The role of G-protein-coupled receptor kinase 2 in diabetic mechanical hyperalgesia in rats.

BACKGROUND: Previous studies have indicated a negative correlation between GRK2 expression and pain development and transmission. Here, we investigated whether G-protein-coupled receptor kinase 2 (GRK2) was involved in regulating diabetic mechanical hyperalgesia (DMH). METHODS: The adeno-associated viral vectors containing the GRK2 gene (AAV-GRK2) were used to up-regulate GRK2 protein expression. The expression of GRK2 and exchange protein directly activated by cyclic adenosine monophosphate 1 (Epac1) in the dorsal root ganglion (DRG) of lumbar 4-6 was detected via immunoblotting and immunohistochemistry, and the transfection of the GRK2 gene was detected by immunofluorescence. RESULTS: Low levels of GRK2 were able to sustain STZ-induced pain in DMH rats. Intrathecal injection of AAV-GRK2 vector up-regulated GRK2 expression, providing pain rain to rats with DMH. With an increase in DMH duration, there was a decrease in paw withdrawal threshold (PWT) value, aggravating the pain, resulting in a decreasing pattern in GRK2 protein expression over time, whereas Epac1 protein expression showed an opposite trend. CONCLUSION: GRK2 expression regulated DMH progression and is expected to play a role in the development of targeted therapy for DMH. GRK2 and Epac1 expressions play a vital role in maintaining pain in DMH rats.

Role and mechanism of uncoupling protein 2 on the fatty acid-induced dysfunction of pancreatic alpha cells in vitro.

BACKGROUND: Uncoupling protein (UCP) 2 is related to the dysfunction of beta cells induced by fatty acids. However, whether UCP2 has similar effects on alpha cell is still not clear. This study aimed to investigate the effects of UCP2 and its possible mechanisms in lipotoxicity-induced dysfunction of pancreatic alpha cells. METHODS: The alpha TC1-6 cells were used in this study to evaluate the effects of palmitate and/or UCP2 inhibit factors on the glucagon secretory function, glucagon content, the glucagon mRNA level and the nitrotyrosine level in the supernatant. Meantime, the expression levels of UCP2 and peroxisome proliferator-activated receptor-γ coactivator-1 alpha (PGC-1 alpha) were measured by real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blotting. Furthermore, the possible relationship between UCP2 and insulin signal transduction pathway was analyzed. RESULTS: Palmitate stimulated alpha cell glucagon secretion and the expression of UCP2 and PGC-1 alpha, which could be partially decreased by the inhibition of UCP2. Palmitate increased nitrotyrosine level and suppressed insulin signal transduction pathway in alpha cells. Inhibition of UCP2 influenced the effects of free fatty acid on alpha cells and may relate to glucagon secretion. CONCLUSION: UCP2 played an important role on alpha cell dysfunction induced by free fatty acid in vitro, which may be related to its effects on oxidative stress and insulin signal transduction pathway.

Sequence of fat partitioning and its relationship with whole body insulin resistance.

BACKGROUND: Currently it is unclear whether lipid accumulation occurs in a particular sequence and its relationship with whole body insulin resistance (IR). This study aimed to answer this question. METHODS: Male Sprague-Dawley (SD) rats were fed on a normal or a high-fat diet for 20 weeks. Serum triglycerides (TG), serum free fatty acids (FFA), fasting plasma glucose (FPG), and liver and skeletal muscle TG were measured. The glucose infusion rate (GIR) and mRNA levels of acetyl-CoA carboxylase (ACC) and carnitine palmitoyltransferase-1 (CPT-1) in the liver and skeletal muscle were determined at different stages. RESULTS: Compared with rats fed on the normal diet, serum FFA was not significantly increased in rats fed on the high-fat diet until 20 weeks. In contrast, liver TG was significantly increased by the high-fat diet by four weeks (20-fold; P < 0.01), and remained elevated until the end of the study. However, skeletal muscle TG was not significantly increased by the high-fat diet until 20 weeks (10.6-fold; P < 0.01), and neither was the FPG. The GIR was significantly reduced (1.6-fold; P < 0.01) by the high-fat diet after 8 weeks. The mRNA levels of ACC gradually increased over time and CPT-1 decreased over time, in both the liver and skeletal muscle in rats fed the high-fat diet. CONCLUSIONS: Lipid accumulation in the liver occurs earlier than lipid accumulation in the skeletal muscle. Fatty liver may be one of the early markers of whole body IR. Changes in the gene expression levels of ACC and CPT-1 may have important roles in the process of IR development.

