Exposure to extremely low frequency magnetic fields affects insulin-secreting cells

To evaluate the effects of extremely low frequency magnetic field (ELFMF) on beta-cell survival and function, we cultured a hamster-derived insulin-secreting cell line (HIT-T15), which exhibits responsiveness to glucose in a semi-physiological range, under exposure to sham and ELFMF conditions, and assessed cell survival and function. We used our previously developed ELFMF exposure unit (a sinusoidal magnetic field at a frequency of 60 Hz, 5 mT) to culture cells under exposure to ELFMF conditions. We found that exposure to ELFMF for 5 days in the absence of glucose increased cell number, exposure for 2 days in the absence of glucose and for 5 days with 100 mg/dl glucose increased the insulin secretion to the culture medium, and exposure for 2 and 5 days with 40 and 100 mg/dl glucose increased intracellular insulin concentration in HIT-T15 cells. The increase in cell number under apoptotic culture conditions by exposure to ELFMF could lead to new therapeutic concepts in the treatment of diabetes. The ELFMF-induced increase in intracellular insulin concentration could be utilized to develop culture conditions to enhance intracellular insulin concentration in insulin-secreting cells that would be useful for cell transplantation to cure diabetes mellitus.     

Postinhibitory overshoot of insulin release by gastric inhibitory polypeptide

Following intravenous infusion of somatostatin $\text{in vivo}$ occasionally there is a large rebound overshoot of insulin release. An $\text{in vitro}$ model to simulate this phenomenon was made by perfusing rat pancreas with gastric inhibitory polypeptide (GIP) during simultaneous perfusion with somatostatin. Adding GIP (100 ng/ml) to the perfusate for 2 minutes beginning either 3 or 9 minutes before terminating the somatostatin perfusion produced a large overshoot in insulin release. The magnitude of overshoot was greater when medium contained 300 mg/dl glucose that when it contained 150 mg/dl glucose. Perfusion with GIP for 2 minutes beginning 9 minutes before increasing the glucose concentration of the medium from 30 to 300 mg/dl elicited a large increase in both the acute and second-phase release of insulin. These suggest that post-inhibitory overshoot of insulin release after somatostatin may be produces $\text{in vitro}$ by the suppressed action of stimulatory hormones such as GIP. Prior infusion with GIP can also potentiate glucose-stimulated insulin increase.     

Regulation of somatostatin and growth hormone-releasing factor by gonadal steroids in fetal rat hypothalamic cells in culture.

The mechanism underlying the sexually dimorphic pattern of growth hormone (GH) secretion in the rat has not been clearly elucidated. In the present study, we assayed the possible direct effect of gonadal steroids on both somatostatin (SS) and growth hormone-releasing factor (GRF) in fetal rat hypothalamic cells in culture. Hypothalamic cells, obtained by mechanical dispersion, were maintained as monolayer cultures in serum-supplemented medium. After 20 days in culture, cells were incubated with serum free medium containing testosterone (T, 10, 20, 40 ng/dl) or estradiol (E, 0.1, 1, 10 ng/dl) for 48 h. At the end of the experiments, immunoreactive SS (IR-SS) and immunoreactive GRF (IR-GRF) were measured by specific radioimmunoassays (RIAs) in media and cell extracts. After 48 h of incubation with testosterone, somatostatin in both media and cells was significantly reduced. On the contrary, this treatment lead to a dose-dependent increase in media and cell GRF content. When cells were incubated with estradiol for 48 h, a significant inhibition in medium SS release was observed, whereas intracellular SS slightly increased at the highest concentration of 10 ng/dl. Estradiol treatment resulted in an inconsistent decrease in media and cells IR-GRF. Our results indicate that both SS and GRF are under the influence of testosterone and estradiol acting at the hypothalamic level, and furthermore suggest that at this stage of brain development, gonadal steroids may regulate GH secretion through their ability to modulate hypothalamic SS and GRF.     

