Changes induced by sucrose administration on glucose metabolism in pancreatic islets in normal hamsters

We correlated the changes in glucose-induced insulin secretion with those observed in glucose metabolism and hexokinase/glucokinase activity in islets from normal sucrose-fed hamsters. Blood glucose and insulin levels were measured in normal male hamsters fed with (S5) or without (C5) 10% sucrose in the drinking water for 5 weeks. Isolated islets (collagenase digestion) from both groups of animals were used to study insulin secretion, 14CO2 and 3H2O production from D-[U-14C]-glucose and D-[5-3H]-glucose respectively, with 3.3 or 16.7 mM glucose in the medium, and hexokinase/glucokinase activity (fluorometric assay) in islet homogenates. Whereas S5 and C5 animals had comparable normal blood glucose levels, S5 showed higher insulin levels than C5 hamsters (2.3 ± 0.1 vs 0.6 ± 0.03 ng/ml, P14CO2 and 3H2O at both glucose concentrations (14CO2: 3.3 mM glucose: 0.27 ± 0.01 vs 0.18 ± 0.01, P3H2O: 3.3 mM glucose: 0.31 ± 0.02 vs 0.15 ± 0.01, Pm and Vmax values from S5 animals were significantly higher than those from C5 ones (Km: 100.14 ± 7.01 vs 59.90 ± 3.95 ♂M, Pmax: 0.010 ± 0.0005 vs 0.008 ± 0.0006 pmol glucose/ng DNA/min, Pm value from S5 animals was significantly lower than in C5 animals (Km: 15.31 ± 2.64 vs 35.01 ± 1.65 mM, Pmax figures were within a comparable range in both groups (Vmax: 0.048 ± 0.009 vs 0.094 ± 0.035 pmol glucose/ng DNA/min, not significant). The glucose phosphorylation ratio measured at 1 and 100 mM (hexokinase/glucokinase ratio) was significantly higher in S5 (0.26 ± 0.02) than in C5 animals (0.11 ± 0.01, P<0.005), and it was attributable to an increase in the hexokinase activity in S5 animals. In conclusion, sucrose administration increased the hexokinase/glucokinase activity ratio in the islets, which would condition the increase in glucose metabolism by β-cells, and in β-cell sensitivity and responsiveness to glucose. These results support the concept that increased hexokinase rather than glucokinase activity causes the β-cell hypersensitivity to glucose, hexokinase being metabolically more active than glucokinase to up-regulate β-cell function.Facultad de Ciencias MédicasCentro de Endocrinología Experimental y Aplicad

Changes induced by sucrose administration on glucose metabolism in pancreatic islets in normal hamsters

We correlated the changes in glucose-induced insulin secretion with those observed in glucose metabolism and hexokinase/glucokinase activity in islets from normal sucrose-fed hamsters. Blood glucose and insulin levels were measured in normal male hamsters fed with (S5) or without (C5) 10% sucrose in the drinking water for 5 weeks. Isolated islets (collagenase digestion) from both groups of animals were used to study insulin secretion, 14CO2 and 3H2O production from D-[U-14C]-glucose and D-[5-3H]-glucose respectively, with 3.3 or 16.7 mM glucose in the medium, and hexokinase/glucokinase activity (fluorometric assay) in islet homogenates. Whereas S5 and C5 animals had comparable normal blood glucose levels, S5 showed higher insulin levels than C5 hamsters (2.3 ± 0.1 vs 0.6 ± 0.03 ng/ml, P14CO2 and 3H2O at both glucose concentrations (14CO2: 3.3 mM glucose: 0.27 ± 0.01 vs 0.18 ± 0.01, P3H2O: 3.3 mM glucose: 0.31 ± 0.02 vs 0.15 ± 0.01, Pm and Vmax values from S5 animals were significantly higher than those from C5 ones (Km: 100.14 ± 7.01 vs 59.90 ± 3.95 ♂M, Pmax: 0.010 ± 0.0005 vs 0.008 ± 0.0006 pmol glucose/ng DNA/min, Pm value from S5 animals was significantly lower than in C5 animals (Km: 15.31 ± 2.64 vs 35.01 ± 1.65 mM, Pmax figures were within a comparable range in both groups (Vmax: 0.048 ± 0.009 vs 0.094 ± 0.035 pmol glucose/ng DNA/min, not significant). The glucose phosphorylation ratio measured at 1 and 100 mM (hexokinase/glucokinase ratio) was significantly higher in S5 (0.26 ± 0.02) than in C5 animals (0.11 ± 0.01, P<0.005), and it was attributable to an increase in the hexokinase activity in S5 animals. In conclusion, sucrose administration increased the hexokinase/glucokinase activity ratio in the islets, which would condition the increase in glucose metabolism by β-cells, and in β-cell sensitivity and responsiveness to glucose. These results support the concept that increased hexokinase rather than glucokinase activity causes the β-cell hypersensitivity to glucose, hexokinase being metabolically more active than glucokinase to up-regulate β-cell function.Facultad de Ciencias MédicasCentro de Endocrinología Experimental y Aplicad

