|
The islet transcription factor pancreatic duodenal homeobox-1 (PDX-1, also known as insulin promoter factor-1 or IPF-1) is an orphan homeodomain protein that plays an important role in the development, proliferation, differentiation and maturation of pancreatic cells. It is initially detected in the part of the dorsal and ventral primitive gut epithelium that later develops into the pancreas in embryonic period. In adults, its expression is found predominantly in the differentiated islet beta-cells. A recent study shows that PDX-1 is a major islet transcription factor which activates insulin gene transcription.1 Glucose-induced insulin biosynthesis is concerned with some motifs in insulin gene promoter, and PDX-1 activates insulin gene transcription and biosynthesis through binding to these motifs. Targeted inactivation of PDX-1 gene in the mice as well as its mutation in humans2 results in agenesis of the pancreas. Although PDX-1 gene expression does not appear to be required for pancreatic determination of the endoderm, it is crucial for the development of endocrine and exocrine cell types. Differentiation and maintenance of the beta-cell phenotype also require PDX-1. In mice, beta-cell-selective disruption of PDX-1 led to the development of diabetes with increasing age and was associated with reduced insulin and GLUT2 expression.3 Indeed, mice heterozygous for PDX-1 were found to be glucose intolerant.3,4 In transgenic mice expressing an antisense ribozyme specific for mouse PDX-1 in the beta-cells, the expression of PDX-1 gene was decreased and followed by impaired glucose tolerance and elevated glycated hemoglobin levels.5
In humans, a subpopulation of type 2 diabetes is monogenic and carries mutations in genes important for normal beta-cell function. Heterozygous individuals carrying one of the mutant genes develop a form of maturity-onset diabetes of the young (MODY). MODY4 has been linked to heterozygosity for mutations in PDX-1.6
In normal pancreatic beta-cells, glucose is the main physiological regulator of acute insulin secretion and biosynthesis, but supraphysiologic concentration glucose has inhibitive effects on beta-cell secretion and sensitivity of peripheral tissue to insulin, known as glucose toxicity. Indeed, exposure to high glucose levels causes impairment of beta-cell function in experimental systems, reducing glucose-induced insulin release and depleting the islet insulin content. Whether these glucose toxicity act through affecting the expression of PDX-1 remains to be clarified. We thus studied effects of supraphysiologic concentration glucose on the expression of PDX-1 and insulin secretion.
METHODS
Experimental animals and reagents
Mature male SD rats were supplied by the Experimental Animal Centre of Guangxi Medical University. Collagenase V and hexadecanoic acid were supplied by Sigma Corporation (USA), RPMI 1640 was supplied by Gibco Corporation (USA) and new-born calf serum by Hyclone Corporation (USA). Bovine serum albumin was purchased from Amresco Corporation (USA). Anti-PDX-1 polyclonal antibody was purchased from Chemicon Corporation (USA) and S/P immunohistochemistry kit, phosphate buffered solution (PBS) and polylysine slide were purchased from Maixin-Bio Corporation (Shanghai, China). Kit for rat in situ hybridization of PDX-1 was purchased from Boster Biotechnology Corporation (Wuhan, China). Insulin radioimmunoassay kit was purchased from Institute of Biological Products (Beijing, China).
Islet isolation
The method to separate islet cells described by Shen et al7 was applied in this study. Pancretic tissues were isolated from mature male SD rats weighting 200-250 g, which were anesthetized by 10% chloral hydrate through intraperitoneal injection. Pancretic tissue was washed with Hank solution 2-3 times, then was minced into 1-3 m3 pieces. Islet pieces were digested with mild shaking for 10-15 minutes at 37˚C by collagenase method (0.5 mg/ml collagenase V in Hank's solution containing Ca 7.5 mmol/L, pH 7.8 ), then the digestion was ceased by dropping iced Hank's solution (at 4˚C) containing 10% fetal bovine serum albumin, followed by standing for several minutes. The pellet was resuspended and the above mentioned procdure was repeated 2-3 times until the pellet was digested fully. Finally, the pellet was centrifugated at 200×g for 5 minutes, discarding supernatant, washed 3 times with iced Hank's solution containing 10% fetal bovine serum albumin. Then collected islet cells were cultured in RPMI 1640 containing 10% fetal bovine serum albumin (containing sodium pyruvate 1 mmol/L, L-glutamine 5 mmol/L, Heps 2 mmol/L, sodium bicarbonate 24 mmol/L, penicillin 100 IU/ml and streptomycin 100 µg/ml, pH 7.2-7.4), incubated for 24 hours at 37˚C (5% CO2 and 95% O2).
Experimental design
Experimental islet cells were incubated in bottles (1×107 cells/bottle) and divided into five groups randomly as follows: (1) Control group (LG3d): islet cells were incubated for 3 days in the media containing glucose 5.6 mmol/L and RPMI 1640. (2) LG6d group: islet cells were incubated for 6 days in the medium containing glucose 5.6 mmol/L and RPMI 1640. (3) HG3d group: islet cells were incubated for 3 days in the media containing glucose 33 mmol/L and RPMI 1640. (4) HG6d group: islet cells were incubated for 6 days in the medium containing glucose 33 mmol/L and RPMI 1640. (5) HG-LG group: islet cells were incubated for 3 days in the media containing glucose 33 mmol/L and RPMI 1640, followed by another 3 days incubation in the media containing glucose 5.6 mmol/L.
