Induction Of Fatty Acid Synthase Research Articles

Insulin-Induced AKR1C3 Induces Fatty Acid Synthase in a Model of Human PCOS Adipocytes.

Polycystic ovary syndrome (PCOS) is the most common endocrinopathy in women. In PCOS, insulin resistance and hyperandrogenism could drive the increased risk for cardiometabolic disease. Aldo-keto reductase family 1 member C3 (AKR1C3) is induced by insulin in PCOS adipocytes and is the predominant enzyme for potent androgen formation causing ligand-dependent androgen receptor (AR) activation. AR induces fatty acid synthase (FASN), a central enzyme for de novo lipogenesis. To investigate how insulin signaling induces AKR1C3 to promote lipid overload through induction of FASN, we used differentiated human Simpson-Golabi-Behmel syndrome adipocytes as a model for PCOS adipocytes. Induction of AKR1C3 and FASN was shown to be dependent on phosphoinositide 3-kinase/protein kinase B/ mammalian target of rapamycin/nuclear factor-erythroid 2-related factor 2 using pharmacological and genetic manipulation. FASN induction was shown to be AKR1C3 and AR dependent. Monofunctional AKR1C3 inhibitors, which competitively inhibit AKR1C3, did not block FASN induction, whereas bifunctional inhibitors, which competitively inhibit AKR1C3 and attenuate AR signaling by increasing AR degradation and ubiquitination, did suggesting a nonenzymatic role for AKR1C3 to stabilize AR. AKR1C3 and AR interacted as seen by co-immunoprecipitation, proximity ligation assay, and co-occupancy on FASN locus using chromatin immunoprecipitation-quantitative polymerase chain reaction assays in a ligand-dependent and ligand-independent manner. In the absence of androgens, bifunctional inhibitors prevented lipid droplet formation, whereas monofunctional inhibitors did not. We propose that AKR1C3 has 2 roles in PCOS: to catalyze potent androgen formation in adipocytes promoting hyperandrogenism and to induce FASN by stabilizing AR in the absence of androgens. AKR1C3 may be a therapeutic target for bifunctional inhibitors to reduce cardiometabolic disease in PCOS women.

Insulin Signaling Induces AKR1C3 to Promote Lipid Overload through FASN in SGBS Cells, as a Model of PCOS Adipocytes

Polycystic ovary syndrome (PCOS) is the most common endocrinopathy in women of reproductive age, affecting 8‐13% of women. The characteristic androgen excess (AE) of PCOS is believed to promote comorbidities, such as obesity and type 2 diabetes. AE correlates with insulin resistance (IR) in these women and may be the driving factor for lipid overload. Aldo‐keto reductase family 1 member C3 (AKR1C3) is induced by insulin in PCOS adipocytes and may be central in driving the AE of PCOS. AKR1C3 predominantly catalyzes formation of androgens such as testosterone and 5α‐dihydrotestosterone in peripheral tissues, which are potent agonists for the androgen receptor (AR) and upregulate de novo lipogenesis. Fatty acid synthase (FASN) is induced by androgens and may be responsible for an altered lipid profile in mature adipocytes. Nuclear factor erythroid 2‐related factor 2 (NRF2) is a known transcription factor for AKR1C3 and is regulated by insulin. AKR1C3 overexpression in adipocytes by insulin activation of NRF2 could increase androgen receptor signaling and promote de novo lipogenesis. The resulting lipid overflow could exacerbate insulin resistance through a feedforward mechanism and potentiate the cardio‐metabolic risk seen in PCOS. We hypothesize that insulin signaling induces AKR1C3 to promote lipid overload through FASN in PCOS adipocytes. Using differentiated human Simpson‐Golabi‐Behmel Syndrome (SGBS) adipocytes as a model for PCOS adipocytes, we have confirmed that AKR1C3 is induced >600 fold by insulin. Using pharmacological inhibition, we found that the insulin induction of AKR1C3 and FASN is dependent on PI3K/AKT/mTOR measured by RT‐qPCR and immunoblot. Using NRF2 siRNA and ML‐385, a NRF2 inhibitor, we have determined that AKR1C3 and FASN induction are NRF2 dependent, indicating NRF2 may be one transcription factor that binds to the AKR1C3 promotor region in adipocytes. We also determined that FASN induction is both AKR1C3 and AR dependent using pharmacological inhibition/antagonism and siRNA, where its effect was measured by RT‐qPCR and immunoblot. Pharmacological inhibition with NRF2 or AKR1C3 inhibitors additionally downregulated de novo lipogenesis measured by Oil Red O staining, which is a phenotypic readout for FASN activity. These studies elucidate the signaling mechanism by which AKR1C3 and FASN are induced by insulin in adipocytes. We conclude that upregulation of AKR1C3 may promote lipid overload in PCOS adipocytes by induction of FASN, and that AKR1C3 is a possible therapeutic target to prevent the risk of cardio‐metabolic disease in PCOS women.

