| Part I The construction and evaluation of T2DM rat model and duodenal-jejunal bypass modelBackgroundDiabetes is one of the most common chronic diseases, and more than90%patients are type2diabetes mellitus (T2DM). The global prevalence of T2DM is rising dramatically, driven by the economic development and the changes of the lifestyle. There were366million people with diabetes in2011, and this is set to escalate to552million by2030. Most people with diabetes live in low-and middle-income countries, and these countries will also see the greatest increase in the coming future. Although diet, exercise, oral hypoglycemic drugs and insulin injection can control the hyperglycemia and other metabolic parameters in short time, however, the compliance rate is very low for these treatments are life-long. Bariatric surgery can achieve significant and long-term weight loss. The long-term glycemic control is not ideal with the conventional therapies. Along with the sustained weight loss, bariatric surgery leads to significant improvement of hypertension, hyperlipidemia, T2DM and other complications of morbid obesity. Previous researches indicated that the mechanisms of T2DM resolution after bariatric surgery involved both weight dependent and independent mechanisms. The sustained and significant improvement of T2DM after bariatric surgery are confirmed by many clinical research and animal experiments. Although "Foregut hypothesis" and "Hindgut hypothesis" could partly reveal the mechanisms, the exact biomolecular mechanisms are still to be elucidated.ObjectiveIn this study, we aimed to develop a new T2DM rat model, which could resemble the history and pathologic characteristics of human T2DM. Duodenal-jejunal bypass (DJB) will be performed, the glycemic control and the associated mechanisms will be evaluated and further studied. The present study was conducted to investigate:(1) To develop a new T2DM rat model that could resemble the development and metabolic characteristics of human T2DM;(2) To evaluate the glucose homeostasis after DJB and confirm the therapeutic effect of DJB on T2DM in the new rat model;(3) To evaluate the change of lipid parameters after DJB;(3) To evaluate the changes of the gastrointestinal hormones associated with glucose metabolism.MethodsThe insulin resistance was induced by high fat diet (HFD) for one month in SD rats. Then low dose of streptozotocin (STZ,35mg/kg) was intraperitoneally injected to induce hyperglycemia. The rat model well resembled the development and metabolic characteristics of human T2DM. DJB, a stomach-preserving model of RYGB, has been proven to provide significant and durable diabetes control without changing food intake or body weight. So DJB is a good model to study the weight-independent mechanisms of T2DM resolution after bariatric surgery. The following parameters are determined in rats that underwent DJB, sham DJB (S-DJB) and normal SD rats (CON):(1) The fasting blood glucose and non-fasting blood glucose were determined every week;(2) The glucose tolerance was evaluated by oral glucose tolerance test (OGTT) at2,4,8, and12weeks after surgery;(3) The insulin sensitivity was evaluated by insulin tolerance test (ITT) before surgery and at2and12weeks after surgery;(4) The glucose stimulated insulin secretion was determined before surgery,2and12weeks after surgery to evaluate the function of pancreatic β cells;(5) The glucose stimulated glucagon-like peptide-1(GLP-1), peptide YY (PYY) and glucose dependent insulinotropic peptide (GIP) were determined at2and12weeks after surgery;(6) The lipid parameters were determined at12weeks after surgery.Results:(1) The T2DM rat model was successfully induced by HFD and low dose of STZ. The T2DM rat model showed characteristics of insulin resistance (the glucose stimulated insulin secretion was increased and the AUCITT was also increased) and hyperglycemia (the random blood glucose was instantly and significantly increased, however, the fasting blood glucose was increased gradually), which resembled the development and metabolic characteristics of human T2DM.(2) No significant difference was observed between rats in the DJB and S-DJB groups in terms of body weight and caloric intake at any observational time (*P>0.05).(3) The non-fasting blood glucose was significantly and markedly decreased after DJB as early as2weeks after the surgery (*P<0.05); the fasting blood glucose of rats in S-DJB group was increased gradually and maintained stable and normal in the DJB group, a significant difference was observed6weeks after surgery (*P<0.05).