Interactions Between Nuclear Receptor SHP and FOXA1 Maintain Oscillatory Homocysteine Homeostasis in Mice.

BACKGROUND & AIMS: Hyperhomocysteinemia is often associated with liver and metabolic diseases. We studied nuclear receptors that mediate oscillatory control of homocysteine homeostasis in mice. METHODS: We studied mice with disruptions in Nr0b2 (called small heterodimer partner [SHP]-null mice), betaine-homocysteine S-methyltransferase (Bhmt), or both genes (BHMT-null/SHP-null mice), along with mice with wild-type copies of these genes (controls). Hyperhomocysteinemia was induced by feeding mice alcohol (National Institute on Alcohol Abuse and Alcoholism binge model) or chow diets along with water containing 0.18% DL-homocysteine. Some mice were placed on diets containing cholic acid (1%) or cholestyramine (2%) or high-fat diets (60%). Serum and livers were collected during a 24-hour light-dark cycle and analyzed by RNA-seq, metabolomic, and quantitative polymerase chain reaction, immunoblot, and chromatin immunoprecipitation assays. RESULTS: SHP-null mice had altered timing in expression of genes that regulate homocysteine metabolism compared with control mice. Oscillatory production of S-adenosylmethionine, betaine, choline, phosphocholine, glyceophosphocholine, cystathionine, cysteine, hydrogen sulfide, glutathione disulfide, and glutathione, differed between SHP-null mice and control mice. SHP inhibited transcriptional activation of Bhmt and cystathionine γ-lyase by FOXA1. Expression of Bhmt and cystathionine γ-lyase was decreased when mice were fed cholic acid but increased when they were placed on diets containing cholestyramine or high-fat content. Diets containing ethanol or homocysteine induced hyperhomocysteinemia and glucose intolerance in control, but not SHP-null, mice. In BHMT-null and BHMT-null/SHP-null mice fed a control liquid, lipid vacuoles were observed in livers. Ethanol feeding induced accumulation of macrovesicular lipid vacuoles to the greatest extent in BHMT-null and BHMT-null/SHP-null mice. CONCLUSIONS: Disruption of Shp in mice alters timing of expression of genes that regulate homocysteine metabolism and the liver responses to ethanol and homocysteine. SHP inhibits the transcriptional activation of Bhmt and cystathionine γ-lyase by FOXA1.

Small heterodimer partner/neuronal PAS domain protein 2 axis regulates the oscillation of liver lipid metabolism.

UNLABELLED: In mammals, circadian rhythms are essential for coordinating the timing of various metabolic processes. The Clock gene regulates diurnal plasma triglyceride fluctuation through nuclear receptor small heterodimer partner (Shp; Nr0b2). Given that SHP is a critical regulator of metabolism in the liver, it is unknown whether SHP is necessary to coordinate metabolism and circadian rhythms. Shp(+/+) and Shp(-/-) mice on a C57BL/6 background (n = 3-5/group) were fed a standard chow diet and water ad libitum. Serum and livers were collected at zeitgeber time 2, 6, 10, 14, 18, and 22. In vivo and in vitro assays included RNA sequencing, quantitative polymerase chain reaction, very-low-density lipoprotein production, adenovirus overexpression and small interfering RNA knockdown, serum parameters, circadian locomotor activity, Oil Red O staining, transient transfection, luciferase reporter assay, chromatin immunoprecipitation assay, gel-shift assay, coimmunoprecipitation, and western blottings. Shp deficiency had a robust global impact on major liver metabolic genes. Several components of the liver clock, including peroxisome proliferator-activated receptor-γ, coactivator 1 (Pgc-1α), neuronal PAS domain-containing protein 2 (Npas2), and retinoic acid-related orphan receptor (Ror)α/γ were sharply induced in Shp(-/-) liver. At the molecular level, SHP inhibited Npas2 gene transcription and promoter activity through interaction with Rorγ to repress Rorγ transactivation and by interacting with Rev-erbα to enhance its inhibition of Rorα activity. Conversely, Npas2 controlled the circadian rhythm of Shp expression by binding rhythmically to the Shp promoter, which was enhanced by nicotinamide adenine dinucleotide, but not nicotinamide adenine dinucleotide phosphate. Phenotypically, Npas2 deficiency induced severe steatosis in Shp(-/-) mice, which was attributed to the dysregulation of lipoprotein metabolism. CONCLUSION: Shp and Npas2 crosstalk is essential to maintain hepatic lipid homeostasis.

Genome-wide transcriptome analysis identifies novel gene signatures implicated in human chronic liver disease.

