| Part 1. The construction and evaluation of Duodenal-jejunal bypass surgeries in type 2 diabetic rat modelsBackgroundDiabetes is a kind of chronic systemic disease that does great harm to people’s health, about 85-95% of which are type 2 diabetes mellitus (T2DM). In 2013, there were 382 million diabetic patients in the world, and about 5.1 million patients died of diabetes and related complications. And the health expenditure on diabetes was more than 548 billion dollars. As the incidence increase, there would be more than 592 million diabetic patients by 2035. If diabetes is not efficiently controlled, the long-term hyperglycemic state would result in microvascular and macrovascular complications, including myocardial infarction, stroke, blindness, neuropathy and renal failure. Gastric bypass surgery can not only lead to rapid amelioration of T2DM, but can also significantly decrease the incidence of complications. Duodenal-jejunal bypass (DJB) is a modified surgical model of gastric bypass, which can lead to rapid and sustained alleviation of T2DM. "Foregut hypothesis" and "hindgut hypothesis" are two prevailing theories to elucidate the anti-diabetic effects of DJB. The "foregut hypothesis" suggests that exclusion of duodenum and proximal jejunum from contact with ingested nutrients plays a dominant role in the amelioration of T2DM, while the "hindgut hypothesis" posits that expedited nutrients flow to the distal gut is the critical point for the diabetes control. The underlying mechanisms of diabetes control after DJB still need further investigation.ObjectiveThis study was aimed at developing a T2DM rat model through high-fat diet (HFD) feeding and intraperitoneal injection of low dose streptozotocin (STZ). Afterwards, we performed conventional DJB and two modified procedures of DJB in the diabetic rats. The objectives of this study include:(1) To compare the effects of three DJB procedures on food intake, body weight, glucose tolerance, insulin tolerance, bile acids, insulin and gut hormones; (2) To explore the anti-diabetic mechanisms of DJB beyond the classical "foregut hypothesis" and "hindgut hypothesis".MethodsIn this study, insulin resistance was firstly induced in male Wistar rats by HFD feeding, then a low dose of STZ (35mg/kg) was injected intraperitoneally to induce a diabetic state. Afterwards, sham surgery, DJB, duodenal-jejunal anastomosis (DJA) and long alimentary limb duodenal-jejunal bypass (LDJB) were conducted in the T2DM rats. DJA and LDJB are two modified procedures of DJB. These three procedures excluded the same length of proximal intestine and identically expedited nutrients flow to the distal gut. So, by comparing the anti-diabetic effects of these three procedure, it facilitated us to reveal the underlying mechanisms of diabetes control after DJB beyond the classical "foregut hypothesis" and "hindgut hypothesis". And we evaluated the body weight, food intake, glucose tolerance, insulin tolerance, insulin resistance, β cells functions, serum total bile acids and gut hormones including Glucagon-like peptide-1 (GLP-1) and Glucose dependent insulinotropic peptide (GIP) of all surgical groups at indicated time points postoperatively.Results1. There was no significant difference in body weight and food intake between sham, DJA, DJB and LDJB groups both preoperatively and at any measured time points postoperatively (P>0.05).2. There was no significant difference in fasting blood glucose between the four groups preoperatively (P>0.05); DJA, DJB, and LDJB groups all exhibited lower values of fasting blood glucose than the sham group at all measured time points between 2 and 12 weeks postoperatively (P<0.05).3. At 2 and 12 weeks postoperatively, the values of AUCOGTT of DJA, DJB and LDJB groups were all statistically lower than that of the sham group (P<0.05). Notably, the AUCOGTT of DJB and LDJB groups were also significantly lower than that of DJA group (P<0.05).4. At 2 and 12 weeks postoperatively, DJA, DJB, and LDJB groups all exhibited lower values of AUCITT than the sham group (P<0.05), indicating improved systemic insulin resistance. And DJA, DJB, and LDJB groups showed comparable values of AUCITT postoperatively (P>0.05).5. At both 2 and 12 weeks postoperatively, the DJB and LDJB groups exhibited higher levels of fasting serum total bile acids than both sham and DJA groups (P<0.05). And the DJA group showed comparable levels of fasting serum total bile acids to the sham group (P>0.05).6. At 2 and 12 weeks postoperatively, there was no significant difference in both fasting and glucose-stimulated insulin secretion between sham, DJA, DJB and LDJB groups (P>0.05).7. At 2 weeks postoperatively, the LDJB group exhibited markedly higher global and peak GLP-1 concentration than the sham and DJA groups (P<0.05). And, the DJB group showed higher GLP-1 than the two groups at 15 min after glucose gavage (P<0.05). At 12 weeks postoperatively, the DJB group