)-induced triglyceride (TG) accumulation in HepG2 cells. Furthermore, in vivo protective effects of GN against hepatic steatosis were assessed in high-fat diet (HFD)-induced obese mice. GN stimulated the phosphorylation of AMPK, acetyl-CoA carboxylase (ACC), and sterol regulatory element-binding protein 1c (SREBP1c) in HepG2 cells. It decreased the expression of lipogenesis genes, but increased the expression of fatty acid oxidation genes. Additionally, GN decreased the expression of lipogenesis genes induced by the LXR agonist T0901317 or PA in HepG2 cells, resulting in reduced intracellular TG content. However, preincubation with compound C, an AMPK inhibitor, prevented GN-mediated effects. Administration of GN to HFD-induced obese mice decreased HFD-induced liver weight, hepatic TG accumulation, and cytoplasmic lipid droplet. These findings demonstrate that GN activates the AMPK pathway and ameliorates HFD-induced hepatic steatosis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Previously, we reported that activating transcription factor 3 (ATF3) downregulates peroxisome proliferator activated receptor (PPARγ) gene expression and inhibits adipocyte differentiation in 3T3-L1 cells. Here, we investigated another role of ATF3 on the regulation of PPARγ activity. ATF3 inhibited PPARγ-stimulated transactivation of PPARγ responsive element (PPRE)-containing reporter or GAL4/PPARγ chimeric reporter. Thus, ATF3 effectively repressed rosiglitazone-stimulated expression of adipocyte fatty acid binding protein (aP2), PPARγ target gene, in 3T3-L1 cells. Coimmunoprecipitation and GST pulldown assay demonstrated that ATF3 interacted with PPARγ. Accordingly, ATF3 prevented PPARγ from binding to PPRE on the aP2 promoter. Furthermore, ATF3 suppressed p300-mediated transcriptional coactivation of PPRE-containing reporter. Chromatin immunoprecipitation assay showed that overexpression of ATF3 blocked both binding of PPARγ and recruitment of p300 to PPRE on aP2 promoter induced by rosiglitazone treatment in 3T3-L1 cells. Taken together, these results suggest that ATF3 interacts with PPARγ and represses PPARγ-mediated transactivation through suppression of p300-stimulated coactivation in 3T3-L1 cells, which may play a role in inhibition of adipocyte differentiation., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Activating transcription factor 3 (ATF3) is a stress-adaptive transcription factor that mediates cellular stress response signaling. We previously reported that ATF3 represses CCAAT/enhancer binding protein α (C/EBPα) expression and inhibits 3T3-L1 adipocyte differentiation. In this study, we explored potential role of ATF3 in negatively regulating peroxisome proliferator activated receptor-γ (PPARγ). ATF3 decreased the expression of PPARγ and its target gene in 3T3-L1 adipocytes. ATF3 also repressed the activity of -2.6Kb promoter of mouse PPARγ2. Overexpression of PPARγ significantly prevented the ATF3-mediated inhibition of 3T3-L1 differentiation. Transfection studies with 5' deleted-reporters showed that ATF3 repressed the activity of -2037bp promoter, whereas it did not affect the activity of -1458bp promoter, suggesting that ATF3 responsive element is located between the -2037 and -1458. An electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrated that ATF3 binds to ATF/CRE site (5'-TGACGTTT-3') between -1537 and -1530. Mutation of the ATF/CRE site abrogated ATF3-mediated transrepression of the PPARγ2 promoter. Treatment with thapsigargin, endoplasmic reticulum (ER) stress inducer, increased ATF3 expression, whereas it decreased PPARγ expression. ATF3 knockdown significantly blocked the thapsigargin-mediated downregulation of PPARγ expression. Furthermore, overexpression of PPARγ prevented inhibition of 3T3-L1 differentiation by thapsigargin. Collectively, these results suggest that ATF3-mediated inhibition of PPARγ expression may contribute to inhibition of adipocyte differentiation during cellular stress including ER stress., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Obesity-associated adipose tissue hypoxia plays a pivotal role in insulin resistance via impaired adipocyte dysfunction including mitochondria dysfunction. In this study, we investigated the involvement of hypoxia-inducible ATF3 in adipocyte hypoxia-mediated mitochondrial dysfunction. While HIF-1α and ATF3 were increased in white adipose tissue of high fat diet (HFD) obese mice compared with control lean mice, mitochondria-related genes were significantly reduced. Treatment with hypoxia mimetics CoCl(2) or incubation with 2% O(2) impaired mitochondria function as demonstrated by decreases in ATP production, NADH dehydrogenase activity, mitochondrial membrane potential, and reduced expression of mitochondria-related genes including NRF-1, PGC-1α, COX1 and SOD in 3T3-L1 adipocyte cells. Furthermore, overexpression of ATF3 in 3T3-L1 cells also decreased mitochondria function as well as expression of mitochondria-related genes. ATF3 knockdown in 3T3-L1 cells partly prevented the hypoxia-mediated decrease in mitochondria function and expression of mitochondria-related genes. The mitochondria-related genes were decreased in white adipose tissue of ATF3-overexpressing mice compared with wild-type mice. These results suggest that ATF3 may play a role in adipocyte hypoxia-mediated mitochondrial dysfunction in obesity., (Copyright © 2013 Elsevier Inc. All rights reserved.)

