Regulation of hindbrain Pyy expression by acute food deprivation, prolonged caloric restriction, and weight loss surgery in mice.

PYY is a gut-derived putative satiety signal released in response to nutrient ingestion and is implicated in the regulation of energy homeostasis. Pyy-expressing neurons have been identified in the hindbrain of river lamprey, rodents, and primates. Despite this high evolutionary conservation, little is known about central PYY neurons. Using in situ hybridization, PYY-Cre;ROSA-EYFP mice, and immunohistochemistry, we identified PYY cell bodies in the gigantocellular reticular nucleus region of the hindbrain. PYY projections were present in the dorsal vagal complex and hypoglossal nucleus. In the hindbrain, Pyy mRNA was present at E9.5, and expression peaked at P2 and then decreased significantly by 70% at adulthood. We found that, in contrast to the circulation, PYY-(1-36) is the predominant isoform in mouse brainstem extracts in the ad libitum-fed state. However, following a 24-h fast, the relative amounts of PYY-(1-36) and PYY-(3-36) isoforms were similar. Interestingly, central Pyy expression showed nutritional regulation and decreased significantly by acute starvation, prolonged caloric restriction, and bariatric surgery (enterogastroanastomosis). Central Pyy expression correlated with body weight loss and circulating leptin and PYY concentrations. Central regulation of energy metabolism is not limited to the hypothalamus but also includes the midbrain and the brainstem. Our findings suggest a role for hindbrain PYY in the regulation of energy homeostasis and provide a starting point for further research on gigantocellular reticular nucleus PYY neurons, which will increase our understanding of the brain stem pathways in the integrated control of appetite and energy metabolism.

Basic helix-loop-helix transcription factors and enteroendocrine cell differentiation.

For over 30 years it has been known that enteroendocrine cells derive from common precursor cells in the intestinal crypts. Until recently little was understood about the events that result in commitment to endocrine differentiation or the eventual segregation of over 10 different hormone-expressing cell types in the gastrointestinal tract. Enteroendocrine cells arise from pluripotent intestinal stem cells. Differentiation of enteroendocrine cells is controlled by the sequential expression of three basic helix-loop-helix transcription factors, Math1, Neurogenin 3 (Neurog3) and NeuroD. Math1 expression is required for specification and segregation of the intestinal secretory lineage (Paneth, goblet,and enteroendocrine cells) from the absorptive enterocyte lineage. Neurog3 expression represents the earliest stage of enteroendocrine differentiation and in its absence enteroendocrine cells fail to develop. Subsequent expression of NeuroD appears to represent a later stage of differentiation for maturing enteroendocrine cells. Enteroendocrine cell fate is inhibited by the Notch signalling pathway, which appears to inhibit both Math1 and Neurog3. Understanding enteroendocrine cell differentiation will become increasingly important for identifying potential future targets for common diseases such as diabetes and obesity.

Identification of a transcriptional enhancer important for enteroendocrine and pancreatic islet cell-specific expression of the secretin gene.

It is well established that the gene encoding the hormone secretin is expressed in a specific enteroendocrine cell, the S cell. We now show that the secretin gene is transiently expressed in insulin-producing B cells of the developing pancreatic islets in addition to the intestine. Furthermore, secretin is produced by most established islet cell lines. In order to identify and characterize the regulatory elements within the secretin gene that control tissue-specific expression, we have introduced secretin reporter gene constructions into the secretin-producing HIT and STC-1 cell lines as well as the nonexpressing INR1-G9 glucagonoma line. Analysis of deletion mutants revealed that sequences between 174 and 53 bp upstream from the transcriptional start site are required for maximal expression in secretin-producing cells. This positive element functioned independently of position and orientation. Further deletions into the enhancer resulted in a stepwise loss of transcriptional activity, suggesting the presence of several discrete control elements. The sequence CAGCTG within the secretin enhancer closely resembles that of the core of the B-cell-specific enhancer in the insulin gene. Point mutations introduced into this putative element led to greater than 85% reduction in transcriptional activity. Gel mobility shift assays suggested that a factor in B cells closely related or identical to proteins that bind to the insulin enhancer interacts with the CAGCTG motif in the secretin gene.

Isolation, characterization, and developmental expression of the rat peptide-YY gene.