exhibited statistically higher global and peak GLP-1 concentration than the sham group (P<0.05), while the LDJB group displayed higher global and peak GLP-1 concentration than both sham and DJA groups (P<0.05). GLP-1 also trended to increase in the DJA group, however, it did not reach statistical difference (P>0.05).8. There was no significant difference in glucose-stimulated GIP secretion between the four groups (P>0.05).9. At 12 weeks postoperatively, the LDJB group exhibited significantly lower values of fasting serum total cholesterol than the sham group (P<0.05); DJA, DJB and LDJB groups all exhibited lower values of fasting serum triglycerides and free fatty acids than the sham group (P<0.05).Conclusions1. DJA, DJB and LDJB procedures could all achieve rapid and remarkable improvements of glucose tolerance independently of weight loss and food restriction. The glucose-lowering effects of DJB and LDJB were better than those of DJA.2. DJA, DJB and LDJB procedures could all result in rapid improvement of insulin sensitivity, while they could not significantly increase insulin secretion shortly after surgery.3. GLP-1 and bile acids were elevated after DJB surgery, which might be associated with the improved glucose tolerance and insulin sensitivity.4. Though DJA, DJB and LDJB surgery excluded the same length of proximal intestine and identically expedited nutrients flow to the distal gut, they showed differential anti-diabetic effects, which means that the classical "foregut hypothesis" and "hindgut hypothesis" are not sufficient to elucidate the anti-diabetic effects of DJB. We speculate that expedited bile acids flow to the distal gut might be another potent contributor.1. DJA, DJB and LDJB procedures could all achieve rapid and remarkable improvements of glucose tolerance independently of weight loss and food restriction. The glucose-lowering effects of DJB and LDJB were better than those of DJA.2. DJA, DJB and LDJB procedures could all result in rapid improvement of insulin sensitivity, while they could not significantly increase insulin secretion shortly after surgery.3. GLP-1 and bile acids were elevated after DJB surgery, which might be associated with the improved glucose tolerance and insulin sensitivity.4. Though DJA, DJB and LDJB surgery excluded the same length of proximal intestine and identically expedited nutrients flow to the distal gut, they showed differential anti-diabetic effects, which means that the classical "foregut hypothesis" and "hindgut hypothesis" are not sufficient to elucidate the anti-diabetic effects of DJB. We speculate that expedited bile acids flow to the distal gut might be another potent contributor.Part 2. The effects of duodenal-jejunal bypass on hepatic fat accumulation and insulin resistance in type 2 diabetic ratsBackgroundT2DM is a kind of chronic metabolic disease that is characterized by insulin resistance and impaired insulin secretion, and insulin resistance is the most important feature. Insulin resistance is defined by attenuated response to insulin in liver, skeletal muscle and adipose tissues, which would result in increased hepatic glucose output and as well as decreased glucose uptake by peripheral tissues. DJB can lead to dramatic improvement of insulin resistance, however, the underlying mechanisms are still elusive. The etiology of insulin resistance is complicated, and many studies indicate that fat accumulation in liver and skeletal muscle plays an important role in the development of insulin resistance. The hepatic fat can suppress insulin-mediated phosphorylation of tyrosine residual in IRS-1 and IRS-2 molecules and impair hepatic insulin signal transduction by activating PKC-ε and JNK. No matter in healthy subjects or in obese and T2DM patients, the liver fat content is always positively correlated with systemic insulin resistance. Clinical studies showed that T2DM patients have increased hepatic fat content compared with the body weight matched healthy individuals. And treatments targeting at decreasing liver fat accumulation can significantly improve insulin resistance and T2DM. Bariatric procedures could dramatically alleviate hepatic fat accumulation, while as a non-bariatric procedure, the effects of DJB on liver fat deposition are still unclear.ObjectiveWe conducted sham and DJB operations on T2DM rat models induced by HFD feeding and low dose of STZ injection. Then effects of surgeries on liver and skeletal muscle fat deposition, adipose tissues distribution, serum lipid profiles and liver function parameters, and as well as hepatic and systemic insulin resistance in T2DM rats were evaluated.MethodsThe T2DM rats randomly underwent sham and DJB surgeries, and after operation, the following studies were conducted:(1) Body weight and food intake of rats were monitored preoperatively and once a week postoperatively; (2) At 2 and 8 weeks postoperatively, insulin tolerance test (ITT) and intraperitoneal pyruvate