Effect Of Free Fatty Acid On Insulin Resistance And β Cell Function

Obesity is associated with insulin resistance mainly due to the release of free fatty acids(FFA) from the expanded adipose tissue mass and cytokines.Numerous studies have shown that FFA,which are often elevated in obese individuals have been implicated as an important causative link in the association between obesity,insulin resistance and type 2 diabetes mellitus.An elevation of plasma FFA has been shown to impair insulin action,and to be a risk factor for the development of type 2 diabetes. Contrary to muscle insulin action and glucose metabolism,the effects of FFA on hepatic insulin action and glucose metabolism have not been extensively investigated.Glucose is produced through the pathway of glycogenolysis and gluconeogenesis. The main substrates for gluconeogenesis are free fatty acid(FFAs),glycerol,alanine and lactate,and liver glycogen is the substrate for glycogenolysis.Endogenous plasma FFAs are elevated and are correlated with the rates of basal hepatic glucose production(HGP) in type 2 diabetes.A large number of studies have demonstrated that infusion of intralipid + heparin(IH) increases endogenous glucose production (EGP) during euglycemic clamps.The increase of HGP could be partly attributed to a breakdown of hepatic autoregulation.In addition,FFA may decrease the ability of insulin to suppress HGP(i.e.hepatic insulin resistance) by impairing hepatic insulin action(signaling).This speculation is supported by recent studies in which high fat feeding decreased hepatic IRS-2 associated PI3-Kinase activity.The mechanism of elevation of HGP in type 2 diabetes is still unclear.Previous studies have demonstrated that insulin acts directly at the liver to suppress HGP by acutely inhibiting glycogenolysis(i.e.inhibition breakdown of glycogen.Insulin also indirectly suppresses HGP via its peripheral actions,by reducing the level of gluconeogenic precursors and FFA.Insulin binds to its receptor at the surface of the hepatocytes to initiate its action through signaling molecules. Upon insulin binding to its receptor,insulin receptor tyrosine kinase activity is activated,which results in receptor autophosphorylation.The activated insulin receptor tyrosine kinase also phosphorylates insulin receptor substrates,which include insulin receptor substrate(IRS) 1-4.IRS-2 has been shown to play an important role in hepatic insulin action.IRS-2 knockout mice have impaired hepatic insulin signaling (i.e.impaired PI3-Kinase activity,impaired insulin suppression of HGP),Enhanced insulin-stimulated PI3-kinase activity associated with IRS-1 and IRS-2 was observed in liver from rats fed a high-saturated-fat diet(58%lard,wt/wt) for 2 wk.Oxidative stress is present in type 2 diabetes patients,and can directly induce insulin resistance in vivo.Purpose1.TO determine whether the prolonged elevation of FFA-induced IR is associated with evidence of increased FFA oxidation and/or PKC activation and or decreased GluT-42.Determine whether the prolonged elevation of FFA affect insulin gene expression.Materials and Methods1.Animal modelsNormal Male Wistar rats,weighing 250-300g,were used for experiments.2.Surgical proceduresAfter 3-5 days of adaptation to the facility,rats were anesthetized and indwelling catheters were inserted into the right internal jugular vein for infusions and the left carotid artery for sampling.The venous catheter was extended to the level of the right atrium,and the arterial catheter was advanced to the level of the aortic arch.Both catheters were tunneled subcutaneously and exteriorized.The catheters were filled with a mixture of 60%polyvinylpyrrolidone and heparin(1,000 U/ml) to maintain patency and were closed at the end with a metal pin.The rats were allowed a minimum 3-4 days period of post-surgery recovery before experiments.3.Experimental designThe rats were fasted overnight and randomized to two groups,each of which received Intralipid plus Heparin(IH) infusion(20%Intralipid + 20U/ml heparin,5μl/min) and saline(SAL) was infused respectively with equivolume.The duration of different solution was 48h,and experimental determinations were made in the basal fasting state and during hyperinsulinemic euglycernic clamp.For the basal protocol,the different solution was infused throughout the 48-hour experiments.The Clamp protocols were similar to the Basal protocol with the additional infusion of insulin(5 mU.kg-1.min-1) during the last two hours of the experiments,resulting in plasma insulin levels in the postprandial range.To maintain euglycemia during insulin infusion,a variable infusion of 20%glucose was given intravenously through the jugular catheter and adjusted according to frequent glycemic determination(every 5-10 min).The glucose infusion was adjusted to sustain the normal glucose level.Blood samples for glucose,insulin,FFA were taken during the last 30 min(every 10 min) of each experiment.At the end of the experiments the rats were anesthetized with intraarterial pentobarbital,and the liver was freeze-clamped with pre-cooled aluminum tongs within 45 s of anesthetic injection while infusions were maintained through the jugular vein.4.Laboratory methods(1) Plasma