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C R Barnett

Publications and source records attributed to C R Barnett.

At least 37 records · Page 2Linked to original sources
PubMed1997

Characterization of insulin glycation in insulin-secreting cells maintained in tissue culture.

Characteristics of cellular insulin glycation were examined in the pancreatic B-cell line, BRIN-BD11. The extent of insulin glycation increased stepwise during 72 h of culture at 5.6-33.3 mmol/l glucose, attaining levels up to 27%. Glycation of insulin at 33.3 mmol/l glucose was rapid, reaching maximal values within 2 h, and not readily reversible during 2 to 24 h of subsequent exposure to 5.6 mmol/l glucose. Glycated insulin was readily secreted by BRIN-BD11 cells upon active stimulation with glucose and other secretagogues. Cellular insulin glycation was decreased by 66-80% by inhibitors of protein glycation, vitamin C, aminoguanidine or acetylsalicylic acid. Modulation of insulin-secretory activity of BRIN-BD11 cells by co-culture at high glucose with diazoxide. L-alanine or glibenclamide indicated that long-term stimulation of secretion was associated with a decrease in the extent of insulin glycation. Glycation of insulin in vitro was substantially less extensive than in BRIN-BD11 cells, although glucose-6-phosphate and glyceraldehyde-3-phosphate were 1.4- to 2.0-fold more reactive than glucose per se. These observations indicate that insulin is readily glycated and secreted from insulin-secreting cells under hyperglycaemic conditions in culture.

Ascorbic Acid↗
PubMed1996

Effect of vitamin C supplementation on hepatic cytochrome P450 mixed-function oxidase activity in streptozotocin-diabetic rats.

The effect of vitamin C supplementation on hepatic cytochrome P450 expression was investigated in streptozotocin (STZ) diabetic male Wistar Albino rats. STZ-treated rats displayed the usual characteristics of diabetes including; hyperphagia, polydipsia, decreased body weight gain and also the increased expression and activity of hepatic CYP1A, 2B, 2E and 4A proteins. Vitamin C administration in drinking water (2% w/v) was associated with significant decreases in the levels of hyperglycaemia (P < 0.05), glycosylated haemoglobin (P < 0.05), hyperlipidaemia (P < 0.001), and hyperketonaemia (P < 0.001) associated with STZ-diabetes. Vitamin C-treatment selectively reduced the activity and expression of CYP2E proteins (P < 0.001). These effects on CYP2E expression may be mediated by the reduced levels of circulating ketone bodies, however, a direct effect on CYP2E expression in diabetes cannot be discounted.

Animals↗
PubMed1996

Hexose recognition by insulin-secreting BRIN-BD11 cells.

Clonal BRIN-BD11 cells were produced by electrofusion of NEDH rat islet B-cells with immortal RINm5F cells. Western blotting analysis revealed that unlike RINm5F, novel BRIN-BD11 cells expressed high levels of the glucose transport protein GLUT-2, coupled with a rapid and sustained uptake of D-glucose, significantly greater than RINm5F after only 5 min (p < 0.05). Whereas BRIN-BD11 cells expressed a high glucokinase:hexokinase ratio with 1.4-2.0 fold and 1.4-1.7 fold stepwise stimulation of insulin secretion with 4.2-16.7 mM D-glucose and D-mannose respectively, RINm5F had a lower glucokinase:hexokinase ratio (p < 0.001) and were notably unresponsive to D-glucose and D-mannose. Unlike RINm5F cells, BRIN-BD11 were unresponsive to other hexoses, with RINm5F only responding to D-galactose (p < 0.05). BRIN-BD11 cells should be useful for studies of nutrient-induced insulin secretion.

Animals↗
PubMed1996

D-glucose metabolism in BRIN-BD11 islet cells.

A novel insulin-secreting cell line, BRIN-BD11, was recently established following electrofusion of RINm5F cells with NEDH rat pancreatic islet cells. In the present study, D-glucose metabolism was compared in BRIN-BD11 and RINm5F cells. The concentration dependency of D[5-3H]glucose utilization displayed a comparable pattern in the two cell lines, but the absolute values were lower in BRIN-BD11 than RINm5F cells. Except in the case of D-[1-14C]glucose, the ratio between 14C labeled D-glucose oxidation and D-[5-3H]glucose utilization was higher, however, in BRIN-BD11 than RINm5F cells. Moreover, BRIN-BD11 cells were less affected than RINm5F cells by a rise in D-glucose concentration, in terms of the inhibitory action of the hexose upon oxidative variables, such as oxidative glycolysis, pyruvate decarboxylation, and oxidation of glucose-derived acetyl residues in the Krebs cycle. The total energy yield from D-glucose catabolism appeared similar, however, in BRIN-BD11 and RINm5F cells. These findings extend the knowledge that BRIN-BD11 cells display an improved metabolic and secretory behavior, when considering the difference otherwise found between normal and tumoral islet cells.

