We report 10 heterozygous mutations in the human insulin gene in 16 probands with neonatal diabetes. A combination of linkage and a candidate gene approach in a family with four diabetic members led to the identification of the initial INS gene mutation. The mutations are inherited in an autosomal dominant manner in this and two other small families whereas the mutations in the other 13 patients are de novo. Diabetes presented in probands at a median age of 9 weeks, usually with diabetic ketoacidosis or marked hyperglycemia, was not associated with ␤ cell autoantibodies, and was treated from diagnosis with insulin. The mutations are in critical regions of the preproinsulin molecule, and we predict that they prevent normal folding and progression of proinsulin in the insulin secretory pathway. The abnormally folded proinsulin molecule may induce the unfolded protein response and undergo degradation in the endoplasmic reticulum, leading to severe endoplasmic reticulum stress and potentially ␤ cell death by apoptosis. This process has been described in both the Akita and Munich mouse models that have dominant-acting missense mutations in the Ins2 gene, leading to loss of ␤ cell function and mass. One of the human mutations we report here is identical to that in the Akita mouse. The identification of insulin mutations as a cause of neonatal diabetes will facilitate the diagnosis and possibly, in time, treatment of this disorder.endoplasmic reticulum stress ͉ insulin biosynthesis ͉ disulfide bonds ͉ unfolded protein response

Two-thirds partial hepatectomy (PH) induces differentiated cells in the liver remnant to proliferate and regenerate to its original size. The proliferation-specific HNF-3/fork head homolog-11B protein (HFH-11B; also known as Trident and Win) is a family member of the winged helix/fork head transcription factors and in regenerating liver its expression is reactivated prior to hepatocyte entry into DNA replication (S phase). To examine whether HFH-11B regulates hepatocyte proliferation during liver regeneration, we used the ؊3-kb transthyretin (TTR) promoter to create transgenic mice that displayed ectopic hepatocyte expression of HFH-11B. Liver regeneration studies with the TTR-HFH-11B mice demonstrate that its premature expression resulted in an 8-h acceleration in the onset of hepatocyte DNA replication and mitosis. This liver regeneration phenotype is associated with protracted expression of cyclin D1 and C/EBP␤, which are involved in stimulating DNA replication and premature expression of M phase promoting cyclin B1 and cdc2. Consistent with the early hepatocyte entry into S phase, regenerating transgenic livers exhibited earlier expression of DNA repair genes (XRCC1, mHR21spA, and mHR23B). Furthermore, in nonregenerating transgenic livers, ectopic HFH-11B expression did not elicit abnormal hepatocyte proliferation, a finding consistent with the retention of the HFH-11B transgene protein in the cytoplasm. We found that nuclear translocation of the HFH-11B transgene protein requires mitogenic signalling induced by PH and that its premature availability in regenerating transgenic liver allowed nuclear translocation to occur 8 h earlier than in wild type.The mammalian liver is one of the few adult organs capable of completely regenerating itself in response to cellular injury from toxins, viral infections, or tissue removal (15,38,46). Liver regeneration after two-thirds partial hepatectomy (PH) represents a balance between hepatocyte proliferation and the maintenance of hepatocyte-specific gene expression required for liver homeostasis (22,46). A potent activation of hepatocyte immediate early transcription factors is observed during liver regeneration and includes c-Jun, c-Fos, c-Myc, NF-B, signal transducers, and activators of transcription 3 (stat3) and the CCAAT/enhancer protein ␤ (C/EBP␤) genes (7,9,25,46). Furthermore, maintenance of hepatocyte-specific gene transcription is coincident with sustained expression of hepatocyte nuclear factor genes (16,20,41). More recent genetic data demonstrated that the cytokine interleukin-6 (IL-6) plays an important role in establishing responsiveness of hepatocytes to growth factors which are released after liver injury (8,54). In a PH model of liver regeneration, homozygous null interleukin-6 (IL-6) or type 1 tumor necrosis factor receptor (TNFR-I) mice exhibited a 70% reduction in hepatocyte replication and this proliferation defect was eliminated by an intraperitoneal injection of IL-6 prior to surgery (8,43,54). This proliferation defect was accompanied by a failur...