In the present study we describe the isolation, structural characterization, and developmental expression of the gene encoding the intestinal hormone peptide-YY. Examination of the nucleotide sequence of the peptide-YY gene reveals that each of the four exons encodes a functional domain of its mRNA that is analogous to the corresponding exons of the genes encoding two closely related peptides neuropeptide-Y and pancreatic polypeptide. The highly conserved structural organization of the genes encoding this family of three peptides suggests that each gene arose from the duplication of a common ancestral gene. Developmental studies reveal that the peptide-YY gene exhibits a complex pattern of tissue-specific expression in the gastrointestinal tract. Unlike many gastrointestinal hormones, peptide-YY mRNA levels are highest before birth. The pancreas appears to be the major site of peptide-YY gene expression in the fetus, exceeding colonic expression by 7-fold. The abundance of peptide-YY mRNA in the pancreas declines rapidly after birth, in contrast to the colon, where mRNA levels are maintained throughout development into adulthood. Expression of the peptide-YY gene before birth antedates the presence of known enteral secretagogues for this hormone, suggesting alternate mechanisms that control its biosynthesis during development.

Review article: transcriptional events controlling the terminal differentiation of intestinal endocrine cells.

Secretin-producing enteroendocrine cells arise from a multipotential endocrine progenitor in the crypts of the small intestine. As these cells migrate up the crypt-villus axis, they produce secretin and stop dividing as they terminally differentiate and die. Transcription of the secretin gene is controlled by a complex enhancer binding to multiple transcription factors. The basic helix-loop-helix protein, BETA2, binds to an E box sequence and associates with the p300 coactivator to activate transcription of the secretin gene. Basic helix-loop-helix proteins appear to play a pivotal role in the control of cellular differentiation. BETA2 induces cell cycle arrest and apoptosis in addition to activating secretin gene expression. Thus BETA2 may function as a master regulatory gene to coordinate terminal differentiation of secretin cells.

Different splice site utilization generates diversity between the rat and human pancreatic polypeptide precursors.

Pancreatic polypeptide is derived from a polyprotein precursor molecule. Although the amino acid sequences specifying the signal peptide and pancreatic polypeptide are well conserved between the rat and the human, the carboxy-terminal amino acid sequences of the precursors are highly divergent. To better understand the molecular basis of the divergence between the rat and human C-terminal peptides, we have determined the nucleotide sequence of the rat pancreatic polypeptide gene. A comparison between the primary structures of the rat and human genes reveals that the heterogeneity of the C-terminal peptides can be explained in large part by a frameshift mutation and the utilization of an alternative splice donor site in the third exon of the rat gene. As a consequence of the displaced splice site, part of the third exon of the rate gene is homologous to the sequence in the third intron of the human gene. Our results suggest that the rat and human pancreatic polypeptide genes arose from a common ancestral gene, and that differences in the C-terminal domains of the precursor reflect less strict evolutionary constraints than those imposed upon the amino-terminal domains of the precursor.

Structure of a precursor to human pancreatic polypeptide.

We have isolated mRNA from a human pancreatic islet cell tumor and have identified among the cell-free translation products a precursor of pancreatic polypeptide with an approximate Mr = 11,000. Recombinant DNA molecules encoding this precursor were selected from a cDNA library prepared from the islet tumor mRNA. From the nucleotide sequences of cDNAs encoding the precursor, we have deduced the complete amino acid sequence of pre-propancreatic polypeptide. These sequences encode a protein consisting of 95 amino acid residues with a Mr = 10,432. The sequence of human pancreatic polypeptide occurs in the middle of the precursor and is flanked at its carboxyl terminus by a 27-amino acid sequence which is similar to a peptide previously isolated from canine pancreatic islets. At the amino terminus of the precursor is a probable leader sequence which is rich in hydrophobic residues. A smaller pancreatic polypeptide-related protein was generated in cell-free translations of mRNA supplemented with microsomal membranes. Sequential Edman degradations of this smaller peptide indicate that the sequence of pancreatic polypeptide is located at the amino terminus of the prohormone.

Secretin: structure of the precursor and tissue distribution of the mRNA.

