Elevated Free Fatty Acids Synergize With Hyperglycemia To Cause Pancreatic β-cell Failure

Part one: The establishment of modified technique of perfusion of isolated rat pancreasObjective: The isolated rat pancreas perfusion is a standardized method for evaluating islet cell insulin secretion function in vitro, which presents consistent, quantitative and repetitive characteristics. The aim of the study was to set up and validate a perfused rat pancreas model suitable for further studies.Materials and Methods: 10 six-week-old male Wistar rats were presented with a highly-palatable high-fat cafeteria-style diet (66% calories as fat) for 12 weeks. After 12 weeks,an isolated rat pancreas perfusion technique was applied into the study of insulin secretion in these high-fat diet-induced obese Wistar rats. The pancreata were dissected away from adjacent tissues, as described in the donor surgical procedures of pancreas transplantation.In the isolated pancreas preparation, which included an attached segment of duodenum, the splenic veins were ligated, and the left gastric arteries and veins were ligated and cut; the abdominal aorta was carefully dissected and tightly ligated just above the celiac artery and then cannulated just below the superior mesenteric artery to direct perfusate; the portal vein was also cannulated to drain perfusate. A multi-channel peristaltic pump was used to administer perfusion medium via the cannula inserted in the abdominal aorta.The basal perfusate was a Krebs-Ringer bicarbonate buffer with the following components: 118 mmol/l NaCl, 4 mmol/l KCl, 2.5 mmol/l CaCl₂, 1.2 mmol/l MgSO₄, 1.2 mmol/l KH₂PO₄, 25 mmol/l NaHCO₃, 1.25 g/l fatty acid-free bovine serum albumin, and 40 g/l dextran T70. As required, it was supplemented with 5.6 mM glucose, 16.7 mM glucose, and a combination of 19 mM L-arginine and 5.6 mM glucose.The resulting pH was close to 7.4.The perfusate was continually oxygenated in an atmosphere of 95% O₂ and 5% CO₂ 1 h before and during the whole experiments, and maintained at 37℃.The perfused pancreas was placed in a temperature-controlled experimental chamber at 37℃.The chamber was also supplied with the basal perfusate and oxygenated with 95% O₂-5% CO₂. All the pancreata were first perfused with the basal medium for a 20-min equilibration period which followed the end of the surgery. After this, each pancreas was then perfused for 10 minutes with 5.6 mM glucose. This was followed by a 20-minute test with 16.7 mM glucose, a 10-minute interval with 5.6 mM glucose, and finally a 20-minute test with 19 mM arginine. The flow rate was maintained constant throughout at 3 ml/min.The perfusion pressure was kept at 60-80mmHg.The complete effluent was collected from the carmula in the portal vein at 1-min intervals in chilled tubes and frozen for storage at -20℃until assayed for immunoreactive insulin. After the experiment, the duodenal peristaltic activity was observed.Results: The functional integrity of the pancreata was assessed by (1) the constancy of perfusion pressure over the whole experiment time(60-80mmHg);(2) presence of duodenal peristaltic activity; (3) presence of a rebound insulin secretion following a bolus of 19 mM arginine at the end of the experiment.In experiments of isolated perfused rat pancreata, the isolated pancreata of 6 rats met all the criteria for the assessment of the functinal integrity of the perfused pancreata: (1)The constancy of perfusion pressure was kept over the whole experiment time ((70.17±5.04)mmHg) .(2) It was observed that the duodenal peristaltic activity was present after the experiment.