| ObjectivesObesity is a public health problem,and it increases the risk of metabolic disease including obesity,diabetes,fatty liver and so on.Leptin or leptin receptor dysfunction in both animals and humans is associated with profound metabolic abnormalities which include obesity,altered energy homeostasis,dyslipidemia,and hyperglycemia.The leptin receptor gene mutant db/db mouse displays obesity,infertility,and varying degrees of diabetes.Both ob/ob mice with a point mutation in leptin gene and leptin knockout rats display obesity,glucose intolerance,and hyperinsulinemia.Moreover,leptin affects insulin action in astrocytes and impairs insulin-mediated physical activity.Shi et al showed that immunization against leptin mimicked loss of leptin bioactivity and stimulated fat growth in certain types of animals.In addition,immunization against leptin receptor extracellular domain caused reductions in body weight in chickens(Z.D.Shi,unpublished data).However,the effects of intramuscular injection of leptin or its receptor immunogen on leptin-insulin feedback regulation and molecular mechanism are still unknown.The present study aimed to investigate the effects of intramuscular injection of exogenous leptin or its receptor on fat deposition,leptin-insulin feedback regulation and the molecular mechanism of leptin-associated signaling pathway.MethodsAnimals and ethical approvalThe present study was carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.The experimental protocol was approved by the Institutional Animal Care and Use Committee(IACUC)of the Southern Medical University of China(IACUC Number:R-20120928-01).Forty-five female SD rats(40 days old)were purchased from the Laboratory Animal Center of Southern Medical University of China and used for immunization.Collection of orbital venous plexus blood was performed under xylazine anesthesia(intramuscular injection,0.13 mg/kg body weight).Rats were anesthetized(intraperitoneal injection,60 mg/kg body weight)and euthanized by exsanguination prior to tissue collection.One rat per cage was allowed with free access to water and food.Preparation of immunogenRecombinant Tibet minipig's leptin(Lep)and the extracellular near-transmembrane domain of long form leptin receptor(LepR)were prepared and purified as described by previously.The Lep or LepR fusion proteins were adjusted to a concentration of 6 mg/mL and mixed with mineral oil adjuvant at a ratio of 1:2(v:v)and homogenized to produce the Lep or LepR immunogens containing a final concentration of 2 mg/mL for immunization.The immunogen for control group was prepared similarly but with keyhole limpet hemocyanin(KLH,a control protein used for immune competence testing).Animal grouping and samplingForty-eight female SD rats at 40 days of age were weighed and randomLy divided into three groups,including KLH(n=15),LepR(n=17)and Lep(n=16);rats in each group were intramuscularly injected with 1mL Lep or LepR or KLH immunogens(The concentration was 2mg/mL)on Day 1.Booster immunizations were given on Day 21 and 42.Lee's index,body weight,food intake,and body length were determined,and blood(orbital venous plexus,9:00 am)samples were collected on Day 0(1 day before experiment began),7,14,21,28,35,42,49,and 52.After centrifugation at 2000g,serum was separated and stored at-80? until analyses of antibody titer,serum chemistry(glucose,TC,HDL,LDL,TG,Ca and Pi),and serum hormones(FSH,LH,E2,PRL,P,T,GH,cortisol,CCK,GC,IGF-1,FT3,FT4,leptin and insulin).At Day 52,rats were euthanized and tissues were collected including abdominal fat,subcutaneous fat,liver,hypothalamus,leg muscle and pancreas.Abdominal fat(individual mesenteric,inguinal,and perirenal white tissues)and liver were removed,cleaned,and weighed.Abdominal fat(%)=abdominal fat weight(g)/body weight(g)*100.Liver index(%)=liver weight(g)/body weight(g)*100.Liver fat was extracted using Folch method,and the fatty acids were determined as previously reported.Lee's index was used to evaluate the degree of obesity of mature rats.Lee's index was calculated as follows:Lee's index=body weight(g)1/3/nasal-anal length(cm)*1000.Measurement of blood antibody titerA standard enzyme-linked immunosorbent assay(ELISA)method was adapted to measure anti-leptin titer in rat plasma.Recombinant Lep or LepR(5 mg/well in 250 mL)was used to coat 96-well microtiter plates(BIO-RAD,Shang Hai,China)for capturing anti-leptin or anti-leptin receptor antibody in the plasma(diluted 160 times with 5%skimmed