Secretin is a 27-amino acid gastrointestinal hormone that stimulates the secretion of bicarbonate-rich pancreatic fluid. The unusually high number of serine, leucine, and arginine residues in secretin has precluded the use of oligonucleotides to screen cDNA libraries to isolate a secretin cDNA. In the present study, a short cDNA encoding porcine secretin was amplified from duodenal mucosal first-strand cDNA template by using 16,384- and 4096-fold degenerate primers in the DNA polymerase chain reaction. From the sequence of the amplified cDNA, an unambiguous oligonucleotide probe was designed to screen a cDNA library. Here we report the sequences of cDNAs encoding the porcine and rat secretin precursors. The predicted amino acid sequences reveal that each precursor consists of a signal peptide, an N-terminal peptide, secretin, and a 72-amino acid C-terminal peptide. Secretin has been highly conserved through evolution. Rat secretin differs from its porcine counterpart by a single glutamine-for-arginine substitution at position 14. In contrast, the amino acid sequences of the C-terminal peptides are only 39% conserved between the two species, suggesting that the C-terminal peptide does not have an essential physiologic function. RNA blot hybridizations reveal that the rat secretin gene is expressed throughout the small intestine. Although secretin immunoreactivity has been localized in the central nervous system by some laboratories, we are unable to detect secretin mRNA in tissues of the central nervous system by Northern blot hybridization.

Expression of peptide YY in all four islet cell types in the developing mouse pancreas suggests a common peptide YY-producing progenitor.

The islets of Langerhans contain four distinct endocrine cell types producing the hormones glucagon, insulin, somatostatin and pancreatic polypeptide. These cell lineages are thought to arise from a common, multipotential progenitor cell whose identity has not been well established. The pancreatic and intestinal hormone, peptide YY, has been previously identified in glucagon-producing cells in islets; however, transgenic mice expressing Simian Virus 40 large T antigen under the control of the peptide YY gene expressed the oncoprotein in beta, delta and pancreatic polypeptide cells, and occasionally developed insulinomas, suggesting relationships between peptide YY-producing cells and several islet cell lineages. The four established pancreatic islet cell types were examined for coexpression of peptide YY in islets of normal and transgenic mice throughout development. Peptide YY immunoreactivity was identified in the earliest endocrine cells in the fetal pancreas and was coexpressed in each islet cell type during development. Peptide YY showed a high degree of co-localization with glucagon- and insulin-producing cells in early pancreatic development, but by adulthood, peptide YY was expressed in less than half of the alpha cells and was no longer expressed in beta cells. Peptide YY was also coexpressed with somatostatin and pancreatic polypeptide when these cell types first appeared, but most delta and pancreatic polypeptide cells continued to express peptide YY throughout development. The use of conditions that distinguish peptide YY from the related peptides, pancreatic polypeptide and neuropeptide Y, as well as the ability of the peptide YY gene to direct expression of a reporter gene in islets of transgenic mice, establishes expression of peptide YY in the earliest pancreatic endocrine cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of the amino-terminal domain of simian virus 40 early region in inducing tumors in secretin-expressing cells in transgenic mice.

BACKGROUND & AIMS: The early region of simian virus 40 (SV40) encodes 2 transforming proteins, large T (Tag) and small t antigen, that produce neuroendocrine tumors in the intestine and the pancreas when expressed in secretin cells of transgenic mice. METHODS: Two SV40 early-region transgenes containing a deletion that eliminated expression of the small t antigen were expressed in transgenic mice under control of the secretin gene. The 2 lines of mice, one expressing the native large T antigen and the other T antigen with a mutation in its N-terminal J domain, were examined to determine which biological activities of the SV40 early region were required for tumorigenesis. RESULTS: Most animals expressing wild-type large T antigen developed pancreatic insulinomas and lymphomas and died between 3 and 6 months of age. However, small intestinal neoplasms were extremely rare in the absence of small t antigen expression. Transgenic lines expressing the J domain mutant failed to develop tumors. CONCLUSIONS: Transformation of secretin-producing enteroendocrine cells by SV40 requires functional cooperation between intact large T and small t oncoproteins. In contrast, large T antigen alone is sufficient to induce tumors in the endocrine pancreas and thymus.

Peptide YY expression is an early event in colonic endocrine cell differentiation: evidence from normal and transgenic mice.

