On-Chip Reconstitution of Uniformly Shear-Sensing 3D Matrix-embedded Multicellular Blood Microvessel.

Preclinical human-relevant modeling of organ-specific vasculature offers a unique opportunity to recreate pathophysiological intercellular, tissue-tissue, and cell-matrix interactions for a broad range of applications. Here, we present a reliable, and simply reproducible process for constructing user-controlled long rounded extracellular matrix (ECM)-embedded vascular microlumens on-chip for endothelization and co-culture with stromal cells obtained from human lung. We demonstrate the critical impact of microchannel cross-sectional geometry and length on uniform distribution and magnitude of vascular wall shear stress, which is key when emulating in vivo-observed blood flow biomechanics in health and disease. In addition, we provide an optimization protocol for multicellular culture and functional validation of the system. Moreover, we show the ability to finely tune rheology of the three-dimensional natural matrix surrounding the vascular microchannel to match pathophysiological stiffness. In summary, we provide the scientific community with a matrix-embedded microvasculature on-chip populated with all-primary human-derived pulmonary endothelial cells and fibroblasts to recapitulate and interrogate lung parenchymal biology, physiological responses, vascular biomechanics, and disease biogenesis in vitro. Such a mix-and-match synthetic platform can be feasibly adapted to study blood vessels, matrix, and ECM-embedded cells in other organs and be cellularized with additional stromal cells.

Long non-coding RNA G23Rik attenuates fasting-induced lipid accumulation in mouse liver.

Peroxisome proliferator-activated receptor α (PPARα) is a key mediator of lipid metabolism and metabolic stress in the liver. A recent study revealed that PPARα-dependent long non-coding RNAs (lncRNAs) play an important role in modulating metabolic stress and inflammation in the livers of fasted mice. Here hepatic lncRNA 3930402G23Rik (G23Rik) was found to have active peroxisome proliferator response elements (PPREs) within its promoter and is directly regulated by PPARα. Although G23Rik RNA was expressed to varying degrees in several tissues, the PPARα-dependent regulation of this lncRNA was only observed in the liver. Pharmacological activation of PPARα induced PPARα recruitment at the G23Rik promoter and a pronounced increase in hepatic G23Rik lncRNA expression. A G23Rik-null mouse line was developed to further characterize the function of this lncRNA in the liver. G23Rik-null mice were more susceptible to hepatic lipid accumulation in response to acute fasting. Histological analysis further revealed a pronounced buildup of lipid droplets and a significant increase in neutral triglycerides and lipids as indicated by enhanced oil red O staining of liver sections. Hepatic cholesterol, non-esterified fatty acid, and triglyceride levels were significantly elevated in G23Rik-null mice and associated with induction of the lipid-metabolism related gene Cd36. These findings provide evidence for a lncRNA dependent mechanism by which PPARα attenuates hepatic lipid accumulation in response to metabolic stress through lncRNA G23Rik induction.

Gene repression through epigenetic modulation by PPARA enhances hepatocellular proliferation.

Peroxisome proliferator-activated receptor α (PPARA) is a key mediator of lipid metabolism and inflammation. Activation of PPARA in rodents causes hepatocyte proliferation, but the underlying mechanism is poorly understood. This study focused on genes repressed by PPARA and analyzed the mechanism by which PPARA promotes hepatocyte proliferation in mice. Activation of PPARA by agonist treatment was autoregulated, and induced expression of the epigenetic regulator UHRF1 via activation of the newly described PPARA target gene E2f8, which, in turn, regulates Uhrf1. UHRF1 strongly repressed the expression of CDH1 via methylation of the Cdh1 promoter marked with H3K9me3. Repression of CDH1 by PPARA activation was reversed by PPARA deficiency or knockdown of E2F8 or UHRF1. Furthermore, a forced expression of CDH1 inhibited expression of the Wnt signaling target genes such as Myc after PPARA activation, and suppressed hepatocyte hyperproliferation. These results demonstrate that the PPARA-E2F8-UHRF1-CDH1 axis causes epigenetic regulation of hepatocyte proliferation.

Feedback repression of PPARα signaling by Let-7 microRNA.