milk),and biotinylated goat anti-rat antibody(Gene Co.,Guangzhou,China)was used to bind the captured antibody with the streptavidin labeled horseradish peroxidase(Dingguo Biotechnology Co.,Ltd.,China).Development of color was allowed to proceed in darkness upon addition of chromogen tetramethyl benzidine(Sigma Chemical Co.,St Louis,USA),in the presence of 0.03%hydrogen peroxide and terminated as appropriate with the addition of 2%sulphuric acid.Absorbance was read at 450nm(Model 680 micro plate×reader,BIO-RAD,california,USA)and the results represented anti-leptin or anti-leptin receptor antibody titers of both immunized and control rats.Glucose tolerance test(GTT)Six rats from each group(KLH,Lep,and LepR)were randomLy selected for GTT.The GTT was performed on Days 0,28,and 50.Rats were fasted for 6 h before intraperitoneal injection of D-glucose(2 g/kg body weight),and blood was collected by tail vein puncture.Fasting glucose levels at 0,15,30,60,and 120min were measured using glucose meter(Abbott Diabetes Care,FreeStyle Freedom,USA).Serum chemistry,serum leptin and insulin levelsSerum Ca,serum Pi,serum glucose,total cholesterol,HDL,LDL,triglycerides,sex hormones and reproductive-related hormones were determined by Nanjing Jiancheng Bioengineering Institute(Nanjing,China).CCK were measured with rat CCK enzyme-linked immunosorbent assay(ELISA)kit(Groundwork Biotechnology Diagnosticate,San Diego,CA,USA).Serum leptin level was measured using a rat leptin ELISA kit(Minneapolis Corporation,Billerica,USA).Serum insulin level was measured using an ELISA kit(American Laboratory Products Co.Diagnostics,Salem,USA).Histological analysis of fat tissue and liver and immunohistochemical analysis of pancreasTissues were collected in the same size and position from six rats of each group.Frozen sections of liver(right lobe,0.3×0.3x4.3 cm)were stained with Oil Red 0(Sigma,ST.LOUIS,MO,USA).Paraffin sections of subcutaneous or abdominal fat(right hip,0.5×0.5×0.5cm)were stained with hematoxylin and eosin.For immunohistochemical analyses,paraffin sections of pancreas(0.8×0.8×0.8 cm)were incubated with insulin antibody(Abcam,Cambridge,England).Microscopic images were observed,captured,and analyzed using Eclipse Ti-U inverted microscope(Nikon),NIS-Elements software(Nikon),and Image-Pro Plus,Version 6.0 software.Real-time polymerase chain reaction(RT-PCR)analysis of genes messenger ribonucleic acid(mRNA)expression in hypothalamus or fat tissue or musclePost homogenization,total ribonucleic acid(RNA)was extracted from hypothalamus using TRIzol reagent(Invitrogen,Guangzhou,China).Extracted RNAs were reverse transcribed to synthesize first-strand complementary deoxy ribonucleic acid(cDNA)using First Strand cDNA Synthesis Kit(TOYOBO,Osaka,Japan).The cDNA was stored at-20? for subsequent analysis.Primers were purchased from Invitrogen(Guangzhou,China).(?-actin sequences were 5'-CACCCGCGAGTACAACCTTC-3'(forward)and 5'-CCCATACCCACCATCACACC-3'(reverse);NPY sequences were 5'-CTGACCCTCGCTCTATCC-3'(forward)and 5'-GGTCTTC A AGCCTTGTTCT-3'(reverse);and POMC sequences were 5'-CCTCCTGCTTCAGACCTCCA'(forward)and 5'-GGCTGTTCATCTCCGTTGC-3'(reverse);gonadotropin-releasing hormone(GnRH)sequences were 5 '-ATTCTACTGACTTGGTGCGTG-3'(forward)and 5'-GGAATATGTGCAACTTGGTGT-3'(reverse);gonadotropin-releasing hormone receptor(GnRHR)sequences were 5'-GTATGCTGGAGAGTTACTCTGCA-3'(forward)and 5'-GGATGATGAAGAGGCAGCTGAAG-3'(reverse);leptin(Lep)sequences were 5'-CACACGCAGTCGGTATCC-3'(forward)and 5'-CGGCTACCACATCCAAGGAA-3'(reverse);SREBP-1 sequences were 5'-CCATCGACTACATCCGCTTCTT-3'(forward)and 5'-TGGGCTTTTCACCTGGTTATCCTC-3'(reverse).RT-PCR analysis was performed per the manufacturer's instructions:2min at 50?,2min at 950C,followed by 40 two-temperature cycles[15s at 95? and 30s at 60?(?-actin)or 63?(POMC)or 57?(NPY)or 62?(GnRH)or 68?(GnRHR)or 54?(Lep)or 55?(SREBP-1)](TOYOBO,RT-PCR Master Mix(SYBR Green),Osaka,Japan).Gene expressions were normalized against ?