glucose was measured with a Biosen Glucose Analyzer 5030(2) Insulin level was decided by radio-immune method(3) PKC-δtranslocation:Liver samples(250mg) were homogenized by hand-held glass homogenizer in buffer A,the homogenates were centrifuged at 100,000 x g for 1 h at 4oC,and the supernatants were retained as the cytosolic fraction.The pellet was resuspended in buffer B,homogenized by passing through a 23-gauge needle three times,incubated for 15 min on ice,and centrifuged at 100,000 x g for 1 h at 4oC.The supernatant provided the solubilized membrane fraction.The protein concentration in all samples was determined by the detergent-compatible modified Lowry microassay (BioRad),using serum albumin as the standard.Fiftyμg of protein in all samples were mixed with equal volumes of 3 x sample-loading buffer(6.86 M urea,4.29% sodium dodecyl sulphate(SDS),300 mM dithiothreitol,43 mM Tris-HCl(pH 6.8) and left at room temperature for 30 min.The mixture was then vortexed and subjected to SDS-PAGE(10%polyacrylamide).Following electrophoretic separation,proteins were transferred to Immobilon-P membranes.The membranes were then incubated for lh at 4oC in Tris-buffered saline-Tween(TBST) containing 5%non-fat dried milk, pH 7.4.After the blocking step,membranes were washed in rinsing solution(TBST, pH 7.4) and then incubated overnight at a concentration of 1:1000 with an affinity-purified polyclonal antibody specific for PKC-δ.The translocation of the DAG-sensitive isoform of PKC-δ,from cystosol to membrane reflects its activation, and was assessed by comparing immunoblots of the cytosolic and membrane-associated fractions.(4)Carbonyl protein:Liver tissue was homogenized at 4℃in a solution containing HEPES,cell debris was removed by centrifugation.Protein concentrations were determined using a standardized assay kiat with bovine serum albumine solution as the standard.Oxidative protein damage,assessed by the formation of carbonyl groups,was measured as described by Levine et al.1 mg of protein was precipitated by addition supernatant discarded.Protein pellets were incubated with and without 2, 4-dinitrophenylhydrazine(DNPH,10 mM in 2 HC1) and were allowed to stand at room temperature for 1 hour,vortexing every 15 minutes.Following incubation, protein was re-precipitated using 20%TCA and the pellet was obtained by centrifugation(8500g) for 3 minutes.The pellets were washed 3 times with ethanol-ethyl acetate(1:1) to remove free DNPH,allowing the samples to stand for 10 minutes each time before discarding the supernatant.The pellet was then re-dissolved in guanidine solution for 1 hour at 37℃.Insoluble material was removed by centrifugation(8500g) for 3 minutes.Carbonyl content was calculated from the sample absorbance at 365 nm compared to their complementary HCl-treated blanks, using a molar absorption coefficient of 22000M-1cm-1.Insulin expression was tested by immunohistochemistry method:1:500 on rat pancreas with overnight incubation and Cy labeled secondary antibody.Suggested tissue fixative is 10%formalin or 4% paraformaldehyde.It is best to cut unfixed frozen tissue and then fix the sections for 10 minutes in 4%paraformaldehyde in PBS followed by 4 x 5 minute rinses in PBS. Suggested permeabilization is 10 minutes in ice cold MeOH.Blocking buffer should contain serum from the same host as the secondary antibody.Optimal working dilutions must be determined by the end user.Result1.After 48h infusion of Intralipid + Heparin,plasma FFA level increased 2.1 fold of SAL,Plasma glucose levels higher with IH vs.SAL infusion in the basal experiments but were maintained at similar levels during the hyperinsulinemic clamps, and GIR of IH decreased 58.6%vs SAL.(P＜0.001).2.To investigate the mechanisms of FFA-induced impairment in hepatic glucose metabolism,we measured carbonyl protein content(to indirectly assess FFA oxidation),IH increased carbonyl protein by 3.2 fold of SAL(P＜0.001).3.IH induced hepatic PKC-δtranslocation from the cytosolic to the membrane fraction in basal study(Membrane/Cytosolic ratio:1.12+0.12 in SAL,4.60+0.36 in IH, P＜0.001).hepatic insulin resistance was associated with oxidative stress along with activation of PKC-δand subsequently with impairment of insulin signaling pathway.4.IH decreased hepatic GluT-4 translocation from the cytosolic to the membrane fraction in basal study,IH decreased GluT-4 translocation by 4.74 fold of SAL. (P＜0.001).5.48h infusion of Intralipid + Heparin affected insulin gene expression,in IH insulin(+) cell decreased 55.60%of SAL,and the level of PDX-1 are 0.78±0.04, 1.10±0.07 respectively in IH and SAL(P＜0.05).Conclusion1.Glucose and FFA levels were both elevated by IH,and IR was significant in IH.2.Oxidative stress has very important function on IR lead by the elevation of FFA.3.PKC-δtranslocation was observed in IH,it indicates that PKC-δplays a key role in insulin resistance induced by prolonged infusion of IH.4.The expression of GluT-4 on membrane decreased by Western Blot in IH,it leads to decreased effects of insulin.5.The level of PDX-1mRNA and insulin measured by RT-PCR and immune histochemistry respectively decreased in IH,insulin gene expression was affected by prolonged infusion of IH.