Inhibition of Y1 receptor signaling improves islet transplant outcome

Failure to secrete sufficient quantities of insulin is a pathological feature of type-1 and type-2 diabetes, and also reduces the success of islet cell transplantation. Here we demonstrate that Y1 receptor signaling inhibits insulin release in β-cells, and show that this can be pharmacologically exploited to boost insulin secretion. Transplanting islets with Y1 receptor deficiency accelerates the normalization of hyperglycemia in chemically induced diabetic recipient mice, which can also be achieved by short-term pharmacological blockade of Y1 receptors in transplanted mouse and human islets. Furthermore, treatment of non-obese diabetic mice with a Y1 receptor antagonist delays the onset of diabetes. Mechanistically, Y1 receptor signaling inhibits the production of cAMP in islets, which via CREB mediated pathways results in the down-regulation of several key enzymes in glycolysis and ATP production. Thus, manipulating Y1 receptor signaling in β-cells offers a unique therapeutic opportunity for correcting insulin deficiency as it occurs in the pathological state of type-1 diabetes as well as during islet transplantation.Islet transplantation is considered one of the potential treatments for T1DM but limited islet survival and their impaired function pose limitations to this approach. Here Loh et al. show that the Y1 receptor is expressed in β- cells and inhibition of its signalling, both genetic and pharmacological, improves mouse and human islet function.info:eu-repo/semantics/publishe

Characterization of the distal promoter of the human pyruvate carboxylase gene in pancreatic beta cells.

Pyruvate carboxylase (PC) is an enzyme that plays a crucial role in many biosynthetic pathways in various tissues including glucose-stimulated insulin secretion. In the present study, we identify promoter usage of the human PC gene in pancreatic beta cells. The data show that in the human, two alternative promoters, proximal and distal, are responsible for the production of multiple mRNA isoforms as in the rat and mouse. RT-PCR analysis performed with cDNA prepared from human liver and islets showed that the distal promoter, but not the proximal promoter, of the human PC gene is active in pancreatic beta cells. A 1108 bp fragment of the human PC distal promoter was cloned and analyzed. It contains no TATA box but possesses two CCAAT boxes, and other putative transcription factor binding sites, similar to those of the distal promoter of rat PC gene. To localize the positive regulatory region in the human PC distal promoter, 5'-truncated and the 25-bp and 15-bp internal deletion mutants of the human PC distal promoter were generated and used in transient transfections in INS-1 832/13 insulinoma and HEK293T (kidney) cell lines. The results indicated that positions -340 to -315 of the human PC distal promoter serve as (an) activator element(s) for cell-specific transcription factor, while the CCAAT box at -71/-67, a binding site for nuclear factor Y (NF-Y), as well as a GC box at -54/-39 of the human PC distal promoter act as activator sequences for basal transcription

Oligonucleotides used in EMSA.

*<p>3′ labeled with biotin.</p

The human PC P2 promoter sequence and its alignment with the rat PC P2 promoter.

<p>Boxes represent the putative transcription factor binding sites for Sp1, FoxA2/HNF3β, USF1/2, and CBF. Identical nucleotides between human and rat sequence are symbolized by an asterisk.</p

Identification of positive regulatory element(s) located between −114 and −39 of the human PC P2 promoter.

