Biomedical subjects

A Giaccari

Publications and source records attributed to A Giaccari.

At least 19 recordsLinked to original sources

PubMed2003

Transgenic mice with dominant negative PKC-theta in skeletal muscle: a new model of insulin resistance and obesity.

Protein kinase C theta (PKC-theta) is the PKC isoform predominantly expressed in skeletal muscle, and it is supposed to mediate many signals necessary for muscle histogenesis and homeostasis, such as TGFbeta, nerve-dependent signals and insulin. To study the role of PKC-theta in these mechanisms we generated transgenic mice expressing a "kinase dead" mutant form of PKC-theta (PKC-thetaK/R), working as "dominant negative," specifically in skeletal muscle. These mice are viable and fertile, however, by the 6-7 months of age, they gain weight, mainly due to visceral fat deposition. Before the onset of obesity (4 months of age), they already show increased fasting and fed insulin levels and reduced insulin-sensitivity, as measured by ipITT, but normal glucose tolerance, as measured by ipGTT. After the 6-7 months of age, transgenic mice develop hyperinsulinemia in the fasting and fed state. The ipGTT revealed in the transgenic mice both hyperglycemia and hyperinsulinemia. At the molecular level, impaired activation of the IR/IRS/PI3K pathway and a significant decrease both in the levels and in insulin-stimulated activation of the serine/threonine kinase Akt were observed. Taken together these data demonstrate that over-expression of dominant negative PKC-theta in skeletal muscle causes obesity associated to insulin resistance, as demonstrated by defective receptor and post-receptorial activation of signaling cascade.

Animals↗

PubMed2001

Acute hyperglycemia and acute hyperinsulinemia decrease plasma fibrinolytic activity and increase plasminogen activator inhibitor type 1 in the rat.

Decreased plasma fibrinolysis may contribute to accelerated atherothrombosis in diabetes. To observe whether hyperglycemia and hyperinsulinemia, common findings in type 2 diabetes, acutely affect plasma fibrinolysis in vivo, we evaluated plasma fibrinolysis (lysis of fibrin plates, free PAI-1 activity and t-PA activity) in the rat after a hyperglycemic euinsulinemic clamp (n=8), an euglycemic hyperinsulinemic clamp (n=7) or a saline infusion (n=15). Plasma fibrinolytic activity was sharply reduced after both the hyperglycemic and hyperinsulinemic clamps as compared to the respective controls (mean lysis areas on the fibrin plate, 139+/-21 vs. 323+/-30 mm2, p<0.001; 78+/-27 vs. 312+/-27 mm2 p<0.001, respectively). Plasma PAI-1 activity was greater after both hyperglycemic and hyperinsulinemic clamps as compared to saline infusion (6.6+/-2.6 vs. 1.6+/-0.6 IU/ml, p<0.001; 26+/-4 vs. 1.3+/-0.7 IU/ml, p<0.0001, respectively). Plasma t-PA activity was significantly reduced both after the hyperglycemic (0.36+/-0.15 vs. 2.17+/-0.18 IU/ml in controls, p<0.001) and the hyperinsulinemic (0.3+/-0.1 vs. 2.3+/-0.3 IU/ml in control, p<0.001) clamps. These data show that in vivo both acute hyperglycemia and acute hyperinsulinemia can decrease plasma fibrinolytic potential and that this is due to increased plasma PAI-1 and decreased free t-PA activities.

Animals↗

PubMed2000

Effects of glucosamine infusion on insulin secretion and insulin action in humans.