Aerobiosis↗
PubMed1996

Identification of the site of glycation of human insulin.

This study evaluates the nature of glycated human insulin formed following exposure to hyperglycemic conditions in vitro. Glycated insulin was purified by RP-HPLC and its molecular mass (5971.3 Da) determined by plasma desorption mass spectrometry (MS). The difference in mass (163.7 Da) from nonglycated insulin (5807.6 Da) corresponds to a single reduced glucose (glucitol) residue. Following reduction of insulin disulfide bridges, MS confirmed that the B-chain was glycated. Enzymatic digestions with trypsin, endoproteinase Glu-C, and thermolysin, followed by RP-HPLC and identification of fragments by MS, localized glycation to the B-chain (1-5) region. Electrospray tandem MS identified the site of glycation as the B-chain NH2-terminal Phe1 residue. This was confirmed by automated Edman degradation with glycated human insulin.

Amino Acid Sequence↗
PubMed1996

Characteristics of BRIN-BG5 and BRIN-BG7, two novel glucose-responsive insulin-secreting cell lines produced by electrofusion.

Two hybrid insulin-secreting cell lines (BRIN-BG5 and BRIN-BG7) were established by the novel approach of electrofusing RINm5F cells with New England Deaconess Hospital rat pancreatic islet cells. Cells were selected from the fusion mixture on the basis of insulin output. Wells showing five to ten times greater insulin output than parental RINm5F cells were selected, subcultured and cloned. Clonal BRIN-BG5 and BRIN-G7 cells grow as monolayers with epithelial morphology. The differences in doubling time of 28 and 20 h respectively were associated with morphological differences; the growth pattern and insulin content of each cell line remaining stable for over 50 passages. In acute 20-min tests, both cell lines showed peak secretory responses (1.9- and 1.8-fold respectively) to 8.4 mmol/l glucose. Membrane depolarization with 25 mmol/l K+ evoked 3.7- and 3.9-fold increases in insulin output. L-Alanine (10 mmol/l) also served to promote 2.4- and 1.6-fold increases in insulin release respectively. Increasing the Ca2+ concentration from 1.28 to 7.68 mmol/l potentiated this effect by 1.8- and 1.5-fold. Incubation with forskolin (25 mumol/l) or phorbol-12-myristate 13-acetate (10 nmol/l), in the presence of L-alanine, similarly enhanced the secretory effect on BRIN-BG5 and BRIN-BG7 cells by 1.3- to 2.1-fold and 1.2- to 1.5-fold respectively. The presence of a functional glucose-sensing mechanism in both cell lines was confirmed by the demonstration of the glucose transporter GLUT-2 and measurement of glucokinase activity. These functional properties suggest that insulin-secreting BRIN-BG5 and BRIN-BG7 cells represent two useful glucose-responsive cell lines for future studies of the function of the pancreatic B-cell.

Animals↗
PubMed1996

Mechanisms of amino acid-induced insulin secretion from the glucose-responsive BRIN-BD11 pancreatic B-cell line.

The effects of different classes of amino acids known to be transported and utilized by pancreatic B-cells were examined using the novel glucose-responsive pancreatic B-cell line, BRIN-BD11. Amino acids tested included alpha-aminoisobutyric acid, L-alanine, L-arginine, L-glutamine, glycine, L-leucine, L-lysine, L-proline and L-serine. At non-stimulatory (1.1 mmol/l) glucose, acute incubations with either 1 or 10 mmol/l amino acid evoked 1.3- to 4.7-fold increases of insulin release. Raising glucose to 16.7 mmol/l enhanced the effects of all amino acids except L-glutamine, and increased insulin output at 10 mmol/l compared with 1 mmol/l amino acid. Glyceraldehyde (10 mmol/l) also served to promote 10 mmol/l amino acid-induced insulin secretion with the exceptions of L-arginine, glycine, L-lysine and L-proline. At 16.7 mmol/l glucose, diazoxide (300 mumol/l) significantly decreased the secretory response to all amino acids except L-glutamine. Likewise, verapamil (20 mumol/l) or depletion of extracellular Ca2+ reduced insulin output indicating the importance of Ca2+ influx in the actions of amino acids. These data indicate that BRIN-BD11 cells transport and utilize amino acids, acting in association with glycolysis, K(+)-ATP channels and/or voltage-dependent Ca2+ channels to promote Ca2+ influx and insulin secretion. The response of BRIN-BD11 cells to glucose and amino acids indicates that this is a useful cell line for future research on the mechanisms of nutrient regulation of insulin secretion.

