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When amino acids are plentiful in the diet, the liver upregulates most enzymes responsible for amino acid degradation. In particular, the activity of urea cycle enzymes increases in response to high-protein diets to facilitate the excretion of excess nitrogen. KLF15 has been established as a critical regulator of amino acid catabolism including ureagenesis and we have recently identified FoxO transcription factors as an important upstream regulator of KLF15 in the liver. Therefore, we explored the role of FoxOs in amino acid metabolism under high-protein diet. Our findings revealed that the concentrations of two urea cycle-related amino acids, arginine and ornithine, were significantly altered by FoxOs knockdown. Additionally, using KLF15 knockout mice and an in vivo Ad-luc analytical system, we confirmed that FoxOs directly regulate hepatic Ass1 expression under high-protein intake independently from KLF15. Moreover, ChIP analysis showed that the high-protein diet increased FoxOs DNA binding without altering the nuclear protein amount. Therefore, FoxOs play a direct role in regulating ureagenesis via a KLF15-independent pathway in response to high-protein intake., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The pancreatic islet vasculature is of fundamental importance to the β-cell response to obesity-associated insulin resistance. To explore islet vascular alterations in the pathogenesis of type 2 diabetes, we evaluated two insulin resistance models: ob/ob mice, which sustain large β-cell mass and hyperinsulinemia, and db/db mice, which progress to diabetes due to secondary β-cell compensation failure for insulin secretion. Time-dependent changes in islet vasculature and blood flow were investigated using tomato lectin staining and in vivo live imaging. Marked islet capillary dilation was observed in ob/ob mice, but this adaptive change was blunted in db/db mice. Islet blood flow volume was augmented in ob/ob mice, whereas it was reduced in db/db mice. The protein concentrations of total and phosphorylated endothelial nitric oxide synthase (eNOS) at Ser1177 were increased in ob/ob islets, while they were diminished in db/db mice, indicating decreased eNOS activity. This was accompanied by an increased retention of advanced glycation end-products in db/db blood vessels. Amelioration of diabetes by Elovl6 deficiency involved a restoration of capillary dilation, blood flow, and eNOS phosphorylation in db/db islets. Our findings suggest that the disability of islet capillary dilation due to endothelial dysfunction impairs local islet blood flow, which may play a role in the loss of β-cell function and further exacerbate type 2 diabetes., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

High protein diet (HPD) is an affordable and positive approach in prevention and treatment of many diseases. It is believed that transcriptional regulation is responsible for adaptation after HPD feeding and Kruppel-like factor 15 (KLF15), a zinc finger transcription factor that has been proved to perform transcriptional regulation over amino acid, lipid and glucose metabolism, is known to be involved at least in part in this HPD response. To gain more insight into molecular mechanisms by which HPD controls expressions of genes involved in amino acid metabolism in the liver, we performed RNA-seq analysis of mice fed HPD for a short period (3 days). Compared to a low protein diet, HPD feeding significantly increased hepatic expressions of enzymes involved in the breakdown of all the 20 amino acids. Moreover, using KLF15 knockout mice and in vivo Ad-luc analytical system, we were able to identify Cth (cystathionine gamma-lyase) as a new target gene of KLF15 transcription as well as Ast (aspartate aminotransferase) as an example of KLF15-independent gene despite its remarkable responsiveness to HPD. These findings provide us with a clue to elucidate the entire transcriptional regulatory mechanisms of amino acid metabolic pathways., Competing Interests: Declaration of competing interest The authors declare no competing financial and non-financial interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

While molecular oxygen is essential for aerobic organisms, its utilization is inseparably connected with generation of oxidative insults. To cope with the detrimental aspects, cells evolved antioxidative defense systems, and insufficient management of the oxidative insults underlies the pathogenesis of a wide range of diseases. A battery of genes for this antioxidative defense are regulated by the transcription factors nuclear factor-erythroid 2-like 1 and 2 (NRF1 and NRF2). While the regulatory steps for the activation of NRFs have been investigated with particular emphasis on nuclear translocation and proteosomal degradation, unknown redundancy may exist considering the indispensable nature of these defense systems. Here we unraveled that C-terminal binding protein 2 (CtBP2), a transcriptional cofactor with redox-sensing capability, is an obligate partner of NRFs. CtBP2 forms transcriptional complexes with NRF1 and NRF2 that is required to promote the expression of antioxidant genes in response to oxidative insults. Our findings illustrate a basis for understanding the transcriptional regulation of antioxidative defense systems that may be exploited therapeutically., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest regarding the publication of this short communication., (Copyright © 2021 Elsevier Inc. All rights reserved.)

