Effectiveness And Mechanism Of Bariatric Surgery On Pancreatic And Renal Function In A Type 2 Diabetic Rats

Part 1 Effect and Mechanism of Bariatric Surgery on Islet Function in Type 2 Diabetic Rats.BackgroundDiabetes mellitus(DM)is a multi-etiological metabolic disease that seriously endangers human life and health.In 2017,the total number of diabetic patients around the world has reached 425 million.China has the heaviest diabetes burden,with 114 million patients,accounting for nearly 27%of the global total.The incidence of type 2 diabetes mellitus(T2DM)has shown a rapid upward trend in the past few decades.Most of the new patients are concentrated in low-and middle-income developing countries.The treatment and prevention of diabetes has brought tremendous economic and social burden,which has become an urgent public health problem.T2DM is a slowly progressive disease,and its natural history often manifests itself as normal glucose tolerance?impaired glucose tolerance? type 2 diabetes.The central aspect of T2DM development is the progressive decline of pancreatic?-cell function and insulin resistance,among which,the impaired ?-cell function is the basis of its pathogenesis and progression,and often exists in the early stage of disease,showing as impaired first phase insulin secretion and compensatory second phase insulin.Impaired glucose tolerance will further progress to T2DM if no intervention is implemented.An increasing number of studies have confirmed that chronic inflammation combined with glucose toxicity as well as lipotoxicity is an important pathogenesis of the progressive decline of ?-cell function.And NLRP3 inflammasome may play an important role in this pathological process.NLRP3(Nod-like receptor family pyrin domain containing 3)belongs to the nucleotide-binding oligomerization domain-like receptor(NLR)family pattern recognition receptor.The NLRP3 inflammasome is a protein complex composed of NLRP3,apoptosis-associated speck-like protein(ASC)and caspase-1.Once activated,the inflammasome activates caspase-1 rapidly and further cleaves IL-1? and IL-18 precursor molecules and promotes their secretion,which contributes to the inflammatory response.Recent studies have shown that NLRP3 inflammasome has played an important role in the occurrence and progression of T2DM.Bariatric surgery,also known as metabolic surgery,was originally designed to treat morbid obesity.However,long-term studies suggest that bariatric surgery can achieve rapid and lasting treatment effect of obesity-associated T2DM beyond weight loss.At present,bariatric surgery has been formally included in the treatment guidelines by the American Diabetes Association(ADA),the International Diabetes Federation(IDF)and the Diabetes Branch of the Chinese Medical Association.Some scholars reported that the ?-cell function of T2DM patients after bariatric surgery has been improved,but the specific mechanism is unknown.ObjectivesBy using a high fat diet(HFD)combined with low dose streptozotocin(STZ)induced T2DM rat model,this study aims to verify the following hypothesis:(1)Duodenal-jejunal bypass(DJB)can effectively improve the impaired pancreatic islet function in T2DM rats;(2)NLRP3 inflammasome in infiltrating macrophages plays an important role in the modulation of ? cell function after duodenal-jejunal bypass surgery.Methods In this study,high fat diet was used to induce insulin resistance in SD rats(last for 4 weeks),and then a low dose of streptozotocin(35 mg/kg)was injected intraperitoneally to induce hyperglycemia,which simulates the natural process and metabolism characters of human T2DM.Then the T2DM rats were randomly divided into sham operation group(SHAM)and DJB surgery group.DJB and sham surgery were performed respectively.DJB is similar to Roux-en-Y Gastric Bypass(RYGB)but retains the integrity of the stomach.DJB surgery model has been widely used to study the weight-independent antidiabetic mechanisms of bariatric surgery.The main indexes of the two groups were detected after operation.(1)Body weight,food intake,and fasting blood glucose:the corresponding parameters of each group were recorded at the corresponding time points.(2)Glucose tolerance:Oral glucose tolerance test(OGTT)and intraperitoneal glucose tolerance test(IPGTT)were performed at different time points after operation.(3)Islet P-cell function:Glucose-stimulated insulin secretion(GSIS)test in vivo and in vitro,p-cell mass measurement and pancreatic insulin content were evaluated.(4)Islet inflammation Response status:The islet macrophage infiltration and NLRP3 inflammasome pathway were evaluated by immunohistochemistry,PCR,Western Blot and other techniques.(5)Incretin:the level of Glucagon-like peptide 1(GLP-1)at each time point was measured.(6)Islet cell apoptosis:TUNEL(TdT-mediated dUTP Nick-End Labeling)method was used to detect apoptosis.At 10 weeks after operation,the rats in DJB group were randomly divided into three groups and macrophage reconstruction experiments were performed.One group received wild-type macrophages by intraperitoneal injection.The second group was intraperitoneally injected with NLRP3 knockout macrophages,the third group was injected with the same amount of saline as a control.The above six experimental parameters were re-evaluated in DJB rats reconstituted with macrophages.Results(I)The type 2 diabetic SD rat model was successfully established by high-fat diet feeding combined with low-dose STZ intraperitoneal injection.This model successfully simulates the natural course of human T2DM.