Interaction of caerulein, glucose, and amino acids on insulin secretion from the perfused rat pancreas

The effect of caerulein on insulin response to graded amounts of glucose from the isolated perfused rat pancreas was investigated in the presence or absence of an amino acids mixture. Caerulein at a concentration of 0.1 ng/ml which is a submaximal concentration for an effect on exocrine pancreatic secretion potentiated insulin responses to glucose concentrations less than 200 mg/dl, but produced no further increase when added to a glucose stimulus over a 200 mg/dl. However, in the presence of amino acids the insulin response to 200 mg/dl glucose was significantly potentiated by the stimulation of 0.1 ng/ml caerulein. The effectiveness of caerulein as an insulinotropic agent depended on the glucose concentration only when amino acids were present. These results indicate that caerulein, at a concentration which stimulate pancreatic exocrine secretion, has a synergistic effect on insulin response to glucose and amino acids and therefore raises the possibility that endogenously released CCK may contribute to the entero-insular axis.     

Carbon Source Requirements for Exopolysaccharide Production by Lactobacillus casei CG11 and Partial Structure Analysis of the Polymer

Exopolysaccharide production by Lactobacillus casei CG11 was studied in basal minimum medium containing various carbon sources (galactose, glucose, lactose, sucrose, maltose, melibiose) at concentrations of 2, 5, 10, and 20 g/liter. L. casei CG11 produced exopolysaccharides in basal minimum medium containing each of the sugars tested; lactose and galactose were the poorest carbon sources, and glucose was by far the most efficient carbon source. Sugar concentrations had a marked effect on polymer yield. Plasmid-cured Muc^- derivatives grew better in the presence of glucose and attained slightly higher populations than the wild-type strain. The values obtained with lactose were considerably lower for both growth and exopolysaccharide yield. The level of specific polymer production per cell obtained with glucose was distinctively lower for Muc^- derivatives than for the Muc⁺ strain. The polymer produced by L. casei CG11 in the presence of glucose was different from that formed in the presence of lactose. The polysaccharide produced by L. casei CG11 in basal minimum medium containing 20 g of glucose per liter had an intrinsic viscosity of 1.13 dl/g. It was rich in glucose (76%), which was present mostly as 2- or 3-linked residues along with some 2,3 doubly substituted glucose units, and in rhamnose (21%), which was present as 2-linked or terminal rhamnose; traces of mannose and galactose were also present.     

Effect of 2-deoxy-D-glucose on hypothalamic-pituitary-thyroid axis in rats

The intraperitoneal administration to rats of 500 mg/kg body weight of 2-deoxy-D-glucose, an analog of glucose which produces intracellular glucopenia with rise in extracellular fluid glucose concentration, is followed by a significant though transient reduction of hypothalamic TRH content, observed at 15 and 25 minutes after drug administration. A subsequent increase in serum thyrotropin followed by that of triiodothyronine concentration was also observed. These findings indicate that the neuroglucopenia induced by 2-deoxy-D-glucose may play a role in the regulating the hypothalamic-pituitary-thyroid axis.     

β‐Cell Function and Insulin Sensitivity in Adolescents From an OGTT

Given the increase in the incidence of insulin resistance, obesity, and type 2 diabetes in children and adolescents, it would be of paramount importance to assess quantitative indices of insulin secretion and action during a physiological perturbation, such as a meal or an oral glucose‐tolerance test (OGTT). A minimal model method is proposed to measure quantitative indices of insulin secretion and action in adolescents from an oral test. A 7 h, 21‐sample OGTT was performed in 11 adolescents. The C‐peptide minimal model was identified on C‐peptide and glucose data to quantify indices of β‐cell function: static φ_s and dynamic φ_d responsivity to glucose from which total responsivity φ was also measured. The glucose minimal model was identified on glucose and insulin data to estimate insulin sensitivity, S_I, which was compared to a reference measure, S_I^ref, provided by a tracer method. Disposition indices, which adjust insulin secretion for insulin action, were then calculated. Indices of β‐cell function were φ_s = 51.35 ± 8.89 × 10^?9min^?1, φ_d = 1,392 ± 258 × 10^?9, and φ = 82.09 ± 17.70 × 10^?9min^?1. Insulin sensitivity was S_I = 14.19 ± 2.73 × 10^?4, not significantly different from S_I^ref = 14.96 ± 3.04 × 10^?4 dl/kg·min per µU/ml, and well correlated: r = 0.98, P < 0.0001, thus indicating that S_I can be accurately measured from an oral test. Disposition indices were DI_s = 1,040 ± 201 × 10^?14 dl/kg/min² per pmol/l, DI_d = 33,178 ± 10,720 × 10^?14 dl/kg/min per pmol/l, DI = 1,844 ± 522 × 10^?14 dl/kg/min² per pmol/l. Virtually the same minimal model assessment was obtained with a reduced 3 h, 9‐sample protocol. OGTT interpreted with C‐peptide and glucose minimal model has the potential to provide novel insight regarding the regulation of glucose metabolism in adolescents, and to evaluate the effect of obesity and interventions such as diet and exercise.     

