Construction of a mutation due to a fourteen base-pair insertion in HNF-1α gene causing maturity-onset diabetes of the young (MODY) in Thai patients.

Objective: The aim of this study is to generate a mutation causing maturity-onset diabetes of the young (MODY) in Thai patients by insertion of a fourteen base-pair (bp) into a HNF-1a gene using a modified site-directed ligase-independent mutagenesis (SLIM) method. Methods: Two pairs of long- and short-tailed primers were designed to amplify a plasmid construct containing a HNF-1a and to insert a 14-bp at a desired position. Long-tailed primers contained the overhanging 14-nucleotide (nt) insert at their termini which were complementary to each other. Polymerase chain reactions (PCR) were performed in two separated tubes using different pairs of primers. After amplifications, PCR products from both tubes were pooled together, denatured and then re-annealed to allow formation of double stranded DNA molecules containing the 14-bp insert within HNF-1a. The pooled and reannealed PCR products without ligation were transformed into competent E.coli cells to generate a ligated recombinant plasmid with a 14-bp insertion. Results: Five of 14 bacterial colonies contained the desired recombinant plasmid with a 14-bp insertion within HNF-1a The efficiency of the method for generation of recombinant plasmid was about 36 percent. Conclusion: This method is simple and rapid to insert a long stretch of nucleotides into a plasmid construct containing a gene of interest at a desired position. A recombinant plasmid containing an insertion mutation in a HNF-1a gene was successfully generated, allowing an opportunity to perform functional study of the mutated gene.

Glycemic and lipid responses to glucomannan in Thais with type 2 diabetes mellitus.

OBJECTIVE: To evaluate the benefits of glucomannan supplement on glycemic and lipid controls in type 2 diabetic patients. MATERIAL AND METHOD: A single-blind, placebo-controlled, crossover trial with two treatments separated by a 2-week washout period was performed in 10 men and 10 women with type 2 diabetes mellitus. Two separated protocols of experiments were sequentially followed. Initially, purified glucomannan (1 g) or placebo was ingested 30 min before 75-g glucose load to evaluate their effects on glucose absorption and insulin secretion in oral glucose tolerance test (OGTT). Later, the glycemic and lipid changes after 4-week intervention with 3 g/day glucomannan comparing to the placebo were determined. The standard OGTT was performed before and after ending of each intervention. RESULTS: Glucomannan taken before performing the OGTT can lower the rise of blood glucose and insulin from 1 to 2 hour in comparison with the placebo, though a statistically significance of insulin was not achieved. Long-term glucomannan supplement significantly reduced the 120-min glucose area under the curve of OGTT. Glucomannan also decreased the rise of low-density lipoprotein cholesterol (LDL-C). Reductions of HOMA-insulin resistance index and body mass index were detected in glucomannan-treated group though the former was shown only in females. No within- and between-group differences of insulin, fructosamine, and other lipids were observed in glucomannan- nor placebo- treated groups. CONCLUSION: In type 2 diabetes, pre-prandial glucomannan ingestion attenuated a rise of blood glucose without significantly affecting insulin levels. Long-term supplement of glucomannan to the regular diabetic regimen lessened post challenge glucose AUC and impeded the rise of LDL-C. Supplement of glucomannan may be beneficial to the glycemic and lipid controls in type 2 diabetes mellitus.

Estrogen reduced blood glucose in high fat-fed mice: An animal model of type 2 diabetes.

Objective: This study aims to produce a mouse model of type 2 diabetes by using high fat diet. The C57BL/6J mouse strain can develop type 2 diabetes by putting on high fat diet. Methods: A group of C57BL/6J male mice were fed with a high fat diet (53% energy by fat) while another group was fed with normal diet (4.5% energy by fat). Results: At the 16th week of feeding study, the high fat-fed mice developed type 2 diabetes and had higher fat-pad weight than the normal diet-fed mice. However, plasma triglyceride (TG) levels of the two groups were not different. High fat-induced diabetic mice were administered 0.2 ตg/g body weight of 17-β estradiol for 2 weeks. Their fasting blood levels were reduced to become lesser than the levels in high-fat fed mice without estrogen. A trend of decrease in plasma TG level of 17β estradiol treated mice was observed. Conclusion: This study demonstrated that high fat diet could induce type 2 diabetes in a mouse model and that estrogen could reduce the fasting blood glucose in these mice.

Estrogen increases glucose-induced insulin secretion from mouse pancreatic islets cultured in a prolonged high glucose condition.

BACKGROUND: It is known that males are more susceptible to develop type 2 diabetes than females. Estrogen has a protective effect on pancreatic islet against toxic agent such as amyloid. The role of estrogen in protection pancreatic islet against high glucose is still unknown. OBJECTIVE: Administration of estrogen in an ovariectomised animal shows a protective effect against type 2 diabetes. The present study aimed to determine the direct effect of estrogen on the islet function after prolonged culture in high glucose. MATERIAL AND METHOD: Estrogen (10-1 M in ethanol) was co-cultured with mouse pancreatic islets in normal glucose medium (11.1 mM) for 3 hours or with normal and high glucose medium (40 mM) for 10 days. RESULTS: Estrogen increased glucose-induced insulin secretion in islet culture in normal glucose medium for both 3-hour and 10-day culture. Prolonged exposure of pancreatic islet to high glucose generated impaired glucose-induced insulin secretion, which was partially abrogated by the presence of 10(-5) M estrogen. CONCLUSION: These results indicated a direct effect of estrogen on improving insulin secretion from mouse pancreatic islets that has been impaired by prolonged exposure to high glucose.

In vitro study of insulin secretion from mouse pancreatic islets-siriraj experiences.

Pancreatic islet isolation and culture technique are tools for direct investigation of the effects of substances on insulin secretion. Glucose is a well known insulin stimulating substance from the pancreatic islet. This study aims to demonstrate the first success (in Thailand) in islet isolation with intact insulin secretion from a mouse pancreas using collagenase P enzyme and histopaque separation. Isolated islets were cultured in RPMI 1640 for 24 hours before undergoing a glucose stimulation test. Glucose at five different concentrations (2.8, 5.6, 10, 15 and 20 mM glucose was higher than with 2.8mM basal glucose concentration. Insulin secretion increased about 1.7 to 3.5 fold from a basal level of 2.8 mM glucose without any difference in insulin content at any glucose concentrations used. To our knowledge, these data demonstrate the first success in mouse pancreatic islet isolation and culture in Thailand. This technique can be used as a tool for further investigation of the in vitro effects of substances such as plants or new drugs on insulin secretion.