The hormone peptide YY is produced by endocrine cells in the pancreas, ileum and colon. We have previously shown that peptide YY is coexpressed in all four islet cell types in the murine pancreas when they first appear, suggesting a common peptide YY-producing progenitor. In the colon, peptide YY has been frequently identified in glucagon-expressing L-type endocrine cells. Characterization of colonic endocrine tumors in transgenic mice expressing simian virus 40 large T antigen under the control of the peptide YY gene 5' flanking region revealed tumor cells producing not only peptide YY and glucagon, but also neurotensin, cholecystokinin, substance P, serotonin, secretin, and gastrin. This suggested that multiple enteroendocrine lineages were related to peptide YY-producing cells. Subsequent examination of the ontogeny of colonic endocrine differentiation in nontransgenic mice revealed that peptide YY was the first hormone to appear during development, at embryonic day 15.5. Between embryonic days 16.5 and 18.5, cells expressing glucagon, cholecystokinin, substance P, serotonin, secretin, neurotensin, gastrin and somatostatin first appeared and peptide YY was coexpressed in each cell type at this time. Peptide YY coexpression continued in a significant fraction of most enteroendocrine cell types throughout fetal and postnatal development and into adulthood, with the exception of serotonin-producing cells. This latter population of cells expanded dramatically after birth with rare coexpression of peptide YY. These studies indicate that expression of peptide YY is an early event in colonic endocrine differentiation and support the existence of a common progenitor for all endocrine cells in the colon.

Exons of the human pancreatic polypeptide gene define functional domains of the precursor.

Pancreatic polypeptide is a 36-amino acid peptide which inhibits pancreatic exocrine function. We have previously determined from the nucleotide sequence of a cDNA that pancreatic polypeptide is derived from a 95-amino acid precursor, prepropancreatic polypeptide. Pulse-chase studies have suggested that the precursor is cleaved to produce three peptides: pancreatic polypeptide, an icosapeptide, and a smaller peptide. In the present study, we have used the cloned cDNA as a hybridization probe to isolate the pancreatic polypeptide gene from a human bacteriophage genomic library. The nucleotide sequence of 2.8 kilobases of DNA representing the entire human pancreatic polypeptide gene was determined. The gene contains four exons and three introns. Exon 1 encodes the 5'-untranslated region of the mRNA, exon 2 encodes the signal sequence and the sequence of pancreatic polypeptide, exon 3 encodes the icosapeptide, and exon 4 encodes a carboxyl-terminal heptapeptide and the 3'-untranslated region of the mRNA. By Southern blot analysis, the gene detected in a pancreatic polypeptide-producing islet cell tumor was indistinguishable from that in normal human leukocytes. The structure of the human pancreatic polypeptide gene is consistent with the hypothesis that prepropancreatic polypeptide generates three distinct peptides, each encoded by a separate exon. Increased expression of pancreatic polypeptide in the islet cell tumor does not appear to be correlated with major alterations in pancreatic polypeptide gene structure.

Targeted ablation of secretin-producing cells in transgenic mice reveals a common differentiation pathway with multiple enteroendocrine cell lineages in the small intestine.

The four cell types of gut epithelium, enteroendocrine cells, enterocytes, Paneth cells and goblet cells, arise from a common totipotent stem cell located in the mid portion of the intestinal gland. The secretin-producing (S) cell is one of at least ten cell types belonging to the diffuse neuroendocrine system of the gut. We have examined the developmental relationship between secretin cells and other enteroendocrine cell types by conditional ablation of secretin cells in transgenic mice expressing herpes simplex virus 1 thymidine kinase (HSVTK). Ganciclovir-treated mice showed markedly increased numbers of apoptotic cells at the crypt-villus junction. Unexpectedly, ganciclovir treatment induced nearly complete ablation of enteroendocrine cells expressing cholecystokinin and peptide YY/glucagon (L cells) as well as secretin cells, suggesting a close developmental relationship between these three cell types. In addition, ganciclovir reduced the number of enteroendocrine cells producing gastric inhibitory polypeptide, substance-P, somatostatin and serotonin. During recovery from ganciclovir treatment, the enteroendocrine cells repopulated the intestine in normal numbers, suggesting that a common early endocrine progenitor was spared. Expression of BETA2, a basic helix-loop-helix protein essential for differentiation of secretin and cholecystokinin cells was examined in the proximal small intestine. BETA2 expression was seen in all enteroendocrine cells and not seen in nonendocrine cells. These results suggest that most small intestinal endocrine cells are developmentally related and that a close developmental relationship exists between secretin-producing S cells and cholecystokinin-producing and L type enteroendocrine cells. In addition, our work shows the existence of a multipotent endocrine-committed cell type and locates this hybrid multipotent cell type to a region of the intestine populated by relatively immature cells.