Autophagy activation and SREBP-1 induction contribute to fatty acid metabolic reprogramming by leptin in breast cancer cells.

Leptin, a hormone predominantly derived from adipose tissue, is well known to induce growth of breast cancer cells. However, its underlying mechanisms remain unclear. In this study, we examined the role of reprogramming of lipid metabolism and autophagy in leptin-induced growth of breast cancer cells. Herein, leptin induced significant increase in fatty acid oxidation-dependent ATP production in estrogen receptor-positive breast cancer cells. Furthermore, leptin induced both free fatty acid release and intracellular lipid accumulation, indicating a multifaceted effect of leptin in fatty acid metabolism. These findings were further validated in an MCF-7 tumor xenograft mouse model. Importantly, all the aforementioned metabolic effects of leptin were mediated via autophagy activation. In addition, SREBP-1 induction driven by autophagy and fatty acid synthase induction, which is mediated by SREBP-1, plays crucial roles in leptin-stimulated metabolic reprogramming and are required for growth of breast cancer cell, suggesting a pivotal contribution of fatty acid metabolic reprogramming to tumor growth by leptin. Taken together, these results highlighted a crucial role of autophagy in leptin-induced cancer cell-specific metabolism, which is mediated, at least in part, via SREBP-1 induction.

Dietary intake of probiotic kimchi ameliorated IL-6-driven cancer cachexia.

Cancer cachexia is a syndrome accompanying weight loss, skeletal muscle atrophy, and loss of adipose tissue in patients with advanced cancer. Since interleukin-6 (IL-6) is one of core mediators causing cancer cachexia and kimchi can modulate IL-6 response, we hypothesized dietary intake of kimchi can ameliorate cancer cachexia. In this study, we studied preemptive administration of kimchi can ameliorate mouse colon carcinoma cells colon (C26) adenocarcinoma-induced cancer cachexia and explored anti-cachexic mechanisms of kimchi focused on the changes of muscle atrophy, cachexic inflammation, and catabolic catastrophe. As results, dietary intake of kimchi significantly attenuated the development of cancer cachexia, presented with lesser weight loss, higher muscle preservation as well as higher survival from cancer cachexia in mice. Starting from significant inhibition of IL-6 and its signaling, kimchi afforded significant inhibition of muscle specific ubiquitin-proteasome system including inhibition of atrogin-1 and muscle ring finger protein-1 (MuRF-1) with other muscle related genes including mitofusin-2 (Mfn-2) and PGC-1α. Significant inhibition of lipolysis gene such as adipose triglyceride lipase (ATGL) and hormone-sensitive ligase (HSL) accompanied with significant induction of fatty acid synthase (FAS) and sterol response element binding protein 1 (SREBP1) was achieved with kimchi. As gene regulation, IL-6 and their receptor as well as Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) were significantly attenuated with kimchi. In conclusion, dietary intake of cancer preventive kimchi can be an anticipating option to ameliorate cancer cachexia via suppressive action of IL-6 accompanied with decreased muscle atrophy and lipolysis.

Histone modifications in fatty acid synthase modulated by carbohydrate responsive element binding protein are associated with non‑alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) is a manifestation of metabolic syndrome in the liver and is closely associated with diabetes; however, its pathogenesis remains to be elucidated. Carbohydrate responsive element binding protein (ChREBP), the hub of glucolipid metabolism, regulates the induction of fatty acid synthase (FASN), the key enzyme of de novo lipogenesis, by directly binding to carbohydrate response element (ChoRE) in its promoter. Investigations of histone modifications on NAFLD remain in their infancy. In the present study, by using ChIP, the association between histone modifications and FASN transcription was investigated and histone modifications in FASN modulated by ChREBP were measured. It was demonstrated that ChREBP induced FASN ChREBP-ChoRE binding to accelerate the expression of FASN, leading to hepatocellular steatosis by facilitating H3 and H4 acetylation, H3K4 trimethylation and the phosphorylation of H3S10, but inhibiting the trimethylation of H3K9 and H4K20 in FASN promoter regions of HepG2 and L02 cells. It was also found that ChREBP-ChoRE binding of FASN relied on histone acetylation and that the transcriptional activity of ChREBP on FASN is required, based on the premise that histone acetylation causes conformational changes in FASN chromatin. This indicated histone acetylation as a crucial mechanism involved in the transcription of FASN modulated by ChREBP. Consequently, the present study provides further insight into the pathophysiology and a novel therapeutic potential of NAFLD based on epigenetic mechanisms.