[Relationship between oxidative stress and beta cell lipoapoptosis in rats].

OBJECTIVE: To investigate the effect of beta cell lipoapoptosis after long term high-fat feeding in rats, and to investigate the relationship between oxidative stress, gene expression and beta cell lipoapoptosis. METHODS: Forty-one SD male rats were randomly divided into 2 groups: high-fat diet group (HF group) and control group (NC group). At the end of 28 weeks, the levels of malondialdehyde (MDA) and glutamylcysteinylglycine (GSH) in plasma and pancreatic tissue,the early-phase insulin secretion in beta cells, the beta cell apoptosis (TUNEL technology) and the uncoupling protein 2 (UCP2) gene expression in islets were measured. RESULT: The concentrations of MDA both in plasma and pancreatic tissue were higher in HF group than those in NC group.In contrast, The contents of GSH both in plasma and pancreatic tissue were lower in HF group. Insulin secretion response to glucose load was significantly decreased in HF group (3.0 fold Compared with 5.7 fold, P<0.01). Blood glucose levels at 3 min, 5 min and 10 min during IVGTT were significantly higher in HF group than those in NC group (P<0.05). The frequency of beta cell apoptosis was increased by 40.0% in HF group (P<0.01). The gene expression of UCP2 in islets was increased by 22.4% in HF group (P<0.01). CONCLUSION: The frequency of beta cell apoptosis in high-fat feeding rats is affected by oxidative stress, which results in increasing UCP2 gene expression.

[Relationship between islet beta cell dysfunction and gene expression of uncoupling protein-2 and possible mechanism thereof].

OBJECTIVE: To study the effects of high fat diet on the functions of islet beta cells and the role of uncoupling protein-2 (UCP2) therein and possible mechanism. METHODS: Forty SD rats were randomly divided into two equal groups: high-fat-(HF) diet group, fed with HF diet for 20 weeks, and normal diet control (NC) group, fed with normal diet. At the end of the twentieth week blood samples were collected from the heart to determine the serum fasting blood glucose (FBG) and fasting insulin (FINS), and plasma nitrotyrosine, malondialdehyde (MDA), and glutamylcysteinylglycine (GSH), indicators of oxidative stress. Glucose infusion rate (GIR) was measured using euglycemic hyperinsulinemic clamp test to evaluate the peripheral insulin resistance. Pancreatic islets were isolated and collected. Islet perfusion was conducted to evaluate the insulin secretion in the islet beta cells. Real-time PCR was used to detect the expression of insulin receptor substrate-1 (IRS-1), IRS-2, and uncoupling protein 2 (UCP2) genes in the islet. Immunohistochemistry was used to detect the protein expression of IRS-1 and IRS-2. RESULTS: (1) The concentrations of plasma nitrotyrosine and MDA of the HF group were both significantly higher than those of the NC group (both P < 0.05). However, the plasma GSH of the HF group was significantly lower than that of the NC group (P < 0.01). (2) The blood glucose of both groups became stable since 60 min after the experiment and the GIR of the HF group was (5.25 +/- 1.2) mg x min(-1) x kg(-1), significantly lower r than that of the NC group [(13.6 +/- 1l.7) mg x min(-1) x kg(-1), P < 0.01). (3) The peak of glucose-stimulated insulin secretion (GSIS) of the HF group was significantly lower than that of the NC group; and the GSIS peak increase In comparison with the NC group. (4) In comparison with the NC group, the mRNA expression levels of IRS-1 and IRS-2 genes of the HF group were significantly lower, by 42.3% and 28.1% respectively (both P < 0.05), and the expression of UCP2 was significantly higher, by 32.5% (P < 0.05). (5) Compared with the NC group, the protein expression levels of IRS-1 and IRS-2 in the islets of the HF group were lower, by 26.3% and 11.2% respectively, however not significantly (both P > 0.05). (6) There was a significantly negative correlation between the UCP2 and IRS-1/IRS-2 gene expression in islet beta cells in the HF group (r = -0.621 and r = -0.436, both P < 0.05). CONCLUSION: High-fat-diet impairs the expression of insulin signal transduction molecules and the function of islet beta cells that may be correlated with overexpression of UCP2. The basic insulin secretion of HF group was significantly higher than that of the NC group; but the glucose-stimulated insulin secretion (GSIS) peak decreased in comparison with the NC group. Compared with the NC group, the protein expression levels of IRS-1 and IRS-2 in the islets of the HF group were lower, by 26.3% (P < 0.05) and 11.2% (P > 0.05) respectively.