In Japan, a new opportunistic community-based walk-in HbA1c testing program at pharmacies was enabled in 2014. An economic evaluation of this program from societal perspective has previously been published. This study examines the effect of a subsidy program for walk-in HbA1c-testing at community pharmacies in Japan on public health care expenditure by conducting a budget impact analysis from payer's perspective. The study focused on Adachi Ward in Tokyo, where a pioneering subsidy program was implemented. It examined the budget impact of the subsidy program over a 15 years. The total subsidy paid by the local authority during the first year was 2909 USD. Public expenditure remained positive for the first five years before becoming negative in the sixth year, eventually resulting in savings of 221,000 USD in the 15th year. The cost of treating type 2 diabetes that is detected early is offset by a reduction in expensive treatments for complicated cases. Subsidizing walk-in fingertip HbA1c testing in community pharmacies is likely to result in a significant reduction in public expenditure over the mid-to long term. Our result suggests that a similar strategy in other areas could also have a potentially favorable budget impact., (Copyright © 2020. Published by Elsevier Inc.)

The epithelial to mesenchymal transition (EMT) is a cell intrinsic program controlling cellular morphological and phenotypic remodeling in a wide range of biological processes. Despite the accumulating evidence, the transcriptional networks regulating EMT still remain to be elucidated. In this study, we demonstrate that C-terminal binding protein 2 (CtBP2), a critical transcriptional co-repressor harboring pyridine nucleotide sensing capability, orchestrates the EMT program at least in part through a novel transcriptional interaction with an octamer transcription factor, OCT1 (POU2F1, POU class 2 homeobox 1). We identified novel interactions of CtBP2 with several octamer transcription factors, and CtBP2 exhibits a direct interaction with OCT1 in particular. OCT1 accelerates the EMT program as reported, which is diminished by the mutation of the CtBP-binding motif in OCT1, suggesting OCT1 represses epithelial gene expression through recruiting the co-repressor CtBP2. In accordance with these findings, a canonical EMT activator transforming growth factor-β (TGF-β) promotes the formation of the CtBP2/OCT1 complex. Our observations illustrate the role of CtBP2 to orchestrate the EMT program through the interaction with OCT1 and highlight the potential of therapeutic exploitation of this new transcriptional system for a wide range of diseases., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest regarding the publication of this short communication., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Sodium-glucose cotransporter 2 (SGLT2) inhibitors have both anti-diabetic and anti-obesity effects. However, the precise mechanism of the anti-obesity effect remains unclear. We previously demonstrated that the glycogen depletion signal triggers lipolysis in adipose tissue via liver-brain-adipose neurocircuitry. In this study, therefore, we investigated whether the anti-obesity mechanism of SGLT2 inhibitor is mediated by this mechanism. Diet-induced obese mice were subjected to hepatic vagotomy (HVx) or sham operation and loaded with high fat diet containing 0.015% tofogliflozin (TOFO), a highly selective SGLT2 inhibitor, for 3 weeks. TOFO-treated mice showed a decrease in fat mass and the effect of TOFO was attenuated in HVx group. Although both HVx and sham mice showed a similar level of reduction in hepatic glycogen by TOFO treatment, HVx mice exhibited an attenuated response in protein phosphorylation by protein kinase A (PKA) in white adipose tissue compared with the sham group. As PKA pathway is known to act as an effector of the liver-brain-adipose axis and activate triglyceride lipases in adipocytes, these results indicated that SGLT2 inhibition triggered glycogen depletion signal and actuated liver-brain-adipose axis, resulting in PKA activation in adipocytes. Taken together, it was concluded that the effect of SGLT2 inhibition on weight loss is in part mediated via the liver-brain-adipose neurocircuitry., (Copyright © 2017 Elsevier Inc. All rights reserved.)

