David Q. Shih

Hepatocyte nuclear factor-1α is an essential regulator of bile acid and plasma cholesterol metabolism

Maturity-onset diabetes of the young type 3 (MODY3) is caused by haploinsufficiency of hepatocyte nuclear factor-1α (encoded by TCF1). Tcf1−/− mice have type 2 diabetes, dwarfism, renal Fanconi syndrome, hepatic dysfunction and hypercholestrolemia. Here we explore the molecular basis for the hypercholesterolemia using oligonucleotide microchip expression analysis. We demonstrate that Tcf1−/− mice have a defect in bile acid transport, increased bile acid and liver cholesterol synthesis, and impaired HDL metabolism. Tcf1−/− liver has decreased expression of the basolateral membrane bile acid transporters Slc10a1, Slc21a3 and Slc21a5, leading to impaired portal bile acid uptake and elevated plasma bile acid concentrations. In intestine and kidneys, Tcf1−/− mice lack expression of the ileal bile acid transporter (Slc10a2), resulting in increased fecal and urinary bile acid excretion. The Tcf1 protein (also known as HNF-1α) also regulates transcription of the gene (Nr1h4) encoding the farnesoid X receptor-1 (Fxr-1), thereby leading to reduced expression of small heterodimer partner-1 (Shp-1) and repression of Cyp7a1, the rate-limiting enzyme in the classic bile acid biosynthesis pathway. In addition, hepatocyte bile acid storage protein is absent from Tcf1−/− mice. Increased plasma cholesterol of Tcf1−/− mice resides predominantly in large, buoyant, high-density lipoprotein (HDL) particles. This is most likely due to reduced activity of the HDL-catabolic enzyme hepatic lipase (Lipc) and increased expression of HDL-cholesterol esterifying enzyme lecithin:cholesterol acyl transferase (Lcat). Our studies demonstrate that Tcf1, in addition to being an important regulator of insulin secretion, is an essential transcriptional regulator of bile acid and HDL-cholesterol metabolism.

β-cell–specific deletion of the Igf1 receptor leads to hyperinsulinemia and glucose intolerance but does not alter β-cell mass

Regulation of glucose homeostasis by insulin depends on the maintenance of normal β-cell mass and function. Insulin-like growth factor 1 (Igf1) has been implicated in islet development and differentiated function, but the factors controlling this process are poorly understood. Pancreatic islets produce Igf1 and Igf2, which bind to specific receptors on β-cells. Igf1 has been shown to influence β-cell apoptosis, and both Igf1 and Igf2 increase islet growth; Igf2 does so in a manner additive with fibroblast growth factor 2 (ref. 10). When mice deficient for the Igf1 receptor (Igf1r+/−) are bred with mice lacking insulin receptor substrate 2 (Irs2−/−), the resulting compound knockout mice show a reduction in mass of β-cells similar to that observed in pancreas of Igf1r−/− mice (ref. 11), suggesting a role for Igf1r in growth of β-cells. It is possible, however, that the effects in these mice occur secondary to changes in vascular endothelium or in the pancreatic ductal cells, or because of a decrease in the effects of other hormones implicated in islet growth. To directly define the role of Igf1, we have created a mouse with a β-cell–specific knockout of Igf1r (βIgf1r−/−). These mice show normal growth and development of β-cells, but have reduced expression of Slc2a2 (also known as Glut2) and Gck (encoding glucokinase) in β-cells, which results in defective glucose-stimulated insulin secretion and impaired glucose tolerance. Thus, Igf1r is not crucial for islet β-cell development, but participates in control of differentiated function.

Pancreatic β Cell-specific Transcription of thepdx-1 Gene THE ROLE OF CONSERVED UPSTREAM CONTROL REGIONS AND THEIR HEPATIC NUCLEAR FACTOR 3β SITES

To identify potential transactivators ofpdx-1, we sequenced approximately 4.5 kilobases of the 5′ promoter region of the human and chicken homologs, assuming that sequences conserved with the mouse gene would contain criticalcis-regulatory elements. The sequences associated with hypersensitive site 1 (HSS1) represented the principal area of homology within which three conserved subdomains were apparent: area I (−2694 to −2561 base pairs (bp)), area II (−2139 to −1958 bp), and area III (−1879 to −1799 bp). The identities between the mouse and chicken/human genes are very high, ranging from 78 to 89%, although only areas I and III are present within this region in chicken. Pancreatic β cell-selective expression was shown to be controlled by mouse and human area I or area II, but not area III, from an analysis of pdx-1-driven reporter activity in transfected β- and non-β cells. Mutational and functional analyses of conserved hepatic nuclear factor 3 (HNF3)-like sites located within area I and area II demonstrated that activation by these regions was mediated by HNF3β. To determine if a similar regulatory relationship might exist within the context of the endogenous gene, pdx-1 expression was measured in embryonic stem cells in which one or both alleles of HNF3β were inactivated. pdx-1 mRNA levels induced upon differentiation to embryoid bodies were down-regulated in homozygous null HNF3β cells. Together, these results suggest that the conserved sequences represented by areas I and II define the binding sites for factors such as HNF3β, which control islet β cell-selective expression of the pdx-1 gene.

