Cloning genomic INGAP: a Reg-related family member with distinct transcriptional regulation sites

Journal of Endocrinology

Bowman²,

Hamblet³

et al. 2006

Islet neogenesis associated protein (INGAP) is a protein factor that can stimulate new islet mass from adult pancreatic progenitor cells. In models of islet neogenesis, INGAP expression is elevated in pancreatic acinar cells. Using a transgenic model to drive a sustained expression of INGAP in pancreatic acinar cells, we have identified a protection to chemical-induced hyperglycemia. A sustained expression of INGAP during development did not perturb islet development or basal blood glucose homeostasis, although b-cell mass and pancreatic insulin content were significantly increased in the INGAP transgenic mice. When challenged with a diabetogenic dose of streptozotocin (STZ), mice carrying the INGAP transgene did not become hyperglycemic. In contrast, wild-type mice became and remained hyperglycemic, blood glucoseO 550 mg/dl. The serum insulin levels and islet morphology were preserved in the transgenic mice after STZ treatment. These data suggest that the sustained expression of INGAP in the acinar pancreas confers resistance to a diabetogenic insult. The INGAP transgenic mouse provides a new model to uncover factors that are protective to diabetes onset and biomarkers to track b-cell pathology.

Section: Introductionmentioning

confidence: 99%

Islet neogenesis associated protein transgenic mice are resistant to hyperglycemia induced by streptozotocin

Journal of Endocrinology

Bowman²,

Hamblet³

et al. 2006

“…Recent cloning of the genomic sequence for INGAP has identified promoter activity that is responsive to defined biochemical stimuli (Taylor-Fishwick et al 2003). Potential binding sites for PDX-1, a homeobox transcription factor, for PAN-1 a ubiquitous class A basic helix-loophelix transcription factor and for NeuroD a class B basic helix-loop-helix transcription factor were identified upstream to the transcriptional start site for the INGAP gene.…”

Section: Resultsmentioning

confidence: 99%

“…Expression of the factor was confirmed by Western blot analysis (data not shown). The human embryonic kidney epithelial cell line, 293, has no detectable expression of endogenous INGAP and can support INGAP-promoter reporter gene expression in response to defined exogenous stimuli (Taylor-Fishwick et al 2003). The ability of each transcription factor to activate the INGAP promoter was measured by the production of the reporter gene, -galactosidase using a luminescence-based assay.…”

Section: Resultsmentioning

confidence: 99%

“…Sequential deletions of the INGAP promoter were constructed as previously described (Taylor-Fishwick et al 2003). All fragments were digested with XmaI and BglII (New England Biolabs, Beverly, MA, USA) and subcloned into the p gal-basic vector (Clontech), a promoterless -galactosidase expression vector.…”

Section: Plasmids and Antibodymentioning

confidence: 99%

“…The molecular events regulating endogenous expression of INGAP are unclear and insight into these pathways has come from the recent cloning of the INGAP 5-prime regulatory region (Taylor-Fishwick et al 2003). The regulatory region of INGAP has, in addition to transcriptional elements resulting from conventional signaling pathways including AP-1 and STAT, a number of predicted interaction sites for transcription factors associated with pancreas development.…”

Section: Introductionmentioning

confidence: 99%

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PDX-1 can repress stimulus-induced activation of the INGAP promoter

Journal of Endocrinology

Shi²,

Pittenger³

et al. 2006

Self Cite

Islet neogenesis associated protein (INGAP) promotes the generation of new islet mass in adult animal models. It is not understood what factors control the expression of INGAP. In this study, factors that regulate the expression of INGAP promoter activity are reported. To determine factors that regulate INGAP expression, we previously cloned the promoter region for INGAP. Analysis of the INGAP promoter suggested that candidate regulators of INGAP expression include the transcription factors PDX-1, NeuroD, PAN-1, STAT and AP-1. Using gene addition experiments in the 293 cell line the activity of these transcription factors on an INGAP-promoter construct linked to the -galactosidase reporter has been determined. Induction of AP-1 activity or STAT activity using PMA or LIF stimulation respectively, or direct expression of PAN-1 specifically up-regulates INGAP promoter activity. In contrast, co-expression of PDX-1 but not NeuroD inhibits activation of the INGAP-promoter driven by PAN-1, PMA or LIF stimulation. PDX-1 binds directly to the INGAP promoter as determined in electromobility shift and antibody supershift assays. Expression of the INGAP-promoter-reporter construct in the HIT-T15 beta-cell line, a cell line that expresses endogenous PDX-1, did not reveal PMA-mediated stimulation of INGAP promoter activity. HIT-T15 cells however did efficiently transfect (>68%) and respond (2-fold) to PMA-induced signal transduction to a transfected AP-1-CAT reporter. Partial reduction of PDX-1 expression in HIT-T15 cells was associated with recovery of PMA induced INGAP promoter activity. These data suggest that expression of PDX-1 is associated with a repression of stimulus-induced INGAP promoter activity that appears to be mediated by a direct DNA interaction. These findings implicate PDX-1 in a possible feedback loop to block unbridled islet expansion.

Transplantation and beyond

Drug Development Research

Pittenger²,

Vinik³

2008

Self Cite

The pathophysiology of diabetes, either type 1 or type 2, involves the loss of b cell mass to a point where there is insufficient insulin secretion to meet the body's needs. The overall loss of b cells results from b cell destruction in conjunction with a failure of physiological regulators to restore or regenerate the b cell population. Historically, therapy for diabetes has been limited to efforts to control hyperglycemia and to prevent and/or ameliorate the complications associated with diabetes. Multiple approaches to repopulate the diabetic pancreas with functional islets have been proposed, including whole pancreas transplantation with or without a kidney, direct islet transplantation from cadaveric donors, generation of artificial islets, and development of new islets from stem cells. Ex vivo differentiation of embryonic stem cells offers great attraction as an alternative islet source but, to date, fully functional islets have not been developed. An alternative approach is to target stem cells present in the adult pancreas. Stimulation of these cells with various factors offers the hope of promoting differentiation to functional islets. Islet neogenesis-associated peptide (INGAP) is one such factor. INGAP treatment has reversed insulin-dependent diabetes in a mouse model. INGAP treatment has elicited production of C-peptide in insulin-dependent diabetic patients. Future approaches may combine INGAP with immunosuppression in an attempt to reverse type 1 diabetes. Drug Dev Res 69: [165][166][167][168][169][170][171][172][173][174][175][176] 2008