The molecular mechanisms behind human liver disease progression to cirrhosis remain elusive. Nuclear receptor small heterodimer partner (SHP/Nr0b2) is a hepatic tumor suppressor and a critical regulator of liver function. SHP expression is diminished in human cirrhotic livers, suggesting a regulatory role in human liver diseases. The goal of this study was to identify novel SHP-regulated genes that are involved in the development and progression of chronic liver disease. To achieve this, we conducted the first comprehensive RNA sequencing (RNA-seq) analysis of Shp(-/-) mice, compared the results with human hepatitis C cirrhosis RNA-seq and nonalcoholic steatohepatitis (NASH) microarray datasets, and verified novel results in human liver biospecimens. This approach revealed new gene signatures associated with chronic liver disease and regulated by SHP. Several genes were selected for validation of physiological relevance based on their marked upregulation, novelty with regard to liver function, and involvement in gene pathways related to liver disease. These genes include peptidoglycan recognition protein 2, dual specific phosphatase-4, tetraspanin 4, thrombospondin 1, and SPARC-related modular calcium binding protein-2, which were validated by qPCR analysis of 126 human liver specimens, including steatosis, fibrosis, and NASH, alcohol and hepatitis C cirrhosis, and in mouse models of liver inflammation and injury. This RNA-seq analysis identifies new genes that are regulated by the nuclear receptor SHP and implicated in the molecular pathogenesis of human chronic liver diseases. The results provide valuable transcriptome information for characterizing mechanisms of these diseases.

Aminopyridine analogs selectively target metastatic pancreatic cancer.

Metastatic outgrowth is supported by metabolic adaptations that may differ from the primary tumor of origin. However, it is unknown if such adaptations are therapeutically actionable. Here we report a novel aminopyridine compound that targets a unique Phosphogluconate Dehydrogenase (PGD)-dependent metabolic adaptation in distant metastases from pancreatic cancer patients. Compared to structurally similar analogs, 6-aminopicolamine (6AP) potently and selectively reversed PGD-dependent metastatic properties, including intrinsic tumorigenic capacity, excess glucose consumption, and global histone hyperacetylation. 6AP acted as a water-soluble prodrug that was converted into intracellular bioactive metabolites that inhibited PGD in vitro, and 6AP monotherapy demonstrated anti-metastatic efficacy with minimal toxicity in vivo. Collectively, these studies identify 6AP and possibly other 6-aminopyridines as well-tolerated prodrugs with selectivity for metastatic pancreatic cancers. If unique metabolic adaptations are a common feature of metastatic or otherwise aggressive human malignancies, then such dependencies could provide a largely untapped pool of druggable targets for patients with advanced cancers.

The role of SHP/REV-ERBα/CYP4A axis in the pathogenesis of alcohol-associated liver disease.

Alcohol-associated liver disease (ALD) represents a spectrum of histopathological changes, including alcoholic steatosis, steatohepatitis, and cirrhosis. One of the early responses to excessive alcohol consumption is lipid accumulation in the hepatocytes. Lipid ω-hydroxylation of medium- and long-chain fatty acid metabolized by the cytochrome P450 4A (CYP4A) family is an alternative pathway for fatty acid metabolism. The molecular mechanisms of CYP4A in ALD pathogenesis have not been elucidated. In this study, WT and Shp-/- mice were fed with a modified ethanol-binge, National Institute on Alcohol Abuse and Alcoholism model (10 days of ethanol feeding plus single binge). Liver tissues were collected every 6 hours for 24 hours and analyzed using RNA-Seq. The effects of REV-ERBα agonist (SR9009, 100 mg/kg/d) or CYP4A antagonist (HET0016, 5 mg/kg/d) in ethanol-fed mice were also evaluated. We found that hepatic Cyp4a10 and Cyp4a14 expression were significantly upregulated in WT mice, but not in Shp-/- mice, fed with ethanol. ChIP quantitative PCR and promoter assay revealed that REV-ERBα is the transcriptional repressor of Cyp4a10 and Cyp4a14. Rev-Erbα-/- hepatocytes had a marked induction of both Cyp4a genes and lipid accumulation. REV-ERBα agonist SR9009 or CYP4A antagonist HET0016 attenuated Cyp4a induction by ethanol and prevented alcohol-induced steatosis. Here, we have identified a role for the SHP/REV-ERBα/CYP4A axis in the pathogenesis of ALD. Our data also suggest REV-ERBα or CYP4A as the potential therapeutic targets for ALD.

Pancreatic cancers suppress negative feedback of glucose transport to reprogram chromatin for metastasis.

Although metastasis is the most common cause of cancer deaths, metastasis-intrinsic dependencies remain largely uncharacterized. We previously reported that metastatic pancreatic cancers were dependent on the glucose-metabolizing enzyme phosphogluconate dehydrogenase (PGD). Surprisingly, PGD catalysis was constitutively elevated without activating mutations, suggesting a non-genetic basis for enhanced activity. Here we report a metabolic adaptation that stably activates PGD to reprogram metastatic chromatin. High PGD catalysis prevents transcriptional up-regulation of thioredoxin-interacting protein (TXNIP), a gene that negatively regulates glucose import. This allows glucose consumption rates to rise in support of PGD, while simultaneously facilitating epigenetic reprogramming through a glucose-fueled histone hyperacetylation pathway. Restoring TXNIP normalizes glucose consumption, lowers PGD catalysis, reverses hyperacetylation, represses malignant transcripts, and impairs metastatic tumorigenesis. We propose that PGD-driven suppression of TXNIP allows pancreatic cancers to avidly consume glucose. This renders PGD constitutively activated and enables metaboloepigenetic selection of additional traits that increase fitness along glucose-replete metastatic routes.