Glucose toxicity (i.e., glucose-induced reduction in insulin secretion and action) may be mediated by an increased flux through the hexosamine-phosphate pathway. Glucosamine (GlcN) is widely used to accelerate the hexosamine pathway flux, independently of glucose. We tested the hypothesis that GlcN can affect insulin secretion and/or action in humans. In 10 healthy subjects, we sequentially performed an intravenous glucose (plus [2-3H]glucose) tolerance test (IVGTT) and a euglycemic insulin clamp during either a saline infusion or a low (1.6 micromol x min(-1) x kg(-1)) or high (5 micromol x min(-1) x kg(-1) [n = 5]) GlcN infusion. Beta-cell secretion, insulin (SI*-IVGTT), and glucose (SG*) action on glucose utilization during the IVGTT were measured according to minimal models of insulin secretion and action. Infusion of GlcN did not affect readily releasable insulin levels, glucose-stimulated insulin secretion (GSIS), or the time constant of secretion, but it increased both the glucose threshold of GSIS (delta approximately 0.5-0.8 mmol/l, P < 0.03-0.01) and plasma fasting glucose levels (delta approximately 0.3-0.5 mmol/l, P < 0.05-0.02). GlcN did not change glucose utilization or intracellular metabolism (glucose oxidation and glucose storage were measured by indirect calorimetry) during the clamp. However, high levels of GlcN caused a decrease in SI*-IVGTT (delta approximately 30%, P < 0.02) and in SG* (delta approximately 40%, P < 0.05). Thus, in humans, acute GlcN infusion recapitulates some metabolic features of human diabetes. It remains to be determined whether acceleration of the hexosamine pathway can cause insulin resistance at euglycemia in humans.

Adult↗

PubMed1999

Evidence for glucose/hexosamine in vivo regulation of insulin/IGF-I hybrid receptor assembly.

Hybrid receptors composed of an insulin alphabeta-hemireceptor and a type 1 IGF alphabeta-hemireceptor are formed in tissues expressing both molecules. We recently reported an increased hybrid receptor expression in skeletal muscle of type 2 diabetic patients that is inversely correlated with in vivo insulin sensitivity. It is unclear whether these changes were due to primary abnormalities or to secondary derangements acting in vivo, such as hyperglycemia. To address this, we determined abundance of hybrids in skeletal muscle from three groups of rats: controls, diabetic (90% pancreatectomy), and diabetic treated with phlorizin to normalize plasma glucose levels. We found that the abundance of hybrid receptors was higher in diabetic rats compared with control and phlorizin-treated diabetic rats (percentage of 125I-insulin bound versus total added radioactivity [B/T] = 1.8+/-0.11, 0.4+/-0.01, and 0.32+/-0.04, respectively; P < 0.0001). Fasting plasma glucose levels were positively correlated with hybrids abundance (r = 0.77, P < 0.002). Hybrid receptor protein content, assessed by immunoblotting, was 2.4-fold higher in diabetic rats as compared with control and phlorizin-treated diabetic rats. Because it has been shown that some of the regulatory effects of glucose may be mediated by the glucosamine pathway, we subsequently determined the effect of an in vivo glucosamine infusion on hybrid receptor formation. We found that abundance of hybrids was significantly higher in muscle from glucosamine-treated rats compared with control rats (B/T = 0.17+/-0.02 and 0.11+/-0.01, respectively; P < 0.009). Quantitation of hybrid content by immunoblotting revealed that their abundance was 1.9-fold higher in glucosamine-treated rats. The results demonstrate that 1) elevated glucose levels in diabetic rats are associated with increased expression of hybrid receptors in muscle, 2) correction of hyperglycemia with phlorizin completely reverses increased expression of hybrids, and 3) glucosamine infused into control rats mimics the effects of hyperglycemia on hybrid receptor formation. Thus, the results support the hypothesis that glucose acting, at least in part, through the glucosamine pathway may play an important role in regulating hybrid receptor assembly in vivo.

Animals↗

PubMed1998

Relative contribution of glycogenolysis and gluconeogenesis to hepatic glucose production in control and diabetic rats. A re-examination in the presence of euglycaemia.