Role of Foxa-2 in adipocyte metabolism and differentiation

Hepatocyte nuclear factors-3 (Foxa-1-3) are winged forkhead transcription factors that regulate gene expression in the liver and pancreatic islets and are required for normal metabolism. Here we show that Foxa-2 is expressed in preadipocytes and induced de novo in adipocytes of genetic and diet-induced rodent models of obesity. In preadipocytes Foxa-2 inhibits adipocyte differentiation by activating transcription of the Pref-1 gene. Foxa-2 and Pref-1 expression can be enhanced in primary preadipocytes by growth hormone, suggesting that the antiadipogenic activity of growth hormone is mediated by Foxa-2. In differentiated adipocytes Foxa-2 expression leads to induction of gene expression involved in glucose and fat metabolism, including glucose transporter-4, hexokinase-2, muscle-pyruvate kinase, hormone-sensitive lipase, and uncoupling proteins-2 and -3. Diet-induced obese mice with haploinsufficiency in Foxa-2 (Foxa-2+/-) develop increased adiposity compared with wild-type littermates as a result of decreased energy expenditure. Furthermore, adipocytes of these Foxa-2+/- mice exhibit defects in glucose uptake and metabolism. These data suggest that Foxa-2 plays an important role as a physiological regulator of adipocyte differentiation and metabolism.

Profound defects in pancreatic β-cell function in mice with combined heterozygous mutations in Pdx-1, Hnf-1α, and Hnf-3β

Defects in pancreatic β-cell function contribute to the development of type 2 diabetes, a polygenic disease that is characterized by insulin resistance and compromised insulin secretion. Hepatocyte nuclear factors (HNFs) -1α, -3β, -4α, and Pdx-1 contribute in the complex transcriptional circuits within the pancreas that are involved in β-cell development and function. In mice, a heterozygous mutation in Pdx-1 alone, but not Hnf-1α+/−, Hnf-3β+/−, or Hnf-4α+/−, causes impaired glucose-stimulated insulin secretion in mice. To investigate the possible functional relationships between these transcription factors on β-cell activity in vivo, we generated mice with the following combined heterozygous mutations: Pdx-1+/−/Hnf-1α+/−, Pdx-1+/−/Hnf-3β+/−, Pdx-1+/−/Hnf-4α+/−, Hnf-1α+/−/Hnf-4α+/−, and Hnf-3β+/−/Hnf-4α+/−. The greatest loss in function was in combined heterozygous null alleles of Pdx-1 and Hnf-1α (Pdx-1+/−/Hnf-1α+/−), or Pdx-1 and Hnf-3β (Pdx-1+/−/Hnf-3β+/−). Both double mutants develop progressively impaired glucose tolerance and acquire a compromised first- and second-phase insulin secretion profile in response to glucose compared with Pdx-1+/− mice alone. The loss in β-cell function in Pdx-1+/−/Hnf-3β+/− mice was associated with decreased expression of Nkx-6.1, glucokinase (Gck), aldolase B (aldo-B), and insulin, whereas Nkx2.2, Nkx-6.1, Glut-2, Gck, aldo-B, the liver isoform of pyruvate kinase, and insulin expression was reduced in Pdx-1+/−/Hnf-1α+/− mice. The islet cell architecture was also abnormal in Pdx-1+/−/Hnf-3β+/− and Pdx-1+/−/Hnf-1α+/− mice, with glucagon-expressing cells scattered throughout the islet, a defect that may be connected to decreased E-cadherin expression. Our data suggest that functional interactions between key islet regulatory factors play an important role in maintaining islet architecture and β-cell function. These studies also established polygenic mouse models for investigating the mechanisms contributing to β-cell dysfunction in diabetes.

Dissecting the transcriptional network of pancreatic islets during development and differentiation

Maturity onset diabetes of the young (MODY) is a heterogenous, monogenic disease that is characterized by an autosomal dominant inheritance and an early disease onset, usually before 25 years of age (1). In contrast to late-onset forms of type 2 diabetes in which β-cell defects develop as a result of insulin resistance, MODY is caused by a primary defect in pancreatic β-cell function and impaired glucose-stimulated insulin secretion (2). Mutations in five genes are associated with different forms of MODY. The MODY2 gene encodes the glycolytic enzyme glucokinase that acts as the glucose sensor of the β cell and when inactivated, leads to impaired sensing of blood glucose levels (3). However, the majority of MODY forms are caused by mutations in transcription factors that are enriched in pancreatic β cells and include hepatocyte nuclear factors 1α (HNF-1α, MODY3), HNF-4α (MODY1), insulin promoter factor 1 (IPF-1/PDX-1, MODY4), and HNF-1β (MODY5) (see Table 1). These forms of diabetes share many pathophysiological features and are known to regulate the expression of genes such as the glucose transporter 2 (GLUT-2) and glycolytic genes that are essential for normal β-cell function (4–6). Studies performed in extrapancreatic tissues such as the liver, gut, and visceral endoderm have shown that HNFs form a hierarchical transcriptional network that regulates differentiation and metabolism in these cells (7–9). Two recent studies, including the paper by Boj et al. in this issue of PNAS (10), significantly advance our understanding of this regulatory circuit in pancreatic β cells.