tolerance test (ipPTT) were conducted to evaluate systemic and hepatic insulin resistance; (3) At 2 and 8 weeks postoperatively, blood was collected from retrobulbar venous plexus, and lipid profiles and blood hepatic function parameters were measured; (4) At 2 weeks postoperatively,8 mice from each group were euthanized, omentum, mesenteric, retroperitoneal and peri-epididymis adipose tissue were dissected and weighed; Then liver, skeletal muscle and myocardium tissues were collected, and the triglycerides content were measured. At 8 weeks postoperatively, the rest rats were euthanized, and the above parameters were evaluated as what was done in the second week.Results1. There was no significant difference in body weight and daily food intake between the two groups both preoperatively and at any measured time points postoperatively (P>0.05).2. At 2 weeks after operation, there was no significant difference in hepatic, skeletal muscular and myocardial fat content (P>0.05). While at 8 weeks after operation, the DJB group exhibited significantly lower hepatic triglycerides content than the sham group (P<0.05).3. At 8 weeks postoperatively, the DJB group exhibited statistically lower levels of Alanine Aminotransferase (ALT), Aspartate Aminotransferase (AST) and Alkaline Phosphatase (ALP) than the sham group (P<0.05).4. At 8 weeks after operation, the ratios of omentum and mesenteric adipose tissue were significantly lower in the DJB group than in the sham group (P<0.05); and there was no statistical difference in both retroperitoneal and peri-epididymis adipose tissue ratios between the two surgical groups (P>0.05).5. At 2 weeks postoperatively, there was no significant difference in fasting serum triglycerides (TG), free fatty acids (FFAs) and cholesterol between the sham and DJB groups (P>0.05); At 8 weeks after operation, the fasting serum TG and FFAs concentration of the DJB group were both statistically lower than those of the sham group (P<0.05).6. At 2 and 8 weeks postoperatively, the DJB group exhibited significantly lower values of AUCITT and AUCipPTT than the sham group (P<0.05), indicating improved systemic and hepatic insulin sensitivity respectively.Conclusions1. At the early stage after operation, DJB can dramatically suppress hepatic fat accumulation and decrease serum hepatic function parameters independently of weight loss. While DJB surgery has no significant effects on skeletal muscle and myocardium fat accumulation shortly after operation.2. DJB surgery resulted in decreased ratios of omentum and mesenteric adipose tissues, while it had no remarkable influence on the ratios of retroperitoneal and peri-epididymis adipose tissues. Both omentum and mesenteric adipose tissues are visceral adipose tissues, which are important sources of hepatic lipids. So decreased omentum and mesenteric adipose tissues might be associated with decreased hepatic triglycerides.3. DJB surgery dramatically decreased the levels of fasting serum TG and FFAs in the T2DM rats.4. DJB surgery led to dramatic improvements of systemic and hepatic insulin sensitivity in T2DM rats. And decreased hepatic fat content, omentum and mesenteric adipose tissue ratios and serum FFAs might collectively contribute to the alleviation of insulin resistance after DJB. accumulation and decrease serum hepatic function parameters independently of weight loss. While DJB surgery has no significant effects on skeletal muscle and myocardium fat accumulation shortly after operation.2. DJB surgery resulted in decreased ratios of omentum and mesenteric adipose tissues, while it had no remarkable influence on the ratios of retroperitoneal and peri-epididymis adipose tissues. Both omentum and mesenteric adipose tissues are visceral adipose tissues, which are important sources of hepatic lipids. So decreased omentum and mesenteric adipose tissues might be associated with decreased hepatic triglycerides.3. DJB surgery dramatically decreased the levels of fasting serum TG and FFAs in the T2DM rats.4. DJB surgery led to dramatic improvements of systemic and hepatic insulin sensitivity in T2DM rats. And decreased hepatic fat content, omentum and mesenteric adipose tissue ratios and serum FFAs might collectively contribute to the alleviation of insulin resistance after DJB.Part 3. Mechanisms of ameliorated hepatic fat accumulation after duodenal-jejunal bypass in type 2 diabetic ratsBackgroundThe hepatic triglycerides mainly derive from the serum FFAs pool and newly synthesized fatty acids through hepatic de novo lipogenesis pathway. And the serum FFAs pool is comprised of both dietary fatty acids and fatty acids released by white adipose tissue. Hepatic de novo lipogenesis is a metabolic pathway that converts excessive carbohydrates into fatty acids. Sterol-regulatory element binding proteins lc (Srebp-lc) and carbohydrate response element binding protein (ChREBP) and are two transcriptional regulators of this pathway, they