Markus Stoffel

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.

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-1 α ^+/− , Hnf-3 β ^+/− , or Hnf-4 α ^+/− , 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.

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. View this table: Table 1 Genetic classification of MODY subtypes The transcriptional cascade of HNFs was first elucidated in hepatocytes where it was shown that HNF-4α, a member of the steroid hormone receptor superfamily, was an essential positive regulator of HNF-1α (11, 12). This was one of the first transcriptional hierarchies that was characterized in …

Hepatocyte nuclear factor 4α (HNF4α) plays a critical role in regulating the expression of many genes essential for normal functioning of liver, gut, kidney, and pancreatic islets. A nonsense mutation (Q268X) in exon 7 of the HNF4α gene is responsible for an autosomal dominant, early-onset form of non-insulin-dependent diabetes mellitus (maturity-onset diabetes of the young; gene named MODY1 ). Although this mutation is predicted to delete 187 C-terminal amino acids of the HNF4α protein the molecular mechanism by which it causes diabetes is unknown. To address this, we first studied the functional properties of the MODY1 mutant protein. We show that it has lost its transcriptional transactivation activity, fails to dimerize and bind DNA, implying that the MODY1 phenotype is because of a loss of HNF4α function. The effect of loss of function on HNF4α target gene expression was investigated further in embryonic stem cells, which are amenable to genetic manipulation and can be induced to form visceral endoderm. Because the visceral endoderm shares many properties with the liver and pancreatic β-cells, including expression of genes for glucose transport and metabolism, it offers an ideal system to investigate HNF4-dependent gene regulation in glucose homeostasis. By exploiting this system we have identified several genes encoding components of the glucose-dependent insulin secretion pathway whose expression is dependent upon HNF4α. These include glucose transporter 2, and the glycolytic enzymes aldolase B and glyceraldehyde-3-phosphate dehydrogenase, and liver pyruvate kinase. In addition we have found that expression of the fatty acid binding proteins and cellular retinol binding protein also are down-regulated in the absence of HNF4α. These data provide direct evidence that HNF4α is critical for regulating glucose transport and glycolysis and in doing so is crucial for maintaining glucose homeostasis.