ATF3 is a stress-adaptive gene that regulates proliferation or apoptosis under stress conditions. However, the role of ATF3 is unknown in adipocyte cells. Therefore, in this study, we investigated the functional role of ATF3 in adipocytes. Both lentivirus-mediated overexpression of ATF3 and stably-overexpressed ATF3 inhibited adipocyte differentiation in 3T3-L1 cells, as revealed by decreased lipid staining with oil red staining and reduction in adipogenic genes. Thapsigargin treatment and overexpression of ATF3 decreased C/EBPα transcript and repressed the activity of the 3.6-kb mouse C/EBPα promoter, demonstrating that ATF3 downregulates C/EBPα expression. Transfection studies using mutant constructs containing 5'-deletions in the C/EBPα promoter revealed that a putative ATF/CRE element, GGATGTCA, is located between -1921 and -1914. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrated that ATF3 directly binds to mouse C/EBPα promoter spanning from -1928 to -1907. Both chemical hypoxia-mimetics or physical hypoxia led to reduce the C/EBPα mRNA and repress the promoter activity of the C/EBPα gene, whereas increase ATF3 mRNA, suggesting that ATF3 may contribute to the inhibition of adipocyte differentiation in hypoxia through downregulation of C/EBPα expression. Collectively, these results demonstrate that ATF3 represses the C/EBPα gene, resulting in inhibition of adipocyte differentiation, and thus plays a role in hypoxia-mediated inhibition of adipocyte differentiation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Chronic endoplasmic reticulum (ER) stress leads to β-cell failure via reduction of pancreatic and duodenal homeobox-1 (PDX-1) activity, which contributes to the pathogenesis of type 2 diabetes. However, the exact mechanisms by which ER stress reduces PDX-1 activity in pancreatic β-cells are unclear. Previously, we showed that ATF3 downregulates PDX-1 gene expression in MIN6N8 pancreatic β-cells. Here, we investigated another role of ATF3 on the regulation of PDX-1 activity. ATF3 significantly inhibited PDX-1-stimulated transactivation of reporter plasmid containing promoters for PDX-1 binding element and the PDX-1 target gene glucokinase, which is dependent on C-terminal domain of ATF3. ATF3 interacted with PDX-1, and effectively inhibited p300-mediated transcriptional coactivation of the PBE-containing promoter, whereas C-terminal domain-deleted ATF3 did not inhibit the transcoactivation of p300. ATF3 decreased the interaction of p300 with PDX-1 in MIN6N8 cells coexpressing PDX-1 and ATF3. In addition, chromatin immunoprecipitation analysis demonstrated that both tunicamycin treatment and ATF3 overexpression inhibited the recruitment of p300 to PDX-1 on the insulin promoter in MIN6N8 cells. Taken together, these results suggest that ATF3 inhibits PDX-1-mediated transactivation through the inhibition of p300-stimulated coactivation, which may lead to β-cell dysfunction by ER stress., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The downregulation of PDX-1 expression plays an important role in development of type 2 diabetes. However, the negative regulator of PDX-1 expression is not well known. In this study, we analyzed the mouse PDX-1 promoter to characterize the effects of ATF3 on PDX-1 expression in pancreatic β-cells. Both thapsigargin treatment, an inducer of ER stress, and ATF3 expression decreased PDX-1 expression in pancreatic β-cells, MIN6N8. Furthermore, they also repressed the activity of -4.5 Kb promoter of mouse PDX-1 gene. Transfection studies with 5' deleted-reporters showed that ATF3 repressed the activity of 0.9Kb PDX-1 promoter, whereas it did not affect the activity of 0.7 Kb PDX-1 promoter, suggesting that ATF3 responsive element is located between the -903 and -702. An electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrated that ATF3 binds directly to the promoter region spanning from -759 to -738. Moreover, mutation of the putative ATF/CRE site between -752 and -745 abrogated ATF3-mediated transrepression of the PDX-1 promoter. PDX-1 was decreased in MIN6N8 cells treated with high glucose or high palmitate, whereas ATF3 was increased, indicating that ATF3 plays a role in hyperglycemia or hyperlipidemia-mediated downregulation of PDX-1 expression. Collectively, these results demonstrate that ATF3 represses PDX-1 expression via binding to an ATF3-responsive element in its promoter, which plays an important role in suppression of pancreatic