(3) Total insulin response to argine stimulation was significantly increased compared with glucose stimulation (maximum insulin secretion rate:(987.47±99.50)μU/min vs (545.41±50.00)μU/min,P＜0.05;mean insulin secretion rate:(543.92±72.75)μU/min vs (259.99±27.90)μU/min, P＜0.05).Conclusion:The isolated rat pancreas perfusion technique was successfully established in our laboratory.It is suitable for insulin secretion study in vitro. Part two: Effects of glucolipotoxicity on pancreaticβ-cell function from both in vitro and in vivo studiesObjective: We have found in a previous study that obese diabetic patients with unprovoked ketosis have severely insulin resistance and markedly impairedβ-cell insulin secretion. To explore the roles of elevated glucose and free fatty acids (FFAs) in the pathogenesis of ketone overproduction in obese diabetes, here we test the effect of FFAs in the presence or absence of elevated glucose on insulin secretion and ketone production.Materials and Methods: 34 high-fat obese male Wistar rats were assigned to three groups and all underwent 48 hours infusion in jugular vein in each group: Controls (NS, n=11): normal saline alone; FFA group (n=11): 20% intralipid + heparin to maintain circulating FFAs concentration at 1000-2000μM; GS-FFA group (n=12): 25%glucose +20% intralipid +heparin in amounts required to maintain circulating glucose and FFAs concentration at 15-20mM and 1000-2000p.M respectively. Measurement of plasmaβ-hydroxybutyrate (β-HBA) was carried out just before the infusion.and at the end of the infusion. After the infusion,the rats in each group were further separated into two subgroups with an average of five to six rats in each. An IV GTT was performed in one subgroup to evaluate the glucose-stimulated insulin secretion (GSIS) in vivo, and an isolated pancreas perfusion was fulfilled in the other subgroup to test the GSIS in vitro. Pancreata were perfused with a Krebs-Ringer bicarbonate buffer. As required, this basal perfusate was supplemented with 5.6 mM glucose, 16.7 mM glucose, and a combination of 19 mM L-arginine and 5.6 mM glucose.Results: (1) Plasmaβ-HBA levels were similar at baseline in various groups (GS-FFA: 0.218±0.051, FFA: 0.201±0.055, NS: 0.220±0.049; P＞0.05 ) . (2) At the end of the infusion, GS-FFA, FFA and NS rats exhibited increased plasmaβ-HBA levels, of which plasmaβ-HBA level was strongly increased in GS-FFA rats when compared with FFA and NS rats (GS-FFA: 1.257±0.344 mmol/L, FFA: 0.526±0.107 mmol/L, NS: 0.290±0.170 mmol/L; GS-FFA vs. FFA and NS, P＜0.05 ; FFA vs. NS, P＞0.05). (3) After the infusion, the glucose-stimulated insulin secretions by IVGTT were significantly impaired in the GS-FFA rats compared with the FFA and NS rats (GS-FFA vs. FFA and NS, P＜0.05 ; FFA vs. NS, P＞0.05). (4) After the infusion,basal mean rates of insulin secretion from the perfused pancreata were similar in each group (GS-FFA: 106.72±39.64 uU/min, FFA: 122.26±45.93 uU/min, NS: 123.37±52.90 uU/min; P＞0.05). Mean rates of insulin secretion of exposure to 16.7 mM glucose showed a clear-cut decrease in the GS-FFA rats compared with the FFA and NS rats (GS-FFA: 158.27±48.08 uU/min, FFA: 356.41±47.06 uU/min, NS: 386.67±64.80 uU/min; GS-FFA vs. FFA and NS, P＜0.05 ; FFA vs. NS, P＞0.05). Exposure to 19 mM arginine caused an important insulin release in the perfused pancreata that was greater in the FFA and NS groups compared with the GS-FFA group (GS-FFA: 233.14±61.58 uU/min, FFA: 548.47±64.26 uU/min, NS: 601.12±106.18 uU/min; GS-FFA vs. FFA and NS, P＜0.05 ; FFA vs. NS, P＞0.05). Conclusion: A marked synergistic effect of high glucose and FFAs on impaired insulin secretions causes ketone overproduction in high fat-fed obese rats. It is suggested that the pathogenesis of ketone overproduction in obese patients with unprovoked ketosis may be associated with dramatically declinedβ-cell insulin secretion, as a result of synergistic effect of high glucose and FFAs. The insulin deficiency results