SUMMARY Biological membranes are complex, and the mechanisms underlying their homeostasis are incompletely understood. Here, we present a quantitative genetic interaction map (E-MAP) focused on various aspects of lipid biology, including lipid metabolism, sorting, and trafficking. This E-MAP contains ~250,000 negative and positive genetic interaction scores and identifies a molecular crosstalk of protein quality control pathways with lipid bilayer homeostasis. Ubx2p, a component of the endoplasmic-reticulum-associated degradation pathway, surfaces as a key upstream regulator of the essential fatty acid (FA) desaturase Ole1p. Loss of Ubx2p affects the transcriptional control of OLE1, resulting in impaired FA desaturation and a severe shift toward more saturated membrane lipids. Both the induction of the unfolded protein response and aberrant nuclear membrane morphologies observed in cells lacking UBX2 are suppressed by the supplementation of unsaturated FAs. Our results point toward the existence of dedicated bilayer stress responses for membrane homeostasis.

In the developing pancreas, the basic helix-loop-helix (bHLH) protein Neurogenin3 (Ngn3) specifies which precursor cells ultimately will become endocrine cells and initiates the islet differentiation program. NeuroD1, a closely related bHLH protein and a downstream target of Ngn3, maintains the differentiation program initiated by Ngn3. We have developed an in vitro model of Ngn3-dependent differentiation by infecting pancreatic duct cell lines with an Ngn3-expressing adenovirus. We found that both Ngn3 and its downstream target NeuroD1 activated the islet differentiation program in these cells by inducing the expression of genes with early roles in the differentiation cascade, as well as genes characteristic of fully differentiated islet cells. Induction of these genes, as exemplified by the insulin1 gene, involved alteration of the local chromatin structure. Interestingly, the subsets of genes activated by Ngn3 and NeuroD1 were not completely overlapping, indicating that these two bHLH proteins serve specific functions in the development of the endocrine pancreas. In addition, microarray gene expression analysis identified a previously uncharacterized group of Ngn3-induced genes with potentially important roles in islet development and function. These studies demonstrate how Ngn3 initiates islet differentiation and provide us with a model for testing methods for producing islet cells for people with diabetes. During pancreatic development, differentiation of endocrine and exocrine cells from a common endodermal progenitor cell requires the coordinated regulation of specific sets of genes. This process can be envisioned as a hierarchy or cascade of transcription factors that initiate and maintain the distinct gene expression programs that define the various pancreatic cell types (1). Among these factors, the basic helix-loop-helix (bHLH) protein Neurogenin3 (Ngn3) plays a dominant role in the specification of the endocrine͞islet cell lineage.During embryonic development, Ngn3 appears transiently in scattered pancreatic epithelial cells (2, 3). Several lines of evidence indicate that the expression of Ngn3 in these undifferentiated cells directs them to an endocrine cell fate and initiates the program of islet differentiation. First, lineage tracing shows that these transient Ngn3-expressing cells differentiate exclusively into islet cells (4). Second, mice homozygous for a targeted deletion of the ngn3 gene fail to generate any islet cells (5). Third, ectopic expression of Ngn3 drives embryonic endoderm to an endocrine fate (2, 3, 6).Ngn3 may play a similar role in the generation of new islet cells postnatally. It has been suggested that cells along the pancreatic ducts may act as progenitors for new islet cells in the postnatal period, although recent lineage tracing experiments suggest that the bulk of newly generated beta cells in adult mice result from the replication of preexisting beta cells (7).Ngn3 initiates islet cell differentiation, but other factors downstream of ngn3 must complete the task. Genetic...

To investigate the role of the Sry/hydroxymethylglutaryl box (Sox) transcription factors in the development of the pancreas, we determined the expression pattern of Sox factors in the developing mouse pancreas. By RT-PCR, we detected the presence of multiple Sox family members in both the developing pancreas and mature islets and then focused on two factors, Sox2 and Sox4. The expression field of Sox2, which plays a role in the maintenance of some stem cell populations, included the developing duodenum, but Sox2 was specifically excluded from the pancreatic buds. In contrast, Sox4 was detected broadly in the early pancreatic buds and eventually became restricted to the nuclei of all islet cells in the adult mouse. Mice homozygous for a null mutation of the sox4 gene showed normal pancreatic bud formation and endocrine cell differentiation up to embryonic day 12.5. Beyond that date, cultured pancreatic explants lacking sox4 failed to form normal islets. Instead, a markedly reduced number of endocrine cells were found scattered through the explant. We show here that several Sox transcription factors are expressed in the developing pancreas and in the islet, and that one of these factors, Sox4, is required for the normal development of pancreatic islets. Diabetes 54:3402-3409, 2005 S cattered through the exocrine pancreas, the islets of Langerhans are highly organized clusters of endocrine cells comprised of four distinct cell types: the glucagon-secreting ␣-cells, the insulinsecreting ␤-cells, the somatostatin-secreting ␦-cells, and the pancreatic polypeptide-secreting cells. Because of the essential role of the islet hormones in energy metabolism, defects in the development, maintenance, or function of the endocrine pancreas have serious consequences, including diabetes.The pancreas forms from the endoderm in the region of the foregut/midgut junction and is first visible in mice at embryonic day 9.5 (e9.5) (1). The first endocrine cells of the pancreas appear at around e9.5 in the dorsal pancreatic bud. These cells express glucagon; a few insulinexpressing cells appear ϳ1 day later. At ϳe13-14, the pancreas undergoes a distinct change termed the secondary transition, characterized by the appearance of ductal cells, exocrine cells, and delta cells and a rapid expansion of the insulin-expressing cells. The endocrine cells found in the pancreas before the secondary transition have clear differences from mature islet cells and may result from developmental pathways that are distinct from those that produce the more mature endocrine cells that arise following the secondary transition (2-6). By e18, the first pancreatic polypeptide-expressing cells appear, and the endocrine cells organize into distinct islets with the ␤-cells forming the central core. Around the same time, ␤-cell neogenesis declines, but the simultaneous onset of ␤-cell replication continues to expand the ␤-cell population and enlarge the forming islets (7).This process of endocrine cell determination and differentiation depends on the proper sequentia...