β-cells function., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Mitochondrial dysfunction induces apoptosis of pancreatic β-cells and leads to type 2 diabetes, but the mechanism involved in this process remains unclear. Chronic endoplasmic reticulum (ER) stress plays a role in the apoptosis of pancreatic β-cells; therefore, in current study, we investigated the implication of ER stress in mitochondrial dysfunction-induced β-cells apoptosis. Metabolic stress induced by antimycin or oligomycin was used to impair mitochondrial function in MIN6N8 cells, which are mouse pancreatic β-cells. Impaired mitochondria dysfunction increased ER stress proteins such as p-eIF2α, GRP78 and GRP 94, as well as ER stress-associated apoptotic factor, CHOP, and activated JNK. AMP-activated protein kinase (AMPK) was also activated under mitochondria dysfunction by metabolic stress. However, the inhibition of AMPK by treatment with compound C, inhibitor of AMPK, and overexpression of mutant dominant negative AMPK (AMPKK45R) blocked the induction of ER stress, which was consist-ent with the decreased β-cell apoptosis and increase of insulin content. Furthermore, mitochondrial dysfunction increased the expression of the inducible nitric oxide synthase (iNOS) gene and the production of nitric oxide (NO), but NO production was prevented by compound C and mutant dominant negative AMPK (AMPK-K45R). Moreover, treatment with 1400W, which is an inhibitor of iNOS, prevented ER stress and apoptosis induced by mitochondrial dysfunction. Treatment of MIN6N8 cells with lipid mixture, physiological conditions of impaired mitochondria function, activated AMPK, increased NO production and induced ER stress. Collectively, these data demonstrate that mitochondrial dysfunction activates AMPK, which induces ER stress via NO production, resulting in pancreatic β-cells apoptosis.

Adiponectin is an adipocyte-derived hormone that has antidiabetic and antiatherogenic effects through two membrane receptors, adiponectin receptor 1 (AdipoR1) and adiponectin receptor 2 (AdipoR2). Although it has been reported that the expression of AdipoR1 and AdipoR2 is regulated under physiological and pathophysiological states, their regulation is largely unknown. Previously, we demonstrated that endoplasmic reticulum (ER) stress or obesity-inducible ATF3 negatively regulates the expression of adiponectin and AdipoR2. Here, we investigated the regulation of another adiponectin receptor, AdipoR1 by ATF3, to determine if ATF3 may contribute to impairment of adiponectin signaling by repressing the expression of both adiponectin and adiponectin receptors. We found that treatment with thapsigargin, a stimulator of ATF3 expression as an inducer of ER stress, decreased AdipoR1 expression in insulin-sensitive cells (HepG2, C2C12) and insulin secreting cells (MIN6N8). Furthermore, overexpression of lentivirus carrying-ATF3 decreased AdipoR1 expression in those cells, demonstrating that ATF3 downregulates AdipoR1 expression. Next, we investigated the effects of ATF3 on human AdipoR1 promoter activity and identified an ATF3-responsive region in the promoter. Both thapsigargin treatment and ATF3 expression repressed AdipoR1 promoter activity. Transfection studies using mutant constructs containing 5'-deletions in the human AdipoR1 promoter revealed that putative ATF/CRE site is located between the -248 and -224, TGACGCGG. Chromatin immunoprecipitation assay demonstrated that ATF3 directly binds to human AdipoR1 promoter spanning from -248 to -224. Finally, deletion of the putative ATF/CRE site abrogated ATF3-mediated transrepression of the AdipoR1 promoter. Importantly, ATF3 expression was increased in hyperglycemia or TNF-α-treated C2C12 cells in which AdipoR1 expression was decreased, suggesting that ATF3 may contribute to downregulation of AdipoR1 by hyperglycemia and TNF-α. Collectively, these results demonstrate that ATF3 negatively regulates human AdipoR1 expression via binding to an ATF3-responsive region in the promoter, which plays an important role in attenuation of adiponectin signaling and induction of insulin resistance., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Adiponectin is expressed in adipose tissue by adipogenic transcription factors including PPARgamma, C/EBPalpha, and ADD1/SREBP1c. Because cAMP-response element binding protein (CREB) is also a central transcriptional activator of adipocyte differentiation, we evaluated CREB to determine