Hypoxia-inducible factor-1α (HIF-1α) is a transcription factor that regulates cellular stress responses. While the levels of HIF-1α protein are tightly regulated, recent studies suggest that it can be active under normoxic conditions. We hypothesized that HIF-1α is required for normal β cell function and reserve and that dysregulation may contribute to the pathogenesis of type 2 diabetes (T2D). Here we show that HIF-1α protein is present at low levels in mouse and human normoxic β cells and islets. Decreased levels of HIF-1α impaired glucosestimulated ATP generation and β cell function. C57BL/6 mice with β cell-specific Hif1a disruption (referred to herein as β-Hif1a-null mice) exhibited glucose intolerance, β cell dysfunction, and developed severe glucose intolerance on a high-fat diet. Increasing HIF-1α 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 β-Hif1a-null mice. Increasing HIF-1α levels markedly increased expression of ARNT and other genes in human T2D islets and improved their function. Further analysis indicated that HIF-1α was bound to the Arnt promoter in a mouse β cell line, suggesting direct regulation. Taken together, these findings suggest an important role for HIF-1α in β cell reserve and regulation of ARNT expression and demonstrate that HIF-1α is a potential therapeutic target for the β cell dysfunction of T2D. IntroductionThe transcription factor HIF-1α is important for a range of functions, including cellular responses to hypoxia and other stressors, angiogenesis, and fetal development (1-6). It has strong antiapoptotic effects (7-11) and is implicated in the pathogenesis of cardiovascular diseases and some cancers (12)(13)(14)(15)(16)(17)(18)(19)(20).HIF-1α is a member of the bHLH-PAS family (reviewed in refs. 2, 18, 21) and functions as an obligate dimer with other family members, including aryl hydrocarbon receptor (AhR) nuclear translocator (ARNT). We previously reported that ARNT was decreased in islets isolated from patients with type 2 diabetes (T2D) and that decreasing ARNT in Min6 cells or disrupting it in mouse β cells caused changes in gene expression and glucose-stimulated insulin secretion (GSIS) similar to those seen in islets isolated from humans with T2D (22). Recently, we reported a loss of ARNT expression in the livers of people with T2D, affecting dysregulation of gluconeogenesis (23). Though the specific ARNT partner which is important for its actions in β cells (or liver) is not known, candidates include AhR, HIF-1α, HIF-2α, HIF-3α, and circadian rhythm molecules, e.g., BMAL.

Previously, we and others have demonstrated the association of a C/T single nucleotide polymorphism (SNP), in the Kozak sequence of CD40, with Graves' disease (GD). Here, using an expanded data set of patients, we confirm the association of the CD40 SNP with GD (n ¼ 210, P ¼ 0.002, odds ratio (OR) ¼ 1.8). Subset analysis of patients with persistently elevated thyroid peroxidase (TPO) and/or thyroglobulin (Tg) antibodies (Abs), (TPO/Tg Abs), after treatment (n ¼ 126), revealed a significantly stronger association of the SNP with disease (P ¼ 5.2 Â 10 À5 , OR ¼ 2.5) than in GD patients who were thyroid antibodynegative. However, the CD40 SNP was not associated with TPO/Tg Abs in healthy individuals. Next, we tested the CD40 SNP for association with Myasthenia Gravis (MG), which, like GD is an antibody-mediated autoimmune condition. Analysis of 81 MG patients found no association of the SNP with disease. Functional studies revealed significant expression of CD40 mRNA and protein in the thyroid (target tissue in GD) but not in skeletal muscle (target tissue in MG). Combined, our genetic and tissue expression data suggest that the CD40 Kozak SNP is specific for thyroid antibody production involved in the etiology of GD. Increased thyroidal expression of CD40 driven by the SNP may contribute to this disease specificity.