Several studies have suggested that, in non-insulin-dependent diabetes mellitus, augmented gluconeogenesis is responsible for increased endogenous glucose production (EGP) and in the end determines fasting hyperglycaemia. However, human and animal studies have been conducted by comparing euglycaemic control subjects to hyperglycaemic diabetic probands. We measured EGP and hepatic gluconeogenesis comparing control and diabetic rats in the fasting state (with diabetic animals in hyperglycaemia), re-examining them in the presence of identical euglycaemia (with diabetic rats made acutely euglycaemic through i. v. phloridzin) or during a hyperinsulinaemic clamp. All rats were infused with [3-3H]-glucose and [U-14C]-lactate; the ratio between 14C-uridine-diphosphoglucose (reflecting 14C-glucose 6-phosphate) and 2 14C-phosphoenolpyruvate specific activities (both purified by high performance liquid chromatography from liver) measured hepatic gluconeogenesis. In diabetic animals, although overall EGP ( approximately 19.5 mg x kg[-1] x min[-1]) remained unaffected by experimental euglycaemia, the contribution of glycogenolysis largely increased (from 5.4 to 11.7 mg x kg(-1) min(-1), hyper- vs euglycaemia) while gluconeogenesis decreased (from 14.0 to 8.1 mg x kg(-1) x min[-1]); both were responsible for the augmented EGP (control rats, EGP: 12.7 mg x kg(-1) x min(-1); gluconeogenesis: 5.9 mg x kg(-1) x min(-1); glycogenolysis: 6.7 mg x kg[-1] x min[-1]). Finally, during insulin clamp, gluconeogenesis and glycogenolysis were similarly decreased, and both contributed to the hepatic insulin-resistance of diabetic animals. We conclude that, in this model of non-insulin-dependent diabetes, augmented gluconeogenesis is not primarily responsible for fasting hyperglycaemia and hepatic insulin resistance. Finally, failure to accurately match the experimental conditions in which diabetic and control humans or animals are compared affects gluconeogenesis, overestimating its role in determining hyperglycaemia.

Animals↗

PubMed1998

Expression of the two insulin receptor isoforms is not altered in the skeletal muscle and liver of diabetic rats.

Alternative splicing of the 36-base pair exon 11 of the human insulin receptor (IR) gene and of the corresponding domain of the rat IR gene results in the synthesis of two IR isoforms with distinct functional characteristics. Altered expression of these IR isoforms has been previously demonstrated in the skeletal muscle of patients with non-insulin-dependent diabetes mellitus (NIDDM); however, this observation was not confirmed by other studies and is still a matter of debate. To assess whether the reported altered isoform expression is due to the secondary metabolic derangement of diabetes, we examined alternative splicing of IR mRNAs (IR36+ and IR36-, corresponding to human Ex11+ and Ex11-) in the skeletal muscle and liver of 6-hour fasting 90% pancreatectomized insulin-resistant diabetic and control Sprague-Dawley rats, using the reverse transcriptase-polymerase chain reaction (PCR) technique. Both diabetic and control rats showed the same pattern of IR mRNA expression: the liver exclusively expressed IR36+ mRNA, whereas only IR36- mRNA was detected in muscle. In conclusion, diabetes mellitus per se does not alter the expression of IR isoforms in the liver and skeletal muscle, and therefore, at least in this animal model of NIDDM, impaired insulin action develops independently from a relative increase in IR36+ mRNA expression in skeletal muscle.

Animals↗

PubMed1998

Increased OB gene expression leads to elevated plasma leptin concentrations in patients with chronic primary hyperinsulinemia.

Leptin, a hormone secreted by adipocytes, decreases food intake and increases energy expenditure. The role of insulin in the regulation of leptin secretion is poorly understood and is still a topic of debate. Insulin increases leptin mRNA synthesis in rodents, but in humans, the available data are discordant. To investigate the role of chronic hyperinsulinemia in the regulation of plasma leptin concentrations, we studied 13 patients with surgically confirmed insulinoma before and after tumor removal, along with 15 healthy control subjects matched for sex, age, and BMI. Immunoreactive plasma leptin levels were measured by radioimmunoassay; leptin mRNA levels were also determined by reverse transcription-competitive polymerase chain reaction in a subgroup of six patients with insulinoma and six control subjects. All determinations were made with subjects in the fasting state. Plasma leptin concentrations correlated positively with leptin mRNA levels (r = 0.880, P < 0.001). Leptin levels, both plasma protein and mRNA, were significantly higher in the insulinoma patients than in the control subjects (plasma protein: 17.5 +/- 3.6 vs. 2.9 +/- 0.4 ng/ml, respectively, P < 0.001; mRNA: 0.98 +/- 0.33 vs. 0.19 +/- 0.064 amol/microg RNA, respectively, P < 0.05), and they correlated positively with fasting plasma insulin levels in the patients with insulinoma (plasma protein: r = 0.686, P < 0.01; mRNA: 0.796, P < 0.05). Finally, removal of the insulin-secreting tumor was followed by the normalization of plasma leptin levels. In summary, in patients with insulinoma, 1) plasma leptin levels and leptin mRNA are elevated; 2) a direct relationship exists between leptin, both circulating protein and mRNA, and insulin concentrations; and 3) plasma leptin returns to normal levels after tumor removal. These data, therefore, support a role for insulin in the chronic regulation of leptin gene expression.