Transcriptional regulation of secretin gene expression.

Expression of the gene encoding the hormone secretin is restricted to a specific enteroendocrine cell type and to beta-cells in developing pancreatic islets. To characterize regulatory elements in the secretin gene responsible for its expression in secretin-producing cells, we used a series of reporter genes for transient expression assays in transfection studies carried out in secretin-producing islet cell lines. Analysis of the transcriptional activity of deletion mutants identified a positive cis regulatory domain between 174 and 53 base pairs upstream from the transcriptional initiation site which was required for secretin gene expression in secretin-producing HIT insulinoma cells. Within this enhancer were sequences resembling two binding sites for the transcription factor Sp1, as well as a consensus sequence for binding to helix-loop-helix proteins. Analysis of these three elements by site-directed mutagenesis suggests that each is important for full transcriptional activity. The role of proximal enhancer sequences in directing secretin gene expression to appropriate tissues is further supported by studies in transgenic mice revealing that 1.6 kilobases of the secretin gene 5' flanking sequence were sufficient to direct the expression of either human growth hormone or simian virus 40 large T-antigen reporter genes to all major secretin-producing tissues.

Studies in transgenic mice reveal potential relationships between secretin-producing cells and other endocrine cell types.

We have produced transgenic mice expressing fusion genes consisting of 1.6 kilobase pairs of the secretin gene 5' flanking region to direct the expression of human growth hormone (hGH) or simian virus 40 large T antigen to secretin-producing cells. Analysis of different mouse tissues for hGH transcripts revealed expression in each of the major secretin-producing tissues, namely the intestine and endocrine pancrease. Multiple label immunohistochemistry demonstrated that the transgene was correctly directed to secretin cells in the intestinal tract, including a previously unrecognized population of secretin cells in the colon of adult and developing mice. In the small intestine, subpopulations of hGH-containing cells frequently coexpressed substance P, serotonin, and cholecystokinin, whereas in the colon, cells expressing hGH frequently coexpressed glucagon, peptide YY, or neurotensin. Transgenic mice expressing large T antigen in secretin cells developed poorly differentiated neuroendocrine tumors of the small intestine, well differentiated colonic tumors containing glucagon-expressing cells, and insulin-producing tumors in pancreas. These studies indicate that the major cis-regulatory sequences necessary for secretin expression in enteroendocrine cells and fetal islets are localized with 1.6 kilobase pairs of the transcriptional start site. Coexpression of reporter transgenes with several gastrointestinal hormones suggests a potential relationships between secretin cells and other enteroendocrine cell types, as well as pancreatic beta cells.

The basic helix-loop-helix protein BETA2 interacts with p300 to coordinate differentiation of secretin-expressing enteroendocrine cells.

The major epithelial cell types lining the intestine comprise a perpetually self-renewing population of cells that differentiate continuously from a stem cell in the intestinal crypts. Secretin-producing enteroendocrine cells represent a nondividing subpopulation of intestinal epithelial cells, suggesting that expression of the hormone is coordinated with cell cycle arrest during the differentiation of this cell lineage. Here we report that the basic helix-loop-helix protein BETA2 associates functionally with the coactivator, p300 to activate transcription of the secretin gene as well as the gene encoding the cyclin-dependent kinase inhibitor p21. Overexpression of BETA2 in cell lines induces both cell cycle arrest and apoptosis suggesting that BETA2 may regulate proliferation of secretin cells. Consistent with this role, we observed both reentry of normally quiescent cells into the cell cycle and disrupted cell number regulation in the small intestine of BETA2 null mice. Thus, BETA2 may function to coordinate transcriptional activation of the secretin gene, cell cycle arrest, and cell number regulation, providing one of the first examples of a transcription factor that controls terminal differentiation of cells in the intestinal epithelium.

Pyruvate carboxylase deficiency and lactic acidosis in a retarded child without Leigh's disease.