in long-chain faty acyl CoA accumulated in cytosol of hepatocyte transport into mitochondrion in large amount and cause ketone overproduction. Part three: The action of apoptosis and mitochondria signal pathway of apoptosis inβ-cell dysfunction induced by glucolipotoxicity in high fat-fed obese ratsObjective: Type 2 diabetes is characterized by a progressive decline inβ-cell fimction and the major mechanism underlying this is increasedβ-cell apoptosis induced by glucolipotoxicity. Accumulating evidence suggests that mitochondria signal pathway of apoptosis could be crucial for the execution of apoptoticβ-cell death induced by glucolipotoxicity. In the present study, we investigate the possible mechanism ofβ-cell dysfunction induced by glucolipotoxicity in high fat-fed obese rats through apoptosis and mitochondria signal pathway of apoptosis.Materials and Methods: 18 six-week-old male Wistar rats were presented with a highly-palatable high-fat cafeteria-style diet (66% calories as fat) for 12 weeks. After 12 weeks,these high-fat obese male Wistar rats were assigned to three groups and all underwent 48hr infusion in jugular vein in each group: Controls (NS, n=6): normal saline alone; FFA group (n=6): 20% intralipid + heparin to maintain circulating FFAs concentration at 1000-2000μM; GS-FFA group (n=6): 25%glucose +20% intralipid +heparin in amounts required to maintain circulating glucose and FFAs concentration at 15-20mM and 1000-2000μM respectively. Measurement of plasmaβ-hydroxybutyrate (β-HBA) was carried out just before the infusion and at the end of the infusion. After the infusion, an IVGTT was performed in three groups. The rats were sacrificed after the IVGTT and a fragment of the tail of each pancreas was removed and formaldehyde-fixed immediately.The detection of apoptotic islet cells was based on the nick-end labeling (TUNEL) method.Immunohistochemical staining was performed for the detection of cyt C,AIF, caspase-9 and caspase-3 of islet cells.In addition, the tail portion of the pancreas was also fixed in 3% glutaraldehyde at 4℃immediately. Ultrastructural alterations of mitochondria inβ-cells were examined and photographed using a transmission electron microscope linked to a camera.Results: (1) Part two showed increased plasmaβ-HBA levels at the end of the infusion and plasma glucose/insulin levels during IVGTT in NS, FFA, GS-FFA groups. (2) After the infusion,apoptotic percentages of islet cells by the TUNEL assay from the NS, FFA, GS-FFA rats were12.9±4.2% (NS),14.8±3.7% (FFA) and 22.7±8.6% (GS-FFA) respectively. There were more apoptotic cells in GS-FFA rats than in FFA and NS rats (GS-FFA vs. FFA and NS, P＜0.05 ; FFA vs. NS, P＞0.05); At the ultrastructural level, marked abnormalities in theβ-cells of the GS-FFA rats were swelling of the matrix and disruption of the cristae of mitochondria, chromatin clumping suggestive of apoptotic phenomena. In the islets of the FFA and NS rats, by contrast, these changes were less prominent; Immunohistochemistry revealed that the islets had increased levels of Cyt C,AIF, caspase-9 and caspase-3 in the GS-FFA rats compared with the FFA and NS rats (GS-FFA vs. FFA and NS, P＜0.05 ; FFA vs. NS, P＞0.05).Conclusion: The effect of glucolipotoxicity on impaired insulin secretions causes ketone overproduction in high fat-fed obese rats and the mechanism of induction ofβ-cell dysfunction by glucolipotoxicity relates to increasedβ-cell apoptosis. In sequential order,glucolipotoxicity leads to the release of Cyt C and AIF from mitochondria into cytosol, the activation of caspase-9 and caspase- 3 and the mobilisation of mitochondria signal transduction of apoptosis. Mitochondria signal pathway of apoptosis plays a crucial part in the execution of apoptoticβ-cell death induced by glucolipotoxicity.