Myeloid cell deletion of Aryl hydrocarbon Receptor Nuclear Translocator (ARNT) induces non-alcoholic steatohepatitis.

BACKGROUND AND AIM:Non-alcoholic steatohepatitis (NASH) is predicted to become the most common cause of cirrhosis and liver failure. Risk factors include obesity, insulin resistance and diabetes. Macrophages and other myeloid cells play crucial roles in initiating and driving inflammation. Aryl hydrocarbon Receptor Nuclear Translocator (ARNT) is a transcription factor which binds to a range of partners to mediate responses to environmental signals, including the diet. In people with diabetes it is decreased in liver. We hypothesised that myeloid cell ARNT activity may contribute to the development of liver pathology. METHODS:Floxed-ARNT mice were bred with LysM-Cre mice to generate mice with reduced ARNT in myeloid cells. Animals were fed a high fat diet (HFD) and liver pathology was assessed. Histology, mRNA, fat accumulation and metabolism were studied. RESULTS:Animals with reduced myeloid ARNT developed steatohepatitis on a HFD, with additional alterations of metabolism and fat deposition. Steatohepatitis was accompanied by hepatic macrophage infiltration and expression of both M1 and M2 markers. Expression of mRNAs for Cxcl1, Mcp-1, Tnf-α and Tgf-β1 were increased. Human livers from controls and people with NASH were tested; ARNT mRNA was decreased by 80% (p = 0.0004). CONCLUSIONS:Decreased myeloid ARNT may play a role in the conversion from non-alcoholic fatty liver to steatohepatitis. Increasing ARNT may be a therapeutic strategy to reduce NASH

Beta-cell ARNT is required for normal glucose tolerance in murine pregnancy

Aims:Insulin secretion increases in normal pregnancy to meet increasing demands. Inability to increase beta-cell function results in gestational diabetes mellitus (GDM). We have previously shown that the expression of the transcription factor ARNT (Aryl-hydrocarbon Receptor Nuclear Translocator) is reduced in the islets of humans with type 2 diabetes. Mice with a beta-cell specific deletion of ARNT (β-ARNT mice) have impaired glucose tolerance secondary to defective insulin secretion. We hypothesised that ARNT is required to increase beta-cell function during pregnancy, and that β-ARNT mice would be unable to compensate for the beta-cell stress of pregnancy. The aims of this study were to investigate the mechanisms of ARNT regulation of beta-cell function and glucose tolerance in pregnancy.Methods:β-ARNT females were mated with floxed control (FC) males and FC females with β-ARNT males.Results:During pregnancy, β-ARNT mice had a marked deterioration in glucose tolerance secondary to defective insulin secretion. There was impaired beta-cell proliferation in late pregnancy, associated with decreased protein and mRNA levels of the islet cell-cycle regulator cyclinD2. There was also reduced expression of Irs2 and G6PI. In contrast, in control mice, pregnancy was associated with a 2.1-fold increase in ARNT protein and a 1.6-fold increase in cyclinD2 protein, and with increased beta-cell proliferation.Conclusions:Islet ARNT increases in normal murine pregnancy and beta-cell ARNT is required for cyclinD2 induction and increased beta-cell proliferation in pregnancy

Data from: Hepatic Aryl hydrocarbon Receptor Nuclear Translocator (ARNT) regulates metabolism in mice

Background & Aims: Aryl hydrocarbon Receptor Nuclear Translocator (ARNT) and its partners hypoxia-inducible factors (HIF)-1α and HIF-2α are candidate factors for the well-known link between the liver, metabolic dysfunction and elevation in circulating lipids and glucose. Methods: Hepatocyte-specific ARNT-null (LARNT), HIF-1α-null (LHIF1α) and HIF-2α-null (LHIF2α) mice were created. Results: LARNT mice had increased fasting glucose, impaired glucose tolerance, increased glucose production, raised post-prandial serum triglycerides (TG) and markedly lower hepatic ATP versus littermate controls. There was increased expression of G6Pase, Chrebp, Fas and Scd-1 mRNAs in LARNT animals. Surprisingly, LHIF1α and LHIF2α mice exhibited no alterations in any metabolic parameter assessed. Conclusions: These results provide convincing evidence that reduced hepatic ARNT can contribute to inappropriate hepatic glucose production and post-prandial dyslipidaemia. Hepatic ARNT may be a novel therapeutic target for improving post-prandial hypertriglyceridemia and glucose homeostasis

Data from: Hepatic Aryl hydrocarbon Receptor Nuclear Translocator (ARNT) regulates metabolism in mice

