Essential Requirement forPax6 in Control of Enteroendocrine Proglucagon Gene Transcription
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The primary function of islet A cells is the synthesis and secretion of glucagon, an essential hormonal regulator of glucose homeostasis. The proglucagon gene is also expressed in enteroendocrine L cells of the intestinal epithelium, which produce glucagon-like peptide 1 (GLP-1) and glucagon-like peptide 2 (GLP-2), regulators of insulin secretion and intestinal growth, respectively. We show here that Pax6, a critical determinant of islet cell development and proglucagon gene expression in islet A cells, is also essential for glucagon gene transcription in the small and large intestine. Pax6 is expressed in enteroendocrine cells, binds to the G1 and G3 elements in the proglucagon promoter, and activates proglucagon gene transcription. The dominant negative Pax6 allele, SEYNeu, represses proglucagon gene transcription in enteroendocrine cells. Mice homozygous for the SEYNeu mutation exhibit markedly reduced levels of proglucagon mRNA transcripts in the small and large intestine, and GLP-1 or GLP-2-immunopositive enteroendocrine cells were not detected in the intestinal mucosa. These findings implicate an essential role for Pax6 in the development and function of glucagon-producing cells in both pancreatic and intestinal endodermal lineages.Keywords:
Proglucagon
Enteroendocrine cell
PAX6
Alpha cell
Glucagon-like peptide-2
Prohormone convertase
Intestinal epithelium
We have investigated the effects of chronically elevated glucose concentrations on the pancreatic α-cell line αTC1-6. We show that basal glucagon secretion and proglucagon gene expression were increased in response to high glucose levels. The extent of acute stimulated secretion of glucagon was also increased in response to high glucose, as was the transcription of the prohormone processing enzymes PC1/3 and PC2. The secretion of GLP-1, a proglucagon-derived peptide produced by cleavage of proglucagon by PC1/3, was also increased in response to high glucose. Gene expression profiling experiments showed that a number of components of the regulated secretory pathway were up-regulated at high glucose concentrations, including processing enzymes and exocytotic proteins. Immunoblot analysis showed that the expression of the exocytotic SNARE proteins, as well as that of PC1/3, chromogranin A, and 7B2, were all increased after chronic exposure to high glucose levels. Immunocytochemistry showed no changes in the expression of the mature α-cell markers glucagon and brn-4 and no induction of the immature α-cell marker pdx-1. We conclude that chronically elevated glucose concentrations up-regulate the regulated secretory response of the α-cell.
Proglucagon
Prohormone convertase
Chromogranin A
Enteroendocrine cell
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The primary function of islet A cells is the synthesis and secretion of glucagon, an essential hormonal regulator of glucose homeostasis. The proglucagon gene is also expressed in enteroendocrine L cells of the intestinal epithelium, which produce glucagon-like peptide 1 (GLP-1) and glucagon-like peptide 2 (GLP-2), regulators of insulin secretion and intestinal growth, respectively. We show here that Pax6, a critical determinant of islet cell development and proglucagon gene expression in islet A cells, is also essential for glucagon gene transcription in the small and large intestine. Pax6 is expressed in enteroendocrine cells, binds to the G1 and G3 elements in the proglucagon promoter, and activates proglucagon gene transcription. The dominant negative Pax6 allele, SEYNeu, represses proglucagon gene transcription in enteroendocrine cells. Mice homozygous for the SEYNeu mutation exhibit markedly reduced levels of proglucagon mRNA transcripts in the small and large intestine, and GLP-1 or GLP-2-immunopositive enteroendocrine cells were not detected in the intestinal mucosa. These findings implicate an essential role for Pax6 in the development and function of glucagon-producing cells in both pancreatic and intestinal endodermal lineages.