During pancreatic development, the paired homeodomain transcription factor PAX4 is required for the differentiation of the insulin-producing beta cells and somatostatin-producing delta cells. To establish the position of PAX4 in the hierarchy of factors controlling islet cell development, we examined the control of the human PAX4 gene promoter. In both cell lines and transgenic animals, a 4.9-kilobase pair region directly upstream of the human PAX4 gene transcriptional start site acts as a potent pancreas-specific promoter. Deletion mapping experiments demonstrate that a 118-base pair region lying approximately 1.9 kilobase pairs upstream of the transcription start site is both necessary and sufficient to direct pancreas-specific expression. Serial deletions through this region reveal the presence of positive elements that bind several pancreatic transcription factors as follows: the POU homeodomain factor HNF1␣, the orphan nuclear receptor HNF4␣, the homeodomain factor PDX1, and a heterodimer composed of two basic helix-loop-helix factors. Interestingly, mutations in the genes encoding four of these factors cause a dominantly inherited form of human diabetes called Maturity Onset Diabetes of the Young. In addition, PAX4 itself has at least two high affinity binding sites within the promoter through which it exerts a strong negative autoregulatory effect. Together, these results suggest a model in which PAX4 expression is activated during pancreatic development by a combination of pancreas-specific factors but is then switched off once PAX4 protein reaches sufficient levels.Organogenesis of the mammalian pancreas and its differentiation into discrete populations of endocrine and exocrine cells depends on the tightly regulated temporal and spatial expression of an array of transcription factors (for reviews see Refs. 1-3). Some of these factors, such as HNF3␤ (4, 5), PDX1 (6, 7), and HLXb9 (8, 9), are involved in the formation and outgrowth of the dorsal and ventral pancreatic buds. Within the forming pancreas, the basic helix-loop-helix transcription factor neurogenin3 functions as a pro-endocrine gene, initiating the program of endocrine differentiation in selected cells (10, 11). The complete differentiation and maturation of the endocrine cells requires additional factors, including neuroD1/BETA2 (12), NKX2.2 (13), ISL1 (14), and PAX6 (2, 15).Once neurogenin3 expression sets a progenitor cell on a course of endocrine differentiation, additional factors are necessary to determine which of the four endocrine cell types it will become. One of these islet cell-type determination factors is the paired homeodomain transcription factor PAX4. PAX4 functions as a potent transcriptional repressor and is expressed only transiently in the fetal pancreas, peaking during the period of beta and delta cell differentiation (16). Mice with a targeted disruption of the pax4 gene have a marked decrease in beta and delta cells with a commensurate increase in alpha cells (17), suggesting that PAX4 functions by repressing the alpha...

The localization of the two major placental glucose transporter isoforms, GLUTi and GLUT3 was studied in 20-d pregnant rats. Immunocytochemical studies revealed that GLUTi protein is expressed ubiquitously in the junctional zone (maternal side) and the labyrinthine zone (fetal side) of the placenta. In contrast, expression of GLUT3 protein is restricted to the labyrinthine zone, specialized in nutrient transfer. After 19-d maternal insulinopenic diabetes (streptozotocin), placental GLUT3 mRNA and protein levels were increased four-to-fivefold compared to nondiabetic rats, whereas GLUTi mRNA and protein levels remained unmodified. Placental 2-deoxyglucose uptake and glycogen concentration were also increased fivefold in diabetic rats. These data suggest that GLUT3 plays a major role in placental glucose uptake and metabolism.The role of hyperglycemia in the regulation of GLUT3 expression was assessed by lowering the glycemia of diabetic pregnant rats. After a 5-d phlorizin infusion to pregnant diabetic rats, placental GLUT3 mRNA and protein levels returned to levels similar to those observed in nondiabetic rats. Furthermore, a short-term hyperglycemia (12 h), achieved by performing hyperglycemic clamps induced a fourfold increase in placental GLUT3 mRNA and protein with no concomitant change in GLUTi expression.This study provides the first evidence that placental GLUT3 mRNA and protein expression can be stimulated in vivo under hyperglycemic conditions. Thus, GLUT3 transporter isoform appears to be highly sensitive to ambient glucose levels and may play a pivotal role in the severe alterations of placental function observed in diabetic pregnancies. (J. Clin. Invest. 1995.96:309-317.)