Oral Metformin Treatment Prevents Enhanced Insulin Demand and Placental Dysfunction in the Pregnant Rat Fed a Fructose-Rich Diet

The intake of a fructose-rich diet (FRD) in the normal female rat induces features similar to those observed in the human metabolic syndrome phenotype. We studied the impact of FRD administration to mothers on pregnancy outcome. On gestational day (Gd) zero rats were assigned to either group: ad libitum drinking tap water alone (normal diet, ND) or containing fructose (10% w/vol; FRD) through pregnancy; all rats were fed a Purina chow diet ad libitum ND and FRD rats were daily cotreated or not with metformin (60 mg/Kg/day oral; ND + MF and FRD + MF) and submitted to a high glucose load test on Gd 14. Additionally, placentas from different groups were studied on Gd 20. Data indicated that: (1) although FRD rats well tolerated glucose overload, their circulating levels of insulin were significantly higher than in ND rats; (2) the mesometrial triangle blood vessel area was significantly lower in placentas from FRD than ND dams; (3) the detrimental effects of FRD administration to mothers were ameliorated by metformin cotreatment. Our study suggests that excessive intake of fructose during pregnancy enhanced the risk for developing gestational diabetes and subsequent preeclampsia, and that metformin prevented the poor pregnancy outcome induced by FRD.Instituto Multidisciplinario de Biología CelularCentro de Endocrinología Experimental y Aplicad

Islet adaptive changes to fructose-induced insulin resistance: β-cell mass, glucokinase, glucose metabolism, and insulin secretion

β-Cell mass, hexokinase/glucokinase (HK/GK) activity, glucose metabolism and insulin secretion were studied in the islets of rats with fructose-induced insulin resistance (IR). Normal male Wistar rats were fed a standard commercial diet and water without (control, C) or with 10% fructose-rich diet (FRD) for 3 weeks. Blood glucose (strips), triglyceride (commercial kit), and insulin (RIA) levels were measured at the time of death. Glucose-induced insulin release, glucose metabolism (14CO2 and 3H2O production from D-[U-14C]- and D-[5-3H]-glucose) and HK/GK activity (G-6-P production), transcription (RTPCR), protein expression (Western blot), and cellular compartmentalization were measured in isolated islets (collagenase digestion). FRD rats presented normoglycemia but impaired glucose tolerance, hypertriglyceridemia, hyperinsulinemia, and increased HOMA-IR index. In these rats, β-cell mass decreased significantly by 33%, with a 44% increase in the percentage of apoptotic cells. Glucose-induced insulin release and islet glucose metabolism were higher in FRD rats. While GK activity (total and cytosolic fraction) and protein expression were significantly higher in FRD islets, HK showed no change in any of these parameters. Our results demonstrate that the changes induced by dietary-induced IR upon β-cell function and mass are strongly conditional on the nutrient model used. In our model (intact animals with impaired glucose tolerance), GK activity increases through mechanisms previously shown only in vitro or under highly hyperglycemic conditions. Such an increase plays a pivotal role in the adaptive increased release of insulin in response to IR, even in the presence of marked β-cell mass reduction.Centro de Endocrinología Experimental y Aplicad

Pancreatic duodenal homeobox-1 and islet neogenesis-associated protein: A possible combined marker of activateable pancreatic cell precursors