The forkhead box (Fox) family of transcription factors share homology in the winged helix͞forkhead DNA-binding domain and play important roles in regulating cellular proliferation, differentiation, longevity, and cellular transformation. Forkhead box M1B (FoxM1B) is a ubiquitously expressed member of the Fox transcription factor family whose expression is restricted to proliferating cells and that mediates hepatocyte entry into DNA synthesis and mitosis during liver regeneration. Recent cDNA microarray studies indicated that age-related defects in cellular proliferation are associated with diminished expression of the FoxM1B transcription factor. Here, we show that increased levels of FoxM1B in regenerating liver of old transgenic mice restore the sharp peaks in hepatocyte DNA replication and mitosis that are the hallmarks of young regenerating mouse liver. Restoration of the young regenerating liver phenotype is associated with increased expression of numerous cell cycle regulatory genes that include cyclin D1, cyclin A2, cyclin F, cyclin B1, cyclin B2, Cdc25B, and p55cdc. Cotransfection assays in the human hepatoma HepG2 cell line demonstrated that FoxM1B protein stimulated expression of both the cyclin B1 and cyclin D1 promoters, suggesting that these cyclin genes are a direct FoxM1B transcriptional target. These results suggest that FoxM1B controls the transcriptional network of genes that are essential for cell division and exit from mitosis. Our results indicate that reduced expression of the FoxM1B transcription factor contributes to the decline in cellular proliferation observed in the aging process.T he mammalian liver is one of the few adult organs capable of completely regenerating itself in response to injury through the release of growth factors that stimulate reentry of terminally differentiated hepatocytes into the cell cycle (1-3). Liver regeneration induced by a two-thirds partial hepatectomy (PHx) results in synchronous induction of sharp peaks in hepatocyte DNA replication (S phase) and mitosis (M phase), which requires participation of the IL-6-signaling pathway (3-5). The forkhead box (Fox) family of transcription factors (6) shares homology in the winged-helix DNA-binding domain (7), and its members play important roles in regulating transcription of genes involved in cellular proliferation, differentiation, metabolic homeostasis, longevity, and cellular transformation (8-18). The mammalian (human) Fox family member FoxM1B (previously known as HFH-11B or trident) is a ubiquitously expressed transcription factor restricted to proliferating cells of the mouse embryo (including liver) and is essential for embryonic development (19), but its expression diminishes during postnatal cellular differentiation (20). In regenerating liver, FoxM1B expression is reactivated before DNA replication (S phase) and sustained throughout the period of hepatocyte proliferation (20). Liver regeneration studies with transgenic mice, in which the transthyretin (TTR) promoter functioned to prematurely express FoxM1...

Recent studies have established that vitamin D plays multiple biological roles beyond calcium metabolism; however, whether vitamin D is involved in energy metabolism is unknown. To address this question, we characterized the metabolic phenotypes of vitamin D receptor (VDR)-null mutant mice. Under a normocalcemic condition, VDR-null mice displayed less body fat mass and lower plasma triglyceride and cholesterol levels compared with wild-type (WT) mice; when placed on a high-fat diet, VDR-null mice showed a slower growth rate and accumulated less fat mass globally than WT mice, even though their food intake and intestinal lipid transport capacity were the same as WT mice. Consistent with the lower adipose mass, plasma leptin levels were lower and white adipocytes were histologically smaller in VDR-null mice than WT mice. The rate of fatty acid beta-oxidation in the white adipose tissue was higher, and the expression of uncoupling protein (UCP) 1, UCP2 and UCP3 was markedly upregulated in VDR-null mice, suggesting a higher energy expenditure in the mutant mice. Experiments using primary brown fat culture confirmed that 1,25-dihydroxyvitamin D3 directly suppressed the expression of the UCPs. Consistently, the energy expenditure, oxygen consumption, and CO2 production in VDR-null mice were markedly higher than in WT mice. These data indicate that vitamin D is involved in energy metabolism and adipocyte biology in vivo in part through regulation of beta-oxidation and UCP expression.

Murine hepatocyte nuclear factor-3 beta (HNF-3 beta) protein is a member of a large family of developmentally regulated transcription factors that share homology in the winged helix/fork head DNA binding domain and that participate in embryonic pattern formation. HNF-3 beta also mediates cell-specific transcription of genes important for the function of hepatocytes, intestinal and bronchiolar epithelial, and pancreatic acinar cells. We have previously identified a liver-enriched transcription factor, HNF-6, which is required for HNF-3 beta promoter activity and also recognizes the regulatory region of numerous hepatocyte-specific genes. In this study we used the yeast one-hybrid system to isolate the HNF-6 cDNA, which encodes a cut-homeodomain-containing transcription factor that binds with the same specificity as the liver HNF-6 protein. Cotransfection assays demonstrate that HNF-6 activates expression of a reporter gene driven by the HNF-6 binding site from either the HNF-3 beta or transthyretin (TTR) promoter regions. We used interspecific backcross analysis to determine that murine Hnf6 gene is located in the middle of mouse chromosome 9. In situ hybridization studies of staged specific embryos demonstrate that HNF-6 and its potential target gene, HNF-3 beta, are coexpressed in the pancreatic and hepatic diverticulum. More detailed analysis of HNF-6 and HNF-3 beta's developmental expression patterns provides evidence of colocalization in hepatocytes, intestinal epithelial, and in the pancreatic ductal epithelial and exocrine acinar cells. The expression patterns of these two transcription factors do not overlap in other endoderm-derived tissues or the neurotube. We also found that HNF-6 is also abundantly expressed in the dorsal root ganglia, the marginal layer, and the midbrain. At day 18 of gestation and in the adult pancreas, HNF-6 and HNF-3 beta transcripts colocalize in the exocrine acinar cells, but their expression patterns diverge in other pancreatic epithelium. HNF-6, but not HNF-3 beta, expression continues in the pancreatic ductal epithelium, whereas only HNF-3 beta becomes restricted to the endocrine cells of the islets of Langerhans. We discuss these expression patterns with respect to specification of hepatocytes and differentiation of the endocrine and exocrine pancreas.