HMG-CoA reductase (HMGCR) catalyzes the conversion of HMG-CoA to mevalonic acid (MVA); this is the rate-limiting enzyme of the mevalonate pathway that synthesizes cholesterol. Statins, HMGCR inhibitors, are widely used as cholesterol-reducing drugs. However, statin-induced myopathy is the most adverse side effect of statins. To eludicate the mechanisms underlying statin the myotoxicity and HMGCR function in the skeletal muscle, we developed the skeletal muscle-specific HMGCR knockout mice. Knockout mice exhibited postnatal myopathy with elevated serum creatine kinase levels and necrosis. Myopathy in knockout mice was completely rescued by the oral administration of MVA. These results suggest that skeletal muscle toxicity caused by statins is dependent on the deficiencies of HMGCR enzyme activity and downstream metabolites of the mevalonate pathway in skeletal muscles rather than the liver or other organs., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Fatty acid elongase 5 (ELOVL5) is an enzyme involved in the synthesis of polyunsaturated fatty acids. Sterol Regulatory Element-binding Protein (SREBP)-1 activates ELOVL5 and increases polyunsaturated fatty acid synthesis, which in turn negatively affects SREBP-1 expression. Thus, ELOVL5 has been established as an SREBP-1 target gene and an important component of the negative feedback loop of de novo lipogenesis. However, the human ELOVL5 promoter/enhancer has not been fully analyzed and the location of SREBP biding sites around the ELOVL5 gene has yet to be defined. Here we performed a detailed promoter/enhancer analysis of human ELOVL5 gene, and identified two new SREBP binding sites, one in the 10 kb upstream region and one in the exon 1. These two SRE motifs are conserved among mammals and the mechanism found in the present study by which SREBP activates ELOVL5 is considered to be common in mammals. Through these findings, we clarified the molecular mechanism how SREBP activates ELOVL5, an important regulator of de novo lipogenesis., (Copyright © 2015 Elsevier Inc. All rights reserved.)

ELOVL family member 6, elongation of very long-chain fatty acids (Elovl6) is a microsomal enzyme that regulates the elongation of C12-16 saturated and monounsaturated fatty acids and is related to the development of obesity-induced insulin resistance via the modification of the fatty acid composition. In this study, we investigated the role of systemic Elovl6 in the pancreatic islet and β-cell function. Elovl6 is expressed in both islets and β-cell lines. In mice fed with chow, islets of the Elovl6(-/-) mice displayed normal architecture and β-cell mass compared with those of the wild-type mice. However, when fed a high-fat, high-sucrose (HFHS) diet, the islet hypertrophy in response to insulin resistance observed in normal mice was attenuated and glucose-stimulated insulin secretion (GSIS) increased in the islets of Elovl6(-/-) mice compared with those of the wild-type mice. Enhanced GSIS in the HFHS Elovl6(-/-) islets was associated with an increased ATP/ADP ratio and the suppression of ATF-3 expression. Our findings suggest that Elovl6 could be involved in insulin secretory capacity per β-cell and diabetes., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Transcription factor E3 (TFE3) belongs to a basic helix-loop-helix family, and is involved in the biology of osteoclasts, melanocytes and their malignancies. We previously reported the metabolic effects of TFE3 on insulin in the liver and skeletal muscles in animal models. In the present study, we explored a novel role for TFE3 in a skeletal muscle cell line. When TFE3 was overexpressed in C2C12 myoblasts by adenovirus before induction of differentiation, myogenic differentiation of C2C12 cells was significantly inhibited. Adenovirus-mediated TFE3 overexpression also suppressed the gene expression of muscle regulatory factors (MRFs), such as MyoD and myogenin, during C2C12 differentiation. In contrast, knockdown of TFE3 using adenovirus encoding short-hairpin RNAi specific for TFE3 dramatically promoted myoblast differentiation associated with significantly increased expression of MRFs. Consistent with these findings, promoter analyses via luciferase reporter assay and electrophoretic mobility shift assay suggested that TFE3 negatively regulated myogenin promoter activity by direct binding to an E-box, E2, in the myogenin promoter. These findings indicated that TFE3 has a regulatory role in myoblast differentiation, and that transcriptional suppression of myogenin expression may be part of the mechanism of action., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Dicer is a rate-limiting enzyme for microRNA (miRNA) synthesis. To determine the effects of Dicer on adipogenesis, we performed stage-specific knockdown of Dicer using adenovirus encoding short-hairpin RNAi against Dicer in 3T3-L1 cells. When cells were infected with the adenovirus before induction of adipocyte differentiation, Dicer RNAi suppressed the gene expression of inducers of adipocyte differentiation such as PPARγ, C/EBPα, and FAS in 3T3-L1 cells during adipocyte differentiation. Concurrently, both adipocyte differentiation and cellular lipid accumulation were cancelled by Dicer RNAi when compared with control RNAi. Meanwhile, we addressed the roles of Dicer in lipid synthesis and accumulation in the final stages of differentiation. When the differentiated cells at day 4 after induction of differentiation were infected with adenovirus Dicer RNAi, cellular lipid accumulation was unchanged. Consistent with this, Dicer RNAi had no effects on the expression of genes related to cellular lipid accumulation, including PPARγ and FAS. Thus, Dicer controls proadipogenic genes such as C/EBPα and PPARγ in the early, but not in the late, stage of adipogenesis via regulation of miRNA synthesis., (Copyright © 2012 Elsevier Inc. All rights reserved.)