-actin.Western blottingLiver or hypothalamus tissue was homogenized and centrifuged.The supernatants was mixed with 2× LaemmLi buffer(Sigma-Aldrich Corp.,Saint Louis,MO),denatured,resolved by a denaturing polyacrylamide gel electrophoresis,and transferred to nitrocellulose membranes.The anti-JAK2,anti-stat3,anti-stat5,anti-PI3K,and anti-beta-actin primary antibodies(Cell Signaling Technology,Inc.,Boston,MA)were used at 1:200 dilution.The anti-rabbit secondary antibody(Cell Signaling Technology,Inc.)was used at 1:50,000 dilution.SuperSignal West-Pico Kit(Thermo Fisher Scientific,Waltham,MA)was used to develop the signal.The detail procedure was refer to the previous study.Statistical analysisAll values were reported as mean ± standard error of the mean.All statistical analyses were performed using SPSS software,Version 13.0.Two-way analysis of variance(ANOVA)was used to determine the significance among groups of Lee's index,body weight,food intake,body length,blood antibody titer,TC,HDL,LDL,TG,glucose,and GTT.Significances among groups of abdominal fat weigh,abdominal fat rate,liver weight,liver index,histological analysis,RT-PCR and western blotting were assessed by one-way ANOVA.Least significant difference method was used to compare group means.The P values less than or equal to 0.05 were considered significant.The P values less than or equal to 0.001 were considered extremLy significant.ResultsAnti-leptin antibody titer was high in Lep rats at the end pointBefore the first immunization,the anti-leptin or anti-leptin receptor antibody titers in blood were low in all the three groups of rats.However,seven days after the first inoculation of recombinant Lep or LepR immunogen,the anti-leptin antibody titers were significantly higher in Lep and LepR rats than control rats(P<0.001),which were barely detectable.Serum endogenous circulating leptin in Lep rats(8.98± 1.08ng/mL)was three fold higher than KLH(2.88±0.35ng/mL)or LepR rats(2.28± 0.17ng/mL).Increased food intake and decreased serum CCK in LepR or Lep ratFood intake of Lep or LepR rats was increased after the first injection.The respective p values were significant between control and Lep rats at Days 7,28,and 52.Similarly,the respective p values were significant between LepR and control rats at Days 7,28,49,and 52.The levels of serum CCK decreased in both KLH and Lep rats.The level of CCK was significant lower in Lep rat(0.88±0.07 ng/mL)and just only a quarter of that in KLH rat(3.46 ± 0.56 ng/m L).Lep immunogen increased rat body weight and Lee's indexTo quantify the level of obesity,body weight and nasal-anal length were measured and Lee's index was calculated.The body weight of rats immunized with Lep increased approximately 15%more than those immunized with KLH on Day 52.However,there was no significant differences between groups(P>0.05).Lee's index of Lep rats was highest among the three groups of rats between Day 21 and 52,whereas Lee's index of LepR rats was the lowest between Day 28 and 42.Lep immunogen increased TC,LDL,and TG,while LepR decreased LDL and triglycerides at the end pointObesity and diabetes-associated parameters were measured through serum chemistry.The increases in total cholesterol and LDL levels in Lep rats were statistically significant(P<0.05)after the second immunization.HDL levels were similar(P>0.05)among all three groups.Lep rats had the highest triglyceride level among the three groups of rats with a two-fold increase compared to control rats.Glucose intolerance,hyperglycemia,hypoinsulinemia,and decreased beta islet cell area in Lep ratsPost glucose challenge,Lep rats had a high baseline glucose level.Plasma glucose levels at 60 and 120 min on experimental Day 28 and at all-time points on experimental Day 50 were significantly increased in Lep rats compared to KLH and LepR rats,indicating glucose intolerance in Lep rats.Lep rats showed fasting hyperglycemia after 14 days.Meanwhile,Fasting serum insulin levels in Lep rats were significantly decreased(to less than half)than those in KLH and LepR rats.Decreased insulin levels in Lep rats were associated with decreased beta islet cell area compared to KLH rats,indicating impaired insulin secretion.Lep immunogen increased fat deposition,whereas LepR inhibited fat depositionLep rats maintained excess adiposity,steatotic liver,and leptin resistance.Conversely,immunization with LepR reduced fat deposition in abdominal and subcutaneous tissues and liver.At the end of the experiment,Lep rats had 45%more abdominal fat(5.95 ± 0.35g),while LepR rats(3.90±0.36g)had approximately 7%fatter,which was less than the KLH rats(4.18±0.40g).Both the absolute fat mass and relative fat/body weight ratio were significantly different(P<0.05)between KLH and Lep rats.Histology analysis revealed that subcutaneous per adipocyte cell area of Lep rats was 1.8-fold higher than KLH rats,while LepR rats had 0.79-fold increase than KLH rats;Subcutaneous adipocyte cell numbers of Lep rats were 0.7-fold higher than KLH