Proglucagon
Enteroendocrine cell
PAX6
Alpha cell
Glucagon-like peptide-2
Prohormone convertase
Intestinal epithelium
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Proglucagon is processed differentially in the pancreatic alpha cells and the intestinal L cells to yield either glucagon or glucagon-like peptide 1, respectively, structurally related hormones with opposing metabolic actions. Here, we have studied the processing of proglucagon in alpha TC1-6 cells, an islet-cell line transformed by simian virus 40 large tumor (T) antigen, a model of the pancreatic alpha cell. We found that these cells process proglucagon at certain dibasic cleavage sites to release glucagon and only small amounts of glucagon-like peptide 1, as demonstrated by both continuous and pulse-chase labeling experiments. Both normal islet alpha cells and alpha TC1-6 cells were shown to express the prohormone convertase PC2 at high levels, but not the related protease PC3. Expression of PC2 antisense RNA in alpha TC1-6 cells inhibited both PC2 production and proglucagon processing concomitantly. We conclude that PC2 is the key endoprotease responsible for proglucagon processing in cells with the alpha-cell phenotype.
Proglucagon
Prohormone convertase
Alpha cell
Enteroendocrine cell
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Proglucagon
Prohormone convertase
Glucagon-like peptide-2
Chelerythrine
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Glucagon, which raises blood glucose levels by stimulating hepatic glucose production, is produced in alpha-cells via cleavage of proglucagon by prohormone convertase (PC)-2. In the enteroendocrine L-cell, proglucagon is differentially processed by the alternate enzyme PC1/3 to yield glucagon-like peptide (GLP)-1, GLP-2, and oxyntomodulin, which have blood glucose-lowering effects. We hypothesized that alteration of PC expression in alpha-cells might convert the alpha-cell from a hyperglycemia-promoting cell to one that would improve glucose homeostasis.We compared the effect of transplanting encapsulated PC2-expressing alpha TC-1 cells with PC1/3-expressing alpha TCDeltaPC2 cells in normal mice and low-dose streptozotocin (STZ)-treated mice.Transplantation of PC2-expressing alpha-cells increased plasma glucagon levels and caused mild fasting hyperglycemia, impaired glucose tolerance, and alpha-cell hypoplasia. In contrast, PC1/3-expressing alpha-cells increased plasma GLP-1/GLP-2 levels, improved glucose tolerance, and promoted beta-cell proliferation. In GLP-1R(-/-) mice, the ability of PC1/3-expressing alpha-cells to improve glucose tolerance was attenuated. Transplantation of PC1/3-expressing alpha-cells prevented STZ-induced hyperglycemia by preserving beta-cell area and islet morphology, possibly via stimulating beta-cell replication. However, PC2-expressing alpha-cells neither prevented STZ-induced hyperglycemia nor increased beta-cell proliferation. Transplantation of alpha TCDeltaPC2, but not alpha TC-1 cells, also increased intestinal epithelial proliferation.Expression of PC1/3 rather than PC2 in alpha-cells induces GLP-1 and GLP-2 production and converts the alpha-cell from a hyperglycemia-promoting cell to one that lowers blood glucose levels and promotes islet survival. This suggests that alteration of proglucagon processing in the alpha-cell may be therapeutically useful in the context of diabetes.
Proglucagon
Alpha cell
Prohormone convertase
Alpha (finance)
Enteroendocrine cell
Glucagon-like peptide-2
Homeostasis
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Proglucagon contains the sequence of two glucagonlike peptides, GLP-1 and GLP-2, secreted from enteroendocrine cells of the small and large intestine. GLP-1 lowers blood glucose in both NIDDM and IDDM patients and may be therapeutically useful for treatment of patients with diabetes. GLP-1 regulates blood glucose via stimulation of glucose-dependent insulin secretion, inhibition of gastric emptying, and inhibition of glucagon secretion. GLP-1 may also regulate glycogen synthesis in adipose tissue and muscle; however, the mechanism for these peripheral effects remains unclear. GLP-1 is produced in the brain, and intracerebroventricular GLP-1 in rodents is a potent inhibitor of food and water intake. The short duration of action of GLP-1 may be accounted for in part by the enzyme dipeptidyl peptidase 4 (DPP-IV), which cleaves GLP-1 at the NH2-terminus; hence GLP-1 analogs or the lizard peptide exendin-4 that are resistant to DPP-IV cleavage may be more potent GLP-1 molecules in vivo. GLP-2 has recently been shown to display intestinal growth factor activity in rodents, raising the possibility that GLP-2 may be therapeutically useful for enhancement of mucosal regeneration in patients with intestinal disease. This review discusses recent advances in our understanding of the biological activity of the glucagon-like peptides.
