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TIRF imaging of docking and fusion of single insulin granule motion in primary rat pancreatic beta-cells: different behaviour of granule motion between normal and Goto-Kakizaki diabetic rat beta-cells.

ABSTRACT: We imaged and analysed the motion of single insulin secretory granules near the plasma membrane in live pancreatic beta-cells, from normal and diabetic Goto-Kakizaki (GK) rats, using total internal reflection fluorescence microscopy (TIRFM). In normal rat primary beta-cells, the granules that were fusing during the first phase originate from previously docked granules, and those during the second phase originate from 'newcomers'. In diabetic GK rat beta-cells, the number of fusion events from previously docked granules were markedly reduced, and, in contrast, the fusion from newcomers was still preserved. The dynamic change in the number of docked insulin granules showed that, in GK rat beta-cells, the total number of docked insulin granules was markedly decreased to 35% of the initial number after glucose stimulation. Immunohistochemistry with anti-insulin antibody observed by TIRFM showed that GK rat beta-cells had a marked decline of endogenous insulin granules docked to the plasma membrane. Thus our results indicate that the decreased number of docked insulin granules accounts for the impaired insulin release during the first phase of insulin release in diabetic GK rat beta-cells.

SUBMITTER: Ohara-Imaizumi M

PROVIDER: S-EPMC1133756 | biostudies-literature | 2004 Jul

REPOSITORIES: biostudies-literature

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TIRF imaging of docking and fusion of single insulin granule motion in primary rat pancreatic beta-cells: different behaviour of granule motion between normal and Goto-Kakizaki diabetic rat beta-cells.

Ohara-Imaizumi Mica M Nishiwaki Chiyono C Kikuta Toshiteru T Nagai Shintaro S Nakamichi Yoko Y Nagamatsu Shinya S

The Biochemical journal 20040701 Pt 1

We imaged and analysed the motion of single insulin secretory granules near the plasma membrane in live pancreatic beta-cells, from normal and diabetic Goto-Kakizaki (GK) rats, using total internal reflection fluorescence microscopy (TIRFM). In normal rat primary beta-cells, the granules that were fusing during the first phase originate from previously docked granules, and those during the second phase originate from 'newcomers'. In diabetic GK rat beta-cells, the number of fusion events from pr ...[more]

PMID: 15128287

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Project description:BackgroundPatients with diabetes are at a high risk for developing cardiac dysfunction in the absence of coronary artery disease or hypertension, a condition known as diabetic cardiomyopathy. Contributing to heart failure is the presence of diabetic kidney disease. The Goto-Kakizaki (GK) rat is a non-obese, non-hypertensive model of type 2 diabetes that, like humans, shares a susceptibility locus on chromosome 10. Herein, we perform a detailed analysis of cardio-renal remodeling and response to renin angiotensin system blockade in GK rats to ascertain the validity of this model for further insights into disease pathogenesis.MethodsStudy 1: Male GK rats along with age matched Wistar control animals underwent longitudinal assessment of cardiac and renal function for 32 weeks (total age 48 weeks). Animals underwent regular echocardiography every 4 weeks and at sacrifice, early (~24 weeks) and late (~48 weeks) timepoints, along with pressure volume loop analysis. Histological and molecular characteristics were determined using standard techniques. Study 2: the effect of renin angiotensin system (RAS) blockade upon cardiac and renal function was assessed in GK rats. Finally, proteomic studies were conducted in vivo and in vitro to identify novel pathways involved in remodeling responses.ResultsGK rats developed hyperglycaemia by 12 weeks of age (p<0.01 c/w Wistar controls). Echocardiographic assessment of cardiac function demonstrated preserved systolic function by 48 weeks of age. Invasive studies demonstrated left ventricular hypertrophy, pulmonary congestion and impaired diastolic function. Renal function was preserved with evidence of hyperfiltration. Cardiac histological analysis demonstrated myocyte hypertrophy (p<0.05) with evidence of significant interstitial fibrosis (p<0.05). RT qPCR demonstrated activation of the fetal gene program, consistent with cellular hypertrophy. RAS blockade resulted in a reduction blood pressure(P<0.05) cardiac interstitial fibrosis (p<0.05) and activation of fetal gene program. No significant change on either systolic or diastolic function was observed, along with minimal impact upon renal structure or function. Proteomic studies demonstrated significant changes in proteins involved in oxidative phosp4horylation, mitochondrial dysfunction, beta-oxidation, and PI3K/Akt signalling (all p<0.05). Further, similar changes were observed in both LV samples from GK rats and H9C2 cells incubated in high glucose media.ConclusionBy 48 weeks of age, the diabetic GK rat demonstrates evidence of preserved systolic function and impaired relaxation, along with cardiac hypertrophy, in the presence of hyperfiltration and elevated protein excretion. These findings suggest the GK rat demonstrates some, but not all features of diabetes induced "cardiorenal" syndrome. This has implications for the use of this model to assess preclinical strategies to treat cardiorenal disease.