We have recently cloned the murine glucagon receptor (GR) gene and shown that it is expressed mainly in liver. In this organ, the glucagon-GR system is involved in the control of glucose metabolism as it initiates a cascade of events leading to release of glucose into the blood stream, which is a main feature in several physiological and pathological conditions. To better define the metabolic regulators of GR expression in liver we analyzed GR mRNA concentration in physiological conditions associating various glucose metabolic pathways in vivo and in vitro in the rat and in the mouse. First, we report that the concentration of the GR mRNA progressively increased from the first day of life to the adult stage. This effect was abolished when newborn rodents were fasted. Second, under conditions where intrahepatic glucose metabolism was active such as during fasting, diabetes, and hyperglycemic clamp, the concentration of GR mRNA increased independent of the origin of the pathway that generated the glucose flux. These effects were blunted when hyperglycemia was corrected by phlorizin treatment of diabetic rats or not sustained during euglycemic clamp.In accordance with these observations, we demonstrated that the glycolytic substrates glucose, mannose, and fructose, as well as the gluconeognic substrates glycerol and dihydroxyacetone, increased the concentration of GR mRNA in primary cultures of hepatocytes from fed rats. Glucagon blunted the effect of glucose without being dominant. The stimulatory effect of those substrates was not mimicked by the nonmetabolizable carbohydrate L-glucose or the glucokinase inhibitor glucosamine or when hepatocytes were isolated from starved rats. In addition, inhibitors of gluconeogenesis and lipolysis could decrease the concentration of GR mRNA from hepatocytes of starved rats. Combined, these data strongly suggest that glucose flux in the glycolytic and gluconeogenic pathways at the level of triose intermediates could control expression of GR mRNA and participate in controlling its own metabolism. The glucagon receptor (GR)1 is a 63,000-Da plasma membrane protein that belongs to a subfamily of peptide hormone receptors (1, 2). All members of this family contain seven transmembrane domains and are coupled with GTP-binding proteins. Upon binding to its receptor, glucagon initiates its action by activating several GTP-binding proteins which are ratelimiting steps in various signal transduction cascades (3-12).The GR gene is expressed mainly in liver (2,8,13,14) where it initiates a cascade of events leading to synthesis and release of glucose into the blood stream (15). Hepatic glucose production represents a major process in several physiological and pathological conditions. In newborns, glucagon is secreted within an hour of parturition and initiates several processes leading to hepatic glucose production (16) from glycogenolysis and gluconeogenesis (17). Then, during suckling glucagon stimulation ensures hepatic synthesis of glucose, which is otherwise poorly provided by mother's...

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The effects of high glucose on insulin-receptor tyrosine kinase activity and gene expression were investigated in 3T3-HIR cells. Cells incubated for 48 h in the presence of 25 mM glucose showed a 5-fold increase in the amount of insulin receptors per cell, receptor autophosphorylation and phosphorylation of the exogenous substrate poly(Glu/Tyr) compared with cells grown in the absence of glucose but in the presence of 25 mM fructose. These effects were associated with a 4-fold stimulation in steady-state levels of insulin-receptor mRNA. Significant cellular glucose utilization and lactate production were observed in the presence of high glucose in the culture medium, indicating a functional glycolytic pathway in glucose-treated cells, but not in cells treated with fructose. Such a differential response to hexoses favours the hypothesis of a carbohydrate regulation via a glycolytic intermediate. This was further supported by a similar glucose-induced increase in mRNA levels of the enzyme glyceraldehyde-3-phosphate dehydrogenase. To test the hypothesis that the stimulatory effect of glucose on amount of insulin receptors and phosphorylation state could result from post-transcriptional modifications, cells exposed to glucose were incubated with actinomycin D, a potent inhibitor of gene transcription. In cells challenged with high glucose plus inhibitor, insulin-receptor mRNA half-life was increased from 1 to 3 h, indicating that posttranscriptional mechanisms are involved in these processes of glucose regulation. Inhibition of protein synthesis by cycloheximide induced an overexpression of insulin-receptor mRNA levels in the presence of glucose, suggesting that labile repressor protein(s) could be implicated in the effects of glucose. We conclude that (1) long-term culture with high glucose increases the amount of insulin receptors and their tyrosine kinase activity and (2) the glucose-induced increase in insulin-receptor mRNA levels can be accounted for, at least in part, by posttranscriptional events.