Adipose Tissue↗

PubMed1997

Inhibition of the expression of ornithine decarboxylase and c-Myc by cell-permeant ceramide in difluoromethylornithine-resistant leukaemia cells.

Ceramide has emerged as a novel lipid mediator in cell growth and apoptosis. In difluoromethylornithine-resistant L1210 cells stimulated to growth from quiescence, the cell-permeant analogues of ceramide N-acetylsphingosine (C2-ceramide) and N-hexanoylsphingosine (C6-ceramide) inhibited the induction of ornithine decarboxylase (ODC) activity with IC50 of 8.3 and 1.5 microM respectively. This effect was strictly related to the ability to inhibit cell growth and [3H]thymidine incorporation. The suppression of cell growth was also associated with apoptosis. The addition of bacterial sphingomyelinase resulted in a significant, but limited, reduction of ODC induction and [3H]thymidine incorporation. Bacterial lipopolysaccharide, which may act as a ceramide analogue, also inhibited the induction of the enzyme. Moreover, C6-ceramide largely prevented the accumulation of ODC mRNA and its precursor, ODC heterogeneous nuclear RNA, that accompanied the induction of ODC activity. A slight increase in ODC turnover was also observed. The DNA-binding activity of some transcription factors known to bind and transactivate the ODC gene was investigated by gel mobility-shift assay under the same experimental conditions. However, only the binding of Myc/Max was negatively affected by the treatment with C6-ceramide. Furthermore, the amount of immunoreactive c-Myc, which increased after stimulation of the cells to growth, was strongly reduced by C6-ceramide. These results suggest that the inhibition of c-Myc and ODC expression may be early events in the response of leukaemia cells to ceramide.

Animals↗

PubMed1997

Lactate infusion to normal rats during hyperglycemia enhances in vivo muscle glycogen synthesis.

Both hyperglycemia and hyperinsulinemia stimulate whole body and muscle glucose disposal. To define the impact of increased lactate concentration (4-5 mM) on muscle glucose disposal during hyperglycemia, we studied anesthetized normal rats infused with either sodium lactate or sodium bicarbonate as control. Animals were studied under hyperglycemic clamp (13 mM) using [3-3H]glucose (study 1) and 2-deoxy-[1-3H]glucose (study 2) to assess glucose rate of disappearance (Rd), glycolytic flux (GF), glycogen synthesis, and glucose utilization index by different tissues. Moreover, in study 3, the effect of lactate on the pattern of plasma insulin response to hyperglycemia was evaluated. In study 1, lactate infusion resulted in an increased Rd (38.7 +/- 1.7 vs. 32.3 +/- 1.3 mg.min-1.kg-1; P < 0.01), which was explained by an enhanced rate of glycogen synthesis (23.0 +/- 1.7 vs. 14.7 +/- 1.2 mg.min-1.kg-1; P < 0.001), whereas GF was unchanged. In study 2, lactate-infused animals showed an increased 2-deoxy-glucose disposal and a stimulated glycogen synthase activity as well as an increased glycogen accumulation at the end of the study in several skeletal muscles. In study 3, lactate did not induce any change in either early or late insulin response to hyperglycemia. In conclusion, our results show that muscle glycogen deposition may be enhanced by elevated lactate levels under hyperglycemic conditions and support a role for lactate in the regulation of glucose homeostasis.

Animals↗

PubMed1996

Inhibition of the expression of ornithine decarboxylase by haloperidol in difluoromethylornithine-resistant leukemia cells.

In difluoromethylornithine-resistant L1210 cells stimulated to grow from quiescence, haloperidol caused an early and dose-dependent inhibition of the induction of ornithine decarboxylase (ODC) activity, with an IC50 of 3.5 microM. This effect was accompanied by a reduction in the ODC mRNA level and inhibition of cell growth. Other sigma ligands of different chemical classes inhibited the induction of ODC activity, whereas sulpiride, a dopamine antagonist devoid of sigma-binding affinity, was ineffective. These results indicate that the inhibition of ODC expression may be an early event involved in the antiproliferative response of leukemia cells to haloperidol.