Peroxisome proliferator-activated receptor α (PPARα) controls hepatic lipid homeostasis and is the target of lipid-lowering fibrate drugs. PPARα activation represses expression of let-7 microRNA (miRNA), but the function of let-7 in PPARα signaling and lipid metabolism is unknown. In the current study, a hepatocyte-specific let-7b/c2 knockout (let7b/c2ΔHep) mouse line is generated, and these mice are found to exhibit pronounced resistance to diet-induced obesity and fatty liver. Let-7 inhibition by hepatocyte-specific let-7 sponge expression shows similar phenotypes as let7b/c2ΔHep mice. RNA sequencing (RNA-seq) analysis reveals that hepatic PPARα signaling is repressed in let7b/c2ΔHep mice. Protein expression of the obligate PPARα heterodimer partner retinoid X receptor α (RXRα) is reduced in the livers of let7b/c2ΔHep mice. Ring finger protein 8 (Rnf8), which is a direct target of let-7, is elevated in let7b/c2ΔHep mouse liver and identified as a E3 ubiquitin ligase for RXRα. This study highlights a let-7-RNF8-RXRα regulatory axis that modulates hepatic lipid catabolism.

Long non-coding RNA Gm15441 attenuates hepatic inflammasome activation in response to PPARA agonism and fasting.

Exploring the molecular mechanisms that prevent inflammation during caloric restriction may yield promising therapeutic targets. During fasting, activation of the nuclear receptor peroxisome proliferator-activated receptor α (PPARα) promotes the utilization of lipids as an energy source. Herein, we show that ligand activation of PPARα directly upregulates the long non-coding RNA gene Gm15441 through PPARα binding sites within its promoter. Gm15441 expression suppresses its antisense transcript, encoding thioredoxin interacting protein (TXNIP). This, in turn, decreases TXNIP-stimulated NLR family pyrin domain containing 3 (NLRP3) inflammasome activation, caspase-1 (CASP1) cleavage, and proinflammatory interleukin 1ß (IL1B) maturation. Gm15441-null mice were developed and shown to be more susceptible to NLRP3 inflammasome activation and to exhibit elevated CASP1 and IL1B cleavage in response to PPARα agonism and fasting. These findings provide evidence for a mechanism by which PPARα attenuates hepatic inflammasome activation in response to metabolic stress through induction of lncRNA Gm15441.

Metabolomic profiling of metoprolol hypertension treatment reveals altered gut microbiota-derived urinary metabolites.

INTRODUCTION: Metoprolol succinate is a long-acting beta-blocker prescribed for the management of hypertension (HTN) and other cardiovascular diseases. Metabolomics, the study of end-stage metabolites of upstream biologic processes, yield insight into mechanisms of drug effectiveness and safety. Our aim was to determine metabolomic profiles associated with metoprolol effectiveness for the treatment of hypertension. METHODS: We performed a prospective pragmatic trial (NCT02293096) that enrolled patients between 30 and 80 years with uncontrolled HTN. Patients were started on metoprolol succinate at a dose based upon systolic blood pressure (SBP). Urine and blood pressure measurements were collected weekly. Individuals with a 10% decline in SBP or heart rate (HR) were considered responsive. Genotype for the CYP2D6 enzyme, the primary metabolic pathway for metoprolol, was evaluated for each subject. Unbiased metabolomic analyses were performed on urine samples using UPLC-QTOF mass spectrometry. RESULTS: Urinary metoprolol metabolite ratios are indicative of patient CYP2D6 genotypes. Patients taking metoprolol had significantly higher urinary levels of many gut microbiota-dependent metabolites including hydroxyhippuric acid, hippuric acid, and methyluric acid. Urinary metoprolol metabolite profiles of normal metabolizer (NM) patients more closely correlate to ultra-rapid metabolizer (UM) patients than NM patients. Metabolites did not predict either 10% SBP or HR decline. CONCLUSION: In summary, urinary metabolites predict CYP2D6 genotype in hypertensive patients taking metoprolol. Metoprolol succinate therapy affects the microbiome-derived metabolites.

Dietary Intake Regulates the Circulating Inflammatory Monocyte Pool.

Caloric restriction is known to improve inflammatory and autoimmune diseases. However, the mechanisms by which reduced caloric intake modulates inflammation are poorly understood. Here we show that short-term fasting reduced monocyte metabolic and inflammatory activity and drastically reduced the number of circulating monocytes. Regulation of peripheral monocyte numbers was dependent on dietary glucose and protein levels. Specifically, we found that activation of the low-energy sensor 5'-AMP-activated protein kinase (AMPK) in hepatocytes and suppression of systemic CCL2 production by peroxisome proliferator-activator receptor alpha (PPARα) reduced monocyte mobilization from the bone marrow. Importantly, we show that fasting improves chronic inflammatory diseases without compromising monocyte emergency mobilization during acute infectious inflammation and tissue repair. These results reveal that caloric intake and liver energy sensors dictate the blood and tissue immune tone and link dietary habits to inflammatory disease outcome.

