Glucose, metformin, and AICAR regulate the expression of G protein-coupled receptor members in INS-1 beta cell.

Glucagon-like peptide-1 receptor (GLP-1R), glucose-dependent insulinotropic polypeptide receptor (GIPR), and G protein-coupled receptor 40 (GPR40) are members of G protein-coupled receptors (GPCR) family. They are abundantly expressed in islet beta cells, and mediate effects of incretins and fatty acids in beta cells. Glucose and 5-AMP-activated protein kinase (AMPK) are known to be involved in the regulation of beta cell function. Metformin and the potential therapeutic drug for type 2 diabetes, 5-amino-4-imidazolecarboxamide riboside (AICAR), are both known activators of AMPK. Here we studied the effects of glucose, metformin, and AICAR on the expression of GPCR in INS-1 beta cell. INS-1 beta cells were supplemented with different concentrations of glucose, metformin, or AICAR. The expressions of GLP-1R, GIPR, GPR40, and a nuclear transcription factor - peroxisome-proliferator activated receptor alpha (PPARalpha) - were analyzed by real-time RT-PCR and immunoblotting. The time-course of the mRNA degradation of these receptors was also monitored by applying actinomycin D to cells. We demonstrated that the expressions of GLP-1R, GIPR, and PPARalpha were downregulated when INS-1beta cells were treated with glucose, while their expressions were upregulated when treated with metformin or AICAR. Glucose, metformin, or AICAR treatment had no obvious effect on the expression of GPR40. These results indicate that glucose, metformin, and AICAR regulated the expressions of incretin receptors and PPARalpha, but not GPR40 in beta cells. Whether AMPK is a key regulator of these factors mediated receptor regulation remains to be investigated further.

Does P-glycoprotein play a pivotal role in the drug resistance of an MDR variant, K562/Dox?

A multidrug-resistant (MDR) variant, K562/Dox, was selected from repeated exposure of human erythroleukemia cell line K562 to doxorubicin (Dox). K562/Dox displayed typical MDR features with respect to its cross-resistance to a variety of functionally and structurally unrelated compounds: vincristine (Vin), Dox, mitomycin C, reduced steady-state intracellular anthracycline accumulation, and elevated P-glycoprotein expression/mdr1 mRNA transcription/mdr1 gene amplification. Nevertheless, by incubation of cells with Dox/epirubicin (Epi)/daunorubicin (Dau) (5-80 micrograms/ml), the initial drug uptake was similar (p > 0.05) in K562/Dox and K562 cells, suggesting P-glycoprotein-mediated drug efflux would not occur unless a relatively high cellular drug concentration was reached. After 8 h incubation of cells with 50 ng/ml Dox (5 times higher than its IC50 to K562 cells), there were only slight differences (p > 0.05) in intracellular drug levels between K562/Dox and K562 cells, clearly indicating that K562/Dox, circumventing drug toxicity in this case, was irrelevant to reduced drug accumulation caused by P-glycoprotein. Similar results were obtained when Epi or Dau was applied. Despite complete restoration of anthracycline accumulation in K562/Dox cells in the presence of 6 mumol/l verapamil, the reversal of their drug resistance was incomplete. These results suggest that P-glycoprotein-mediated drug efflux possibly did not play a primary role in the drug resistance of K562/Dox cells.

Relationship between vas occlusion techniques and recanalization.

PIP: In southwest China between April, 1988, and March, 1990, providers at 5 family planning centers in Sichuan, Guangxi, Yunnan, and Guizhou provinces performed 2713 vasectomies using 7 occlusion techniques. They all used the no-scalpel vasectomy technique to expose, isolate, and divide the vas deferens. Researchers compared the follow-up outcomes of the various techniques during 1988 and 1992. The techniques included open-ended technique with the fascia interposition (group A); removal of vas segment and ends ligated with no. 1 silk suture (group B); vasectomy with fascia interposition on testicular ends (group C); same as group B plus cautery and washing the ends with phenol or 95% ethanol and normal saline (group D); folding and ligating prostatic ends with no. 1 silk suture (group E); electrocoagulation on ends of vas (group F), and open-ended without fascial interposition (group G). In the 2 years postvasectomy, 61 wives (2.57%) had at least 1 pregnancy. Sperm were still present in the semen of 27 of their vasectomized husbands at 2 years postvasectomy. Recanalization had occurred in 78 (3.29%) men overall. Among the 78 recanalization cases, 27 wives (34.6%) became pregnant (1.14% for the entire population). Group D cases whose vas ends were washed with phenol had a significantly higher reappearance rate of sperm than did those whose ends were washed with 95% ethanol (5.88% vs. 0; p .01). A significant difference in the reappearance rates of sperm in various technique groups occurred with group A having the lowest rate and group G having the highest rate (0.55% vs. 7.53%; p .0001). The reappearance rates for the 2 interposition groups were not statistically different (0.55% for group A and 2.63% for group C). Differences in the complication rates occurred (e.g., 0 for group A vs. 2.17% for group G; p .01). Based on these results, vasectomy plus interposition is the best vas occlusion technique.^ieng