Animals↗

PubMed1996

Inhibition of the expression of ornithine decarboxylase by some kappa-opioidergic receptor ligands in difluoromethylornithine-resistant L1210 cells.

In difluoromethylornithine resistant L1210 cells stimulated to growth from quiescence, the selective kappa-opioidergic agonist trans-(+/-)-3,4-dichloro-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneaceta mid e (U-50488H) caused a dose dependent inhibition of the induction of ODC activity, with a half-maximal effect at about 1 microM. U-50488H also provoked reduction of ODC mRNA level and increase of ODC turnover, as well as inhibition of cell growth. U-69593, another kappa-selective agonist, was only slightly effective. The action of U-50488H on ODC induction was not blocked by naloxone, beta-chlornaltrexamine or by the kappa-selective opioid antagonists Mr1452 and nor-binaltorphimine (nBNI). Actually Mr1452 and nBNI exerted some inhibitory effect. Furthermore, the separated enantiomers (+) and (-) of U-50488H were similarly effective. The (-)cis-(1S,2R)-U50488 stereoisomer, exhibiting low affinity for kappa and high affinity for sigma receptors and carbetapentane, another sigma ligand, also inhibited ODC induction, although less effectively than U-50488H. None of several other opioid ligands tested had significant effects on ODC induction. In conclusion, the inhibition of ODC expression by U-50488H does not involve classical, enantiospecific opioid receptors; rather, these results suggest the involvement of a distinct site of action linked to inhibition of lymphoid cell proliferation.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

PubMed1996

Chronic primary hyperinsulinaemia is associated with altered insulin receptor mRNA splicing in muscle of patients with insulinoma.

Alternative splicing of the 36-base pair exon 11 of the human insulin receptor gene results in the synthesis of two insulin receptor isoforms with distinct functional characteristics (the isoform containing exon 11 has lower insulin binding affinity and lower internalization rate). Altered expression of these insulin receptor isoforms has been previously demonstrated in skeletal muscle of patients with non-insulin-dependent diabetes mellitus (NIDDM). However, this observation was not confirmed by other studies and is still a matter of controversy; furthermore, it is not known whether it represents a primary event or is secondary to hyperinsulinaemia and insulin resistance. In order to address this issue in patients with pure non-genetically determined hyperinsulinaemia, we examined the alternative splicing of insulin receptor mRNAs in skeletal muscle of eight patients with surgically confirmed insulinoma and insulin resistance and in eight healthy subjects, using the reverse transcriptase-polymerase chain reaction technique. The insulinoma patients displayed a significant increase in the expression of the insulin receptor isoform containing exon 11 (75.7 +/- 2.3%) when compared with normal subjects (57.9 +/- 1.5%); furthermore, this increase was positively correlated with plasma insulin concentration and negatively correlated with in vivo insulin sensitivity (glucose clamp). In conclusion, the increased expression of the insulin receptor isoform with lower insulin binding affinity in patients with primary non-genetically determined hyperinsulinaemia supports a role for insulin in the regulation of alternative splicing of insulin receptor pre-mRNA and suggests that in NIDDM an altered receptor isoform distribution might be secondary to the ambient hyperinsulinaemia rather than representing a primary defect.

3T3 Cells↗

PubMed1996

Presence of a DNA-4236 bp deletion and 8-hydroxy-deoxyguanosine in mouse cardiac mitochondrial DNA during aging.

A particular mitochondrial DNA (mtDNA) deletion, the so-called "common deletion", accumulates progressively with age in human post-mitotic cells. We investigated the presence of age-related mtDNA deletions in mouse heart and, according to the free-radical theory of aging, the potential involvement of reactive oxygen species (ROS). Hearts from young, adult and old male Balb/c mice were homogenized and centrifuged in order to discard nuclear DNA. The supernatant was then utilized to prepare mtDNA by SDS-proteinase K digestion. The presence of a mtDNA4236 deletion was estimated by PCR analysis, by separating the amplificated segment on agarose gel. The incidence of the mtDNA4236 deletion was 16%, 28% and 78% in young, adult and old mice, respectively. 8-hydroxy-2'-deoxyguanosine (8-OH-dG), a marker of DNA oxidation, was also determined by HPLC-electrochemical analysis. 8-OH-dG was not detectable in young mice, while its concentrations (moles 8-OH-dG/10(6) moles dG; mean +/- SD) were 59.0 +/- 1.41 and 31.0 +/- 4.24 (p < 0.02) in adult and old mice, respectively. These data indicate that a mtDNA4236 deletion is progressively associated with aging in mouse hearts, and that oxidative damage to mtDNA is greater in middle-aged than senescent animals.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed1996

Increased nonoxidative glucose metabolism in idiopathic reactive hypoglycemia.