Hepatocyte peroxisome proliferator-activated receptor α regulates bile acid synthesis and transport.

Peroxisome proliferator-activated receptor alpha (PPARα) controls lipid homeostasis through regulation of lipid transport and catabolism. PPARα activators are clinically used for hyperlipidemia treatment. The role of PPARα in bile acid (BA) homeostasis is beginning to emerge. Herein, Ppara-null and hepatocyte-specific Ppara-null (Ppara∆Hep) as well as the respective wild-type mice were treated with the potent PPARα agonist Wy-14,643 (Wy) and global metabolomics performed to clarify the role of hepatocyte PPARα in the regulation of BA homeostasis. Levels of all serum BAs were markedly elevated in Wy-treated wild-type mice but not in Ppara-null and Ppara∆Hep mice. Gene expression analysis showed that PPARα activation (1) down-regulated the expression of sodium-taurocholate acid transporting polypeptide and organic ion transporting polypeptide 1 and 4, responsible for the uptake of BAs into the liver; (2) decreased the expression of bile salt export pump transporting BA from hepatocytes into the bile canaliculus; (3) upregulated the expression of multidrug resistance-associated protein 3 and 4 transporting BA from hepatocytes into the portal vein. Moreover, there was a notable increase in the compositions of serum, hepatic and biliary cholic acid and taurocholic acid following Wy treatment, which correlated with the upregulated expression of the Cyp8b1 gene encoding sterol 12α-hydroxylase. The effects of Wy were identical between the Ppara∆Hep and Ppara-null mice. Hepatocyte PPARα controlled BA synthesis and transport not only via direct transcriptional regulation but also via crosstalk with hepatic farnesoid X receptor signaling. These findings underscore a key role for hepatocyte PPARα in the control of BA homeostasis.

Intestinal PPARα Protects Against Colon Carcinogenesis via Regulation of Methyltransferases DNMT1 and PRMT6.

BACKGROUND & AIMS: Many genetic and environmental factors, including family history, dietary fat, and inflammation, increase risk for colon cancer development. Peroxisome proliferator-activated receptor alpha (PPARα) is a nuclear receptor that regulates systemic lipid homeostasis. We explored the role of intestinal PPARα in colon carcinogenesis. METHODS: Colon cancer was induced in mice with intestine-specific disruption of Ppara (PparaΔIE), Pparafl/fl (control), and mice with disruption of Ppara that express human PPARA (human PPARA transgenic mice), by administration of azoxymethane with or without dextran sulfate sodium (DSS). Colons were collected from mice and analyzed by immunoblots, quantitative polymerase chain reaction, and histopathology. Liquid chromatography coupled with mass spectrometry-based metabolomic analyses were performed on urine and colons. We used molecular biology and biochemical approaches to study mechanisms in mouse colons, primary intestinal epithelial cells, and colon cancer cell lines. Gene expression data and clinical features of patients with colorectal tumors were obtained from Oncomine, and human colorectal-tumor specimens and adjacent normal tissues were collected and analyzed by immunohistochemistry. RESULTS: Levels of Ppara messenger RNA were reduced in colon tumors from mice. PparaΔIE mice developed more and larger colon tumors than control mice following administration of azoxymethane, with or without DSS. Metabolomic analyses revealed increases in methylation-related metabolites in urine and colons from PparaΔIE mice, compared with control mice, following administration of azoxymethane, with or without DSS. Levels of DNA methyltransferase 1 (DNMT1) and protein arginine methyltransferase 6 (PRMT6) were increased in colon tumors from PparaΔIE mice, compared with colon tumors from control mice. Depletion of PPARα reduced the expression of retinoblastoma protein, resulting in increased expression of DNMT1 and PRMT6. DNMT1 and PRMT6 decreased expression of the tumor suppressor genes Cdkn1a (P21) and Cdkn1b (p27) via DNA methylation and histone H3R2 dimethylation-mediated repression of transcription, respectively. Fenofibrate protected human PPARA transgenic mice from azoxymethane and DSS-induced colon cancer. Human colon adenocarcinoma specimens had lower levels of PPARA and retinoblastoma protein and higher levels of DNMT1 and PRMT6 than normal colon tissues. CONCLUSIONS: Loss of PPARα from the intestine promotes colon carcinogenesis by increasing DNMT1-mediated methylation of P21 and PRMT6-mediated methylation of p27 in mice. Human colorectal tumors have lower levels of PPARA messenger RNA and protein than nontumor tissues. Agents that activate PPARα might be developed for chemoprevention or treatment of colon cancer.

