Yatrik M. Shah

Intestinal Hypoxia Inducible Transcription Factors are Essential for Iron Absorption Following Iron Deficiency

Iron deficiency and iron overload are among the most prevalent nutritional disorders worldwide. Duodenal cytochrome b (DcytB) and divalent metal transporter 1 (DMT1) are regulators of iron absorption. Their expression is increased during high systemic requirements for iron, but the molecular mechanisms that regulate DcytB and DMT1 expression are undefined. Hypoxia-inducible factor (HIF) signaling was induced in the intestine following acute iron deficiency in the duodenum, resulting in activation of DcytB and DMT1 expression and an increase in iron uptake. DcytB and DMT1 were demonstrated as direct HIF-2alpha target genes. Genetic disruption of HIF signaling in the intestine abolished the adaptive induction of iron absorption following iron deficiency, resulting in low systemic iron and hematological defects. These results demonstrate that HIF signaling in the intestine is a critical regulator of systemic iron homeostasis.

Peroxisome Proliferator-Activated Receptor α Regulates a MicroRNA-Mediated Signaling Cascade Responsible for Hepatocellular Proliferation

ABSTRACT Activation of peroxisome proliferator-activated receptor α (PPARα) leads to hepatocellular proliferation and liver carcinomas. The early events mediating these effects are unknown. A novel mechanism by which PPARα regulates gene expression and hepatocellular proliferation was uncovered. MicroRNA (miRNA) expression profiling demonstrated that activated PPARα was a major regulator of hepatic miRNA expression. Of particular interest, let-7C, an miRNA important in cell growth, was inhibited following 4-h treatment and 2-week and 11-month sustained treatment with the potent PPARα agonist Wy-14,643 in wild-type mice. let-7C was shown to target c-myc via direct interaction with the 3′ untranslated region of c-myc. The PPARα-mediated induction of c-myc via let-7C subsequently increased expression of the oncogenic mir-17-92 cluster; these events did not occur in Pparα-null mice. Overexpression of let-7C decreased c-myc and mir-17 and suppressed the growth of Hepa-1 cells. Furthermore, using the human PPARα-expressing mouse model, which is responsive to Wy-14,643 effects on β-oxidation and serum triglycerides but resistant to hepatocellular proliferation and tumorigenesis, we demonstrated a critical role for let-7C in liver oncogenesis. Wy-14,643 treatment did not inhibit let-7C or induce c-myc and mir-17 expression. These observations reveal a let-7C signaling cascade critical for PPARα agonist-induced liver proliferation and tumorigenesis.

The role of peroxisome proliferator-activated receptors in carcinogenesis and chemoprevention

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that are involved in regulating glucose and lipid homeostasis, inflammation, proliferation and differentiation. Although all of these functions might contribute to the influence of PPARs in carcinogenesis, there is a distinct need for a review of the literature and additional experimentation to determine the potential for targeting PPARs for cancer therapy and cancer chemoprevention. As PPAR agonists include drugs that are used for the treatment of metabolic diseases, a more complete understanding of the roles of PPARs in cancer will aid in determining any increased cancer risk for patients undergoing therapy with PPAR agonists.

Disruption of Hypoxia-Inducible Factor 1 in Adipocytes Improves Insulin Sensitivity and Decreases Adiposity in High-Fat Diet–Fed Mice

OBJECTIVE Obesity, insulin resistance, and type 2 diabetes form a tightly correlated cluster of metabolic disorders in which adipose is one of the first affected tissues. The role of hypoxia and hypoxia-inducible factor 1 (HIF1) in the development of high-fat diet (HFD)–induced obesity and insulin resistance was investigated using animal models. RESEARCH DESIGN AND METHODS Mice with adipocyte-specific targeted disruption of the genes encoding the HIF1 obligatory subunits Hif1α or Arnt (Hif1β) were generated using an aP2-Cre transgene with the Cre/LoxP system. The mice were fed an HFD for 12 weeks and their metabolic phenotypes were determined. Gene expression patterns in adipose tissues were also determined by microarray and quantitative PCR. RESULTS On an HFD, adipocyte-specific ARNT knockout mice and adipocyte-specific HIF1α knockout mice exhibit similar metabolic phenotypes, including reduced fat formation, protection from HFD-induced obesity, and insulin resistance compared with similarly fed wild-type controls. The cumulative food intake remained similar; however, the metabolic efficiency was lower in adipocyte-specific HIF1α knockout mice. Moreover, indirect calorimetry revealed respiratory exchange ratios were reduced in adipocyte-specific HIF1α knockout mice. Hyperinsulinemic-euglycemic clamp studies demonstrated that targeted disruption of HIF1α in adipocytes enhanced whole-body insulin sensitivity. The improvement of insulin resistance is associated with decreased expression of Socs3 and induction of adiponectin. CONCLUSIONS Inhibition of HIF1 in adipose tissue ameliorates obesity and insulin resistance. This study reveals that HIF1 could provide a novel potential therapeutic target for obesity and type 2 diabetes.