Idiopathic reactive hypoglycemia (IRH) is responsible for postprandial hypoglycemia. Normal insulin secretion and reduced response of glucagon to acute hypoglycemia, but mostly increased insulin sensitivity, represent the metabolic features of this syndrome- The present study has two aims: first, to investigate the fate of glucose utilization inside the cells to assess whether increased glucose disposal in IRH is due to the oxidative and/or nonoxidative pathway; and second, to evaluate glucagon response to prolonged insulin-induced hypoglycemia. In eight patients with IRH and eight normal (N) subjects, we performed two studies on different days: (1) 120-minute euglycemic-hyperinsulinemic (1.0 mU . kg-1 . min-1 regular human insulin) clamp associated with indirect calorimetry; and (2) 180-minute hypoglycemic (2.22 to 2.49 mmo/L achieved through 0.85 mU . kg-1 . min-1 intravenous [IV] regular human insulin) clamp. The results showed an increased insulin-mediated glucose uptake in IRH (9.10 +/- 0.19 v 6.78 +/- 0.18 mg kg-1 . min-1, P < .005). Glucose oxidation was similar in IRH subjects and controls both in basal conditions (1.39 +/- 0.16 v 1.42 +/- 0.15 mg . kg-1 . min-1 and during the clamp studies (2.57 +/- 0.21 v 2.78 +/- 0.26 mg . kg-1 . min-1. In contrast, nonoxidative glucose disposal was significantly higher in IRH than in N subjects (6.53 +/- 0.30 v 4.00 +/- 0.21 mg . kg-1 . min-1, P < .001). During insulinization, fat oxidation was reduced slightly more in IRH than in control subjects. During the hypoglycemic clamp, a significant (P < .01) increase in plasma glucagon concentrations was observed in normal subjects as compared with baseline, whereas no change occurred in IRH patients. In conclusion, in IRH: (1) increased insulin-mediated glucose disposal is due to the increase of nonoxidative glucose metabolism; and (2) glucagon secretion has been confirmed to be inadequate. The increase of insulin sensitivity associated with a deficiency in glucagon secretion can widely explain the occurrence of hypoglycemia in the late postprandial phase.

Adult↗

PubMed1995

Protective effect of spermine on DNA exposed to oxidative stress.

Pathological conditions that cause oxidative stress can affect DNA integrity. The aim of this research was to study the protective effect of spermine against DNA damage induced by an oxygen-radical generating system. Deoxyguanosine and DNA were separately dissolved in phosphate buffer and incubated for 1 h at 40 degrees C in the presence of 50 mM H2O2/10 mM ascorbic acid. Single nucleosides and their products of oxidation were then obtained by enzymatic digestion of DNA. The compounds were separated by micellar electrokinetic capillary chromatography (MECC) with SDS-modified mobile phase and detected at 254 nm. Two major products of DNA oxidation have been identified as derivatives of deoxyguanosine with electrophoretic properties different from 8-hydroxy-2'-deoxyguanosine. When the oxidation of DNA was carried out in the presence of 0.1 mM spermine, the formation of the two by-products of deoxyguanosine was markedly reduced. On the contrary, spermine did not prevent the oxidation of deoxyguanosine alone, suggesting that the polyamine should be bound to the DNA strands to exert its antioxidative effect.

Ascorbic Acid↗

PubMed1995

In vivo effects of glucosamine on insulin secretion and insulin sensitivity in the rat: possible relevance to the maladaptive responses to chronic hyperglycaemia.

