Palmitic Acid's Toxicity On Beta Cell And Ameliorating Effect Mechanism Of Fenofibrate

Both Glucotoxicity and Lipotoxicity are two important etiologic theories of diabetes mellitus. It has been proved by clinical data in vivo or in vitro that abnormal high concentrations of free fatty acids, as one independent risk factor of diabetes mellitus, impair insulin secretion function of islet of Langerhans, so called lipotoxicity. Professor McGarry, one famous Banting science fund winner even regarded diabetes mellitus as 'Diabetes Mellipitus' in order to emphasize the important role of lipotoxicity effect on the pathogenesis of diabetes. But the relation between free fatty acids concentrations and function impairment of islet is reported sparsely. So the study about mechanism of lipotoxicity and novel drug of anti-lipotoxicity come into light. Fenofibrate (FF) is a representative drug of the fibrates used in therapies of hyperlipidemias and diabetes mellitus and also an agonist of peroxisome proliferator-activated receptor alpha (PPARα). There has been still two opposite arguments against PPAR α and lipotoxity. Some studies supported the idea that PPARα activated by the fibrates is one way of lipitoxicity. But Yoshikawa H reported PPAR α was inhibited by high level free fatty acids; and the function defect of beta cell induced by fatty acids was improved by treated with the fibrates. It was further testified that the fibrates have much beneficial to diabetic patients including not only increase basic insulin secretion and high concentration stimulated insulin secretion but improve insulin resistance in target tissue such as muscle and liver. So the mostimportant question is what's the direct effect of the fibrates on islet?Insulin secretion is a very complicated process. The extracellular glucose must be transferred into cytoplasm by glucose transport protein 2(GLUT2), and then phosphated by glucose kinase (GK). ATP is a metabolic production of glucose and enhances the ATP/ADP ratio accompanied by K+-ATP channel shutting, and then membrane depolarization induces secretion of insulin granules. Uncoupling protein-2(UCP-2) is a regulator of proton transfer across the mitochondrial membrane and uncouple ATP with oxidation metabolism. UCP-2 can inhibit the ATP sensitive K+ channel protein shutting and was regarded as a negative regulator of insulin. Pancreas-duodenum homeobox-1 (PDX-1) is a highly conserved 284 amino acids homedomain protein expressed selectively in pancreatic precursor cells and in mature pancreatic beta cell. It is essential for pancreas development, since loss of PDX-1 function results in agenesis of the organ both in mice and humans. PDX-1 is also needed for normal function of mature pancreatic beta cells. PDX-1 bonds with insulin promoter Al and A3/A4 box which characterized by containing core TAAT sequence, and directs insulin mRNA translocation from nuclear to cytoplasm, and then activates translation of insulin gene. Palmitic acid (PA) is main free fatty acids. So, we are interested in is, how effect of fenofibrate or palmitic acid on insulin secretion process?Objectives: The aim of this study was to explore several questions as followed: 1. What are the effects of different concentration PA on the basic insulin secretion (BIS), and on the high level glucose stimulated insulin secretion (GSIS) increasing compared with BIS (GSIS/BIS ratio)? Is beta cell function impairment correlated with PA dose? 2. If FF intervene insulin secretion function induced by PA in islets or INS-1 cell? 3. The effects of PA and/or FF on expression of insulin, PDX-1, GLUT2, UCP-2 and PPARa mRNA. 4. The influence of PA and/or FF on PDX-1, GLUT2 protein, and the activity of PDX-1 regulating insulin promoter.Methods: 1. Islet isolation and insulin release. Islets were isolated by digested with collagenase IV from Wistar rat pancreas and were free cultured with RPMI1640 containing 20% new calf serum. The islets were first cultured for 24 hours in medium added with 0.2mM or 0.4mM PA, and then transferred into Krebs Ringerbicarbonate (KRB) buffer containing 3 raM or 20 mM glucose for 20 minutes. Aliquots of the incubation media were retrieved for assay of insulin concentration and calculated BIS, GSIS/BIS ratio. In medium containing 0.2mM PA, different concentration FF(10"3M, lO^IVL 10"5M, 5X 10"6N^ 10"6M, 10'7M> 10"8M)were added and islets were cultured separately for 24 hours, and then did insulin assay.The rat insulinoma cell line INS-1 was cultured in RPMI1640 medium containing 10% FCS, 50 uM (3 - mercaptoethanol. INS-1 cells were routinely seeded in 48-well-plate for insulin secretion studies and used on day 4 at 60 ~70% confluence. INS-1 cell were divided into 4 groups through the whole experiment: control group, PA group (with 0.2mM PA), FF group (with 5xl0"6M fenofibrate) and PF group. After cultured for 24 hours the insulin release experiment was done just like islets.2. Cell morphology. INS-1 cell of control group, PA group, FF group and PF group were cultured for 24 hours and then dyed with typan blue to observe cell morphology changes. Glucose concentrations of mediums in the parallel cell were determined after cultured for 24 hours.3. Measurement of mRNA level. The islets were divided into 6 groups: control group, PA0.2 group, PA0.4 group, FF group (with 5xl0"6M fenofibrate), PA0.2 +FF group and PA0.4 +FF group and cultured for 24 hours. Total RNA were extracted by TRIzol isolation method from about 200~300 isolated islets of each group. 