Autoimmune thyroid diseases (AITD) arise from complex interactions between genetic, epigenetic, and environmental factors. Whole genome linkage scans and association studies have established thyroglobulin (TG) as a major AITD susceptibility gene. However, the causative TG variants and the pathogenic mechanisms are unknown. Here, we describe a genetic/ epigenetic mechanism by which a newly identified TG promoter single-nucleotide polymorphism (SNP) variant predisposes to AITD. Sequencing analyses followed by case control and familybased association studies identified an SNP (؊1623A3 G) that was associated with AITD in the Caucasian population (p ‫؍‬ 0.006). We show that the nucleotide substitution introduced by SNP (؊1623A/G) modified a binding site for interferon regulatory factor-1 (IRF-1), a major interferon-induced transcription factor. Using chromatin immunoprecipitation, we demonstrated that IRF-1 binds to the 5 TG promoter motif, and the transcription factor binding correlates with active chromatin structure and is marked by enrichment of mono-methylated Lys-4 residue of histone H3, a signature of active transcriptional enhancers. Using reporter mutations and siRNA approaches, we demonstrate that the disease-associated allele (G) conferred increased TG promoter activity through IRF-1 binding. Finally, treatment of thyroid cells with interferon ␣, a known trigger of AITD, increased TG promoter activity only when it interacted with the disease-associated variant through IRF-1 binding. These results reveal a new mechanism of interaction between environmental (IFN␣) and genetic (TG) factors to trigger AITD.Autoimmune thyroid diseases (AITD), 4 including Graves disease (GD) and Hashimoto thyroiditis (HT), are characterized by infiltration of the thyroid by T and B cells that react with local antigens leading to immune destruction of the thyroid in HT and production of thyroid-stimulating hormone receptor (TSHR) antibodies in GD. These result in the clinical manifestations of hypothyroidism in HT and hyperthyroidism in GD (1, 2). There is solid evidence that interactions between susceptibility genes and environmental triggers activate the sequence of cellular and humoral immune responses to thyroid antigens that cause AITD (1, 3, 4). Several environmental factors, including exposure to excess iodine, selenium deficiency, various infectious diseases, certain drugs, and pollutants have been associated with AITD (5, 6). Among these factors, interferon ␣ (INF␣), a therapeutic agent widely used for the treatment of chronic hepatitis C infection, has recently emerged as a major factor that triggers AITD (7,8).To date, several gene loci have been associated with AITD, including immune genes (HLA-DR, CTLA-4, CD40, FOXP3, and CD25) and thyroid-specific genes (TSHR and TG). Whole genome linkage screens, performed by our group (9) and others (10), have shown that the thyroglobulin (TG) locus on chromosome 8q24 is strongly linked with AITD. Moreover, TG has emerged as the only thyroid-specific gene that confers susceptibili...

Aim: To evaluate the efficacy and safety of an annual intramuscular injection of cholecalciferol for vitamin D deficiency. Design: Prospective open‐label study. Participants: Five men and 45 women (mean age 66.3 years) with vitamin D deficiency who were given a single therapeutic intramuscular injection of 600 000 IU (15 mg) cholecalciferol (vitamin D3). Outcome measures: Serum levels of calcium, creatinine, 25‐hydroxyvitamin D3 (25OHD3) and parathyroid hormone, as well as early morning 2‐hour urine calcium/creatinine excretion index. Specimens were collected at baseline and after 4 and 12 months of therapy. Data are reported as mean ± 1 SD. Results: Vitamin D deficiency was severe (< 12.5 nmol/L) in one participant, moderate (12.5–24 nmol/L) in 14, and mild (25–49 nmol/L) in 35. Twenty‐four participants (48%) had secondary hyperparathyroidism. Following intramuscular cholecalciferol injection, serum 25OHD3 levels normalised in all participants and remained above 50 nmol/L throughout the study. Serum 25OHD3 levels were significantly higher at 4 months (114 ± 35 nmol/L), and 12 months (73 ± 13 nmol/L) compared with baseline (32 ± 8 nmol/L) (P < 0.001), increasing by an average of 128% over the 12 months. There was a corresponding decrease in serum parathyroid hormone levels at 4 months (6 ± 3 pmol/L) and at 12 months (5.2 ± 3 pmol/L), with a 30% decrease at 12 months from baseline (7.4 ± 4 pmol/L) (P < 0.01). Primary hyperparathyroidism was unmasked in one participant at 4 months and mild hypercalcaemia (serum calcium, < 2.70 mmol/L) was noted in two participants (4%) at 12 months. Serum creatinine levels remained normal in all participants throughout the study, while increases in 2‐hour urine calcium/creatinine excretion index were seen in 10 participants (20%) at 12 months, three of whom had had elevated values at baseline. Conclusions: Once‐yearly intramuscular cholecalciferol injection (600 000 IU) is effective therapy for vitamin D deficiency. While this therapy appears to be safe, the potential for developing hypercalciuria needs to be examined in a large randomised controlled trial.