[Effects of fenofibrate on gene expression of carnitine palmitoyltransferase 1 in liver and skeletal muscle and its influence on insulin sensitivity].

OBJECTIVE: To study the effects of fenofibrate (FF), a peroxisome proliferator activated receptor (PPAR) alpha activator, on the expression of carnitine palmitoyltransferase 1 (CPT-1) mRNA in liver and muscle and its influence on insulin sensitivity. METHODS: Thirty-two normal 8 week-old male SD rats were randomly divided into 3 groups: normal control group, fed with normal food for 3 weeks (NC group, n = 10), high fat diet group, fed with high fat food (HF group, n = 10), and high fat diet supplemented with FF group, fed with high fat food and given with gastric perfusion of FF (50 mg x kg(-1) x d(-1)) (FF group, n = 12). Fast serum triglyceride (TG) level was tested by automatic biochemical analyzer after 8-10 h fasting. Euglycemic-hyperinsulinemic clamp method was used to calculate the glucose infusion rate (GIR) so as to evaluate the insulin sensitivity. By the end of experiment the rats were killed with their internal organs taken out. The triglyceride (TG) contents of liver and skeletal muscle were measured using Folch method. Real-time PCR was used to detect the mRNA expression. of CPT-1 in the liver and skeletal muscles. RESULTS: As compared with the NC group, the TG levels of serum, liver, and skeletal muscle of the HF group were higher than those of the NC group by 0.45-fold, 2.14-fold, and 10.64-fold respectively. The GIR of the HF group was (6.2 +/- 0.8) mg x kg(-1) x min(-1), significantly lower than that of the NC group (15.8 +/- 2.1) mg x kg(-1) x min(-1), (P < 0.01), and that of the P < 0.01. The CPT-1 mRNA expression in liver of the HF group was not significantly different from that of the NC group (P > 0.05); the expression of CPT-1 mRNA in skeletal muscle of the HF group was lower than that of the NC group by 71% (P < 0.01). The CPT-1 mRNA expression in liver and skeletal muscle of the FF group were significantly higher than those of the HF group by 1.00 and 1.05 times respectively (both P < 05). The GIR was negatively correlated with the levels in the liver (r = -0.87, P < 0.01) and in the skeletal muscle (r = -0.78, P < 0.01). CONCLUSION: Fenofibrate promotes the oxygenation of fatty acids by up-regulating the CPT-1 mRNA expression in the liver and skeletal muscles, thus improving the insulin sensitivity.

[The mechanism of and relationship between lipid metabolism genes expression and insulin resistance in high fat-fed mice].