Peroxisome proliferator activated receptor γ in colonic epithelial cells protects against experimental inflammatory bowel disease

Introduction: Peroxisome proliferator activated receptor γ (PPARγ) is expressed in epithelial cells, macrophage, and T and B lymphocytes. Ligand induced activation of PPARγ was reported to attenuate colitis activity but it is not clear whether this protection is mediated by epithelial or leucocyte PPARγ. Methods: Mice with targeted disruption of the PPARγ gene in intestinal epithelial cells, generated using a villin-Cre transgene and floxed PPARγ allele and designated PPARγΔIEpC, were compared with littermate mice having only the PPARγ floxed allele with no Cre transgene that expressed PPARγ in the gut, designated PPARγF/F. Colitis was induced by administering dextran sodium sulphate (DSS) and the two mouse lines compared for typical symptoms of disease and expression of inflammatory cytokines. Results: PPARγΔIEpC mice displayed reduced expression of the PPARγ target genes ADRP and FABP in the gut but were otherwise normal. Increased susceptibility to DSS induced colitis, as defined by body weight loss, colon length, diarrhoea, bleeding score, and altered histology, was found in PPARγΔIEpC mice in comparison with PPARγF/F mice. Interleukin (IL)-6, IL-1β, and tumour necrosis factor α mRNA levels in colons of PPARγΔIEpC mice treated with DSS were higher than in similarly treated PPARγF/F mice. The PPARγ ligand rosiglitazone decreased the severity of DSS induced colitis and suppressed cytokine production in both PPARγF/F and PPARγΔIEpC mice. Conclusions: These studies reveal that PPARγ expressed in the colonic epithelium has an endogenous role in protection against DSS induced colitis and that rosiglitazone may act through a PPARγ independent pathway to suppress inflammation.

Hypoxia-inducible factor-1α regulates β cell function in mouse and human islets

Hypoxia-inducible factor-1alpha (HIF-1alpha) is a transcription factor that regulates cellular stress responses. While the levels of HIF-1alpha protein are tightly regulated, recent studies suggest that it can be active under normoxic conditions. We hypothesized that HIF-1alpha is required for normal beta cell function and reserve and that dysregulation may contribute to the pathogenesis of type 2 diabetes (T2D). Here we show that HIF-1alpha protein is present at low levels in mouse and human normoxic beta cells and islets. Decreased levels of HIF-1alpha impaired glucose-stimulated ATP generation and beta cell function. C57BL/6 mice with beta cell-specific Hif1a disruption (referred to herein as beta-Hif1a-null mice) exhibited glucose intolerance, beta cell dysfunction, and developed severe glucose intolerance on a high-fat diet. Increasing HIF-1alpha levels by inhibiting its degradation through iron chelation markedly improved insulin secretion and glucose tolerance in control mice fed a high-fat diet but not in beta-Hif1a-null mice. Increasing HIF-1alpha levels markedly increased expression of ARNT and other genes in human T2D islets and improved their function. Further analysis indicated that HIF-1alpha was bound to the Arnt promoter in a mouse beta cell line, suggesting direct regulation. Taken together, these findings suggest an important role for HIF-1alpha in beta cell reserve and regulation of ARNT expression and demonstrate that HIF-1alpha is a potential therapeutic target for the beta cell dysfunction of T2D.

Serum Metabolomics Reveals Irreversible Inhibition of Fatty Acid β-Oxidation through the Suppression of PPARα Activation as a Contributing Mechanism of Acetaminophen-Induced Hepatotoxicity

Metabolic bioactivation, glutathione depletion, and covalent binding are the early hallmark events after acetaminophen (APAP) overdose. However, the subsequent metabolic consequences contributing to APAP-induced hepatic necrosis and apoptosis have not been fully elucidated. In this study, serum metabolomes of control and APAP-treated wild-type and Cyp2e1-null mice were examined by liquid chromatography-mass spectrometry (LC-MS) and multivariate data analysis. A dose-response study showed that the accumulation of long-chain acylcarnitines in serum contributes to the separation of wild-type mice undergoing APAP-induced hepatotoxicity from other mouse groups in a multivariate model. This observation, in conjunction with the increase of triglycerides and free fatty acids in the serum of APAP-treated wild-type mice, suggested that APAP treatment can disrupt fatty acid beta-oxidation. A time-course study further indicated that both wild-type and Cyp2e1-null mice had their serum acylcarnitine levels markedly elevated within the early hours of APAP treatment. While remaining high in wild-type mice, serum acylcarnitine levels gradually returned to normal in Cyp2e1-null mice at the end of the 24 h treatment. Distinct from serum aminotransferase activity and hepatic glutathione levels, the pattern of serum acylcarnitine accumulation suggested that acylcarnitines can function as complementary biomarkers for monitoring the APAP-induced hepatotoxicity. An essential role for peroxisome proliferator-activated receptor alpha (PPARalpha) in the regulation of serum acylcarnitine levels was established by comparing the metabolomic responses of wild-type and Ppara-null mice to a fasting challenge. The upregulation of PPARalpha activity following APAP treatment was transient in wild-type mice but was much more prolonged in Cyp2e1-null mice. Overall, serum metabolomics of APAP-induced hepatotoxicity revealed that the CYP2E1-mediated metabolic activation and oxidative stress following APAP treatment can cause irreversible inhibition of fatty acid oxidation, potentially through suppression of PPARalpha-regulated pathways.