The aim of this work was to study the possible relationship between pancreatic duodenal homeobox-1 (Pdx-1) and islet neogenesis-associated protein (INGAP) during induced islet neogenesis. Pregnant hamsters were fed with (S) and without (C) sucrose, and glycemia, insulin secretion in vitro, and pancreas immunomorphometric parameters were measured in their 7-day-old offspring. S offspring had significantly lower glycemic levels than C animals. Insulin release in response to increasing glucose contrations in the incubation medium (2-16 mM glucose) did not increase in pancreata from either C or S offspring. However, pancreata from S offspring released more insulin than those from C animals. In S offspring, β-cell mass, β-cell replication rate and islet neogenesis increased significantly, with a simultaneous decrease in β-cell apoptotic rate. INGAP- and Pdx-1-positive cell mass also increased in the islets and among acinar and duct cells. We found two subpopulations of Pdx-1 cells: INGAP-positive and INGAP-negative. Pdx-1/INGAP-positive cells did not stain with insulin, glucagon, somatostatin, pancreatic polypeptide, or neurogenin 3 antibodies. The increment of Pdx-1/INGAP-positive cells represented the major contribution to the Pdx-1 cell mass increase. Such increments varied among pancreas subsectors: ductal>insular>extrainsular. Our results suggested that INGAP participates in the regulation of islet neogenesis, and Pdx-1/INGAP-positive cells represent a new stem cell subpopulation at an early stage of development, highly activateable in neogenesis.Centro de Endocrinología Experimental y AplicadaFacultad de Ciencias Médica

Changes induced by sucrose administration on glucose metabolism in pancreatic islets in normal hamsters

We correlated the changes in glucose-induced insulin secretion with those observed in glucose metabolism and hexokinase/glucokinase activity in islets from normal sucrose-fed hamsters. Blood glucose and insulin levels were measured in normal male hamsters fed with (S5) or without (C5) 10% sucrose in the drinking water for 5 weeks. Isolated islets (collagenase digestion) from both groups of animals were used to study insulin secretion, 14CO2 and 3H2O production from D-[U-14C]-glucose and D-[5-3H]-glucose respectively, with 3.3 or 16.7 mM glucose in the medium, and hexokinase/glucokinase activity (fluorometric assay) in islet homogenates. Whereas S5 and C5 animals had comparable normal blood glucose levels, S5 showed higher insulin levels than C5 hamsters (2.3 ± 0.1 vs 0.6 ± 0.03 ng/ml, P14CO2 and 3H2O at both glucose concentrations (14CO2: 3.3 mM glucose: 0.27 ± 0.01 vs 0.18 ± 0.01, P3H2O: 3.3 mM glucose: 0.31 ± 0.02 vs 0.15 ± 0.01, Pm and Vmax values from S5 animals were significantly higher than those from C5 ones (Km: 100.14 ± 7.01 vs 59.90 ± 3.95 ♂M, Pmax: 0.010 ± 0.0005 vs 0.008 ± 0.0006 pmol glucose/ng DNA/min, Pm value from S5 animals was significantly lower than in C5 animals (Km: 15.31 ± 2.64 vs 35.01 ± 1.65 mM, Pmax figures were within a comparable range in both groups (Vmax: 0.048 ± 0.009 vs 0.094 ± 0.035 pmol glucose/ng DNA/min, not significant). The glucose phosphorylation ratio measured at 1 and 100 mM (hexokinase/glucokinase ratio) was significantly higher in S5 (0.26 ± 0.02) than in C5 animals (0.11 ± 0.01, P<0.005), and it was attributable to an increase in the hexokinase activity in S5 animals. In conclusion, sucrose administration increased the hexokinase/glucokinase activity ratio in the islets, which would condition the increase in glucose metabolism by β-cells, and in β-cell sensitivity and responsiveness to glucose. These results support the concept that increased hexokinase rather than glucokinase activity causes the β-cell hypersensitivity to glucose, hexokinase being metabolically more active than glucokinase to up-regulate β-cell function.Facultad de Ciencias MédicasCentro de Endocrinología Experimental y Aplicad

Oral Metformin Treatment Prevents Enhanced Insulin Demand and Placental Dysfunction in the Pregnant Rat Fed a Fructose-Rich Diet

The intake of a fructose-rich diet (FRD) in the normal female rat induces features similar to those observed in the human metabolic syndrome phenotype. We studied the impact of FRD administration to mothers on pregnancy outcome. On gestational day (Gd) zero rats were assigned to either group: ad libitum drinking tap water alone (normal diet, ND) or containing fructose (10% w/vol; FRD) through pregnancy; all rats were fed a Purina chow diet ad libitum ND and FRD rats were daily cotreated or not with metformin (60 mg/Kg/day oral; ND + MF and FRD + MF) and submitted to a high glucose load test on Gd 14. Additionally, placentas from different groups were studied on Gd 20. Data indicated that: (1) although FRD rats well tolerated glucose overload, their circulating levels of insulin were significantly higher than in ND rats; (2) the mesometrial triangle blood vessel area was significantly lower in placentas from FRD than ND dams; (3) the detrimental effects of FRD administration to mothers were ameliorated by metformin cotreatment. Our study suggests that excessive intake of fructose during pregnancy enhanced the risk for developing gestational diabetes and subsequent preeclampsia, and that metformin prevented the poor pregnancy outcome induced by FRD.Instituto Multidisciplinario de Biología CelularCentro de Endocrinología Experimental y Aplicad

