Reproducible High Yield of Rat Islets by Stationary in Vitro Digestion Following Pancreatic Ductal or Portal Venous Collagenase Injection1

Gotoh, Mitsukazu; Maki, Takashi; Satoh, Susumu; Porter, J; Bonner-Weir, Susan; O’Hara, Carl; Monaco, Anthony P.

doi:10.1097/00007890-198705000-00024

Cited by 212 publications

(117 citation statements)

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“…Islets were isolated from 200 g male Sprague-Dawley rats (Taconic, Germantown, N. Y., USA) using an adaptation of the method of Gotoh et al [16]: pancreatic duct infiltration with collagenase (Serva, Heidelberg, Germany), Histopaque gradient separation (Sigma, St. Louis, Mo., USA), and hand picking. They were cultured overnight in RPMI 1640 medium containing 5.5 mmol/l glucose, 2 mmol/l glutamine, 10 % newborn calf serum, 100 units/ml penicillin, and 0.1 mg/ml streptomycin (all from Gibco, Grand Island, N. Y., USA), followed by an additional 24 h of culture with 0.25 mmol/l palmitate (sodium salt) in 2 % ethanol or 2 % ethanol alone.…”

Section: Methodsmentioning

confidence: 99%

Beta-cell hypersensitivity to glucose following 24-h exposure of rat islets to fatty acids

1997

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Section: Methodsmentioning

confidence: 99%

Beta-cell hypersensitivity to glucose following 24-h exposure of rat islets to fatty acids

1997

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“…Primary pancreatic islets were isolated as previously reported [14,15] . In brief, rats were anesthetized with intramuscular pentobarbital injection, 8-10 mL icecold Hanks' balanced salt solution (HBSS) containing 0.75 mg/mL type Ⅴ collagenase (Sigma, St Louis, MO, USA) was injected via pancreatic duct, extended pancreas was removed and digested in a 38℃ water bath for 8-10 min.…”

Section: Islet Isolation and Culture Conditionsmentioning

confidence: 99%

Facilitating effects of berberine on rat pancreatic islets through modulating hepatic nuclear factor 4 alpha expression and glucokinase activity

Wang¹,

Lu²,

Leng³

et al. 2008

WJG

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AIM:To observe the effect of berberine on insulin secretion in rat pancreatic islets and to explore its possible molecular mechanism. METHODS: Primary rat islets were isolated from male Sprague-Dawley rats by collagenase digestion and treated with different concentrations (1, 3, 10 and 30 μmol/L) of berberine or 1 μmol/L Glibenclamide (GB) for 24 h. Glucose-stimulated insulin secretion (GSIS) assay was conducted and insulin was determined by radioimmunoassay. 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to evaluate cytotoxicity. The mRNA level of hepatic nuclear factor 4 alpha (HNF4α ) was determined by reverse transcription polymerase chain reaction (RT-PCR). Indirect immunofluorescence staining and Western blot analysis were employed to detect protein expression of HNF4α in the islets. Glucokinase (GK) activity was measured by spectrophotometric method. RESULTS: Berberine enhanced GSIS rather than basal insulin secretion dose-dependently in rat islets and showed no significant cytotoxicity on islet cells at the concentration of 10 μmol/L. Both mRNA and protein expressions of HNF4α were up-regulated by berberine in a dose-dependent manner, and GK activity was also increased accordingly. However, GB demonstrated no regulatory effects on HNF4α expression or GK activity. CONCLUSION: Berberine can enhance GSIS in rat islets, and probably exerts the insulinotropic effect via a pathway involving HNF4α and GK, which is distinct from sulphonylureas (SUs).

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“…Islets were isolated by hand-picking after collagenase digestion of pancreas (32) and were maintained overnight in RPMI 1640 medium supplemented with 10% FCS, 10 mM HEPES, pH 7.4, 1 mM sodium pyruvate, 100 U/ml penicillin-streptomycin, 50 µM β-mercaptoethanol, and 11 mM glucose.…”

Section: Insulin Secretion and Camp Accumulation In Isolated Pancreatmentioning

confidence: 99%

Gluco-incretins control insulin secretion at multiple levels as revealed in mice lacking GLP-1 and GIP receptors

Preitner¹,

Ibberson²,

Franklin³

et al. 2004

J. Clin. Invest.

115

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The role of the gluco-incretin hormones GIP and GLP-1 in the control of β cell function was studied by analyzing mice with inactivation of each of these hormone receptor genes, or both. Our results demonstrate that glucose intolerance was additively increased during oral glucose absorption when both receptors were inactivated. After intraperitoneal injections, glucose intolerance was more severe in double-as compared to single-receptor KO mice, and euglycemic clamps revealed normal insulin sensitivity, suggesting a defect in insulin secretion. When assessed in vivo or in perfused pancreas, insulin secretion showed a lack of first phase in Glp-1R -/-but not in Gipr -/-mice. In perifusion experiments, however, first-phase insulin secretion was present in both types of islets. In double-KO islets, kinetics of insulin secretion was normal, but its amplitude was reduced by about 50% because of a defect distal to plasma membrane depolarization. Thus, gluco-incretin hormones control insulin secretion (a) by an acute insulinotropic effect on β cells after oral glucose absorption (b) through the regulation, by GLP-1, of in vivo first-phase insulin secretion, probably by an action on extra-islet glucose sensors, and (c) by preserving the function of the secretory pathway, as evidenced by a β cell autonomous secretion defect when both receptors are inactivated.

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Reproducible High Yield of Rat Islets by Stationary in Vitro Digestion Following Pancreatic Ductal or Portal Venous Collagenase Injection1

Cited by 212 publications

References 0 publications

Beta-cell hypersensitivity to glucose following 24-h exposure of rat islets to fatty acids

Beta-cell hypersensitivity to glucose following 24-h exposure of rat islets to fatty acids

Facilitating effects of berberine on rat pancreatic islets through modulating hepatic nuclear factor 4 alpha expression and glucokinase activity

Gluco-incretins control insulin secretion at multiple levels as revealed in mice lacking GLP-1 and GIP receptors

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