Autophagy Ablation in Adipocytes Induces Insulin Resistance and Reveals Roles for Lipid Peroxide and Nrf2 Signaling in Adipose-Liver Crosstalk.

Autophagy is a homeostatic cellular process involved in the degradation of long-lived or damaged cellular components. The role of autophagy in adipogenesis is well recognized, but its role in mature adipocyte function is largely unknown. We show that the autophagy proteins Atg3 and Atg16L1 are required for proper mitochondrial function in mature adipocytes. In contrast to previous studies, we found that post-developmental ablation of autophagy causes peripheral insulin resistance independently of diet or adiposity. Finally, lack of adipocyte autophagy reveals cross talk between fat and liver, mediated by lipid peroxide-induced Nrf2 signaling. Our data reveal a role for autophagy in preventing lipid peroxide formation and its transfer in insulin-sensitive peripheral tissues.

Hepatocytes Are the Principal Source of Circulating RBP4 in Mice.

RBP4 is produced mainly by hepatocytes. In type 2 diabetes and obesity, circulating RBP4 is increased and may act systemically to cause insulin resistance and glucose intolerance. Observations that adipocyte RBP4 mRNA increases in parallel with circulating RBP4 in these conditions, whereas liver RBP4 mRNA does not, led to a widely held hypothesis that elevated circulating RBP4 is a direct result of increased production by adipocytes. To test this, we generated mice with hepatocyte-specific deletion of RBP4 (liver RBP4 knockout or LRKO mice). Adipose tissue RBP4 expression and secretion remained intact in LRKO mice and increased as expected in the setting of diet-induced insulin resistance. However, circulating RBP4 was undetectable in LRKO mice. We conclude that adipocyte RBP4 is not a significant source of circulating RBP4, even in the setting of insulin resistance. Adipocyte RBP4, therefore, may have a more important autocrine or paracrine function that is confined within the adipose tissue compartment.

Bcl2 is a critical regulator of bile acid homeostasis by dictating Shp and lncRNA H19 function.

Bile acid (BA) metabolism is tightly controlled by nuclear receptor signaling to coordinate regulation of BA synthetic enzymes and transporters. Here we reveal a molecular cascade consisting of the antiapoptotic protein BCL2, nuclear receptor Shp, and long non-coding RNA (lncRNA) H19 to maintain BA homeostasis. Bcl2 was overexpressed in liver of C57BL/6J mice using adenovirus mediated gene delivery for two weeks. Hepatic overexpression of Bcl2 caused drastic accumulation of serum BA and bilirubin levels and dysregulated BA synthetic enzymes and transporters. Bcl2 reactivation triggered severe liver injury, fibrosis and inflammation, which were accompanied by a significant induction of H19. Bcl2 induced rapid SHP protein degradation via the activation of caspase-8 pathway. The induction of H19 in Bcl2 overexpressed mice was contributed by a direct loss of Shp transcriptional repression. H19 knockdown or Shp re-expression largely rescued Bcl2-induced liver injury. Strikingly different than Shp, the expression of Bcl2 and H19 was hardly detectable in adult liver but was markedly increased in fibrotic/cirrhotic human and mouse liver. We demonstrated for the first time a detrimental effect of Bcl2 and H19 associated with cholestatic liver fibrosis and an indispensable role of Shp to maintain normal liver function.

Molecular analysis of colorectal tumors within a diverse patient cohort at a single institution.

PURPOSE: African American colorectal cancer patients have worse survival outcomes than Caucasian patients. To determine whether differences exist in the molecular mechanisms driving colorectal cancer between African Americans and Caucasians, we characterized patient tumors from a single institution by assessing genetic alterations involved in colorectal cancer progression and response to treatment. EXPERIMENTAL DESIGN: We retrospectively examined 448 African Americans and Caucasians diagnosed with colorectal cancer at The University of Chicago Medical Center between 1992 and 2002. Microsatellite instability (MSI) status was determined by genotyping the BAT25, BAT26, BAT40, D5S346, and BAX loci. Mutations in KRAS codons 12 and 13 and BRAF codon 600 were identified by direct sequencing. MSI and detected mutations were correlated with clinicopathologic features. RESULTS: Overall, no difference existed in MSI or BRAF mutation frequencies between African Americans and Caucasians. However, African Americans with microsatellite stable (MSS)/MSI-low (MSI-L) tumors had a higher proportion of KRAS mutations than Caucasians (34% vs. 23%, P = 0.048) that was isolated to proximal colon cancers and primarily driven by mutations in codon 13. There was no racial difference in receipt of chemotherapy, but African Americans with MSS/MSI-L tumors had a 73% increased risk of death over Caucasians that could not be explained by known prognostic factors. CONCLUSIONS: The significantly higher risk of death among African Americans with MSS/MSI-L tumors may be related to differences in the distribution of factors influencing response to standard therapies. These data underscore the need for further research into the molecular mechanisms driving colorectal cancer progression in underserved and understudied populations.