Genome-wide association studies (GWAS) have consistently implicated noncoding variation within the TCF7L2 locus with type 2 diabetes (T2D) risk. While this locus represents the strongest genetic determinant for T2D risk in humans, it remains unclear how these noncoding variants affect disease etiology. To test the hypothesis that the T2D-associated interval harbors cis-regulatory elements controlling TCF7L2 expression, we conducted in vivo transgenic reporter assays to characterize the TCF7L2 regulatory landscape. We found that the 92-kb genomic interval associated with T2D harbors longrange enhancers regulating various aspects of the spatial-temporal expression patterns of TCF7L2, including expression in tissues involved in the control of glucose homeostasis. By selectively deleting this interval, we establish a critical role for these enhancers in robust TCF7L2 expression. To further determine whether variation in Tcf7l2 expression may lead to diabetes, we developed a Tcf7l2 copy-number allelic series in mice. We show that a null Tcf7l2 allele leads, in a dosedependent manner, to lower glycemic profiles. Tcf7l2 null mice also display enhanced glucose tolerance coupled to significantly lowered insulin levels, suggesting that these mice are protected against T2D. Confirming these observations, transgenic mice harboring multiple Tcf7l2 copies and overexpressing this gene display reciprocal phenotypes, including glucose intolerance. These results directly demonstrate that Tcf7l2 plays a role in regulating glucose tolerance, suggesting that overexpression of this gene is associated with increased risk of T2D. These data highlight the role of enhancer elements as mediators of T2D risk in humans, strengthening the evidence that variation in cis-regulatory elements may be a paradigm for genetic predispositions to common disease.[Supplemental material is available for this article.]Recent GWAS have uncovered a number of loci affecting risk of T2D (Voight et al. 2010). While some of these loci include genes known to play a role in glucose metabolism and diabetes pathogenesis (PPARG, KCNJ11) (Willson et al. 2001;Gloyn et al. 2006), others represent genomic regions with unknown functional roles in disease etiology. Among these, a set of single nucleotide polymorphisms (SNPs) on chromosome 10q25.2 shows strong and consistent association with T2D in virtually every population tested, constituting the greatest effect on risk identified to date, with a cumulative allelic odds ratio of 1.46 (Grant et al. 2006;Cauchi et al. 2007;Lyssenko 2008). These SNPs map to a 92-kb interval within TCF7L2, a gene encoding a transcription factor of the canonical Wnt signaling pathway known to have developmental roles in determining cell fate, survival, proliferation, and movement (Moon et al. 2004;Clevers 2006;MacDonald et al. 2009). Given the complexity of this pathway, establishing a definitive role for TCF7L2 in the etiology of T2D has been challenging (Pearson 2009).While the TCF7L2 T2D-associated interval spans coding sequence, exonic v...