<p>(A) Schematic diagram of 15 bp internal deletions of −114/−39 of the human PC P2 promoter. (B) Transient transfections of a series of 15 bp internal deletion constructs into the INS-1 832/13 and non-beta cell HEK293T cell lines were performed to localize the positive regulatory sequence in the human PC P2 promoter. The luciferase activity of each construct was normalized with the β-galactosidase activity. The normalized reporter activity obtained from each construct is shown as a percent relative to those transfected with the wild type −365 hP2 promoter that was arbitrarily set at 100%. *P value <0.05, **P value <0.01. (C) Gel shift and supershift assays of biotin-labeled probe −78 to −54 region of hP2 promoter (−78/−54 CCAAT-probe) using INS-1 832/13 nuclear extract (Lane 1–5) and non-beta cell HEK293T (Lanes 6–10). The nucleotide sequence of wild type and mutant of the hP2 promoter in the −78 to −54 regions are also shown. Lanes 1 and 5 show probes incubated with nuclear extracts from INS-1 832/13 or HEK293T cells; lanes 2 and 6, 10-fold excess wild-type unlabeled oligonucleotides were incubated with nuclear extracts and probes; lanes 3 and 7, 50-fold excess wild-type unlabeled oligonucleotides were incubated with nuclear extracts and probes; lane 4 and 9, 50-fold excess amount of mutant unlabeled oligonucleotides were incubated with nuclear extracts and probes; lanes 5 and 10, nuclear extracts were pre-incubated with anti-NF-Y antibody before the probes were added to the reactions. Arrow represents CCAAT box–NF-Y, complex.</p

Localization of <i>cis</i>-acting elements of the human PC P2 promoter.

<p>Transient transfections of 8 constructs containing of the 5′-truncated hP2 promoter into INS-1 832/13 cells were performed to identify the regulatory regions of the hP2 promoter. The basal activity of each 5′-truncated hP2 promoter was calculated from the values of luciferase activity which was normalized with the values of β-galactosidase activity to control for transfection efficiency. The normalized luciferase activity of each P2 construct was compared with the activity of the pGL3-basic vector which was arbitrarily set to 1 and presented as the relative luciferase activity. *P value <0.05, **P value <0.01.</p

Oligonucleotides used for generation of 25 bp deletion of −365/−240 hP2, 15 bp deletion of −114/−39 hP2 and 5 bp deletion of −114/−39 hP2 promoter constructs.

<p>Oligonucleotides used for generation of 25 bp deletion of −365/−240 hP2, 15 bp deletion of −114/−39 hP2 and 5 bp deletion of −114/−39 hP2 promoter constructs.</p

Transactivation of a WT −365 human PC P2 luciferase reporter construct and its mutant by Sp1, Sp3, USF1 or USF2.

<p>WT −365 hP2 or −340/−315 hP2 constructs were co-transfected with an empty vector (pcDNA3) or a plasmid overexpressing Sp1, Sp3, USF1 or USF2 into the INS-1 832/13 cell line, and the luciferase activities measured. The luciferase activity was normalized to β-galactosidase activity and expressed as relative luciferase activity. Relative luciferase values obtained from co-transfecting cells with wild type (−365 hP2) or its mutant (−340/−315 hP2) and plasmid overexpressing Sp1, Sp3, USF1 or USF2 were presented as fold change relative to those obtained from those co-transfected with WT with empty vector (pcDNA3) which was arbitrarily set at 1. *p≤0.01.</p

RT-PCR analysis of PC mRNA variants in human liver and human pancreatic islets.

<p>(A) Schematic diagram showing alignment of 3 variants of human PC mRNA (GenBank NM_000920.3, NM_022172.2, BC011617.2). (B) Schematic diagram showing the structure of the human PC gene. Two isoforms of human PC mRNA are initiated by two alternative promoters, the proximal (P1) promoter and the distal (P2) promoter. All PC mRNA variants contain the same coding sequences but differ in their 5′-untranslated regions (UTR) produced from different 5′-UTR exons (UE1/UE2, UE3 and UE4) (C) Examination of human PC mRNA in liver and pancreatic islets using RT-PCR. Two sets of primers were used to amplify two different isoforms of human PC mRNA both in human liver and human islets. The 173 bp fragment PCR product of variant 2 and the 200 bp fragment PCR product of variant 1 were amplified by using Primers set no. 1 and primer set no.2, respectively, Lane 1; 1 kb marker, Lane 2; Negative control for primer set no.1, Lane 3; Negative control for primer set no.2, Lane 4; PCR using primer set no.1 and cDNA prepared from human liver, Lane 5; PCR using primer set no.2 and cDNA prepared from human liver, Lane 6; PCR using primer set no.1 and cDNA prepared from human islets, Lane 7; PCR using primer set no.2 and cDNA prepared from human islets.</p