(4) The glucose tolerance was significantly improved2weeks after DJB, and more markedly improvement was observed4weeks after DJB and comparable to that in the CON group.(5) The insulin sensitivity was significantly improved2weeks after DJB, and more marked improvement was observed at12weeks after surgery, while more drastic deterioration of the insulin sensitivity was observed in the S-DJB group.(6) Hyperinsulinemia was observed after one month of HFD feeding. After STZ injection, the insulin levels decreased. However, the insulin level was comparable to that in the normal SD rats. At2and12weeks after surgery, no significant difference was observed with respect to the glucose stimulated insulin levels among the3groups.(7) At2and12weeks after surgery, the glucose stimulated GLP-1and PYY levels in the DJB groups were much higher than those in the S-DJB group; however, no significant difference was observed among the3groups in terms of the GIP levels.(8) Compared to the S-DJB group, triglycerides and FFAs were significantly lower12weeks after DJB; no significant difference was observed among any of the3groups in terms of total cholesterol levels.Conclusions(1) The T2DM rat model was successfully induced by HFD and low dose of STZ, and the model well resembled the development and metabolic characteristics of human T2DM. (2) DJB could rapidly, markedly and sustainably improve the non-fasting blood glucose in the T2DM rat model, and prevent the increase of the fasting blood glucose.(3) DJB could rapidly and significantly improve the glucose tolerance and insulin sensitivity of T2DM rats; no significant increase of insulin was observed in the present study.(4) GLP-1and PYY, mainly secreted by the hindgut, were significantly increased after DJB in the T2DM rat model; however, the GIP levels were comparable in the three groups;(5) The FFA and triglyceride were significantly decreased after DJB surgery in the T2DM rat model. The present study indicated that DJB could rapidly and sustainably improve glucose homeostasis in the T2DM rat model. The increased GLP-1and PYY may contribute to the improvement of the glucose homeostasis. The bimolecular mechanisms are still unclear, and we will make further research in the following study. Part II The effect of DJB on the inflammatory state of T2DM ratsBackgroundT2DM is characterized by insulin resistance and progressive decline in β-cells, whereas the mechanisms of T2DM is not clear now. In recent years, the inflammation hypothesis in the mechanisms of T2DM attached much concern, especially the effect of inflammation in the development of insulin resistance. A lot of researches indicated that T2DM was a disease associated with innate immunity and low grade chronic inflammation. Obesity is one of the most important risk factors of T2DM. Obesity leads to a state of low grade chronic inflammation, characterized by increased circulating proinflammatory factors, such tumor necrosis factor-a (TNF-a), Interleukin-6(IL-6), C reactive protein (CRP), and et al. Previous researches suggested that the adipose tissue was the origin of the chronic inflammation. As an endocrine organ, the adipose tissue could secret many hormones and cytokines, which play important roles in the regulation of glucose homeostasis, lipid metabolism, coagulation, and et al. The cytokines secrected by adipose tissue are called adipokines or adipocytokines, including leptin, adiponectin, TNF-a, IL-6, monocyte chemotactic protein-1(MCP-1) and others. Currently, bariatric surgery is the most effective treatment for morbid obesity. CRP was reduced after RYGB. Some research found that the expression of proinflammatory cytokines mRNA in the liver and adipose tissue was reduced after bariatric surgery. Most of the studies ascribed the amelioration of proinflammatory state to the weight loss. However, some studies found that the reduction of proinflammatory markers and increases of anti-inflammatory biomarkers were not related to the magnitude of weight loss. Further studies are needed to confirm whether weight-loss independent mechanisms were associated with the decreased inflammatory state, whether the improvement of T2DM was associated with the decreased inflammatory state.ObjectiveAdipose tissue is not exclusively a storage depot for