We have previously demonstrated that neutral cholesterol ester hydrolase 1 (Nceh1) regulates foam cell formation and atherogenesis through the catalytic activity of cholesterol ester hydrolysis, and that Nceh1 and hormone-sensitive lipase (Lipe) are responsible for the majority of neutral cholesterol ester hydrolase activity in macrophages. There are several cholesterol ester-metabolizing tissues and cells other than macrophages, among which adrenocortical cells are also known to utilize the intracellular cholesterol for steroidogenesis. It has been believed that the mobilization of intracellular cholesterol ester in adrenal glands was facilitated solely by Lipe. We herein demonstrate that Nceh1 is also involved in cholesterol ester hydrolysis in adrenal glands. While Lipe deficiency remarkably reduced the neutral cholesterol ester hydrolase activity in adrenal glands as previously reported, additional inactivation of Nceh1 gene completely abrogated the activity. Adrenal glands were enlarged in proportion to the degree of reduced neutral cholesterol ester hydrolase activity, and the enlargement of adrenal glands and the accumulation of cholesterol esters were most pronounced in the Nceh1/Lipe double-deficient mice. Thus Nceh1 is involved in the adrenal cholesterol metabolism, and the cholesterol ester hydrolytic activity in adrenal glands is associated with the organ enlargement., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Sterol-regulatory element binding protein-1c (SREBP-1c) is a transcription factor that controls lipogenesis in the liver. Hepatic SREBP-1c is nutritionally regulated, and its sustained activation causes hepatic steatosis and insulin resistance. Although regulation of SREBP-1c is known to occur at the transcriptional level, the precise mechanism by which insulin signaling activates SREBP-1c promoter remains to be elucidated. Here we show that protein kinase C beta (PKCbeta) is a key mediator of insulin-mediated activation of hepatic SREBP-1c and its target lipogenic genes. Activation of SREBP-1c in the liver of refed mice was suppressed by either adenoviral RNAi-mediated knockdown or dietary administration of a specific inhibitor of protein kinase C beta. The effect of PKCbeta inhibition was cancelled in insulin depletion by streptozotocin (STZ) treatment of mice. Promoter analysis indicated that PKCbeta activates SREBP-1c promoter through replacement of Sp3 by Sp1 for binding to the GC box in the sterol regulatory element (SRE) complex, a key cis-element of SREBP-1c promoter. Knockdown of Sp proteins demonstrated that Sp3 and Sp1 play reciprocally negative and positive roles in nutritional regulation of SREBP-1c, respectively. This new understanding of PKCbeta involvement in nutritional regulation of SREBP-1c activation provides a new aspect of PKCbeta inhibition as a potential therapeutic target for diabetic complications.

It has long been a matter of debate whether the hormone-sensitive lipase (HSL)-mediated lipolysis in pancreatic beta-cells can affect insulin secretion through the alteration of lipotoxicity. We generated mice lacking both leptin and HSL Lep(ob/ob)/HSL(-/-) and explored the role of HSL in pancreatic beta-cells in the setting of obesity. Lep(ob/ob)/HSL(-/-) developed elevated blood glucose levels and reduced plasma insulin levels compared with Lep(ob/ob)/HSL(+/+) in a fed state, while the deficiency of HSL did not affect glucose homeostasis in Lep(+/+) background. The deficiency of HSL exacerbated the accumulation of triglycerides in Lep(ob/ob) islets, leading to reduced glucose-stimulated insulin secretion. The deficiency of HSL also diminished the islet mass in Lep(ob/ob) mice due to decreased cell proliferation. In conclusion, HSL affects insulin secretary capacity especially in the setting of obesity.