Oncogene KRAS activates fatty acid synthase, resulting in specific ERK and lipid signatures associated with lung adenocarcinoma.

KRAS gene mutation causes lung adenocarcinoma. KRAS activation has been associated with altered glucose and glutamine metabolism. Here, we show that KRAS activates lipogenesis, and this activation results in distinct proteomic and lipid signatures. By gene expression analysis, KRAS is shown to be associated with a lipogenesis gene signature and specific induction of fatty acid synthase (FASN). Through desorption electrospray ionization MS imaging (DESI-MSI), specific changes in lipogenesis and specific lipids are identified. By the nanoimmunoassay (NIA), KRAS is found to activate the protein ERK2, whereas ERK1 activation is found in non-KRAS-associated human lung tumors. The inhibition of FASN by cerulenin, a small molecule antibiotic, blocked cellular proliferation of KRAS-associated lung cancer cells. Hence, KRAS is associated with activation of ERK2, induction of FASN, and promotion of lipogenesis. FASN may be a unique target for KRAS-associated lung adenocarcinoma remediation.

Induction of Fatty Acid Synthase is Required for Profibrotic TGFβ Signaling

Alterations in cellular metabolism are known to play critical roles in a variety of normal as well as pathological processes, including (but not limited to) cancer, hepatic steatosis, and cell differentiation. While TGFβ is routinely considered a pleotropic cytokine due to its ability to impact numerous diseases and biological phenotypes, the role of metabolism in profibrotic TGFβ signaling is relatively unexplored/unknown. Fatty Acid Synthase (FASN) is the primary anabolic enzyme responsible for the de novo synthesis of fatty acids. Renewed interest in FASN has been sparked by the recognition of the critical inter‐relation of metabolic dysregulation and tumor progression with the findings that (i) FASN levels are significantly increased in numerous human tumors including breast, prostate, colorectal, and ovarian; and (ii) the efficacy of ErbB receptor inhibitors is enhanced by inhibiting FASN activity. In the current study we show that FASN expression is regulated by TGFβ in multiple murine and human fibroblasts, required for profibrotic TGFβ signaling, and, most importantly, treatment with C75 ((trans‐4‐carboxy‐5‐octyl‐3‐methylenebutyrolactone, a novel cerulenin‐derived semi‐synthetic FASN inhibitor) abrogates pathologic and physiologic changes in a murine model of lung fibrosis.Support or Funding InformationThis work was supported by Public Health Service grants GM‐055816 and GM‐054200 from National Institutes of General Medical Sciences.

Fatty acid remodeling in cellular glycerophospholipids following the activation of human T cells

Changes in fatty acid (FA) and glycerophospholipid (GPL) metabolism associated with cell cycle entry are not fully understood. In this study FA-GPL remodeling was investigated in resting and proliferating primary human T cells. Significant changes were measured in the composition and distribution of FAs in GPLs following receptor activation of human T cells. The FA distribution of proliferating T cells was very similar to that of the human Jurkat T cell line and when the stimulus was removed from proliferating T cells, they stopped proliferating and the FA distribution largely reverted back to that of resting T cells. The cellular content of saturated and monounsaturated FAs was significantly increased in proliferating cells, which was associated with an induction of FA synthase and stearoyl-CoA desaturase-1 gene expression. Additionally, cellular arachidonate was redistributed in GPLs in a distinct pattern that was unlike any other FAs. This redistribution was associated with an induction of CoA-dependent and CoA-independent remodeling. Accordingly, significant changes in the expression of several acyl-CoA synthetases, lysophospholipid acyltransferases, and phospholipase A2 were measured. Overall, these results suggest that metabolic pathways are activated in proliferating T cells that may represent fundamental changes associated with human cell proliferation.