Keratin 23 Is a Peroxisome Proliferator-Activated Receptor Alpha-Dependent, MYC-Amplified Oncogene That Promotes Hepatocyte Proliferation.

Chronic activation of the nuclear receptor peroxisome proliferator-activated receptor alpha (PPARA) promotes MYC-linked hepatocellular carcinoma (HCC) in mice. Recent studies have shown that MYC can function as an amplifier of transcription where MYC does not act as an "on-off" switch for gene expression but rather accelerates transcription rates at active promoters by stimulating transcript elongation. Considering the possibility that MYC may amplify the expression of PPARA target genes to potentiate cell proliferation and liver cancer, gene expression was analyzed from livers of wild-type and liver-specific Myc knockout (MycΔHep ) mice treated with the PPARA agonist pirinixic acid. A subset of PPARA target genes was amplified in the presence of MYC, including keratin 23 (Krt23). The induction of Krt23 was significantly attenuated in MycΔHep mice and completely abolished in Ppara-null mice. Reporter gene assays and chromatin immunoprecipitation confirmed direct binding of both PPARA and MYC to sites within the Krt23 promoter. Forced expression of KRT23 in primary hepatocytes induced cell cycle-related genes. These data indicate that PPARA activation elevates MYC expression, which in turn potentiates the expression of select PPARA target genes involved in cell proliferation. Finally, KRT23 protein is highly elevated in human HCCs. Conclusion: These results revealed that MYC-mediated transcriptional potentiation of select PPARA target genes, such as Krt23, may remove rate-limiting constraints on hepatocyte growth and proliferation leading to liver cancer.

Hepatocyte Peroxisome Proliferator-Activated Receptor α Enhances Liver Regeneration after Partial Hepatectomy in Mice.

Peroxisome proliferator-activated receptor α (PPARα) is a key nuclear receptor involved in the control of lipid homeostasis. In rodents, PPARα is also a potent hepatic mitogen. Hepatocyte-specific disruption of PPARα inhibits agonist-induced hepatocyte proliferation; however, little is known about the exact role of PPARα in partial hepatectomy (PHx)-induced liver regeneration. Herein, using hepatocyte-specific PPARα-deficient (PparaΔHep) mice, the function of hepatocyte PPARα in PHx-induced liver regeneration was investigated. PPARα protein level and transcriptional activity were increased in the liver after PHx. Compared with the Pparafl/fl mice, PparaΔHep mice exhibited significantly reduced hepatocyte proliferation at 32 hours after PHx. Consistently, reduced Ccnd1 and Pcna mRNA and CYCD1 and proliferating cell nuclear antigen protein were observed at 32 hours after PHx in PparaΔHep mice. Furthermore, PparaΔHep mice showed increased hepatic lipid accumulation and enhanced hepatic triglyceride contents because of impaired hepatic fatty acid ß-oxidation when compared with that observed in Pparafl/fl mice. These results indicate that PPARα promotes liver regeneration after PHx, at least partially via regulating the cell cycle and lipid metabolism.

REVERBa couples the circadian clock to hepatic glucocorticoid action.

The glucocorticoid receptor (GR) is a major drug target in inflammatory disease. However, chronic glucocorticoid (GC) treatment leads to disordered energy metabolism, including increased weight gain, adiposity, and hepatosteatosis - all programs modulated by the circadian clock. We demonstrated that while antiinflammatory GC actions were maintained irrespective of dosing time, the liver was significantly more GC sensitive during the day. Temporal segregation of GC action was underpinned by a physical interaction of GR with the circadian transcription factor REVERBa and co-binding with liver-specific hepatocyte nuclear transcription factors (HNFs) on chromatin. REVERBa promoted efficient GR recruitment to chromatin during the day, acting in part by maintaining histone acetylation, with REVERBa-dependent GC responses providing segregation of carbohydrate and lipid metabolism. Importantly, deletion of Reverba inverted circadian liver GC sensitivity and protected mice from hepatosteatosis induced by chronic GC administration. Our results reveal a mechanism by which the circadian clock acts through REVERBa in liver on elements bound by HNF4A/HNF6 to direct GR action on energy metabolism.