lipids, for recently, it was recognized as an endocrine organ. Adipose tissue, especially the visceral adipose tissue, is the main origin site of the proinflammatory cytokines in the morbid obesity. Except for the adipose tissue, the liver is another origin site for the proinflammatory cytokines for fat accumulation and immune cell infilatration. The liver and the adipose tissue play important roles in the glucose metabolism. The present study intended to investigate the effect of DJB on fat distribution, fat accumulation in the liver, macrophage infiltration in the adipose tissue, serial proinflammatory cytokines, mRNA proinflammatory cytokines in the liver and fat, and the association between the inflammatory state and the improvement of insulin sensitivity. The objective of the present study was to elucidate the association between the decreased proinflammatory state and improvement of insulin sensitivity, and the associated mechanisms. MethodsIn the present study, we used the serum and tissue specimens from part I, to determine the following parameters:(1) the change of the inflammatory state:to determine the serial leptin, adiponectin, hsCRP, TNF-a and IL-6with ELISA;(2) the change of fat distribution:weigh the adipose tissue from different parts and calculate the ratio to the body weight;(3) the macrophage infiltration in the adipose tissue:to determine the F4/80positive cells in the adipose tissue with immunohistochemistry; we also got the stromal vascular fraction of the adipose tissue to determine the expression of macrophage markers including Mac-1, F4/80and CD68mRNA with real-time quantitative reverse transcription-PCR (qRT-PCR);(4) the change of the accumulation of fat in the liver;(5) the change of mRNA expression of leptin, adiponectin, TNF-a, IL-6, interleukin-1beta (IL-1β), and monocyte chemoattractant protein-1(MCP-1) in visceral adipose tissue;(6) the change of mRNA expression of TNF-a, IL-6, IL-1β, and MCP-1in the liver;(7) the association between the inflammatory cytokines and improvement of insulin sensitivity.Results(1) Twelve weeks after surgery, the serum leptin, hsCRP and IL-6were significantly decreased in the DJB group, while the TNF-a was statistically comparable in the DJB and S-DJB groups, the adiponectin was significantly increased in the DJB group (*P<0.05).(2) The ratio of epidymal and inguinal adipose tissue to the body weight was not significantly different among the3groups, however, the ratio of weight of omentum and retroperitoneal adipose tissue to body weight was decreased12weeks after DJB (*P<0.05).(3) Compared to the S-DJB group, the fat accumulation in the liver of rats in the DJB group was significantly decreased12weeks after surgery.(4) Compared to the S-DJB group, the F4/80positive cells in the retroperitoneal adipose tissue was decreased12weeks after DJB; the expression of Mac-3, CD68and F4/80mRNA in the stromal vascular fraction was decreased12weeks after DJB (*P<0.05).(5) Compared to the S-DJB group, the mRNA expression of leptin, TNF-a, IL-6, IL-ip and MCP-1in visceral adipose tissue was significantly decreased in the DJB group, while the mRNA expression of adiponectin was significantly increased in the DJB group.(6) Compared to the S-DJB group, the mRNA expression of TNF-a, IL-6, IL-1β and MCP-1in the liver was significantly decreased in the DJB group;(7) At12weeks after surgery, negative correlation was seen between the proinflammatory cytokines and GLP-1levels, positive correlation was seen between the proinflammatory cytokines and AUCITT values.Conclusions(1) DJB could decrease the inflammatory state by decreasing the proinflammatory cytokines and increase the anti-inflammatory cytokines;(2) Twelve weeks after DJB surgery, the ratio of the visceral adipose tissue and the accumulation of fat in the liver were significantly decreased;(3) Twelve weeks after DJB surgery, the macrophage infiltration was significantly decreased in the visceral adipose tissue;(4) Twelve weeks after DJB surgery, the expression of proinflammatory cytokines in the visceral fat were significantly decreased, while the expression of adiponetin was significantly increased;(5) Twelve weeks after DJB surgery, the expression of proinflammatory cytokines in the liver were significantly decreased;(6) Positive correlation was seen between the decrease of the inflammation state and improvement of the insulin sensitivity; the decrease of the inflammation state