1 ug RNA was reverse transcripted (RT) in 20 ul volumes. 2ul RT reaction mixes were amplified with primers for insulin, GLUT2, PPAR a, UCP-2 and GAPDH for 30 cycles. Aliquots of the PCR were tested by 2% agarose gels and gels were stained with ethidium bromide. Signals were quantified by scanning densitometry. PDX-1 was determined with real time PCR.In control group, PA group, FF group and PF group INS-1 cell mRNA levels of GLUT2, PDX-1 > PPARct were determined at the same time.4. GLUT2 protein: After culturing for 24 hours, the INS-1 cells of 4 groups were disrupted in cell lysis buffer. Cell lysates containing 50ug of protein from each group were resolved in 7.5%polyacrylamide gels and transferred to a nitrocellulosemembrane. The membrane was incubated with goat polyclonal antibody of GLUT2. The bounds antibody was further detected with ECL reagent. The GLUT2 proteins in parallel cells were further determined by indirect immuno-fluorescence assay.5. PDX-1 Protein. 50ug total cell protein extracted from Trizol reagent were resolved in gels, the membrane were incubated with goat polyclonal antibody of PDX-1. P actin was examined at the same time.6. Electrophoretic mobility shift assay (EMSA): Biotin was labeled in 5' terminal of synthetic oligonucleotides containing Al element of the rat insulin I gene promoter. Double strand probes were maden by annealing at 95 °C for 3 minutes. Nuclear protein extract (20ug) were incubated with probes for 20 minutes at room temperature, and the complex were resolved by electrophoresis through 6% nondenaturing polyacrylamide gels. Then proteins were transferred to nylon membrane and cross-link DNA to membrane by ultraviolet radiation and obtained the desired signal on the film.Results: 1. Insulin Release: Both PA0.2 and PA0.4 increased BIS and impaired GSIS /BIS ratio as compared with control group (P<0.05). As compared with PA group, high concentration (10"3M, lO^M) FF decreased both BIS and GSIS/BIS ratio; too low concentration FF(10"7, 10"8M) had no effects on PA; but FF of 10"5~10'6M reduced BIS and increased GSIS/BIS which contrasted to the effect of PA. GSIS /BIS ratio of PA0.2 +FF group or PA0.4 +FF group were lower than control or FF group but higher than PA0.2 or PA0.4 group respectively.2. Cell morphology: The control group cell grew well. PA induced about 20% cell come into being little apoptosis body which possessed full membrane structure and didn't dyed by typan blue. FF could lessen the apoptosis body.3. m RNA levels. Both 0.2mM and 0.4 mM PA suppressed expressions of INS, PDX-1 and GLUT2 mRNA as compared with control group; but had no effects on PPARa. Expression of UCP-2 was enhanced in PA group while still higher than control when co-cultured with FF of 5 x 10"6 M. Both PA0.2 +FF group and PA0.4 +FF group enhanced expressions of INS, PDX-1, GLUT2 and PPARa mRNA as compared with PA0.2 or PA0.4 group respectively.The results of mRNA levels of PDX-1, PPARa in INS-1 cell were similar as in islets.4. GLUT2 Protein: Both western blot and immuno-fluorescence assay all showed there were no significant difference among different INS-1 cell groups.5. PDX-1 protein and bounding activity with insulin promoter Al element: INS-1 cell of PA0.2 group decreased PDX-1 protein level and activity with insulin promoter Al as compared with control group. FF group and PA0.2 +FF group had PDX-1 protein level and activity similar as control group. That is to say, FF can activate PDX-1 activity and improve protein concentration which inhibited by PA.Conclusions: ?PA increases BIS abnormally and impairs GSIS/BIS ratio profoundly, and there is a negative correlation between PA dose and beta cell function. (2) FF of 10"5~10'6M reduces BIS and increase GSIS/BIS ratio which contrast to the effect of PA. (3) Expression of UCP-2 is enhanced in PA group accompanied with insulin secretion function defection. It seemed UCP-2 can not interpret the interfering action of FF on PA. ? PA suppresses expressions of GLUT2 mRNA rather than protein levels; FF ameliorates GLUT2 mRNA induced by PA but not protein. (§) PA suppresses expressions of PDX-1 mRNA , protein concentration and activity of regulation insulin gene , while these changes are adjusted by FF.Significances: We explored the effects of different concentration PA on BIS and GSIS/BIS ratio and studied the different effects of FF on abnormal influence on beta cell of PA for the first time, and provide useful theory basis for hyperlipdemia therapy. We study the mechanism of fenofibrate ameliorating toxicity effect of palmitic acid from cell function, gene and protein. Especially we detailed the changes of PDX-1 gene, protein and activity of regulating insulin gene promoter for the first time. This study offer useful data for molecular mechanism of diabetes mellitus.