Mechanisms involved in the β-cell mass increase induced by chronic sucrose feeding to normal rats

The aim of the present study was to clarify the mechanisms by which a sucrose-rich diet (SRD) produces an increase in the pancreatic β-cell mass in the rat. Normal Wistar rats were fed for 30 weeks either an SRD (SRD rats; 63% wt/wt), or the same diet but with starch instead of sucrose in the same proportion (CD rats). We studied body weight, serum glucose and triacylglycerol levels, endocrine tissue and β-cell mass, β-cell replication rate (proliferating cell nuclear antigen; PCNA), islet neogenesis (cytokeratin immunostaining) and β-cell apoptosis (propidium iodide). Body weight (g) recorded in the SRD rats was significantly (P<0.05) larger than that of the CD group (556.0 ± 8.3 vs 470.0 ± 13.1). Both serum glucose and triacylglycerol levels (mmol/l) were also significantly higher (P<0.05) in SRD than in CD rats (serum glucose, 8.11 ± 0.14 vs 6.62 ± 0.17; triacyglycerol, 1.57 ± 0.18 vs 0.47 ± 0.04). The number of pancreatic islets per unit area increased significantly (P<0.05) in SRD rats (3.29 ± 0.1 vs 2.01 ± 0.2). A significant increment (2.6 times) in the mass of endocrine tissue was detected in SRD animals, mainly due to an increase in the β-cell mass (P=0.0025). The islet cell replication rate, measured as the percentage of PCNA-labelled β cells increased 6.8 times in SRD rats (P<0.03). The number of apoptotic cells in the endocrine pancreas decreased significantly (three times) in the SRD animals (P=0.03). The cytokeratin-positive area did not show significant differences between CD and SRD rats. The increase of β-cell mass induced by SRD was accomplished by an enhanced replication of β cells together with a decrease in the rate of β-cell apoptosis, without any evident participation of islet neogenesis. This pancreatic reaction was unable to maintain serum glucose levels of these rats at the level measured in CD animals.Centro de Endocrinología Experimental y AplicadaFacultad de Ciencias Médica

Angiogenesis and VEGF-A pathway are involved in the mechanism by which INGAP_PP increases B-cell mass and function

Background: Islet angiogenesis is needed for embryonic β-cell differentiation and INGAP-PP administration increases Beta-cell mass and function in rats. Aim: To determine the role of islet angiogenesis in the mechanism by which INGAP-PP enhances β-cell mass and function.Facultad de Ciencias Médica

Islet adaptive changes to fructose-induced insulin resistance: β-cell mass, glucokinase, glucose metabolism, and insulin secretion

β-Cell mass, hexokinase/glucokinase (HK/GK) activity, glucose metabolism and insulin secretion were studied in the islets of rats with fructose-induced insulin resistance (IR). Normal male Wistar rats were fed a standard commercial diet and water without (control, C) or with 10% fructose-rich diet (FRD) for 3 weeks. Blood glucose (strips), triglyceride (commercial kit), and insulin (RIA) levels were measured at the time of death. Glucose-induced insulin release, glucose metabolism (14CO2 and 3H2O production from D-[U-14C]- and D-[5-3H]-glucose) and HK/GK activity (G-6-P production), transcription (RTPCR), protein expression (Western blot), and cellular compartmentalization were measured in isolated islets (collagenase digestion). FRD rats presented normoglycemia but impaired glucose tolerance, hypertriglyceridemia, hyperinsulinemia, and increased HOMA-IR index. In these rats, β-cell mass decreased significantly by 33%, with a 44% increase in the percentage of apoptotic cells. Glucose-induced insulin release and islet glucose metabolism were higher in FRD rats. While GK activity (total and cytosolic fraction) and protein expression were significantly higher in FRD islets, HK showed no change in any of these parameters. Our results demonstrate that the changes induced by dietary-induced IR upon β-cell function and mass are strongly conditional on the nutrient model used. In our model (intact animals with impaired glucose tolerance), GK activity increases through mechanisms previously shown only in vitro or under highly hyperglycemic conditions. Such an increase plays a pivotal role in the adaptive increased release of insulin in response to IR, even in the presence of marked β-cell mass reduction.Centro de Endocrinología Experimental y Aplicad