Alanine↗
PubMed1996

Glycation of insulin in the islets of Langerhans of normal and diabetic animals.

The glycation of immunoreactive insulin (IRI) was assessed in extracts of pancreas and islets from control and hyperglycemic animal models. Glycated and nonglycated IRI were separated by affinity chromatography and quantified by radioimmunoassay. Hydrocortisone-treated Wistar rats (80 mg x kg-1 x day-1 and obese hyperglycemic (ob/ob) mice showed significant increases in plasma glucose (P < 0.001), percentage glycated hemoglobin (P < 0.001), plasma IRI (P < 0.01), and total pancreatic IRI content (P < 0.01), compared with their respective controls. These diabetic groups also demonstrated significant increases (P < 0.05) in the percentage of glycated pancreatic IRI above the controls. Streptozotocin-treated (200 mg/kg) Swiss TO mice exhibited significant increases in plasma glucose (P < 0.001), glycated hemoglobin (P < 0.001), and percentage glycated pancreatic IRI (P < 0.05), compared with untreated controls, despite a marked decrease in both plasma IRI (P < 0.001) and total pancreatic IRI content (P < 0.001). Significant elevations in the percentage of glycated IRI were also observed in islets isolated from obese hyperglycemic (ob/ob) mice (P < 0.001), compared with islets from lean controls, and when lean mouse islets were cultured in hyperglycemic media for 24 h (33.3 vs. 5.6 mmol/l D-glucose; P < 0.001). The contribution of glycated plus nonglycated insulin and proinsulin to the total IRI was estimated in lean and obese mouse pancreatic extracts following high-performance liquid chromatography separation. The contribution of proinsulin to the total IRI was approximately 10%. Proinsulin represented 27-28% of the total glycated IRI. These data indicate that the glycation of insulin and proinsulin occurs within the pancreatic islets and is elevated in both insulin-deficient and insulin-resistant diabetic animal models.

Animals↗
PubMed1996

Characterization of a novel glucose-responsive insulin-secreting cell line, BRIN-BD11, produced by electrofusion.

A novel insulin-secreting cell line (BRIN-BD11) was established after electrofusion of RINm5F cells with New England Deaconess Hospital rat pancreatic islet cells. Wells of cell fusion mixture with insulin output 5-10 times greater than parent RINm5F cells were subcultured with eventual establishment of clones, including BRIN-BD11. Morphological studies established that these cells grow as monolayers with epithelioid characteristics, maintaining stability in tissue culture for > 50 passages. Culture of these cells for 24 h at 5.6-33.3 mmol/l glucose revealed a 1.8- to 2.0-fold increase of insulin output compared with 1.4 mmol/l glucose. Dynamic insulin release was recorded in response to 16.7 mmol/l glucose, resulting in a rapid threefold insulin secretory peak followed by a sustained output slightly above basal. In acute 20-min tests, 4.2-16.7 mmol/l glucose evoked a stepwise two- to three-fold stimulation of insulin release. 3-Isobutyl-1-methylxanthine (1 mmol/l) served to increase basal and glucose-stimulated insulin release, shifting the threshold from 4.4 to 1.1 mmol/l glucose. Stimulation of insulin secretion with 16.7 mmol/l glucose was abolished by mannoheptulose or diazoxide (15 or 0.5 mmol/l). In contrast, glyceraldehyde (10 mmol/l) and 25 mmol/l K+ evoked 1.7- to 9.0-fold insulin responses. L-Alanine (10 mmol/l) evoked a twofold secretory response, which was potentiated 1.4-fold by increasing the Ca2+ concentration from 1.28 to 7.68 mmol/l. Forskolin (25 mumol/l) and phorbol 12-myristate 13-acetate (10 nmol/l) both increased insulin secretion in the presence of L-alanine (1.4- and 1.8-fold, respectively). Western blotting confirmed that BRIN-BD11 cells expressed the GLUT2 glucose transporter. This, coupled with a high glucokinase/hexokinase ratio in the cells, confirms an intact glucose sensing mechanism. High-performance liquid chromatography analysis demonstrated that insulin was the major product secreted under stimulatory conditions. Collectively, these data indicate that the BRIN-BD11 cell line represents an important stable glucose-responsive insulin-secreting beta-cell line for future studies.