may correlate with the increased GLP-1levels. Part III The effect of DJB on JNK activity and insulin signaling in T2DM ratsBackgroundInsulin receptor substrates (IRSs) are important regulators in the insulin signaling. In normal conditions, conformal changes after the interaction between insulin and insulin receptors induce tyrosine phosphorylation of special residues of the insulin receptor in the cytoplasm, and then IRSs were recruited. After the interaction between the IRSs and insulin receptor, PI3K was recruited and activated. Tyrosine phosphorylation is essential for the activation of IRSs, while tyrosine phosphorylation will be blocked by serine phosphorylation, which would interfere with the normal insulin signaling. The interaction of TNF-a and its receptors could activate some serine kinase, such as c-Jun N-terminal kinase (JNK) and serine kinase inhibitor kB kinase. These serine kinases can induce insulin resistance by the serine phosphorylation of IRSs which blocks the normal insulin signaling. T2DM is a disease with the excessive activation of innate immunity. The proinflammatory cytokines and free fatty acids can activate the intracellular signaling, such as c-Jun NH2-terminal kinase (JNK) and IkB kinase (IKKβ), et al. These signalings play important roles in the development and progression of insulin resistance and T2DM. JNK can stimulate the serine phosphorylation of IRS at307, while the tyrosine phosphorylation is inhibited, which would inhibit the downstream insulin signaling. RYGB was reported to up-regulate the expression and tyrosine phosphorylation of hepatic insulin signaling pathway in obese diabetic rats. And one of our previous studies has reported that DJB could up-regulate insulin signaling in the liver of T2DM rats, however, no study has investigated the inhibitory phosphorylation of IRS at ser-307and the association with JNK signaling.ObjectiveOur previous study has confirmed that the chronic inflammation state was improved after DJB. The circulating inflammatory cytokines, inflammatory cytokines mRNA expression in both liver and visceral adipose tissue were decreased. And positive correlation was seen between the improvement of inflammation and insulin resistance. For the inflammatory adipokines could activate the intracellular signaling, which play important roles in insulin resistance and T2DM. In the present study, we will investigate the inflammation associated signaling, such as JNK, as well as the insulin signaling. Previous studies indicated that GLP-1had an anti-inflammatory effect and could modulate the related signalings which were important in insulin resistance. In the present study, we will investigate the JNK activity, insulin signaling in liver and adipose tissue.Methods:Using the specimens got from the part I, the following parameters were determined by Western blotting:(1) the expression of JNK and p-JNK in visceral adipose tissue;(2) the expression of IRS-1and IRS-lp"ser307in the visceral adipose tissue;(3)the expression of JNK and p-JNK in the liver;(4) the expression of IRS-2and IRS-2p"ser307in the liver;(5) the IRS-1/2associated PI3K in the visceral adipose tissue and liver.Results(1) The total JNK is not significantly different among the three groups (*P>0.05), while p-JNK/JNK was significantly higher in the S-DJB group compared with the DJB group (*P<0.05).(2) The protein expression of total IRS-1in fat was reduced, but the reduction did not reach the statistical significance (*P>0.05), while the ratio of p-IRS-1at Ser307to total IRS-1was significantly increased in the S-DJB group compared with the DJB and control groups (*P<0.05).(3) Both the JNK and p-JNK was much higher in the S-DJB group than that in DJB group12weeks after surgery(*P<0.05).(4) Compared to the other two groups, the hepatic total IRS-2protein was reduced, while p-IRS-2at Ser307was increased in the S-DJB group (*P<0.05). (5) The IRS-1/2associated PI3K in the visceral adipose tissue and liver were much higher in the DJB group than that in the S-DJB group12weeks after surgery (*P<0.05).Conclusions(1) DJB decreased JNK activity in the visceral adipose tissue12weeks after the surgery.(2) The insulin signaling in the visceral adipose tissue was improved12weeks after DJB.(3) DJB decreased JNK activity in the liver12weeks after DJB.(4) The insulin signaling in the liver was improved12weeks after DJB. |
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