We tested the hypothesis that glucosamine, a putative activator of glucose toxicity in vitro through acceleration of the hexosamine pathway, may determine in vivo the two key features of glucose toxicity in diabetes, namely, peripheral insulin resistance and decreased insulin secretion. Two groups of awake rats were studied either with intraarterial administration of glucosamine (5 mumol.kg-1.min-1) or saline. Insulin secretion was determined after arginine, glucose (hyperglycaemic clamp), and arginine/glucose infusions, while insulin-mediated glucose metabolism was assessed by the euglycaemic hyperinsulinaemic clamp in combination with [3-3H]-glucose infusion. Glucosamine had no effects on arginine-induced insulin secretion both at euglycaemia and hyperglycaemia, but significantly (40-50%) impaired glucose-induced insulin secretion (both first and second phases). During euglycaemic hyperinsulinaemic clamp studies, glucosamine decreased glucose uptake by approximately 30%, affecting glycolysis (estimated from 3H2O rate of appearance) and muscle glycogen synthesis (calculated from accumulation of [3H]-glucosyl units in muscle glycogen) to a similar extent. Muscle glucose 6-phosphate concentration was markedly reduced in the glucosamine-infused rats, suggesting an impairment in glucose transport/phosphorylation. Therefore, an increase in hexosamine metabolism in vivo: 1) inhibits glucose-induced insulin secretion, and 2) reduces insulin stimulation of both glycolysis and glycogen synthesis, thereby mimicking in normal rats the major alterations due to glucose toxicity in diabetes.

Animals↗

PubMed1994

Micellar electrokinetic capillary chromatography of 8-hydroxydeoxyguanosine and other oxidized derivatives of DNA.

8-Hydroxydeoxyguanosine (8-OH-dG) is widely recognized as a marker of DNA oxidation. Until now, 8-OH-dG has been measured by high-performance liquid chromatography or by gas chromatography-mass spectrometry. A method is reported that detects oxidative derivatives of deoxynucleosides by micellar electrokinetic capillary chromatography. Single-stranded DNA was incubated in the presence of 50 mM hydrogen peroxide-10 mM ascorbic acid and hydrolysed by enzymatic digestion. The order of electrophoretic mobilities of deoxynucleosides was dC > dA > T > dG > 8-OH-dG. 8-OH-dG was determined by introducing a laboratory-prepared internal standard. Two additional major oxidative derivatives were identified by comparing the electropherogram of the oxidized DNA with that of the oxidized standard deoxyguanosine.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed1994

Regulation of rat insulin receptor tyrosine kinase by hypoglycemia.

To investigate the effect of hypoglycemia on the regulation of muscle-derived insulin receptor tyrosine kinase activity, four groups of Sprague-Dawley rats were studied: two groups in which either insulin (4 mU/kg.min) or phloridzin (3 mg/kg.min) was infused to acutely reach hypoglycemia (mean, 3.2-3.5 mM); and two control groups in which either saline or phloridzin (3 mg/kg.min) was infused, while maintaining euglycemia. Plasma glucose was maintained constant for 40 min in the hypoglycemic group and for 60 min in the phloridzin-infused euglycemic groups by a variable glucose infusion. Insulin receptors were isolated under conditions designed to preserve their in vivo phosphorylation state, and their tyrosine kinase activity toward poly(Glu-Tyr) was measured in the absence and presence of in vitro exposure to insulin. Insulin infusion resulted in an enhanced in vivo tyrosine kinase activity. Surprising was the finding of a slight increase of the in vivo tyrosine kinase activity in the phloridzin-infused hypoglycemic rats. The in vitro insulin dose-response curves of tyrosine kinase activity showed no significant differences between insulin-infused and control rats. In contrast, there was a marked increase of the insulin-stimulated kinase activity in phloridzin-infused hypoglycemic rats; at 100 nM insulin, tyrosine kinase activity was 1.8-fold more responsive when compared with either insulin-infused rats or control groups. Moreover, in phloridzin-infused hypoglycemic rats, the half-maximal stimulation of tyrosine kinase activity was greater than 10-fold (0.36 +/- 0.01 nM) more sensitive to insulin than both insulin-infused (3.8 +/- 0.03 nM, mean +/- SE) and control groups (4.2 +/- 0.05 and 4.1 +/- 0.04 nM in saline- and phloridzin-infused euglycemic rats, respectively, mean +/- SE). In conclusion, hypoglycemia associated with low plasma insulin concentrations determines a hypersensitization of the intrinsic tyrosine kinase of the insulin receptor.

Animals↗