Fatty Acid Synthase in the Endoscopically Normal Rectal Mucosa as Marker for Obesity-related Colorectal Cancer (CRC) Risk: Gender-specific Implications: Presidential Poster

Purpose: The clinical implications of the profound biological differences in CRC between women and men are largely underappreciated. Our group has noted a “gender paradox” for advanced adenomas and CRC in women and men which may have potential ramifications for screening (JAMA 2009;302:1696-7). We have noted that changes in field carcinogenesis (reviewed in Gastroenterology 2011;140:35-41) may also be gender specific (Cancer Prev Res 2010;3:844-51). With regards to risk factor, the role of obesity is becoming increasingly dominant. Emerging data indicates that men appear to be disproportionately impacted versus women (reviewed by Bardou, et al. Gut 2013;62:933-47). Understanding which obese patients are at elevated risk of CRC is critical. We, therefore, evaluated fatty acid synthase (FASN), a protein implicated in the “lipogenic switch” in early colon carcinogenesis. FASN has been implicated in frank CRCs but little is known about the role of FASN in early events such as field carcinogenesis or its gender predilection. Methods: Two biopsies were obtained from endoscopically normal rectum from 103 patients undergoing screening colonoscopy. Subjects were dichotomized into non-obese and obese using a BMI of 30 as a cut point. Biopsies were subjected to real time PCR analysis for FASN and normalized with beta-actin. Results: Obese patients had an induction of FASN when compared to corresponding non-obese subjects (controls 50%, adenoma-harboring 29%, p<0.05). Rectal FASN was highly overexpressed in adenoma patients versus controls and this was a much more dramatic effect in men than women (see Figure). ANCOVA analysis suggested no significant confounding with age, adenoma size, smoking status, etc.FigureConclusion: We report herein, for the first time, alterations in FASN in patients harboring adenomas. This was accentuated in obese patients suggesting a potential molecular link between obesity and colonic neoplasia. Importantly, the effect was more marked in males than females, consonant with the obesity-CRC epidemiology. These insights into the molecular pathogenesis of obesity-related CRC suggest a role of this “lipogenic switch” in gender differences in CRC. Furthermore, this may be a “druggable” target against obesity-induced CRC and also provide a biomarker for determining at risk patients, thus enabling personalizing CRC prevention strategies.

Abstract A44: Suppression of insulin-induced fatty acid synthase gene expression and colon cancer cell proliferation by members of the Krüppel-like family of transcription factors

Abstract Obesity and the associated hyperinsulinemia are major risk factors for colorectal cancer (CRC). The Fatty Acid Synthase (FASN) gene encodes the protein responsible for de novo synthesis of fatty acids. FASN mRNA and protein are insulin-induced in the colonic epithelium, over-expressed early in CRC, and positively associated with tumor cell proliferation, cell survival and chemo-resistance. FASN and its transcriptional regulators may therefore provide mechanistic links between obesity and CRC predisposition and may be exploitable for CRC prevention. Recent attention has focused on several members of the Krüppel-like family (KLF) of transcription factors in pathogenesis of CRC. Published studies have demonstrated that Krüppel-like Factors 4 and 6 (KLF4, KLF6) are important suppressors of CRC development. Recent work from our laboratory and others has implicated KLF9 as a third potential KLF protein that may be a tumor suppressor in colon and rectum. In the present study, we evaluated the possible actions of KLF9 and KLF4 to oppose the inductions by insulin of colon cancer cell FASN expression and proliferation. Addition of insulin to cell medium to mimic hyperinsulinemia (5nM-25nM, compared to vehicle) led to an increase in proliferation (13–25%, P&amp;lt;0.05, evaluated by MTT assay) of the human colorectal cancer cell line HT-29. An increased insulin level also led to induction of FASN mRNA abundance (P=0.018). Gene expression and cell proliferation were examined after transfection of HT-29 cells with siRNAs targeting KLF4, KLF9, and KLF13 (the latter is highly similar in primary sequence to KLF9). Knockdown of KLF9 caused induction of FASN at both the mRNA (P&amp;lt;0.001) and protein (P=0.01) levels. FASN mRNA levels also increased with siRNA knockdown of KLF13 (P=0.001) and tended (P=0.075) to increase with KLF4 knockdown. Hyperinsulinemia elicited a greater increase in proliferation of cells transfected with KLF9 siRNA than of cells transfected with control (scrambled) siRNA. While transfection of KLF9 expression vector produced a trend of reduced proliferation (P=0.082; compared to empty vector), hyperinsulinemia rescued cells from this effect. A hyperinsulinemic environment coupled with siRNA knockdown of KLF9 augmented FASN mRNA expression, compared to KLF9 knockdown alone. Increased insulin concentrations caused a marked induction of KLF4 but not KLF9 or KLF13, mRNA expression. As KLF4 has been shown to inhibit proliferation of colorectal cancer cells, it is possible that induction of KLF4 by insulin may constitute a counter-regulatory mechanism to control their proliferation. In conclusion, our findings implicate KLF family members KLF9, KLF13 and KLF4 in transcriptional repression of proto-oncogenic FASN in CRC cells. They also highlight the potential cancer-suppressive roles of KLF proteins in normo- and hyperinsulinemic conditions, which clearly warrant further investigation. Supported by NIH RO1 CA136493 and grants from the Arkansas Biosciences Institute and Sturgis Foundation. Citation Information: Cancer Prev Res 2011;4(10 Suppl):A44.