can regulate the transcription of lipogenic genes including acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) by combining with specific sites in the promotors, which would further lead to increased hepatic lipogenesis and fat accumulation. Hepatic expression of Srebp-lc and ChREBP are dramatically increased in some insulin resistant and T2DM rat models. P-oxidation of fatty acids is a metabolic pathway associated with clearance of hepatic lipids, which can decrease hepatic lipid deposition by increasing the utilization of fatty acids. Our study has demonstrated that both GLP-1 and bile acids were elevated after DJB, and recent studies demonstrated that GLP-1 and bile acids can not only suppress hepatic lipogenesis by inhibiting Srebp-lc, but can also augment the β-oxidation pathway by increasing the expression of Carnitine palmitoyl transferase 1 (CPT1). So we speculate that DJB may decrease hepatic fat accumulation by inhibiting hepatic de novo lipogenesis and as well as by increasing the β-oxidation of fatty acids.ObjectiveTo explore the roles of serum FFAs pool, hepatic de novo lipogenesis and β-oxidation of fatty acids in ameliorated hepatic fat accumulation after DJB surgery.MethodsWe evaluated the serum and liver tissue samples acquired in the second part by Elisa, western blot and Real-time PCR (RT-PCR) techniques, and the following studies were conducted:(1) To evaluate the serum FFAs concentration at both fasted state and after glucose administration; (2) To monitor the fasting serum total bile acids of both groups; (3) To assess the glucose-stimulated insulin and GLP-1 secretion in both surgical groups; (4) To evaluate the hepatic expression of key transcriptional regulators (Srebp-1c and ChREBP) and enzymes (ACC and FAS) involved in hepatic de novo lipogenesis by western blot and RT-PCR at 2 and 8 weeks postoperatively; (5) To evaluate the hepatic expression of farnesoid X receptor (FXR) and small heterodimer partner (SHP) by RT-PCR; (6) To evaluate the hepatic expression of CPT1 (the rate-limiting enzyme of β-oxidation) by RT-PCR.Results1. At 2 weeks after operation, the DJB group exhibited significantly lower levels of FFAs at 15 min after glucose gavage (P<0.05); While at 8 weeks postoperatively, the DJB group exhibited decreased serum FFAs concentration than the sham group at both fasted state and after glucose gavage (P<0.05).2. At 8 weeks after operation, the hepatic mRNA expression of Srebp-lc, ChREBP, ACC and FAS were all dramatically down-regulated in the DJB group (P<0.05).3. As shown by western blot, at 2 weeks after operation, the hepatic expression of ChREBP and FAS were down-regulated in the DJB group (P<0.05); and at 8 weeks after operation, the hepatic expression of Srebp-lc, ChREBP, ACC and FAS were all decreased in the DJB group (P<0.05).4. At 2 and 8 weeks postoperatively, the fasting serum total bile acids concentration was much higher in the DJB group than in the sham group (P<0.05).5. At both 2 and 8 weeks postoperatively, there was no significant difference in hepatic mRNA expression of FXR, but the hepatic mRNA expression of SHP was much higher in the DJB group than in the sham group (P<0.05).6. There was no significant difference in glucose-stimulated insulin secretion between groups at both 2 and 8 weeks after operation (P>0.05).7. At 8 weeks after operation, the DJB group demonstrated higher levels of GLP-1 than the sham group after glucose administration (P<0.05).8. At both 2 and 8 weeks after operation, the hepatic mRNA expression of CPT1 was statistically higher in the DJB group than in the sham group (P<0.05).Conclusions1. DJB surgery can decrease the serum FFAs concentration in the T2DM rats.2. DJB surgery can down-regulate the hepatic expression of key transcriptional regulators and enzymes involved in hepatic de novo lipogenesis. Recent research demonstrated that Srebp-lc can directly inhibit hepatic insulin signal transduction by suppressing the transcription of IRS-2. Another study showed that liver specific inhibition of ChREBP could dramatically improve insulin sensitivity. So decreased Srebp-lc and ChREBP per se might be potent contributors of improved insulin sensitivity shortly after DJB.3. DJB up-regulated the hepatic mRNA expression of the rate-limiting enzyme of β-oxidation.4. DJB surgery increased the fasting serum total bile acids concentration and up-regulated the hepatic mRNA expression of SHP, which might be involved in the decreased hepatic de novo lipogenesis and increased β-oxidation after DJB.5. GLP-1 secretion was increased after DJB, which might also contributed to the decreased hepatic de novo lipogenesis and increased β-oxidation.In conclusion, decreased serum FFAs pool, attenuated hepatic de novo lipogenesis and augmented β-oxidation of fatty acids might collectively contributed to the ameliorated hepatic fat accumulation after DJB surgery. |
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