(2)There was no significant difference in the food intake and body weight between DJB group and sham operation group at both preoperative and postoperative time points(p>0.05).(3)Compared with sham operation group,DJB can effectively reduce fasting blood glucose in T2DM rats,and can also significantly improve glucose tolerance in T2DM rats:There was no significant difference in fasting blood glucose and glucose tolerance before operation in all groups(p>0.05).Fasting blood glucose in sham operation group was significantly higher than that in DJB group at 2 weeks after operation(p<0.01).Both OGTT and IPGTT tests showed that the glucose tolerance of DJB group was significantly improved compared with sham operation group 2 weeks post-operation(p<0.01),which was further improved after 4 weeks.(4)The pancreatic islet secretion function of DJB rats was improved:Both in vivo animal experiment and in vitro cell culture experiment showed that the glucose stimulated insulin secretion of DJB group was significantly higher than that of sham operation group(p<0.05).Compared with the sham operation group,the mRNA expression of GLUT2 and GCK,the key genes of pancreatic glucose transport,was also significantly up-regulated in DJB group(p<0.01).(5)The level of serum GLP-1 in DJB group was significantly higher than that in sham operation group(p<0.01).(6)The infiltration of pancreatic macrophages was reduced and the activity of NLRP3 inflammatory pathway was down-regulated after the operation of DJB:The results of immunohistochemistry and PCR showed that the expression of CD68 + in pancreas of DJB group was significantly improved compared with that of sham-operation group;The results of immunohistochemistry,Western Blot and PCR showed that the expression level of NLRP3 in pancreas of DJB rats were significantly lower than those in sham-operation group(p<0.01).The expression level of p65-NF?B,caspase-1 in pancreas of DJB group was also significantly higher in comparison with sham operation group.Compared with sham surgery group,the expression of proinflammatory cytokines such as IL-1? and IL-18 in pancreatic tissue were significantly down-regulated in both protein and mRNA level in DJB group.While the expression level of anti-inflammatory cytokines GFB1,IL-10 and ARG1 were significantly increased(p<0.05).(7)By injecting wild-type macrophages intraperitoneally into DJB rats,the inflammatory state of pancreatic tissue under T2DM condition was reconstructed.However,when injected with NLRP3 knockout macrophages,the pancreas function of DJB rats was not affected:Compared with the control group injected intraperitoneally with saline,the fasting blood glucose of DJB rats injected with wild-type macrophages returned back to a very high level.The secretion of glucose-stimulated pancreas,the volume of islet,the mass of p-cells and the content of pancreatic insulin significantly decreased after macrophage reconstruction.And the expression level of NLRP3 and related pro-inflammatory cytokine expression was also up regulated(p<0.05).However,DJB rats injected NLRP3 knockout macrophages showed no significant difference from the control group.ConclusionsDJB can improve the pancreatic function of T2DM rats by deactivating the NLRP3 inflammasome in infiltrating macrophages.Part 2 Effect and Mechanism of Bariatric Surgery on Renal Function in Type 2 Diabetic Rats.BackgroundDiabetic nephropathy(DN)is the most common microvascular complication of diabetes and it is the main cause of high mortality and morbidity in diabetic patients.Globally,diabetic nephropathy has become the leading cause of end-stage renal disease(ESRD),posing a heavy financial burden to society and the family.In China,at least 50%of type 2 diabetic patients have varying degrees of renal insufficiency.With the increasing incidence of diabetes,diabetic nephropathy has become one of the major chronic diseases that affect the life and health of Chinese people.At present,there is still no specific treatment for diabetic nephropathy.The traditional treatment strategy is comprehensive prevention and treatment,including controlling blood glucose and blood pressure,correcting lipid metabolism disorders,and the application of RAAS(renin-angiotensin-aldosterone system)inhibitors.For end-stage patients,they often need to rely on dialysis or kidney transplant and other alternative treatment.The early prevention and treatment of diabetic nephropathy,as well as specific targeted therapies,has become the focus of recent research.Bariatric surgery has been used frequently in clinical practice as an effective treatment of morbid obesity and diabetes.As early as 2005,Ikramuddin et al reported that the abnormal proteinuria of a DN patient who was insensitive to conservative treatment dropped to a normal level two years after gastric bypass.Then there were some clinical and animal experiments confirmed that bariatric surgery does have certain therapeutic effect on DN.However,the current researches on the efficacy of bariatric surgery on DN were mostly retrospective and small sample studies.There is currently no systematic randomized controlled trial.The mechanism of how bariatric surgery improves DN is not very clear.Long-chain non-coding RNAs(lncRNAs)are a group of RNA molecules with a transcript length of more than 200 nucleotides(nt)which do not participate