Fatty Acid Synthase Modulates Homeostatic Responses to Myocardial Stress

Fatty acid synthase (FAS) promotes energy storage through de novo lipogenesis and participates in signaling by the nuclear receptor PPARα in noncardiac tissues. To determine if de novo lipogenesis is relevant to cardiac physiology, we generated and characterized FAS knockout in the myocardium (FASKard) mice. FASKard mice develop normally, manifest normal resting heart function, and have normal cardiac PPARα signaling as well as fatty acid oxidation. However, they decompensate with stress. Most die within 1 h of transverse aortic constriction, probably due to arrhythmia. Voltage clamp measurements of FASKard cardiomyocytes show hyperactivation of L-type calcium channel current that could not be reversed with palmitate supplementation. Of the classic regulators of this current, Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) but not protein kinase A signaling is activated in FASKard hearts, and knockdown of FAS in cultured cells activates CaMKII. In addition to being intolerant of the stress of acute pressure, FASKard hearts were also intolerant of the stress of aging, reflected as persistent CaMKII hyperactivation, progression to dilatation, and premature death by ∼1 year of age. CaMKII signaling appears to be pathogenic in FASKard hearts because inhibition of its signaling in vivo rescues mice from early mortality after transverse aortic constriction. FAS was also increased in two mechanistically distinct mouse models of heart failure and in the hearts of humans with end stage cardiomyopathy. These data implicate a novel relationship between FAS and calcium signaling in the heart and suggest that FAS induction in stressed myocardium represents a compensatory response to protect cardiomyocytes from pathological calcium flux.

Effect of collagen I and aortic carboxypeptidase-like protein on 3T3-L1 adipocyte differentiation

Aortic carboxypeptidase-like protein (ACLP) is a secreted protein expressed in preadipocytes and down-regulated during adipogenesis. Results from previous studies on the influence of ACLP overexpression on adipogenesis vary from no effect to complete inhibition. We hypothesized that ACLP may modulate adipogenesis in the presence of collagen I, a protein to which it binds. We compared control (pLXSN) 3T3-L1 preadipocytes with 3T3-L1 preadipocytes stably overexpressing ACLP (pLXSN-ACLP) that were grown in standard vs collagen I–coated dishes. Aortic carboxypeptidase-like protein overexpression, via retroviral transduction, resulted in a 3.2-fold increase in ACLP cellular levels and a 2.1-fold increase in ACLP levels released into medium. Aortic carboxypeptidase-like protein overexpression did not inhibit differentiation in standard dishes. In collagen I–coated dishes compared with standard dishes, control preadipocytes, when induced to differentiate, exhibited the same increase in triacylglycerol accumulation, but showed a significantly higher induction of fatty acid synthase (1.6-fold more), peroxisome proliferator-activated receptor γ (1.4-fold more), and CCAAT/enhancer-binding protein α (1.4-fold more). Aortic carboxypeptidase-like protein overexpression significantly reduced this enhanced induction of fatty acid synthase, peroxisome proliferator-activated receptor γ, and CCAAT/enhancer-binding protein α by 65%, 59%, and 66%, respectively, but had no effect on the accumulation of triacylglycerol during differentiation. Finally, studies on proadipogenic insulin signaling in ACLP-overexpressing preadipocytes demonstrated that insulin-stimulated Akt phosphorylation was significantly decreased by 27% in cells cultured in collagen I–coated dishes vs standard dishes. Our data suggest that ACLP inhibits certain aspects of 3T3-L1 adipogenesis in a collagen I–rich environment.