A child with lactic acidosis, severe mental and developmental retardation, and proximal renal tubular acidosis is presented. Biopsy and autopsy studies show severe hepatic, renal cortical, and cerebral deficiencies in pyruvate carboxylase (EC 6.4.1.1) activity. The patient had 1.81 +/- 0.20 units/g fresh weight at biopsy and 0.75 +/- 0.07 units/g fresh weight hepatic pyruvate carboxylase activity at autopsy compared with 10.9, 11.3, and 9.5 units/g fresh weight in two autopsy and one biopsy controls, respectively. The patient's renal cortical pyruvate carboxylase activity at autopsy was 0.008 +/- 0.004 units/g fresh weight compared with 5.05 units/g in the autopsy control. The patient had no detectable (less than 0.018 units/g fresh weight) cerebral pyruvate carboxylase activity at autopsy compared with 0.44, 0.53, and 0.695 units/g in the autopsy cerebrum of one human and two rhesus monkeys, respectively. Pyruvate dehydrogenase complex, phosphoenolpyruvate carboxykinase (PEPCK, EC 4.1.1.32), and fructose-1,6-bisphosphatase (EC 3.1.3.11) activities were in the normal range. The patient's urine pH was above 7.9 when the total serum CO2 was greater than 7.8 mM. However, the patient was able to acidify the urine to pH 5.1 when the total serum CO2 was 1.6 mM. The neuropathologic examination of the brain at autopsy revealed no sign of Leigh's disease, although developmental and degenerative lesions were observed. This is the first reported patient with a primary deficiency in hepatic, renal, and cerebral pyruvate carboxylase deficiency in whom the neuropathologic lesions, distinct from those of Leigh's disease, and proximal renal tubular acidosis have both been documented.

Peptide YY. Structure of the precursor and expression in exocrine pancreas.

Peptide YY is a 36-residue gastrointestinal hormone which inhibits both pancreatic and gastric secretion. We have isolated a cDNA encoding the peptide YY precursor by screening a rat intestinal lambda gt11 cDNA library with an antiserum directed against the porcine hormone. The nucleotide sequence of the cDNA encodes a 98-residue protein (molecular weight, 11, 121) which has an amino acid sequence identical to that of porcine peptide YY. Rat peptide YY is preceded immediately by a signal sequence and followed by a cleavage-amidation sequence Gly-Lys-Arg plus 31 additional amino acids. Thus the peptide YY precursor is similar in structure to that of two related peptides, pancreatic polypeptide and neuropeptide Y. RNA blot hybridizations reveal that the peptide YY gene is much more actively expressed in pancreas than previously realized. In situ hybridizations localized peptide YY cells exclusively to the exocrine pancreas. The abundance of peptide YY in one of its target organs, the pancreas, suggests a paracrine mechanism for peptide YY in regulating pancreatic enzyme secretion.

The basic helix-loop-helix transcription factor BETA2/NeuroD is expressed in mammalian enteroendocrine cells and activates secretin gene expression.

The gene encoding the hormone secretin is expressed only in enteroendocrine S cells and insulin-producing pancreatic beta cells during development. A 120-bp enhancer directs cell-specific expression of the rat secretin gene in secretin-expressing cells. The enhancer includes an E-box sequence, CAGCTG, which is important for transcriptional activity. To further characterize the role of the E box, a consensus binding site for basic helix-loop-helix (bHLH) proteins, we have examined factors that interact with this element in the secretin gene. The results suggest that transcription is activated by a recently isolated tissue-specific bHLH protein, BETA2, heterodimerized to the ubiquitously expressed bHLH proteins, Pan 1 and Pan 2, the rodent homologues of E47 and E12. The importance of BETA2 for transcriptional activation of secretin is further illustrated by antisense experiments inhibiting BETA2 expression in secretin-producing cell lines, which resulted in the inhibition of most E box-dependent transcription. Expression of BETA2 in a nonendocrine cell line conferred the ability to express secretin-reporter genes that are transcribed at minimal levels in the absence of BETA2. Secretin-producing enteroendocrine cells in the murine small intestine showed specific immunostaining with BETA2 antibodies, corroborating observations in cell lines. Thus BETA2 is to our knowledge the first transcription factor identified that specifically activates cell type-specific expression of an intestinal hormone gene.