OBJECTIVE: To observe the relationship between changes of genes expression related to lipid metabolism and insulin resistance induced by high fat diet in SD rats. METHODS: Normal 8-week old male SD rats were randomly divided into 3 groups. They were fed with normal chow (NC, n = 10), high fat diet (HF, n = 10) and high fat diet supplemented with pioglitazone 15 mg x kg(-1) x d(-1) (HP, n = 12). The TG content of liver and skeletal muscle of the rats was measured. Glucose infusion rate (GIR) was used to evaluate the insulin sensitivity by using euglycemic-hyperinsulinemic clamp method. Genes expression was investigated using real-time PCR method. RESULTS: After high fat feeding for 20 weeks, the serum TG, the content of TG in the liver and skeletal muscle of the rats in the HF group increased 0.45, 2.28 and 9.31 fold respectively as compared with those in the NC group. The change of TG content in the liver and skeletal muscle was associated with the reduction of GIR [(6.16 +/- 0.75) mg x kg(-1) x min(-1) vs (15.82 +/- 2.10) mg x kg(-1).min(-1) (P < 0.01)]. As compared with the NC group, the expression of hormone-sensitive lipase (HSL) and fatty-acid synthase (FAS) gene in the HF group was enhanced by 28.2% and 21.3%, respectively (P < 0.05). In addition, the expression of acetyl-CoA carboxylase1 (ACC1) mRNA in the liver increased 48.3% (P < 0.05), increased 101.1% (P < 0.01) and carnitine palmitoyltransferase 1 (CPT-1) decreased 71.0% (P < 0.01) in the skeletal muscle in the rats of HF group. As compared with those in the HF group, GIR increased 1.54 fold in the HP group and on the contrary, serum TG, liver TG and muscle TG decreased about 66%, 64.5% and 59.6% respectively in the HP group (P < 0.05). Accordingly, the expression of FAS and HSL in the adipose tissue and the expression of ACC1 in the liver were reduced (P < 0.05) and the expression of CPT-1 was enhanced and ACC2 was reduced in the muscle (P < 0.01) in the HP group. CONCLUSIONS: The changing expression of genes related to lipid metabolism may play a role in the accumulation of lipids in non-adipose tissue and the induction of insulin resistance in rats fed with high fat diet.

[The relationship between islet alpha cell insulin resistance and inflammatory pathway activation and its mechanism].

OBJECTIVE: To study the changes of inflammatory path molecules in the islet alpha cells in high-fat-diet fed plus beta cell-deleted rat models and the effects of pioglitazone intervention. METHODS: Forty five normal male SD rats, 8 week old, were randomly divided into 3 groups, i.e., a normal diet group (NC), a high fat diet fed group (HF), and a high fat diet fed and pioglitazone treated group (HP, pioglitazone 15 mg kg(-1) d(-1)). At the end of twenty weeks of feeding, fasting serum insulin (FIns), glucagon, free fatty acid (FFA) and high sensitive C reactive protein (hsCRP) were determined. Glucose infusion rate (GIR) was measured by using euglycemic hyperinsulinemia clamp to evaluate the peripheral insulin resistance. The contents of glucagon in perfusion medium during islet cell perfusion was measured with RIA. At the same time, beta cell-deleted rat models were established by injecting large dose streptozocin (100 mg/kg) in 8 rats in each of the three groups, i.e., HF-B group, P-B group and NC-B group. Five days later, the rats were sacrificed and the pancreatic islets were isolated and collected. The expression of NF-kappaB and inhibitor kappaBalpha (IkappaBalpha) gene in the islets was detected with real-time PCR. RESULTS: (1) GIR was decreased significantly in HF group as compared with NC group (P < 0.01). The concentrations of serum FIns, glucagon, FFA and hsCRP in HF group were higher than those in NC group. Pioglitazone intervention could reverse these effects. (2) 16.7 mmol/L glucose could inhibit the glucagon secretion by the islet alpha cells of the NC group rats, but not of the HF group rats. Pioglitazone intervention could reverse these effects. (3) The gene expression of NF-kappaB was significantly increased by 20.5% in the HF-B group than in the NC-B group (P < 0.01). In contrast, the expression of IkappaBalpha was significantly decreased by 24.3% (P < 0.01). The expression of NF-kappaB and IkappaBalpha mRNAs in HP-B group, when compared with that of HF-B group, was improved 78.3% and 58.8%, respectively. CONCLUSIONS: High-fat-diet feeding induces islet alpha cell insulin resistance and activates the mRNA expression of inflammatory path molecules in beta cell-deleted rat models and it may relate with the increased plasma FFA concentration. Pioglitazone intervention can reverse these effects.

[Effect of lipid infusion on the function of islet beta cells and gene expression of insulin signal transduction system].