KLF5 enhances SREBP-1 action in androgen-dependent induction of fatty acid synthase in prostate cancer cells

KLF5 (Krüppel-like factor 5) is a zinc-finger transcription factor that plays a critical role in the regulation of cellular signalling involved in cell proliferation, differentiation and oncogenesis. In the present study, we showed that KLF5 acts as a key regulator controlling the expression of FASN (fatty acid synthase) through an interaction with SREBP-1 (sterol-regulatory-element-binding protein-1) in the androgen-dependent LNCaP prostate cancer cell line. The mRNA level of KLF5 increased when cells were treated with a synthetic androgen, R1881. Furthermore, KLF5 bound to SREBP-1 and enhanced the SREBP-1-mediated increase in FASN promoter activity. The results also demonstrated that the expression of KLF5 in LNCaP prostate cancer cells enhanced FASN expression, whereas silencing of KLF5 by small interfering RNA down-regulated FASN expression. The proximal promoter region and the first intron of the FASN gene contain multiple CACCC elements that mediate the transcriptional regulation of the gene by KLF5. However, other lipogenic and cholesterogenic genes, such as those encoding acetyl-CoA carboxylase, ATP-citrate lyase, the LDL (low-density lipoprotein) receptor, HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) synthase and HMG-CoA reductase are irresponsive to KLF5 expression, owing to the absence of CACCC elements in their promoter regions. Taken together, these results suggest that the FASN gene is activated by the synergistic action of KLF5 and SREBP-1, which was induced by androgen in androgen-dependent prostate cancer cells.

SIRT1 Regulates Hepatocyte Lipid Metabolism through Activating AMP-activated Protein Kinase

Resveratrol may protect against metabolic disease through activating SIRT1 deacetylase. Because we have recently defined AMPK activation as a key mechanism for the beneficial effects of polyphenols on hepatic lipid accumulation, hyperlipidemia, and atherosclerosis in type 1 diabetic mice, we hypothesize that polyphenol-activated SIRT1 acts upstream of AMPK signaling and hepatocellular lipid metabolism. Here we show that polyphenols, including resveratrol and the synthetic polyphenol S17834, increase SIRT1 deacetylase activity, LKB1 phosphorylation at Ser(428), and AMPK activity. Polyphenols substantially prevent the impairment in phosphorylation of AMPK and its downstream target, ACC (acetyl-CoA carboxylase), elevation in expression of FAS (fatty acid synthase), and lipid accumulation in human HepG2 hepatocytes exposed to high glucose. These effects of polyphenols are largely abolished by pharmacological and genetic inhibition of SIRT1, suggesting that the stimulation of AMPK and lipid-lowering effect of polyphenols depend on SIRT1 activity. Furthermore, adenoviral overexpression of SIRT1 stimulates the basal AMPK signaling in HepG2 cells and in the mouse liver. AMPK activation by SIRT1 also protects against FAS induction and lipid accumulation caused by high glucose. Moreover, LKB1, but not CaMKKbeta, is required for activation of AMPK by polyphenols and SIRT1. These findings suggest that SIRT1 functions as a novel upstream regulator for LKB1/AMPK signaling and plays an essential role in the regulation of hepatocyte lipid metabolism. Targeting SIRT1/LKB1/AMPK signaling by polyphenols may have potential therapeutic implications for dyslipidemia and accelerated atherosclerosis in diabetes and age-related diseases.

KLF5 enhances SREBP‐1 action in androgen‐dependent induction of FASN in prostate cancer cell line

The Kruppel like factor 5 (KLF5) is a zinc‐finger transcription factor that plays important roles in regulating all aspects of cellular signaling involved in cell proliferation and oncogenesis. In the present study, KLF5 interacts with sterol regulatory element‐binding protein‐1 (SREBP‐1) in regulating the expression of lipogenic protein, fatty acid synthase (FASN), which plays important roles in androgen‐dependent prostate cancer cells. Genes involved in lipid metabolism, including FASN, are reported to be up‐regulated by androgens in prostate cancer cells through a coordinated cascade controlled by the activation of SREBP‐1. The promoter and intron 1 region of FASN gene contains multiple GC‐boxes that might show response to KLF5. Deletion and mutation analyses indicated that KLF5 binds to the FASN gene and strongly induces its activity. Furthermore, KLF5 protein is able to bind to SREBP‐1 and enhance the SREBP‐1 regulated activation of FASN. Transfection of KLF5 into prostate cancer cells enhanced FASN promoter activity, and conversely, silencing of KLF5 by siRNA down‐regulated FASN expression. The multiple GC motifs are scattered throughout the upstream sequence and first intron of FASN gene whereas these are scarce in the promoters of other lipogenic and cholesterogenic genes such as acetyl‐CoA carboxylase, ATP‐citrate lyase, LDL receptor, HMG‐CoA synthase and HMG‐CoA reductase, which makes FASN gene very unique among them. Taken together, FASN gene could be activated by the synergistic action of KLF5 and SREBP‐1 which is increased by androgen treatment.