Project description:BACKGROUND/OBJECTIVE:Recently, intestinal electrical stimulation (IES) has been reported to result in weight loss; however, it is unclear whether it has a therapeutic potential for diabetes. The aim of the present study was to explore the potential hypoglycemic effects of IES and its possible mechanisms involving β cells in diabetic rats. SUBJECTS/METHODS:Diabetic Goto-Kakizaki (GK) rats were chronically implanted with one pair of electrodes in the duodenum. The oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) were performed with or without IES, and plasma glucagon-like peptide-1 (GLP-1) and insulin level were measured. In the other two OGTT sessions, rats were treated with either Exendin (9-39) (GLP-1 antagonist) or Exendin (9-39) plus IES to investigate the underlying mechanism involving GLP-1. Gastric emptying and small intestinal transit were also measured with or without IES. In a chronic study, GK rats were treated with IES or Sham-IES for 8 weeks. Blood glucose, plasma GLP-1 and insulin level, body weight, and food intake were measured. Pancreas weight, islet β-cell apoptosis, and proliferation were also analyzed. RESULTS:Acute IES reduced blood glucose level from 60 to 120 min during OGTT by 16-20% (all p < 0.05, vs. Sham-IES). GLP-1 antagonist significantly blocked the inhibitory effect of IES on hyperglycemia from 15 to 120 min (all p < 0.05). IES accelerated the small intestinal transit by 15% (p = 0.004). After 8 weeks of chronic stimulation, IES significantly reduced blood glucose (p < 0.05) and body weight (p = 0.02) and increased the plasma GLP-1 concentration (p < 0.05). Furthermore, we observed that chronic IES reduced pancreatic β-cell apoptosis (p = 0.045), but showed no effects on β-cell proliferation. CONCLUSIONS:Our study firstly proved the hypoglycemic effect of IES in a rodent model of type 2 diabetes, possibly attributed to the increasing GLP-1 secretion and improvement in β-cell functions.

Project description:BackgroundThe Goto-Kakizaki (GK) rat is a well-studied non-obese spontaneous type 2 diabetes (T2D) animal model characterized by impaired glucose-stimulated insulin secretion (GSIS) in the pancreatic beta cells. MicroRNAs (miRNAs) are short regulatory RNAs involved in many fundamental biological processes. We aim to identify miRNAs that are differentially-expressed in the pancreatic islets of the GK rats and investigate both their short- and long term glucose-dependence during glucose-stimulatory conditions.Methodology/principal findingsGlobal profiling of 348 miRNAs in the islets of GK rats and Wistar controls (females, 60 days, N = 6 for both sets) using locked nucleic acid (LNA)-based microarrays allowed for the clear separation of the two groups. Significant analysis of microarrays (SAM) identified 30 differentially-expressed miRNAs, 24 of which are predominantly upregulated in the GK rat islets. Monitoring of qPCR-validated miRNAs during GSIS experiments on isolated islets showed disparate expression trajectories between GK and controls indicating distinct short- and long-term glucose dependence. We specifically found expression of rno-miR-130a, rno-miR-132, rno-miR-212 and rno-miR-335 to be regulated by hyperglycaemia. The putative targets of upregulated miRNAs in the GK, filtered with glucose-regulated mRNAs, were found to be enriched for insulin-secretion genes known to be downregulated in T2D patients. Finally, the binding of rno-miR-335 to a fragment of the 3'UTR of one of known down-regulated exocytotic genes in GK islets, Stxbp1 was shown by luciferase assay.Conclusions/significanceThe perturbed miRNA network found in the GK rat islets is indicative of a system-wide impairment in the regulation of genes important for the normal functions of pancreatic islets, particularly in processes involving insulin secretion during glucose stimulatory conditions. Our findings suggest that the reduced insulin secretion observed in the GK rat may be partly due to upregulated miRNA expression leading to decreased production of key proteins of the insulin exocytotic machinery.

Dataset Information

TIRF imaging of docking and fusion of single insulin granule motion in primary rat pancreatic beta-cells: different behaviour of granule motion between normal and Goto-Kakizaki diabetic rat beta-cells.

Publications

TIRF imaging of docking and fusion of single insulin granule motion in primary rat pancreatic beta-cells: different behaviour of granule motion between normal and Goto-Kakizaki diabetic rat beta-cells.

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