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Proglucagon is expressed in pancreatic alpha cells, intestinal L cells and brainstem neurons. Tissue-specific processing of proglucagon yields the peptide hormones glucagon in the alpha cell and glucagon-like peptide (GLP)-1 and GLP-2 in L cells. Both glucagon and GLP-1 are secreted in response to nutritional status and are critical for regulating glycaemia. The sorting of proglucagon to the dense-core secretory granules of the regulated secretory pathway is essential for the appropriate secretion of glucagon and GLP-1. We examined the roles of carboxypeptidase E (CPE), a prohormone sorting receptor, the processing enzymes PC1/3 and PC2 and putative intrinsic sorting signals in proglucagon sorting. In Neuro 2a cells that lacked CPE, PC1/3 and PC2, proglucagon co-localised with the Golgi marker p115 as determined by quantitative immunofluorescence microscopy. Expression of CPE, but not of PC1/3 or PC2, enhanced proglucagon sorting to granules. siRNA-mediated knockdown of CPE disrupted regulated secretion of glucagon from pancreatic-derived alphaTC1–6 cells, but not of GLP-1 from intestinal cell-derived GLUTag cells. Mutation of the PC cleavage site K70R71, the dibasic R17R18 site within glucagon or the alpha-helix of glucagon, all significantly affected the sub-cellular localisation of proglucagon. Protein modelling revealed that alpha helices corresponding to glucagon, GLP-1 and GLP-2, are arranged within a disordered structure, suggesting some flexibility in the sorting mechanism. We conclude that there are multiple mechanisms for sorting proglucagon to the regulated secretory pathway, including a role for CPE in pancreatic alpha cells, initial cleavage at K70R71 and multiple sorting signals.
Proglucagon
Alpha cell
Enteroendocrine cell
Prohormone convertase
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Proglucagon
Prohormone convertase
Glucagon-like peptide-2
Cleavage (geology)
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The proglucagon gene is expressed in a highly restricted tissue-specific manner in the α cells of the pancreatic islet, the hypothalamus, and the small and large intestines. Proglucagon is processed to glucagon and glucagon-like peptides GLP-1 and -2. Glucagon is expressed in α cells and regulates glucose homeostasis. GLP-1 is implicated in the control of insulin secretion, food intake, and satiety signaling, and GLP-2 is implicated in regulating small-bowel growth. Cell-specific expression of the proglucagon gene is mediated by proteins that interact with the proximal G1 promoter element which contains several AT-rich domains with binding sites for homeodomain transcription factors. In an attempt to identify major homeodomain proteins involved in pancreatic α-cell-specific proglucagon expression, we found that the POU domain transcription factor brain 4 is abundantly expressed in proglucagon-producing islet cell lines and rat pancreatic islets. In the latter, brain 4 and glucagon immunoreactivity colocalize in the outer mantle of islets. Electrophoretic mobility shift assays with specific antisera identify brain 4 as a major constituent of nuclear proteins of glucagon-producing cells that bind to the G1 element of the proglucagon gene proximal promoter. Transcriptional transactivation experiments reveal that brain 4 is a major regulator of proglucagon gene expression by its interaction with the G1 element. The finding that a neuronal transcription factor is involved in glucagon gene transcription may explain the presence of proglucagon in certain areas of the brain as well as in pancreatic α cells. Further, this finding supports the idea that the neuronal properties of endodermis-derived endocrine pancreatic cells may find their basis in regulation of gene expression by neuronal transcription factors.
Proglucagon
Alpha cell
Prohormone convertase
PAX4
Enteroendocrine cell
PDX1
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Proglucagon
Prohormone convertase
Glucagon-like peptide-2
Enteroendocrine cell
Alpha cell
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