| Objective:Metabolic Syndrome?MS?,as a high-risk factor of diabetes and cardiocerebral disease,is a cluster of conditions including obesity,hyperglycemia,high blood pressure as well as dyslipidemia,which are often companied by insulin resistance.Central obesity and insulin resistance are accepted as the most important features of MS,yet the details of both pathogeneses remain unclear.The researches of MS are based on appropriate animal models.The commonly used rodent obese models were set up by high fat diet,spontaneous genetic mutation or genetic remodeling.The animal model used in this research is Leptin receptor gene knockout(Lepr-/-)SD rat,which was set up with CRISPR/Cas9 technique by Institute of laboratory animal sciences,CAMS&PUMC in 2015.Functions and morphological changes need to be investigated to assess the validity of this model,though it is characterized by obesity,high food intake and dyslipidemia.As the most characteristic protein of FABPs family,adipocyte fatty acid binding protein?FABP4?is involved in fatty acid transport and lipid metabolism.FABP4 was initially found in adipocytes and macrophages.Recent researches revealed extensive expression of FABP4 as well as its complicated functions.Some of mouse with FABP4 defects manifest defence gene-or obesity-related insulin resistance.Our previous studies showed FABP4 inhibitor BMS309403 reducing fatty acid-induced endoplasmic reticulum stress?ERS?of the human mesangial cells,and FABP4 expressed in pancreatic islets of SD rats.Based on above evidences,we proposed the hypothesis in the present research that Lepr-/-rats might be an ideal and reliable MS model for the pathogenic and pharmacological researches.The hypothesis is supported by the following notions 1.Lepr-/-rats have the features of obesity,hyperglycemia and dyslipidemia;2.Chronic low-grade inflammation and ERS in pancreatic islets of Lepr-/-rats.Another hypothesis waiting to be proved in this research is that 3.Islet function improvement might be gained by ameliorating chronic pancreatic inflammation and ERS via inhibition of FABP4 in Lepr-/-rats.To prove our hypotheses,the research was set up in two parts:first,to investigate the general phenotypes of Lepr-/-rats and their islet function as well as changes of associated target organs to assess the validity of Lepr-/-rats as MS model;second,to assess the effects of FABP4 inhibitor BMS309403 on islet function,ERS and apoptosis in Lepr-/-rats.Methods:1 General phenotypes,islet function and pathological observation of associated organs of Lepr-/-rats.Rats were breed and their genotype were identified by PCR for this research.Rats of wild type?WT?and Lepr-/-at the age of 4 weeks as control and model were respectively picked into experiments.Body weights,daily food intake and blood glucose?random blood glucose,RBG and fasting blood glucose,FBG?of these rats were tested each week till the age of 18 weeks.Glucose tolerance test?GTT?and insulin tolerance test?ITT?were performed on the rats at the age of 10,14,and 18 weeks.Serum lipids were also tested at the same time.8 of WT rats and 10 of Lepr-/-rats?male and female in half?were sacrificed at the both ages of 14 and 18 weeks.Hearts,livers,kidneys,brains,testicles?male?and ovaries?female?from those rats were weighed and the organ coefficients were calculated.Parts of hearts,livers,kidneys and skeletal muscles were dyed with Oli red O.Parts of hearts,livers,kidneys,skeletal muscles,testicles,ovaries,pancreas,visceral adipose and brown adipose were made into paraffin sections and dyed with HE and CD68immunohistochemical?IHC?staining.2 The effects of FABP4 inhibitor on function,inflammation,ERS and apoptosis in pancreatic islet cells of Lepr-/-rats.8 WT rats of 10-week were randomly assigned into WT solvent control group?WT-C;n=5?or WT inhibitor group?WT-T;n=3?.10 Lepr-/-rats of10-week were randomly assigned into KO solvent control group?KO-C;n=5?or KO inhibitor group?KO-T;n=5?.Rats in both inhibitor groups were intragastrically administrated with 40mg/kg/d of BMS309403 for 4 weeks while solvent control groups were administrated with required amount of solvent.Body weights and food intake were kept recorded during experiments.ITT and GTT were performed to test islet function at the end of administration.Blood serum indexes were measured and hearts,livers,kidneys,brains,and testicles were weighed and organ coefficients were recorded.FABP4 protein,ESR related protein?GRP78,ATF6,p-IRE1??and apoptosis relevant protein,Cleaved caspase-3 were tested with Western blots.Immunohistochemistry analysis were performed to evaluate the expressions of Insulin,FABP4,CD68,GRP78,ATF6,p-IRE1?and Cleaved caspase-3.Results:1 The phenotypes of Lepr-/-rats and the pathological changes in their main organs.?1?Breeding and identification:PCR experiments were subjected to pups of 10-day and identified WT rats with band of 622 bp while Lepr-/-rats with band of 368 bp.Pups with both bands were Lepr+/-rats.?2?Lepr-/-rats had increased food intake and body weights from the age of 4 weeks when compared to WT rats.The average body weight of male Lepr-/-rats?807.8±100.0 g?is 1.5 times that of WT rats at the age of 18 weeks?P<0.05?while the average body weight of female