MicroRNAs (miRNAs) are short non-coding RNA that post-transcriptionally regulates gene expression. Some miRNAs have been proposed to be associated with obesity. However, miRNAs, which are related to the development of obesity in vivo remains unknown. Here in, we found the up-regulation of miR-335 in obesity using microarray analysis for miRNA. The expressions of miR-335 in liver and white adipose tissue (WAT) were up-regulated in obese mice including ob/ob, db/db, and KKAy mice. Increased miR-335 expressions were associated with an elevated body, liver and WAT weight, and hepatic triglyceride and cholesterol. Furthermore, miR-335 levels were closely correlated with expression levels of adipocyte differentiation markers such as PPARgamma, aP2, and FAS in 3T3-L1 adipocyte. These findings provide the first evidence that the up-regulated expressions of miR-335 in liver and WAT of obese mice might contribute to the pathophysiology of obesity.

Transgenic mice expressing nuclear sterol regulatory element-binding protein-1a under the control of the insulin promoter were generated to determine the role of SREBP-1a in pancreatic beta-cells. Only low expressors could be established, which exhibited mild hyperglycemia, impaired glucose tolerance, and reduced plasma insulin levels compared to C57BL/6 controls. The islets isolated from the transgenic mice were fewer and smaller, and had decreased insulin content and unaltered glucagon staining. Both glucose- and potassium-stimulated insulin secretions were decreased. The transgenic islets consistently expressed genes for fatty acids and cholesterol synthesis, resulting in accumulation of triglycerides but not cholesterol. PDX-1, BetaEpsilonTauAlpha2, MafA, and IRS-2 were suppressed, partially explaining the loss and dysfunction of beta-cell mass. The transgenic mice on a high fat/high sucrose diet still exhibited impaired insulin secretion and continuous beta-cell growth defect. Therefore, nuclear SREBP-1a, even at a low level, strongly disrupts beta-cell mass and function.

To determine the role of cholesterol synthesis in pancreatic beta-cells, a transgenic model of in vivo activation of sterol-regulatory element binding protein 2 (SREBP-2) specifically in beta-cells (TgRIP-SREBP-2) was developed and analyzed. Expression of nuclear human SREBP-2 in beta-cells resulted in severe diabetes as evidenced by greater than 5-fold elevations in glycohemoglobin compared with C57BL/6 controls. Diabetes in TgRIP-SREBP-2 mice was primarily due to defects in glucose- and potassium-stimulated insulin secretion as determined by glucose tolerance test. Isolated islets of TgSREBP-2 mice were fewer in number, smaller, deformed, and had decreased insulin content. SREBP-2-expressing islets also contained increased esterified cholesterol and unchanged triglycerides with reduced ATP levels. Consistently, these islets exhibited elevated expression of HMG-CoA synthase and reductase and LDL receptor, with suppression of endogenous SREBPs. Genes involved in beta-cell differentiation, such as PDX1 and BETA2, were suppressed, explaining loss of beta-cell mass, whereas IRS2 expression was not affected. These phenotypes were dependent on the transgene expression. Taken together, these results indicate that activation of SREBP-2 in beta-cells caused severe diabetes by loss of beta-cell mass with accumulation of cholesterol, providing a new lipotoxic model and a potential link of disturbed cholesterol metabolism to impairment of beta-cell function.

Hormone-sensitive lipase (HSL) regulates the hydrolysis of acylglycerol and cholesteryl ester (CE) in various organs, including adipose tissues. However, the hepatic expression level of HSL has been reported to be almost negligible. In the present study, we found that mice lacking both leptin and HSL (Lep(ob/ob)/HSL(-/-)) showed massive accumulation of CE in the liver compared with Lep(ob/ob)/HSL(+/+) mice, while triacylglycerol (TG) accumulation was modest. Similarly, feeding with a high-cholesterol diet induced hepatic CE accumulation in HSL(-/-) mice. Supporting these observations, we detected significant expression of protein as well as mRNA of HSL in the liver. HSL(-/-) mice showed reduced activity of CE hydrolase, but not of TG lipase, in the liver compared with wild-type mice. Furthermore, we confirmed the expression of HSL in viable parenchymal cells isolated from wild-type mice. The hepatocytes from HSL(-/-) mice showed reduced activity of CE hydrolase and contained more CE than those from HSL(+/+) mice even without the incubation with lipoproteins. Incubation with LDL further augmented the accumulation of CE in the HSL-deficient hepatocytes. From these results, we conclude that HSL is involved in the hydrolysis of CE in hepatocyes.