OBJECTIVE: To study the changes and mechanism of the function of islet beta cells and insulin signal transduction molecules after lipid infusion. METHODS: Twenty five SD rats were randomly divided into 2 groups, FFA group and NS group. Catheters were implanted under pentobarbital anesthesia in the right atrium via the jugular vein and the left carotid artery. A technique for a 48 h infusion in unrestrained rats was used for triglyceride and heparin or saline infusion. The infusion period started on day 2 after surgery. After 48 h infusion, we determined fasting serum insulin (Ins), free fat acid (FFA) in the blood. The glucose infusion rate (GIR) was measured by hyperinsulinemia euglycemic clamp to evaluate the peripheral insulin resistance. The ivgtt and islet cell perifusion was conducted to evaluate the function of islet beta cells. The rats in the two groups were sacrificed, and the pancreatic islets were isolated and collected. The expressions of insulin receptor substrate-1(IRS-1), insulin receptor substrate-2 (IRS-2) glucose transporter-2 (Glut-2) gene in islets and IRS-1, IRS-2 in muscle were detected by real-time PCR. RESULTS: (1) The serum FFA and insulin concentrations of blood in FFA group were higher than in NS group (P<0.05). (2) The GIR was decreased significantly in FFA group compared with that in NS group (P<0.05). (3) The glucose stimulated insulin secretion increased in the FFA group. (4) The gene expression of IRS-1 in muscle was significantly decreased by 87.7% in FFA group, and the expression of IRS-2 was decreased by 50.7% (all P<0.05). The gene expression of IRS-1 in islets was significantly increased by 29.3% (P<0.05), and the expressions of IRS-2, Glut-2 were increased by 345.1% and 536.4% respectively, in FFA group (all P<0.01). CONCLUSION: Lipid infusion in short time increased the secretion of insulin and impaired expression of insulin signal transduction molecules in muscle but it also increased the expression of insulin signal transduction molecules in islet beta cells.

[The expression of insulin signal transduction molecules and islet alpha cell insulin resistance].

OBJECTIVE: To study the changes of insulin signal transduction molecules in islet alpha cells in high-fat-diet plus beta cell-deleting rat models and its underlying mechanism. METHODS: Thirty SD rats were randomly divided into 2 equal groups and fed with high-fat-diet (HF group) or normal diet (normal control group, NC group) respectively. At the end of twenty-week feeding, the fasting serum insulin (Ins), glucagon (Glc), free fatty acid (FFA), and triglyceride (TG were measured. The glucose infusion rate (GIR) was measured by using euglycemic hyperinsulinemia clamp to evaluate the peripheral insulin resistance. At the same time, large dose streptozocin (100 mg/kg) was injected so as to establish beta cell-deleting rat models, i.e., HF-B group (n = 8) and NC-B group (n = 8). Five days later, the rats of the HF-B and NC-B subgroups were sacrificed, and the pancreatic islets were isolated and collected. The expression of Glc, insulin receptor substrate-1 (IRS-1), IRS-2, and phosphatidylinositol-3-kinase (PI3K) gene in the islets were detected by RT-PCR. RESULTS: (1) The serum FFA, insulin and Glc concentrations of the HF group were 508 (394 - 622) micromol/L, 23.7 (14.0 - 33.4) mIU/L, and 345 (298.6 - 391.4) pg/ml respectively, all significantly higher than those of the NC group [325 (240 - 410) micromol/L, 11.5 (3.6 - 19.4) mIU/L, 256 (226.4 - 285.6) pg/ml; respectively, all P < 0.05]. The GIR of the HF group was 5.25 mgxmin(-1)xkg(-1) +/- 1.2 mgxmin(-1)xkg(-1), significantly lower than that of the NC group (13.6 mgxmin(-1)xkg(-1) +/- 1.7 mgxmin(-1)xkg(-1), P < 0.01)). (2) The gene expression of Glc of the HF-B subgroup was significantly higher than that of the NC-B subgroup by 34.2% +/- 2.1%. In contrast, the expression of IRS-2, and PI3K of the HF-B subgroup was significantly lower than that of the NC-B subgroup by 28.5% +/- 1.8% and 21.3% +/- 1.6% respectively (both P < 0.01). (3) The plasma FFA concentration was asignificantly negatively correlated with GIR (r = -0.675, P < 0.05) and IRS-2 gene expression in islet alpha cells (r = -0.458, P < 0.05) in the HF-B group. CONCLUSION: High-fat-diet feeding plus beta cell-deleting rat model shows an impaired expression of insulin signal transduction molecules in islet alpha cells which may relate with the increased plasma FFA concentration.