Background & Aims: Aryl hydrocarbon Receptor Nuclear Translocator (ARNT) and its partners hypoxia-inducible factors (HIF)-1α and HIF-2α are candidate factors for the well-known link between the liver, metabolic dysfunction and elevation in circulating lipids and glucose. Methods: Hepatocyte-specific ARNT-null (LARNT), HIF-1α-null (LHIF1α) and HIF-2α-null (LHIF2α) mice were created. Results: LARNT mice had increased fasting glucose, impaired glucose tolerance, increased glucose production, raised post-prandial serum triglycerides (TG) and markedly lower hepatic ATP versus littermate controls. There was increased expression of G6Pase, Chrebp, Fas and Scd-1 mRNAs in LARNT animals. Surprisingly, LHIF1α and LHIF2α mice exhibited no alterations in any metabolic parameter assessed. Conclusions: These results provide convincing evidence that reduced hepatic ARNT can contribute to inappropriate hepatic glucose production and post-prandial dyslipidaemia. Hepatic ARNT may be a novel therapeutic target for improving post-prandial hypertriglyceridemia and glucose homeostasis

LARNT data

Data for LARNT pape

Islet histology in ß-ARNT and floxed control mothers.

<p>(A–B) Representative images of insulin immunofluorescence on islets from a floxed control (A) and ß-ARNT (B) mouse in late pregnancy. (C) Beta-cell mass in floxed control (FC) (n = 6) and ß-ARNT (n = 6) females in late pregnancy. p = ns, t-test. (D–E) Representative BrdU (red), insulin (green) and DAPI (blue) immunofluorescence on islets from a (D) floxed control and (E) ß-ARNT mouse in late pregnancy. (F) The BrdU labelling index was significantly decreased in insulin-positive islet cells of ß-ARNT versus floxed control (FC) dams (median 1.86 (1.72–2.55) versus 3.96 (1.86–6.6)) *p<0.05, Mann Whitney test). Data points represent individual mice. (G) The caspase labelling index was similar in insulin-positive islet cells of ß-ARNT versus floxed control dams (median 1.69 (0.48–1.26) versus 0.84 (0.56–3.01) p = NS, Mann Whitney test). Data points represent individual mice.</p

Timelines for the 4 cohorts of pregnant mice.

<p>GTT (glucose tolerance testing) was performed on all female mice prior to pregnancy. After timed mating, GTT was repeated on day 16.5 of gestation in cohort 1, and glucose-stimulated insulin secretion (GSIS) in cohort 2. Newborn pups in these 2 cohorts were weighed at day 1.5. Female offspring from cohort 1 were weighed weekly from weaning and timed mated at 10–12 weeks of age with a GTT at day 16.5. GTT and GSIS were performed prior to pregnancy. In cohort 3, BrdU was administered 16 hours before sacrifice at day 15.5 of gestation. In cohort 4, fetuses and islets were collected at day 16.5 of gestation.</p

Fetal and newborn phenotype.

<p>(A) Weight of floxed control (FC) and ß-ARNT fetuses from diabetic pregnancies (DP) (n = 27 ß-ARNT and 14 FC) and non diabetic pregnancies (NDP) (n = 28 ß-ARNT and 32 FC). **p<0.01, Ŧ = 0.07, t-test. Significant main effect of DP on offspring weight on 2-way ANOVA, p = 0.001. Bars represent mean±SEM. (B) Blood glucose levels in floxed control and ß-ARNT fetuses from diabetic pregnancies and non diabetic pregnancies. Δ = 0.12, t-test. Bars represent mean±SEM. (C) Plasma insulin levels in floxed control and ß-ARNT fetuses from diabetic pregnancies (n = 25 ß-ARNT and 13 FC) and non diabetic pregnancies (n = 14 ß-ARNT and 18 FC). Bars represent mean±SEM. (D) Weight of floxed control and ß-ARNT pups from diabetic pregnancies (for all pups: n = 74 from DP and 41 from NDP, for pups in which genotype was known: n = 19 FC pups from DP, 9 from NDP, n = 9 ß-ARNT pups from DP and 10 from DP). *p<0.05, t-test. Bars represent mean±SEM. For this figure, black bars represent diabetic pregnancies and grey bars represent non diabetic pregnancies.</p

Gene and protein expression: ARNT and cyclinD2.

<p>(A) Gene expression in β-ARNT islets (white bars) expressed as percentage of gene expression in floxed control islets (black bars) (n = 13 β-ARNT and 13 FC). ***p<0.001, **p<0.01, *p<0.05, Ŧ p = 0.06, Δ p = 0.08, t-test. Bars represent mean±SEM. (B) Western blot of ARNT in islets from non pregnant and pregnant wild type females (top) Western blot of cyclinD2 in islets from non pregnant and pregnant floxed control and β-ARNT females (bottom). (C) Quantitation of ARNT protein in wild type female islets, expressed as fold change of non pregnant islets (n = 6 vs. 6). *p = 0.048, t-test. Bars represent mean±SEM. (D) Quantitation of cyclinD2 protein in non pregnant and pregnant β-ARNT (white bars) and floxed control (black bars) islets, expressed as fold change of non pregnant floxed control islets (n = 4 vs. 4). *p = 0.02, **p = 0.002, t-test. Bars represent mean±SEM.</p