Extrahepatic PPARα modulates fatty acid oxidation and attenuates fasting-induced hepatosteatosis in mice.

PPARα (PPARA), expressed in most oxidative tissues, is a major regulator of lipid homeostasis; hepatic PPARA plays a critical role during the adaptive fasting response by promoting FA oxidation (FAO). To clarify whether extrahepatic PPARA activity can protect against lipid overload when hepatic PPARA is impaired, lipid accumulation was compared in WT (Ppara+/+), total body Ppara-null (Ppara-/-), and hepatocyte-specific Ppara-null (PparaΔHep) mice that were fasted for 24 h. Histologic staining indicated reduced lipid accumulation in PparaΔHep versus Ppara-/- mice, and biochemical analyses revealed diminished medium- and long-chain FA accumulation in PparaΔHep mouse livers. Hepatic PPARA target genes were suppressed in both mouse models. Serum FFAs increased in all genotypes after fasting but were highest in Ppara-/- mice. In PparaΔHep mice, FAO genes were increased in brown adipose tissue, heart, and muscle, and total lipase activity was elevated in the muscle and heart, suggesting increased lipid utilization. Thus, extrahepatic PPARA activity reduces systemic lipid load when hepatic lipid metabolism is impaired by elevating FAO and lipase activity in other tissues and, as a result, protects against fasting-induced hepatosteatosis. This has important clinical implications in disease states with impaired hepatic PPARA function, such as nonalcoholic steatohepatitis and nonalcoholic fatty liver disease.

Hepatic peroxisome proliferator-activated receptor alpha mediates the major metabolic effects of Wy-14643.

BACKGROUND AND AIM: Peroxisome proliferator-activated receptor alpha (PPARα) is a molecular target of various fibrate drugs clinically used to lower serum lipids. However, the tissue-specific functions of PPARα remain to be elucidated. This study aimed to explore the tissue-specific functions of PPARα in response to Wy-14643. METHODS: A hepatocyte-specific Ppara knockout mouse line was used to explore the impact of hepatic PPARα activity on the systemic response to treatment with the potent PPARα agonist Wy-14643. RESULTS: Wy-14643 mainly activated hepatic PPARα and regulated the expression of PPARα target genes in liver. Hepatic Ppara disruption abolished the triglyceride lowering effects of Wy-14643, prevented agonist-induced hypophagia, and ablated PPARα target gene response in the liver. CONCLUSIONS: These findings indicate that Wy-14643 treatment mainly activates hepatic PPARα, and the hypolipidemic and hypophagic effects of Wy-14643 are dependent on PPARα activation within hepatocytes.

Metabolic adaptation to intermittent fasting is independent of peroxisome proliferator-activated receptor alpha.

BACKGROUND: Peroxisome proliferator-activated receptor alpha (PPARA) is a major regulator of fatty acid oxidation and severe hepatic steatosis occurs during acute fasting in Ppara-null mice. Thus, PPARA is considered an important mediator of the fasting response; however, its role in other fasting regiments such as every-other-day fasting (EODF) has not been investigated. METHODS: Mice were pre-conditioned using either a diet containing the potent PPARA agonist Wy-14643 or an EODF regimen prior to acute fasting. Ppara-null mice were used to assess the contribution of PPARA activation during the metabolic response to EODF. Livers were collected for histological, biochemical, qRT-PCR, and Western blot analysis. RESULTS: Acute fasting activated PPARA and led to steatosis, whereas EODF protected against fasting-induced hepatic steatosis without affecting PPARA signaling. In contrast, pretreatment with Wy-14,643 did activate PPARA signaling but did not ameliorate acute fasting-induced steatosis and unexpectedly promoted liver injury. Ppara ablation exacerbated acute fasting-induced hypoglycemia, hepatic steatosis, and liver injury in mice, whereas these detrimental effects were absent in response to EODF, which promoted PPARA-independent fatty acid metabolism and normalized serum lipids. CONCLUSIONS: These findings indicate that PPARA activation prior to acute fasting cannot ameliorate fasting-induced hepatic steatosis, whereas EODF induced metabolic adaptations to protect against fasting-induced steatosis without altering PPARA signaling. Therefore, PPARA activation does not mediate the metabolic adaptation to fasting, at least in preventing acute fasting-induced steatosis.

Intermittent Fasting Promotes White Adipose Browning and Decreases Obesity by Shaping the Gut Microbiota.