Cells are programmed to die when critical signaling and metabolic pathways are disrupted. Inhibiting the type 2 ryanodine receptor (RyR2) in human and mouse pancreatic ␤-cells markedly increased apoptosis. This mode of programmed cell death was not associated with robust caspase-3 activation prompting a search for an alternative mechanism. Increased calpain activity and calpain gene expression suggested a role for a calpain-dependent death pathway. Using a combination of pharmacological and genetic approaches, we demonstrated that the calpain-10 isoform mediated ryanodine-induced apoptosis. Apoptosis induced by the fatty acid palmitate and by low glucose also required calpain-10. Ryanodine-induced calpain activation and apoptosis were reversed by glucagon-like peptide or short-term exposure to high glucose. Thus RyR2 activity seems to play an essential role in ␤-cell survival in vitro by suppressing a death pathway mediated by calpain-10, a type 2 diabetes susceptibility gene with previously unknown function.The pancreatic ␤-cell plays a central role in the pathogenesis of diabetes mellitus. A reduction in ␤-cell mass mediated at least in part by an increase in apoptosis is characteristic of the diabetic state (1-3). It is becoming clear that several pathways can lead to ␤-cell apoptosis, including cytokine signaling, excessive Ca 2ϩ influx during chronic hyperglycemia, high levels of free fatty acids, hypoxia or hypoglycemia, endoplasmic reticulum (ER) 1 stress, and loss of growth factor signaling (1, 3-12). Whether various inducers of apoptosis employ distinct molecular mechanisms has not been systematically studied.Intracellular Ca 2ϩ stores play an important role in the regulation of apoptosis in many cell types (13,14). The present study was undertaken to test the hypothesis that alterations in specific intracellular Ca 2ϩ stores may induce apoptosis in pancreatic ␤-cells. There are at least three classes of intracellular Ca 2ϩ stores in ␤-cells, and these are sensitive, respectively, to inositol trisphosphate (IP 3 )/thapsigargin, nicotinic acid adenine dinucleotide phosphate, and cyclic ADP ribose/ryanodine (15-18). In many cell types, ryanodine receptor Ca 2ϩ channels (RyR) transmit Ca 2ϩ signals directly to closely associated mitochondria (19). In the MIN6 ␤-cell line, RyR were shown to regulate ATP production (20). Because of their role in regulating intracellular Ca 2ϩ and mitochondrial function, we focused specifically on RyR as likely mediators of ␤-cell apoptosis. Of the three RyR subtypes, two have been reported to be present in ␤-cells, RyR1 and RyR2. The latter is more abundant and can be distinguished from the former by its insensitivity to dantrolene (21,22). Ryanodine, a plant alkaloid, is the most specific probe for all RyR subtypes, and its activity is lost in RyR-deficient cells (23,24).In the present study, we examined the role of RyR in the survival of human and mouse pancreatic islets. We uncovered a novel apoptosis pathway that is initiated when Ca 2ϩ flux through RyR2 is blocked. The ...

Our previous studies demonstrated a high fat diet-resistant lean phenotype of vitamin D receptor (VDR)-null mutant mice mainly due to increased energy expenditure, suggesting an involvement of the VDR in energy metabolism. Here, we took a transgenic approach to further define the role of VDR in adipocyte biology. We used the aP2 gene promoter to target the expression of the human (h) VDR in adipocytes in mice. In contrast to the VDR-null mice, the aP2-hVDR Tg mice developed obesity compared with the wild-type counterparts without changes in food intake. The increase in fat mass was mainly due to markedly reduced energy expenditure, which was correlated with decreased locomotive activity and reduced fatty acid ␤-oxidation and lipolysis in the adipose tissue in the transgenic mice. Consistently, the expression of genes involved in the regulation of fatty acid transport, thermogenesis, and lipolysis were suppressed in the transgenic mice. Taken together, these data confirm an important role of the VDR in the regulation of energy metabolism.The maintenance of body weight depends on the balance between energy intake and energy utilization. Obesity results when energy consumed exceeds energy utilized. Energy is acquired through diets and can be stored in adipose tissue or utilized by the body to maintain basic cellular functions and physical activities. Energy can also be used for adaptive thermogenesis in response to a cold environment (1). The adipose tissue is unique in that it represents both arms of energy balance. The white adipose tissue (WAT) 2 has the ability to sense the energy state of the body. When energy availability is high, the WAT stores the excess energy as triglyceride in lipid droplets.When energy is needed, triglyceride is broken down to free fatty acids to release into the circulation. This process, known as lipolysis, is regulated by two enzymes, adipose triglyceride lipase (ATGL), and hormone-sensitive lipase (HSL). ATGL initiates the first rate-limiting step of lipolysis by hydrolyzing triglyceride to diacylglyceride (2, 3), which is further broken down to monoglyceride by HSL (4). Monoglyceride lipase cleaves the final ester bond of monoglyceride to release glycerol, and this step is not rate-limiting (5). Lipolysis is activated by catacholamines through the cAMP signaling pathway, leading to protein kinase A activation. Protein kinase A phosphorylates HSL, which promotes HSL translocation to the lipid droplet and access to triglyceride stores (6). HSL and ATGL activity is suppressed by insulin during feeding, as insulin increases the amount of perilipin around the lipid droplets to prevent their access to triglycerides (7).The principal role of the brown adipose tissue (BAT) is to regulate adaptive thermogenesis through the expression of uncoupling proteins (UCPs). UCP1 separates oxidative phosphorylation from ATP production to release energy as heat (8 -10). Studies have shown that increased expression of UCP1 in the BAT or its ectopic expression in the WAT results in increased metabolism...