Elovl-6, a long fatty acid elongase, contributes to de novo synthesis of fatty acids and regulates hepatic insulin sensitivity. Hepatic regulation of Elovl-6 gene expression in various nutritional conditions suggested that, like other lipogenic enzyme genes, Elovl-6 is a target of SREBP-1, a transcription factor governing fatty acid synthesis. Supportively, adenoviral RNAi knockdown of SREBP-1 in mouse liver suppressed Elovl-6 mRNA and fatty acid synthase levels. Therefore, we analyzed mouse Elovl-6 gene promoter to determine its role as an SREBP-1 target. Luciferase reporter assays of 1.4-kb 5' flanking region of mouse Elovl-6 gene in HepG2 cells demonstrated that nuclear SREBPs activated the Elovl-6 promoter, highlighting two SREBP binding sites: proximal SRE-1 and distal SRE-2. EMSA indicated that SRE-1 had higher affinity than SRE-2 for SREBP. ChIP assays confirmed in vivo binding of hepatic nuclear SREBP-1c protein. These data demonstrated that Elovl-6 is regulated directly and primarily by SREBP-1c.

Sterol regulatory element-binding protein (SREBP)-1c is now well established as a key transcription factor for the regulation of lipogenic enzyme genes such as FAS in hepatocytes. Meanwhile, the mechanisms of lipogenic gene regulation in adipocytes remain unclear. Here, we demonstrate that those in adipocytes are independent of SREBP-1c. In adipocytes, unlike in hepatocytes, the stimulation of SREBP-1c expression by liver X receptor agonist does not accompany lipogenic gene upregulation, although nuclear SREBP-1c protein is concomitantly increased, indicating that the activation process of SREBP-1c by the cleavage system is intact in adipocytes. Supportively, transcriptional activity of the mature form of SREBP-1c for the FAS promoter was negligible when measured by reporter analysis. As an underlying mechanism, accessibility of SREBP-1c to the functional elements was involved, because chromatin immunoprecipitation assays revealed that SREBP-1c does not bind to the functional SRE/E-box site on the FAS promoter in adipocytes. Moreover, genetic disruption of SREBP-1 did not cause any changes in lipogenic gene expression in adipose tissue. In summary, in adipocytes, unlike in hepatocytes, increments in nuclear SREBP-1c are not accompanied by transactivation of lipogenic genes; thus, SREBP-1c is not committed to the regulation of lipogenesis.

Squalene synthase (SS) is the first committed enzyme for cholesterol biosynthesis, located at a branch point in the mevalonate pathway. To examine the role of SS in the overall cholesterol metabolism, we transiently overexpressed mouse SS in the livers of mice using adenovirus-mediated gene transfer. Overexpression of SS increased de novo cholesterol biosynthesis with increased 3-hydroxy-3-methyglutaryl-CoA (HMG-CoA) reductase activity, in spite of the downregulation of its own mRNA expression. Furthermore, overexpression of SS increased plasma concentrations of LDL, irrespective of the presence of functional LDL receptor (LDLR). Thus, the hypercholesterolemia is primarily caused by increased hepatic production of cholesterol-rich VLDL, as demonstrated by the increases in plasma cholesterol levels after intravenous injection of Triton WR1339. mRNA expression of LDLR was decreased, suggesting that defective LDL clearance contributed to the development of hypercholesterolemia. Curiously, the liver was enlarged, with a larger number of Ki-67-positive cells. These results demonstrate that transient upregulation of SS stimulates cholesterol biosynthesis as well as lipoprotein production, providing the first in vivo evidence that SS plays a regulatory role in cholesterol metabolism through modulation of HMG-CoA reductase activity and cholesterol biosynthesis.