Experiment in every group was repeated six times. Nutrient solution was refreshed every 24 hours.
Experimental methods
Basic and glucose-induced insulin releasing tests
Separated islet cells were transferred to centrifuge tube of 1 ml (1×106 cells/tube), washed with PBS (pH 7.4) twice, preincubated for 1 hour at 37˚C in the medium containing glucose 5.6 mmol/L and KRBB buffer. Discarding the buffer, the islet cells were incubated for 1 hour at 37˚C in the media containing glucose of 5.6 mmol/L (basic) or 16.7 mmol/L (glucose-induced) and KRBB buffer. Collecting supernate, insulin was detected by RIA-double antibody method.
Assay of islet insulin content
Separated islet cells were transferred to centrifuge tube of 1 ml (1×106 cells／tube), frozen thawing repeatedly and extracted with acid alcohol (0.18 mmol/L hydrogen chloride : dehydrated alcohol = 1:3 volume ratio), incubated for 16-24 hours at 4˚C, insulin was detected by radioimmunoassay-double antibody method.
Assay of PDX-1 protein
Smeared with fresh islet cells, fixed for 20 minutes with 4% paraformaldehyde, washed with PBS (pH 7.4) 3 times and each for 3minutes. Immunohistochemical staining of PDX-1 protein was performed according to instruction manual of S/P immunohistochemistry kit. Positive cell (brown in color) number was counted using a microscope manually.
Assay of the expression of PDX-1 mRNA
Smeared with fresh islet cells, fixed for 20-30 minutes with 4% paraformaldehyde／0.1% DEPC／0.1 mol/L PBS (pH 7.2-7.6), PDX-1 mRNA was detected according to instructions from the manufacturer of the kit for rat in situ hybridization of PDX-1. Positive cell number was counted using a microscope manually.
Statistical analysis
The measurement data were expressed as means± standard deviation and enumerative data as percentage. Comparison within groups was performed by analysis of variance (LSD method and S-N-K method) or non-parameter analysis (Mann-Whitney U method and K-S method, k independent samples), correlation analasis with Spearman method. Statistical differences were computed with the software SPSS 10.0 from the SPSS Institute, USA. A P value less than 0.05 was considered statiscally significant.
RESULTS
Spearman correlation analysis showed that islet insulin content, basic and supraphysiologic glucose-induced insulin secretion were correlated positively with PDX-1 protein, but not with PDX-1 mRNA (Table 1).
 view in a new window |
Table 1. Correlation analysis of insulin with PDX-1 protein and PDX-1 mRNA (r value) |
Non-parameter analysis showed that compared with control group, supraphysiologic concentrtion glucose (HG3d group) inhibited PDX-1 protein synthesis, but did not inhibit PDX-1 mRNA expression (Table 2).
 view in a new window |
Table 2. Non-parameter analysis of high concentration glucose on the expression of PDX-1 (positive cells, %) |
Compared with control group, prolonging of islet cells incubation had no effects on PDX-1 protein synthesis in physiologic concentrtion glucose media, but did strengthen the suppression effects on PDX-1 protein synthesis in supraphysiologic concentrtion glucose media (Table 3).
 view in a new window |
Table 3. Analysis of variance of high concentration glucose on insulin secretion (mIU/106 cells per hour) |
PDX-1 protein in HG-LG group was much higher than that in HG3d group, but lower than that in control group (Table 3).
The results of immunohistochemial staining of PDX-1 protein in every group are shown in Figs. 1-4. The immunohistochemical staining of PDX-1 in HG3d group was weaker than that in LG3d group, the staining in HG6d group was the weakest among all groups, and in HG-LG group was stronger than that in HG3d group.
 view in a new window |
Fig. 1. PDX-1 in LG3d group (SP staining, original magnification ×40).
Fig. 2. PDX-1 in HG3d group (SP staining, original magnification ×40).
Fig. 3. PDX-1 in HG6d group (SP staining, original magnification ×40).
Fig. 4. PDX-1 in HG-LG group (SP staining, original magnify-cation ×40). |
DISCUSSION
Several studies over the past decade have shown that chronic elevation of the blood glucose concentration in humans and in experimental animals reduces insulin secretion as well as insulin action, leading to the concept of glucose toxicity. A finding of clinical relevance is that beta-cells lose their glucose sensitivity when plasma glucose concentration is elevated to 12 mmol/L in normal subjects.8 Conversely, induction of normoglycemia in type 2 diabetes can improve glucose-stimulated insulin secretion. One hypothesis is that increased secretary demand secondary to chronic hyperglycemia induces beta-cell exhaustion, with depletion of insulin storage. Indeed, studies showing that chronic beta- cell stimulation without hyperglycemia can mimic the “diabetic” beta-cell dysfunction support the “overworked beta-cell” hypothesis.9 Robertson and coworkers10 have shown that chronic exposure of insulinoma cell lines to supraphysiological glucose concentrations causes decreased insulin gene expression. The binding activities of the insulin-specific transcription factor PDX-1 were reduced in the HIT-T15 cells, suggesting that these events may provide a mechanism for glucose toxicity on beta-cell function.