Lepr-/-rats?645.5±100.7 g?is 2.3 times that of WT rats?P<0.001?.The average daily food intakes of Lepr-/-rats?male 48.9±3.2 g,female 33.4±6.8 g?were 1.8 and 2.2 times those of WT rats respectively?P<0.001?.?3?Increased RBG was companied by damaged glucose tolerance and insulin resistance while FBG was normal in male Lepr-/-rats.RBG of both male and female Lepr-/-rats fluctuated up to higher levels from 10 weeks to 18weeks,though individual differences showed in Lepr-/-rats.Damaged glucose tolerance was observed in Lepr-/-rats at the age of 14 weeks and worsened to the age of 18 weeks.Blood glucose of male Lepr-/-rats peaked to 20.0 mmol/L and kept at high level?12.6 mmol/L?till 120 minutes later after glucose stimulation.Heavier damage of glucose tolerance occurred in female Lepr-/-rats.Both male and female Lepr-/-rats of age 10-week developed insulin resistance condition,in which blood glucose slightly decreased after intraperitoneal injection with insulin,then went back to high level after 120minutes.Isulin resistance of Lepr-/-rats worsened at the age of 14 weeks.FBG of male Lepr-/-rats showed no signigicant difference when compared to WT rats.?4?Lepr-/-rats showed dyslipidemia and increased nonesterified fatty acids?NEFA?.Significant increases in total cholesterol,triglyceride,NEFA,high-density lipoprotein and low-density lipoprotein in serum were detected in Lepr-/-rats,whose changes were up to 1.5,8.7,2,7,1.5 and 1.8 folds when compared to WT rats?P<0.005?.Dyslipidemia of Lepr-/-rats was kept to 18weeks.?5?The weights of hearts,livers and kidneys of Lepr-/-rats increased.Comparing with WT rats of same age,Lepr-/-rats had increased organ/brain ratio of hearts,livers and kidneys,which went up with age.Male Lepr-/-rats showed an increased testicle/brain ratio with age whereas ovaries/brain ratio showed no significant difference in female Lepr-/-rats.?6?Lepr-/-rats showed islet proliferation,myocardial hypertrophy,fatty liver and obesity-associated kidney disease.The enlarged areas of islets and cell proliferation were observed in pancreatic islets of Lepr-/-rats at the age of14 weeks.Myocardial hypertrophy without lipid accumulation,big lipid droplets in hepatocytes,glomerular capillary dilation?diameter increased when compared to WT group,P<0.05?,subnuclear vacuoles in renal tubular epithelial cells as well as lipid droplets in parts of proximal convoluted tubular cells were also observed in Lepr-/-rats.Cell diameters of visceral adipose and brown adipose increased while multiple vacuoles decreased in brown adipose;no mature sperms were observed in seminiferous tubules while no corpus luteum and mature follicle in overies in Lepr-/-rats.Small lipid droplets occurred in skeletal myocytes in Lepr-/-rats.?7?Chronic low-grade inflammation occurred in multiple organs of Lepr-/-rats.CD68 positive cells in glomeruli,islets and visceral adipose increased in Lepr-/-rats?P<0.005?.2 The effects of FABP4 inhibitor on ERS and apoptosis in islet cells of Lepr-/-rats.?1?BMS309403 decreased body weights of Lepr-/-rats with no effect on their food intake.Treated by BMS309403 4-week,body weights of Lepr-/-rats decreased compared to KO-C group while no difference showed in body weights between WT-C and WT-T group.FABP4 inhibitor had no effect on food intake of WT rats.?2?BMS309403 ameliorated glucose tolerance disorder and insulin resistance of Lepr-/-rats.Compared with KO-C group,blood glucose of Lepr-/-rats administrated with BMS309403 peaked at 30 min in GTT and blood glucose tend to decline after injection of insulin.?3?BMS309403 ameliorated the condition of dyslipidemia in Lepr-/-rats.Serum triglyceride and non-esterified fatty acid of KO-T rats decreased when compared to KO-C group?P<0.05?.?4?BMS309403 had no effect on organ weights of Lepr-/-rats.Hearts,livers and kidneys of Lepr-/-rats after 4-week administration of BMS309403showed no difference in weights from those of rats in KO-C group.?5?BMS309403 decreased inflammatory responses in Lepr-/-rats.The counts of CD68 positive cells in islets of KO-T rats?0.78±0.17?decreased when compared to KO-C group?1.25±0.18??P<0.01?.?6?BMS309403 decreased ERS in islet cells of Lepr-/-rats.Along with the increased apoptotic protein,Cleaved caspase-3,the expressions of ERS associated protein GRP78,ATF6 and p-IRE1?increased in KO-C rat islets compared to WT-C group.Administration of FABP4 inhibitor decreased the expressions of GRP78,ATF6,p-IRE1?and Cleaved caspase-3 in KO-T rat islets.Conclusion:1 Lepr-/-rats have stable phenotypic characters of MS,such as obesity,high food intake,dyslipidemia,insulin resistance,low-grade inflammation in multiple organs hypertrophy and proliferation of islets,fatty livers,obesity associated kidney disease.While keep no different fertility from SD rats,both of which are evidences for Lepr-/-rats as an ideal model of obesity and MS.2 The islet cells in Lepr-/-rats manifested inflammation and ERS.FABP4inhibitor BMS309403 decreased the low-grade pancreatic inflammation in Lepr-/-rats and ameliorated ERS in islet cells as well as their apoptosis.All these results initially imply capability of FABP4 inhibitor BMS309403 as a therapy for MS. |
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