While activation of beige thermogenesis is a promising approach for treatment of obesity-associated diseases, there are currently no known pharmacological means of inducing beiging in humans. Intermittent fasting is an effective and natural strategy for weight control, but the mechanism for its efficacy is poorly understood. Here, we show that an every-other-day fasting (EODF) regimen selectively stimulates beige fat development within white adipose tissue and dramatically ameliorates obesity, insulin resistance, and hepatic steatosis. EODF treatment results in a shift in the gut microbiota composition leading to elevation of the fermentation products acetate and lactate and to the selective upregulation of monocarboxylate transporter 1 expression in beige cells. Microbiota-depleted mice are resistance to EODF-induced beiging, while transplantation of the microbiota from EODF-treated mice to microbiota-depleted mice activates beiging and improves metabolic homeostasis. These findings provide a new gut-microbiota-driven mechanism for activating adipose tissue browning and treating metabolic diseases.

Hepatocyte-specific PPARA expression exclusively promotes agonist-induced cell proliferation without influence from nonparenchymal cells.

Peroxisome proliferator-activated receptor-α (PPARA) is a nuclear transcription factor and key mediator of systemic lipid metabolism. Prolonged activation in rodents causes hepatocyte proliferation and hepatocellular carcinoma. Little is known about the contribution of nonparenchymal cells (NPCs) to PPARA-mediated cell proliferation. NPC contribution to PPARA agonist-induced hepatomegaly was assessed in hepatocyte (Ppara△Hep)- and macrophage (Ppara△Mac)-specific Ppara null mice. Mice were treated with the agonist Wy-14643 for 14 days, and response of conditional null mice was compared with conventional knockout mice (Ppara-/- ). Wy-14643 treatment caused weight loss and severe hepatomegaly in wild-type and Ppara△Mac mice, and histological analysis revealed characteristic hepatocyte swelling; Ppara△Hep and Ppara-/- mice were protected from these effects. Ppara△Mac serum chemistries, as well as aspartate aminotransferase and alanine aminotransferase levels, matched wild-type mice. Agonist-treated Ppara△Hep mice had elevated serum cholesterol, phospholipids, and triglycerides when compared with Ppara-/- mice, indicating a possible role for extrahepatic PPARA in regulating circulating lipid levels. BrdU labeling confirmed increased cell proliferation only in wild-type and Ppara△Mac mice. Macrophage PPARA disruption did not impact agonist-induced upregulation of lipid metabolism, cell proliferation, or DNA damage and repair-related gene expression, whereas gene expression was repressed in Ppara△Hep mice. Interestingly, downregulation of inflammatory cytokines IL-15 and IL-18 was dependent on macrophage PPARA. Cell type-specific regulation of target genes was confirmed in primary hepatocytes and Kupffer cells. These studies conclusively show that cell proliferation is mediated exclusively by PPARA activation in hepatocytes and that Kupffer cell PPARA has an important role in mediating the anti-inflammatory effects of PPARA agonists.

Diabetes-linked transcription factor HNF4α regulates metabolism of endogenous methylarginines and ß-aminoisobutyric acid by controlling expression of alanine-glyoxylate aminotransferase 2.

Elevated levels of circulating asymmetric and symmetric dimethylarginines (ADMA and SDMA) predict and potentially contribute to end organ damage in cardiovascular diseases. Alanine-glyoxylate aminotransferase 2 (AGXT2) regulates systemic levels of ADMA and SDMA, and also of beta-aminoisobutyric acid (BAIB)-a modulator of lipid metabolism. We identified a putative binding site for hepatic nuclear factor 4 α (HNF4α) in AGXT2 promoter sequence. In a luciferase reporter assay we found a 75% decrease in activity of Agxt2 core promoter after disruption of the HNF4α binding site. Direct binding of HNF4α to Agxt2 promoter was confirmed by chromatin immunoprecipitation assay. siRNA-mediated knockdown of Hnf4a led to an almost 50% reduction in Agxt2 mRNA levels in Hepa 1-6 cells. Liver-specific Hnf4a knockout mice exhibited a 90% decrease in liver Agxt2 expression and activity, and elevated plasma levels of ADMA, SDMA and BAIB, compared to wild-type littermates. Thus we identified HNF4α as a major regulator of Agxt2 expression. Considering a strong association between human HNF4A polymorphisms and increased risk of type 2 diabetes our current findings suggest that downregulation of AGXT2 and subsequent impairment in metabolism of dimethylarginines and BAIB caused by HNF4α deficiency might contribute to development of cardiovascular complications in diabetic patients.