Recent studies on the in vivo roles of the sterol regulatory element binding protein (SREBP) family indicate that SREBP-2 is more specific to cholesterogenic gene expression whereas SREBP-1 targets lipogenic genes. To define the molecular mechanism involved in this differential regulation, luciferase-reporter gene assays were performed in HepG2 cells to compare the transactivities of nuclear SREBP-1a, -1c, and -2 on a battery of SREBP-target promoters containing sterol regulatory element (SRE), SRE-like, or E-box sequences. The results show first that cholesterogenic genes containing classic SREs in their promoters are strongly and efficiently activated by both SREBP-1a and SREBP-2, but not by SREBP-1c. Second, an E-box containing reporter gene is much less efficiently activated by SREBP-1a and -1c, and SREBP-2 was inactive in spite of its ability to bind to the E-box. Third, promoters of lipogenic enzymes containing variations of SRE (SRE-like sequences) are strongly activated by SREBP-1a, and only modestly and equally by both SREBP-1c and -2. Finally, substitution of the unique tyrosine residue within the basic helix-loop-helix (bHLH) portion of nuclear SREBPs with arginine, the conserved residue found in all other bHLH proteins, abolishes the transactivity of all SREBPs for SRE, and conversely results in markedly increased activity of SREBP-1 but not activity of SREBP-2 for E-boxes. These data demonstrate the different specificity and affinity of nuclear SREBP-1 and -2 for different target DNAs, explaining a part of the mechanism behind the differential in vivo regulation of cholesterogenic and lipogenic enzymes by SREBP-1 and -2, respectively.

The mammalian enzyme involved in the final elongation of de novo fatty acid biosynthesis following the building of fatty acids to 16 carbons by fatty acid synthase has yet to be identified. In the process of searching for genes activated by sterol regulatory element-binding protein 1 (SREBP-1) by using DNA microarray, we identified and characterized a murine cDNA clone that is highly similar to a fatty acyl-CoA elongase gene family such as Cig30, Sscs, and yeast ELOs. Studies on the cells overexpressing the full-length cDNA indicate that the encoded protein, designated fatty acyl-CoA elongase (FACE), has a FACE activity specific for long-chains; C12-C16 saturated- and monosaturated-fatty acids. Hepatic expression of this identified gene was consistently activated in the livers of transgenic mice overexpressing nuclear SREBP-1a, -1c, or -2. FACE mRNA levels are markedly induced in a refed state after fasting in the liver and adipose tissue. This refeeding response is significantly reduced in SREBP-1 deficient mice. Dietary PUFAs caused a profound suppression of this gene expression, which could be restored by SREBP-1c overexpression. Hepatic FACE expression was also highly up-regulated in leptin-deficient ob/ob mice. Hepatic FACE mRNA was markedly increased by administration of a pharmacological agonist of liver X-activated receptor (LXR), a dominant activator for SREBP-1c expression. These data indicated that this elongase is a new member of mammalian lipogenic enzymes regulated by SREBP-1, playing an important role in de novo synthesis of long-chain saturated and monosaturated fatty acids in conjunction with fatty acid synthase and stearoyl-CoA desaturase.

In the process of seeking sterol regulatory element-binding protein 1a (SREBP-1a) target genes, we identified and cloned a cDNA clone encoding mouse Delta(5)-desaturase (D5D). The hepatic expression of D5D as well as Delta(6)-desaturase (D6D) was highly activated in transgenic mice overexpressing nuclear SREBP-1a, -1c, and -2. Disruption of the SREBP-1 gene significantly reduced the expression of both desaturases in the livers of SREBP-1-deficient mice refed after fasting. The hepatic expression of both desaturases was downregulated by dietary PUFA, which were reported to suppress SREBP-1c gene expression. Sustained expression of hepatic nuclear SREBP-1c protein in the transgenic mice abolished the PUFA suppression of both desaturases. Although these data suggested that SREBP-1c regulates D5D and D6D expression, there was no difference in either the D5D or D6D mRNA level between fasted and refed normal mouse livers, indicating a mechanism for fasting induction of both desaturases. Administration of fibrate, a pharmacological ligand for peroxisome proliferator activating receptor alpha (PPARalpha), caused a significant increase in expression of both desaturases. The data suggested that D5D and D6D expression is dually regulated by SREBP-1c and PPARalpha, two reciprocal transcription factors for fatty acid metabolism, and could be involved in lipogenic gene regulation by producing PUFA.