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Niemann-Pick type C1 (Npc1) protein inactivation results in lipid accumulation in late endosomes and lysosomes, leading to a defect of ATP binding cassette protein A1 (Abca1)-mediated lipid efflux to apolipoprotein A-I (apoA-I) in macrophages and fibroblasts. However, the role of Npc1 in Abca1-mediated lipid efflux to apoA-I in hepatocytes, the major cells contributing to HDL formation, is still unknown. Here we show that, whereas lipid efflux to apoA-I in Npc1-null macrophages is impaired, the lipidation of endogenously synthesized apoA-I by low density lipoprotein-derived cholesterol or de novo synthesized cholesterol or phospholipids in Npc1-null hepatocytes is significantly increased by about 1-, 3-, and 8-fold, respectively. The increased cholesterol efflux reflects a major increase of Abca1 protein in Npc1-null hepatocytes, which contrasts with the decrease observed in Npc1-null macrophages. The increased Abca1 expression is largely post-transcriptional, because Abca1 mRNA is only slightly increased and Lxr␣ mRNA is not changed, and Lxr␣ target genes are reduced. This differs from the regulation of Abcg1 expression, which is up-regulated at both mRNA and protein levels in Npc1-null cells. Abca1 protein translation rate is higher in Npc1-null hepatocytes, compared with wild type hepatocytes as measured by [35 S]methionine incorporation, whereas there is no difference for the degradation of newly synthesized Abca1 in these two types of hepatocytes. Cathepsin D, which we recently identified as a positive modulator of Abca1, is markedly increased at both mRNA and protein levels by Npc1 inactivation in hepatocytes but not in macrophages. Consistent with this, inhibition of cathepsin D with pepstatin A reduced the Abca1 protein level in both Npc1-inactivated and WT hepatocytes. Therefore, Abca1 expression is specifically regulated in hepatocytes, where Npc1 activity modulates cathepsin D expression and Abca1 protein translation rate. Niemann-Pick type C (NPC)2 disease is an autosomal lipid storage disease resulting from mutations in either the Npc1 (95% of families) or Npc2 genes (1-3). It is characterized by progressive hepatosplenomegaly and neurodegeneration, leading to premature death (4, 5). The exact function of both proteins remains unknown, but there is much evidence that they facilitate the transport of lipids, primarily cholesterol, from late endosome to the Golgi apparatus, endoplasmic reticulum (ER), mitochondria, and plasma membrane (6, 7). Thus the storage involves the accumulation of unesterified cholesterol, sphingolipids, and other lipids within the endosomal/lysosomal compartments of cells in almost all body tissues (8). Impaired cholesterol traffic in NPC disease cells prevents the normal down-regulation of endogenous cholesterol synthesis and the low density lipoprotein (LDL) receptor (9, 10), through the interruption of normal cholesterol regulation by sterol regulatory element-binding protein at the ER and the interruption of generation of LDL cholesterol-derived oxysterols in the mitochond...

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