in translation of proteins.LncRNAs are widely involved in almost all physiological and pathological activities in the body.LncRNAs are closely related to various diseases by regulating gene expression at epigenetic level,transcription and post-transcriptional level.Metastasis associated lung adenocarcinoma transcript 1(MALAT1)was originally discovered as a LncRNA associated with tumor and metabolic diseases.Recent studies have confirmed that MALAT1 is involved in the regulation of diabetic microangiopathy.But the specific mechanism is still not very clear.ObjectivesWe look forward to solving the following problems through this research:(1)To evaluate the renal impairment situation of T2DM rat model induced by HFD in combination with low dose STZ;(2)To verify if DJB surgery can improve impaired renal function in T2DM rats;(3)To explore whether long-chain non-coding RNA MALAT1 plays an important role in the improvement of rat renal function after DJB.MethodsAnimal experiment part:The SD rat model of type 2 diabetes was established by high-fat diet(last for 4 weeks)in combination with low-dose STZ injection(35 mg/kg).SD rats were randomly divided into three groups after maintaining in diabetic state for six weeks.They were DJB operation group(DM-DJB),sham operation group(DM-sham),and diabetic control group(DM).Then,DJB and sham surgery were performed respectively.The diabetic control group was treated without any treatment.Another group of healthy SD rats of the same age was selected as the control group(CON).Body weight,food intake,and glucose tolerance levels of the animals in each group were detected and recorded at preoperative and postoperative time points.Urinary albumin excretion rate(UAER)and glomerular filtration rate(GFR)were also measured and evaluated at different time points.The pathological characters of rat renal tissue sections in each group were evaluated.The expression level of MALAT1 and its key regulatory factors were detected by PCR and/or ELISA.In vitro experimental part:We use the renal tubular epithelial cell line HK-2 for cell experiments.First,HK-2 cell line was cultured in medium with different glucose concentration to simulate the high glucose environment in diabetic nephropathy.Then MALAT1 was silenced through siRNA transfection.The expression level ofMALAT1 downstream pathway proteins as well as the inflammatory molecules which are closely related to the pathogenesis and progression of DN was detected afterwards.Results(1)HFD-fed SD rats showed obvious diabetic symptoms such as slowed weight gain,significantly increased urine volume,higher fasting blood glucose,and impaired glucose tolerance etc.after intraperitoneal injection of STZ.Six weeks after STZ injection,glomerular filtration rate and urinary albumin excretion rate in diabetic rats were significantly higher than those in health control group(p<0.05),and obvious renal dysfunction occurred.(2)Compared with diabetic control group and health control group,DJB and sham group had a transient decrease in weight loss and food intake within two weeks after operation.No significant difference was observed at other time points between each group regarding body weight and food intake(p>0.05).(3)Compared with sham group and diabetic control group,the impaired glucose tolerance of rats was significantly improved in DJB group(p<0.05).When it comes to glucose tolerance,there was no significant difference between DJB group and health control group two weeks after operation.The serum level of GLP-1 in DJB group was also significantly higher than that in diabetic control group and sham group(p<0.01).(4)The glomerular filtration rate and urinary albumin excretion rate of DJB rats were significantly lower than those of sham group and diabetic control group(p<0.05).The impaired renal function was alleviated to a certain extent.But the GFR and UAER were still higher in DJB group than the healthy control group.(5)Examination of kidney sections showed that the diabetic group and sham group rats showed a typical pathological manifestation of diabetic nephropathy,with significantly increase glomerular volume and proliferative mesangial matrix.However,the abnormal glomerular volume and mesangial matrix hyperplasia was significantly alleviated in DJB rats.(6)The expression level of MALAT1 and its downstream protein Serum amyloid A protein 3(SAA3)in kidney tissues of DJB rats were significantly lower than those in sham group and diabetic control group(p<0.05).(7)The expression level of interleukin-6(IL-6)and tumor necrosis factor-a(TNF-?),which are closely related to the development of diabetic nephropathy,were significantly increased in the kidney of diabetic rats and sham rats.Compared toshame group,the expression of IL-6 and TNF-a were obviously down-regulated in DJB group(p<0.01).(8)Compared with HK-2 cell line cultured in normal glucose medium(5.5 mM D-glucose)and hypertonic control medium(30 mM L-glucose),the expression level of MALAT1 in HK-2 cells was significantly increased when cultured with high glucose medium(30 mM D-glucose)(p<0.01).(9)The expression level of SAA3,IL-6 and TNF-a in HK-2 cell lines under high glucose conditions were significantly down-regulated by silencing MALAT1 through siRNA transfection.ConclusionsDJB can alleviate the impaired renal function in T2DM rats.LncRNA MALAT1 and its downstream protein SAA3,IL-6,as well as TNF-? play an important role in this improvement mechanism.