if it stimulates adiponectin gene expression. To accomplish this, we evaluated the effects of activated CREB on the promoter activity of the mouse adiponectin gene, and identified the cAMP-response element (CRE) in the promoter. The constitutively active form of CREB increased the promoter activity of the mouse adiponectin gene. In addition, transfection studies using 5' serial deleted promoters revealed the presence of a putative CRE located between the -1250 and -1000bp region. Furthermore, an electrophoresis mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) analysis demonstrated that CREB bound to the region between -1022 and -995 in the adiponectin promoter. Insulin-like growth factor (IGF-1), which activate CREB, increased the adiponectin promoter activity. However, this stimulation was prevented by the dominant negative form of CREB (ACREB) and pretreatment with PD098059, indicating that IGF-1 stimulate adiponectin expression through CREB phosphorylation via the ERK pathway. Importantly, the transactivation of adiponectin expression by CREB was inhibited by ATF3. Coimmunoprecipitation and GST pull-down assay revealed that ATF3 bound to CREB and prevented CREB phosphorylation induced during differentiation of 3T3-L1 adipocytes. Collectively, these findings demonstrate that CREB is a positive regulator of mouse adiponectin gene expression in adipocytes, which play an important role in the regulation of adiponectin expression in response to growth factor., (Copyright 2009 Elsevier Inc. All rights reserved.)

Neuronatin (Nnat) is selectively expressed in the neonatal brain and is involved in neuronal differentiation during brain development. However, Nnat also appears to be abundantly expressed in adipose tissue, and is conspicuously elevated in the adipose tissue of obese Zucker diabetic fatty rats compared with control lean Zucker lean control rats shown in our previous report. Here, we examined the expression of Nnat in adipose tissue and demonstrated that the ectopic expression of Nnat mediated by retroviral infection or stable transfection of 3T3-L1 pre-adipocytes stimulated differentiation into mature adipocytes with early induction of adipogenic transcription factors. Moreover, in 3T3-L1 cells overexpressing Nnat, increased intracellular free calcium levels and enhanced phosphorylation of cAMP-response element-binding protein (CREB) were observed, which appears to potentiate CCAAT/enhancer-binding protein (C/EBP)beta, C/EBPdelta, and C/EBPalpha transcriptional activities. Collectively, the data indicate that Nnat enhances CREB phosphorylation through increasing intracellular free calcium levels, which potentiates expression of adipogenic transcription factors resulting in heightened adipocyte differentiation. These findings contribute to a greater fundamental understanding of obesity, a clinically important risk factor in numerous diseases.

Ornithine decarboxylase (ODC) is the rate-limiting enzyme involved in the biosynthesis of polyamines essential for cell growth and differentiation. Aberrant upregulation of ODC, however, is widely believed to be a contributing factor in tumorigenesis. Antizyme is a major regulator of ODC, inhibiting ODC activity through the formation of complexes and facilitating degradation of ODC by the 26S proteasome. Moreover, the antizyme inhibitor (AZI) serves as another factor in regulating ODC, by binding to antizyme and releasing ODC from ODC-antizyme complexes. In our previous report, we observed elevated AZI expression in tumor specimens. Therefore, to evaluate the role of AZI in regulating ODC activity in tumors, we successfully down-regulated AZI expression using RNA interference technology in A549 lung cancer cells expressing high levels of AZI. Two AZI siRNAs, which were capable to generate a hairpin dsRNA loop targeting AZI, could successively decrease the expression of AZI. Using biological assays, antizyme activity increased in AZI-siRNA-transfected cells, and ODC levels and activity were reduced as well. Moreover, silencing AZI expression decreased intracellular polyamine levels, reduced cell proliferation, and prolonged population doubling time. Our results directly demonstrate that downregulation of AZI regulates ODC activity, intracellular polyamine levels, and cell growth through regulating antizyme activity. This study also suggests that highly expressed AZI may be partly responsible for increased ODC activity and cellular transformation.