1-Methyl-3-isobutylxanthine↗
PubMed1995

Defective expression of cytochrome P450 proteins in the liver of the genetically obese Zucker rat.

The hepatic expression of xenobiotic-metabolising cytochrome P450 isoforms in the genetically obese Zucker rat, a model of obesity, was compared to that of its lean littermate. Cytochrome P450 (CYP) levels were determined using diagnostic substrates and/or immunologically in Western blot analyses. When compared with the lean Zucker rat, the obese animal exhibited hyperglycaemia, hypercholesterolaemia, marked hyperinsulinaemia and hypertriglyceridaemia but was normoketonaemic. CYP3A and CYP1A2 levels were higher in the obese Zucker rat when compared with the lean littermate but, in contrast, a protein recognised by human CYP2D6 and, to a lesser extent, CYP2C11 levels were lower. Pretreatment with acetone, dexamethasone and clofibrate resulted in enhanced p-nitrophenol hydroxylase (CYP2E), erythromycin N-demethylase (CYP3A) and lauric acid hydroxylase (CYP4A) activities respectively in the liver of the lean Zucker rat but, in contrast, the obese Zucker rat was refractive to such treatment; similarly, hepatic apoprotein levels of the CYP2E and CYP4A subfamilies were increased markedly only in the lean Zucker rat. It is concluded that CYP2E, CYP3A and CYP4A subfamilies are poorly expressed in the obese Zucker rat, and this rat strain may serve as a good model for elucidating the molecular mechanisms of induction of these cytochrome P450 proteins.

Acetone↗
PubMed1994

Modulation of the rat hepatic cytochrome P450 composition by long-term streptozotocin-induced insulin-dependent diabetes.

The effects of long-term insulin-dependent diabetes on the enzymatic activities of hepatic cytochrome P450 isozymes were determined in rats rendered diabetic by the administration of streptozotocin and killed 4, 8, and 12 weeks following treatment. The O-dealkylations of ethoxyresorufin and pentoxyresorufin were elevated in the diabetic animals throughout the study, the extent of increase being similar at all three time points. p-Nitrophenol hydroxylase activity was induced in the diabetic animals 4 weeks following treatment with streptozotocin, but the extent of increase became less pronounced with the progress of the disease. A modest increase in ethylmorphine N-demethylase activity was also observed but only in the diabetic animals killed 4 weeks after the induction of diabetes. Finally, lauric acid hydroxylase activity was elevated in the diabetic animals 4 weeks following streptozotocin administration but then declined rapidly with the duration of the disease. It is concluded that the duration of diabetes modulates the hepatic cytochrome P450 profile, with the effect being isoenzyme specific. Mechanisms that may account for these changes are discussed.

Animals↗
PubMed1993

Sex differences in the diabetes-induced modulation of rat hepatic cytochrome P450 proteins.

Sex differences in the diabetes-induced changes in hepatic cytochrome P450 proteins were investigated in rats treated with streptozotocin. Changes in specific cytochrome P450 proteins were monitored using diagnostic substrates and immunologically utilizing specific polyclonal antibodies. When expressed in terms of nmoles of total cytochrome P450, ethoxyresorufin O-deethylase activity was increased by treatment with streptozotocin, the extent of induction being the same in the two sexes. In contrast, lauric acid hydroxylase and ethylmorphine N-demethylase activities were induced only in the male rat. Finally, p-nitrophenol hydroxylase and pentoxyresorufin O-dealkylase were enhanced by the same treatment in both sexes, the effect being more pronounced in the male. These findings indicate that sex-specific changes in certain cytochrome P450 proteins exist in response to insulin-dependent diabetes but these cannot, however, be ascribed to sex differences in the severity of diabetes induced by streptozotocin since the degrees of hyperketonaemia and hyperglycaemia were the same in the two sexes. These are likely to reflect sex-specific differences in growth hormone and triglyceride levels in the diabetic animals.

Animals↗