The present study clearly demonstrated that within 3 days in supraphysiologic concentration glucose circumstances, islet insulin content diminished and basic and glucose-induced insulin release was markedly reduced. When switched from a supraphysiologic concentration glucose back to a physiologic concentration，with a great increase in PDX-1 protein there was a partial increase in islet insulin content, basic and glucose-induced insulin secretion, indicating that PDX-1 protein played a key role in glucose toxicity. PDX-1 regulated pancreatic beta-cell specific gene expression, including insulin, GLUT-2, glucokinase (GCK) and IAPP (islet amyloid polypeptide). Jonsson and coworkers demonstrated that mice lacking PDX-1 gene developed apancrea or pancreatic agenesis, led to death finally. Selective knockout of PDX-1 gene in mice could induce diabetes without insulin resistance, indicating that PDX-1 gene mutation could have relationship with type 2 diabetes. Study on human being also showed the mutation concerned with impaired glucose tolerance, type 4 MODY and type 2 diabetes.
Moran and colleagues11 discovered that when HIT-T15 cell line was incubated for a long time (from 75 to 99 generations) in glucose of 11.1 mmol/L, only immaturation PDX-1 mRNA could be detected, meanwhile insulin secretion decreased obviously. When incubation was continued to 146 generations in glucose of 0.8 mmol/L, insulin secretion recovered a little, but islet insulin content did not change. Our study demonstrated that switching from a supraphysiologic concentration glucose back to a physiologic concentration for 3 days led to increase in islet insulin content and insulin secretion.
Our data also demonstrated that islet insulin content, basic and glucose-induced insulin secretion correlated positively with the expression of PDX-1 protein, but not with level of PDX-1 mRNA in various kinds of glucose concentration. The phenomenon could be explained by regulation of post-transcriptional level. There may be disorder in post-transcriptional modification and splicing, lead to immaturation of mRNA and decrease in PDX-1 protein. Few studies in this regard have been reported.
REFERENCES
1. Hui H, Perfetti R. Pancreas duodenum homeobox-1 regulates pancreas development during embryogenesis and islet cell function in adulthood. Eur J Endocrinol 2002; 146: 129-141. [PubMed]
2. Stoffers DA, Zinkin NT, Stanojevic V, Clarke WL, Habener JF. Pancreatic agenesis attributable to a single nucleotide deletion in the human IPF1 gene coding sequence. Nat Genet 1997; 15: 106-110. [PubMed]
3. Ahlgren U, Jonsson J, Jonsson L, Simu K, Edlund H. Beta-cell-specific inactivation of the mouse Ipf1/Pdx1 gene results in loss of the beta-cell phenotype and maturity onset diabetes. Genes Dev 1998; 12: 1763-1768. [PubMed]
4. Brissova M, Shiota M, Nicholson WE, Gannon M, Knobel SM, Piston PW, et al. Reduction in pancreatic transcription factor PDX-1 impairs glucose-stimulated insulin secretion. J Biol Chem 2002; 277: 11225-11232. [PubMed]
5. Thomas MK, Devon ON, Lee JH, Peter A, Schlosser DA, Tenser MS, et al. Development of diabetes mellitus in aging transgenic mice following suppression of pancreatic homeoprotein IDX-1. J Clin Invest 2001; 108: 319-329. [PubMed]
6. Stoffers DA, Stanojevic V, Habener JF. Insulin promoter factor-1 gene mutation linked to early-onset type 2 diabetes mellitus directs expression of a dominant negative isoprotein. J Clin Invest 1998; 102: 232-241. [PubMed]
7. Shen SX, Dong YH, Feng LN, Bi YJ. Research of islet cell culture technology of SD rats. Chin J Organ Transplant (Chin) 1995; 16: 158-160.
8. Boden G, Ruiz J, Kim CJ, Chen X. Effects of prolonged glucose infusion on insulin secretion, clearance, and action in normal subjects. Am J Physiol 1996; 270: E251-E258.[PubMed]
9. Hosokawa YA, Leahy JL. Parallel reduction of pancreas insulin content and insulin secretion in 48-h tolbutamide-infused normoglycemic rats. Diabetes 1997; 46: 808-813. [PubMed]
10. Robertson RP, Olson LK, Zhang HJ. Differentiating glucose toxicity from glucose desensitization: a new message from the insulin gene. Diabetes 1994; 43: 1085-1089. [PubMed]
11. Moran A, Zhang HJ, Olson LK, Harmon JS, Poitout V, Robertson RP. Differentiation of glucose toxicity from beta cell exhaustion during the evolution of defective insulin gene expression in the pancreatic islet cell line, HIT-T15. J Clin Invest 1997; 99: 534-539. [ PubMed]
|
PDF(1107K)
Rapid Response |