rats(P<0.001),while LepR rats had 1.2-fold increase than KLH rats.Liver weight and liver index were not significantly different among the treatment groups,yet liver triglyceride content of Lep rats was approximately 40%higher than that of KLH rats;liver triglyceride content of LepR rats was not significantly different compared to KLH rats(P>0.05).Oil Red O staining of frozen liver sections showed steatotic liver.Histology analysis revealed that the liver fat area of the Lep rats was increased 8-fold compared to KLH rats(11.21 ±3.59?m2),whereas the liver fat area of LepR rats was just increased 0.34-fold compared to KLH rats.Lep rats showed increased steatotic livers;in contrast,LepR rats showed significantly decreased liver fat deposition compared with KLH rats.Sex hormones and reproductive related hormonesSex hormones in blood,including FSH,LH,P,PRL,E2 and T,were have no significant differences between groups before the first immunization(P>0.05).However,great changes had taken place at the end point of this experiment.Sex hormones in both KLH and Lep rat had increased with age.At 52 day,table 2 showed that FSH in Lep rat was 33%lower than that of KLH rat(6.22±0.45 mIU/mL)(P<0.05);meanwhile,LH and E2 in Lep rats were almost 75%of those in KLH rats(LH:4.42±0.36 mIU/mL;E2:17.38±1.44 mIU/mL)(P<0.05);moreover,the level of T in Lep rat was significantly higher(25%,P<0.05)than that of KLH rat(1148±119 ng/mL).There were no significant differences between KLH and Lep rat P and PRL(P>0.05).The levels of serum cortisolncreased with age,while GH decreased in both KLH and Lep rats.Serum cortisol in Lep rat(8.02±0.53ng/mL)was approximately 40%higher than that of KLH rat.The magnitude of change of GH was less and there was on significant differences between treatments(P>0.05).mRNA expressions of POMC,NPY,PPAR?,GnRH,GnRHR,UCP3,SREBP-1 and leptin in hypothalamus or fat tissue or muscleRT-PCR analysis revealed that the expression of POMC,NPY,two appetite-related genes in hypothalamus,had no significance between groups(P>0.05).However,the POMC/NPY ratio in hypothalamus was significantly(P<0.05)lower in LepR(0.51 ± 0.06)or Lep(0.54±0.05)rats than KLH rats(0.71±0.03)(Figure IF),albeit the differences were not significant(P>0.05)in the levels of UCP3(muscle),OB(abdominal fat),NPY,or POMC mRNA.PPARy in adipose tissue was higher(P<0.05)in the Lep or LepR immunized groups than in the control group.The mRNA expression of GnRH/?-actin in hypothalamus was significantly lower 88%in Lep rat than in KLH rat(0.252±0.020,P<0.05).The mRNA expression of GnRHR/?-actin in hypothalamus was slightly but non-significantly(P>0.05)lower in leptin-immunised(0.492±0.036)than in control rat(0.541±0.031)at the end point of the experiment.Compared to KLH rat,the mRNA expression of SREBP-1/?-actin in liver was significantly higher 40%in Lep rat,wherease lower 100%in LeR rat.Leptin receptor signaling-related protein expressionThe results showed that there was no statistically significant difference among groups,except for p-Stat3 among groups,and p-PI3K between Lep and KLH rat(P>0.05);p-Stat3 in hypothalamus of LepR rat was 4.1 fold higher than of KLH rat;p-Stat3 in liver was different from hypothalamus,both LepR and Lep rat were significantly higher than the control group.Compared with the control group,p-PI3K protein expression in the hypothalamus in Lep rat dropped by nearly 11 times(P<0.000);the expression in the liver was significantly reduced by 42%(P<0.05).ConclusionsImmunization against leptin receptor in SD rats reduced fat deposition and increased FSH,GH,IGF-1,which were correlated with the activated Jak2/Stat3 signaling pathway and accompanied by the up-regulation of PPAR?,GnRHR expression in the hypothalamus and down-regulation SREBP-1 in liver.Immunization against leptin in SD induces hyperphagia,adiposity,fatty liver,type 2 diabetes,sex hormone disorder and calcium loss in rat,all of which were correlated with the decreasing GnRH mRNA expression and POMC/NPY ratio in the hypothalamus,as well as attenuating the Jak2/PI3K signaling.This will be a potential rat model,which can be used in future to explore the treatment options for metabolic diseases.Extensive studies will be needed to determine safety,efficacy,and the mechanism of the effects of intramuscular injection of exogenous leptin or its receptor on the leptin-fat and insulin-glucose axis,hypothalamic-pituitary-gonadal axis and osteoporosis. |
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