Fermented Soy Diet Reduces Weight Gain and Alters Liver Fatty Acid Composition in mice Fed a High Fat Diet

Obesity is now a worldwide problem, yet countries with high intake of soy foods have less incidence of obesity. Fermentation of soy leads to accumulation of aglycone isoflavones. In this study, we compared fermented soy (fsoy, n=12) to soy (n=12) and casein (n=11) diet on weight gain and hepatic lipid composition in diet‐induced obese male C57B16/J mice. Food intake was measured semi‐weekly and weights weekly. After the 17 weeks, necropsy was performed, tissue weights were obtained and livers were snap frozen for measurement of triglycerides, fatty acids, and mRNA levels of lipid metabolizing genes. The fsoy and soy diet groups had increased average daily food intakes and lower weight gain compared to casein group. The fsoy group had a higher feed‐to‐gain ratio. The liver weights and triglyceride level did not differ. The hepatic fatty acid composition was different including a higher omega‐3:omega‐6 ratio in the fsoy group. The fsoy group had a lower fold induction of peroxisome proliferator activated receptor alpha (PPAR ), fatty acid binding protein 1(FABP1), and stearoyl Co‐A desaturase 1 (SCD1) mRNA than the casein group. The fsoy group had a higher fold induction of fatty acid synthase (FAS) and PPAR mRNA than the soy group. Fermented soy decreases weight gain and changes the composition of liver fatty acids in C57B16/J mice. Supported by Ajinomoto Corporation

Tea Polyphenol (−)-Epigallocatechin 3-Gallate Suppresses Heregulin-β1-Induced Fatty Acid Synthase Expression in Human Breast Cancer Cells by Inhibiting Phosphatidylinositol 3-Kinase/Akt and Mitogen-Activated Protein Kinase Cascade Signaling

Tumor-associated fatty acid synthase (FAS) is implicated in tumorigenesis and connected to HER2 (human epidermal growth factor receptor 2) by systemic analyses. Suppression of FAS in cancer cells may lead to growth inhibition and cell apoptosis. Our previous study demonstrated that (-)-epigallocatechin 3-gallate (EGCG), the green tea catechin, could down-regulate FAS expression by suppressing EGFR (epidermal growth factor receptor) signaling and downstream phosphatidylinositol 3-kinase (PI3K)/Akt activation in the MCF-7 breast cancer cell line. Herein, we examined the effects of EGCG on FAS expression modulated by another member of the erbB family, that is, HER2 or HER3. We identified that heregulin-beta1 (HRG-beta1), a HER3 ligand, stimulated dose-dependent FAS expression in breast cancer cell lines MCF-7 and AU565, but not MDA-MB-453. The time-dependent increase in FAS expression after HRG-beta1 stimulation was also observed in MCF-7 cells, and this up-regulation was de novo RNA synthesis dependent. Treatment of MCF-7 cells with EGCG markedly inhibited HRG-beta1-dependent induction of mRNA and protein of FAS. EGCG also decreased the phosphorylation of Akt and extracellular signal-regulated kinase 1/2 that were demonstrated as selected downstream HRG-beta1-responsive kinases required for FAS expression using dominant-negative Akt, PI3K inhibitors (LY294002 and wortmannin), or MEK inhibitor (PD98059). FAS induction by HRG-beta1 was also blocked by AG825, a selective HER2 inhibitor, and by genistein, a selective tyrosine kinase inhibitor, indicating the formation of a heterodimer between HER2 and HER3, and their tyrosine kinase activities are essential for HRG-beta1-mediated elevation of FAS. Additionally, growth inhibition of HRG-beta1-treated cells was parallel to suppression of FAS by EGCG. Taken together, these findings extend our previous study to indicate that EGCG may be useful in the chemoprevention of breast carcinoma in which FAS overexpression results from HER2 or/and HER3 signaling.