Hypoxia-Inducible Factor Augments Experimental Colitis Through an MIF–Dependent Inflammatory Signaling Cascade

BACKGROUND & AIMS Colon epithelial cells are critical for barrier function and contain a highly developed immune response. A previous study has shown hypoxia-inducible factor (HIF) as a critical regulator of barrier protection during colon epithelial injury. However, the role of HIF signaling in colon mucosal immunity is not known. METHODS With the use of cre/loxP technology, intestinal-specific disruption of von Hippel-Lindau tumor suppressor protein (Vhl), hypoxia-inducible factor (Hif)-1alpha, and aryl hydrocarbon nuclear translocator (Arnt) was generated. Colon inflammation was induced using a dextran sulfate sodium (DSS)-induced colitis model, and the mice were analyzed by histologic analysis, Western blot analysis, and quantitative polymerase chain reaction. RESULTS In mice, colonic epithelium disruption of Vhl resulted in constitutive expression of HIF, which initiated an increase in inflammatory infiltrates and edema in the colon. These effects were ameliorated in mice by disruption of both Vhl and Arnt/Hif1beta (which inactivates HIF). In a DSS-induced colitis model, increased HIF expression correlated with more severe clinical symptoms and an increase in histologic damage, while disruption of both Vhl and Arnt in the colon epithelium inhibited these effects. Furthermore, colons with constitutive activation of HIF displayed increased expression of proinflammatory mediators that were synergistically potentiated following DSS administration and reduced by inhibition of the proinflammatory and direct HIF target gene macrophage migration inhibitory factor. CONCLUSIONS The present study shows that a chronic increase in HIF signaling in the colon epithelial cells initiates a hyperinflammatory reaction that may have important implications in developing therapeutic strategies for inflammatory bowel disease.

Hypoxia-Inducible Factor-2α Mediates the Adaptive Increase of Intestinal Ferroportin During Iron Deficiency in Mice

BACKGROUND & AIMS Iron deficiency and iron overload affect over a billion people worldwide. Dietary iron absorption in the small intestine is required for systemic iron homeostasis. Ferroportin (FPN) is the only characterized, mammalian, basolateral iron exporter. Despite the importance of FPN in maintaining iron homeostasis, its in vivo mechanisms of regulation are unclear. METHODS Systemic iron homeostasis was assessed in mice with intestine-specific disruption of genes encoding the von Hippel-Lindau tumor suppressor protein (Vhl), hypoxia-inducible factor (HIF)-1α, HIF-2α, and aryl hydrocarbon nuclear translocator (ARNT). RESULTS We observed biphasic regulation of Fpn during iron deficiency. Fpn was rapidly induced under conditions of low iron, which required the transcription factor HIF-2α. Targeted disruption of HIF-2α in the intestine inhibited Fpn induction in mice with low iron, through loss of transcriptional activation. Analysis of the Fpn promoter and in vivo chromatin immunoprecipitation assays demonstrated that HIF-2α directly binds to the Fpn promoter and induces its expression, indicating a mechanism of transcriptional regulation of Fpn following changes in systemic levels of iron. During chronic iron deficiency, FPN protein levels also increased, via increased stability through a HIF-2α-independent pathway. CONCLUSIONS In mice, expression of the gene that encodes Fpn and its protein levels are regulated by distinct pathways to provide a rapid and sustained response to acute and chronic iron deficiency. Therapies that target FPN might be developed for patients with iron-related disorders.

Metabolomics Reveals that Hepatic Stearoyl-CoA Desaturase 1 Downregulation Exacerbates Inflammation and Acute Colitis

To investigate the pathogenic mechanism of ulcerative colitis, a dextran sulfate sodium (DSS)-induced acute colitis model was examined by serum metabolomic analysis. Higher levels of stearoyl lysophosphatidylcholine and lower levels of oleoyl lysophosphatidylcholine in DSS-treated mice compared to controls led to the identification of DSS-elicited inhibition of stearoyl-CoA desaturase 1 (SCD1) expression in liver. This decrease occurred prior to the symptoms of acute colitis and was well correlated with elevated expression of proinflammatory cytokines. Furthermore, Citrobacter rodentium-induced colitis and lipopolysaccharide treatment also suppressed SCD1 expression in liver. Scd1 null mice were more susceptible to DSS treatment than wild-type mice, while oleic acid feeding and in vivo SCD1 rescue with SCD1 adenovirus alleviated the DSS-induced phenotype. This study reveals that inhibition of SCD1-mediated oleic acid biogenesis exacerbates proinflammatory responses to exogenous challenges, suggesting that SCD1 and its related lipid species may serve as potential targets for intervention or treatment of inflammatory diseases.