Macrophage-conditioned medium inhibits the differentiation of 3T3-L1 and human abdominal preadipocytes

In obesity, a limited adipogenic capacity may promote adipocyte hypertrophy and increase the risk of insulin resistance and type 2 diabetes. Recent data indicate that macrophages reside within adipose tissue in obese rodents and humans. We hypothesised that secreted macrophage factors may inhibit adipogenesis. Conditioned media from cultured murine J774 or human THP-1 macrophages were collected, and added to either murine 3T3-L1 preadipocytes or human abdominal stromal preadipocytes from subcutaneous or omental fat depots. Macrophage-conditioned medium (MacCM) strongly inhibited 3T3-L1 adipogenesis. Dose-response studies with J774-MacCM revealed that 80 and 100% of J774-MacCM completely suppressed triacylglycerol accumulation as well as the induction of fatty acid synthase, peroxisome proliferator-activated receptor gamma, CCAAT/enhancer binding protein alpha, and adiponectin. Similar inhibitory effects on 3T3-L1 preadipocytes were observed with THP-1-MacCM. Differentiation of human abdominal subcutaneous stromal preadipocytes was moderately reduced (subcutaneous>omental) by J744-MacCM. In contrast, the differentiation of both subcutaneous and omental stromal preadipocytes was completely inhibited by THP-1-MacCM, as determined on the basis of morphology and triacylglycerol accumulation, as well as fatty acid synthase and adiponectin protein expression. Secreted macrophage products inhibit the differentiation of 3T3-L1 preadipocytes as well as human abdominal stromal preadipocytes.

Keratinocyte Growth Factor Induces Lipogenesis in Alveolar Type II Cells through a Sterol Regulatory Element Binding Protein-1c–Dependent Pathway

Keratinocyte growth factor (KGF) stimulates fatty acid and phospholipid synthesis in alveolar type II cells in vitro. KGF stimulates lipogenic enzymes, including fatty acid synthase and stearyl-CoA desaturase-1, and transcription factors involved in lipogenesis, such as sterol regulatory element binding protein (SREBP)-1c and CCAAT/enhancer binding protein (C/EBP)alpha and C/EBPdelta. To define the role of SREBP-1c on the induction of lipogenic genes and lipogenesis by KGF in primary cultures of rat type II cells, we used adenoviral vectors to alter levels of SREBP-1c. Overexpression of a dominant-negative form of SREBP-1 decreased lipogenesis and decreased the induction of fatty acid synthase and stearyl coenzyme A desaturase-1 by KGF. Conversely, adenovirus-mediated overexpression of a constitutively active form of SREBP-1c mimicked the effect of KGF on lipogenic enzymes and lipogenesis. These data indicate that SREBP-1c is required for the stimulation of lipogenesis by KGF in the alveolar type II cells and is a key regulator of lung lipid metabolism and that expression of SREBP-1c is sufficient to induce lipogenesis in rat type II cells.

Arachidonic Acid Activates Phosphatidylinositol 3-Kinase Signaling and Induces Gene Expression in Prostate Cancer

Essential fatty acids are not only energy-rich molecules; they are also an important component of the membrane bilayer and recently have been implicated in induction of fatty acid synthase and other genes. Using gene chip analysis, we have found that arachidonic acid, an omega-6 fatty acid, induced 11 genes that are regulated by nuclear factor-kappaB (NF-kappaB). We verified gene induction by omega-6 fatty acid, including COX-2, IkappaBalpha, NF-kappaB, GM-CSF, IL-1beta, CXCL-1, TNF-alpha, IL-6, LTA, IL-8, PPARgamma, and ICAM-1, using quantitative reverse transcription-PCR. Prostaglandin E(2) (PGE(2)) synthesis was increased within 5 minutes of addition of arachidonic acid. Analysis of upstream signal transduction showed that within 5 minutes of fatty acid addition, phosphatidylinositol 3-kinase (PI3K) was significantly activated followed by activation of Akt at 30 minutes. Extracellular signal-regulated kinase 1 and 2, p38 and stress-activated protein kinase/c-Jun-NH(2)-kinase were not phosphorylated after omega-6 fatty acid addition. Thirty minutes after fatty acid addition, we found a significant 3-fold increase in translocation of NF-kappaB transcription factor to the nucleus. Addition of a nonsteroidal anti-inflammatory drug (NSAID) caused a decrease in COX-2 protein synthesis, PGE(2) synthesis, as well as inhibition of PI3K activation. We have previously shown that NSAIDs cause an inhibition of arachidonic acid-induced proliferation; here, we have shown that arachidonic acid-induced proliferation is also blocked (P < 0.001) by PI3K inhibitor LY294002. LY294002 also significantly inhibited the arachidonic acid-induced gene expression of COX-2, IL-1beta, GM-CSF, and ICAM1. Taken together, the data suggest that arachidonic acid via conversion to PGE(2) plays an important role in stimulation of growth-related genes and proliferation via PI3K signaling and NF-kappaB translocation to the nucleus.