Acid glycohydrolase in Chinese hamster with spontaneous diabetes. VII. The lack of short-term glucose-effect in cultured kidney cells

An epithelial cell line, designated CHK-ACE, was established from the kidney of a spontaneously diabetic Chinese hamster from the highly inbred AC line. CHK-ACE was separated into two sublines, CHK-ACE-100 and CHK-ACE-400, by successive passages in 100 and 400 mg/dl glucose respectively. Extra- and intracellular activities of N-acetyl-beta-D-glucosaminidase and beta-D-galactosidase were measured in these cultures after exposure to varying concentrations of glucose (100, 200, 300 and 400 mg/dl) for one passage and 10% heated fetal calf serum for 6.5 h before enzyme measurements were taken; no apparent dependence on medium-glucose concentration was found. In serum-free medium, the time-dependent release of both N-acetyl-beta-D-glucosaminidase and beta-D-galactosidase was sustained for up to 24 h; no significant difference in their activities was found between CHK-ACE-100 cultures grown in 100 and 400 mg/dl glucose for one passage.     

Insulin binding in cultured Chinese hamster kidney epithelial cells the effects of glucose concentration in the medium and tunicamycin

An epithelial cell line established from a Chinese hamster kidney, CHK-AC_E, was separated into two sublines, CHK-AC_E-100 and CHK-AC_E-400, by 18 successive passages in medium containing 100 and 400 mg/dl glucose, respectively. Binding of CHK-AC_E-100 and CHK-AC_E-400 cells to ¹²⁵I-labeled insulin showed similar pH and time dependency; ¹²⁵I-labeled insulin binding as a function of insulin concentration differed in the two sublines, however. Degradation of ¹²⁵I-labeled insulin, as determined by its ability to bind insulin antibody and cells, was more extensive when preincubated with CHK_AC_E-400 cells than with CHK-AC_E-100 cells. When CHK-AC_E-100 cells were grown in 400 mg/dl glucose for six passages, these cells showed more insulin binding sites than cells grown parallel in 100 mg/dl glucose; whereas CHK-AC_E-400 cells grown in 100 mg/dl glucose for six passages showed fewer insulin binding sites than those grown parallel in 400 mg/dl glucose. A slight increase in $\text{K}{}_{\mathrm{<mtext>f</mtext>}}\text{/}\text{K}{}_{\mathrm{<mtext>e</mtext>}}$ ratio was observed in both sublines when grown in 400 mg/dl glucose as compared to 100 mg/dl glucose, indicating attenuated negative cooperativity of the binding sites in cells grown in 400 mg/dl glucose. Tunicamycin, at concentrations from 0.016 to 0.125 μg/ml, showed no direct effect on the assay of ¹²⁵I-labeled insulin binding to CHK-AC_E-100 cells; exposure of CHK-AC_E-100 cells to tunicamycin, at concentrations from 0.01 to 0.2 μg/ml, for 24 h caused a dose-dependent decrease in insulin binding capacity and an increase in $\text{K}{}_{\mathrm{<mtext>f</mtext>}}\text{/}\text{K}{}_{\mathrm{<mtext>e</mtext>}}$ ratio. These data indicate that the number of insulin binding sites in the cultured Chinese hamster kidney epithelial cells increased with high glucose concentrations in the culture medium, whereas tunicamycin, an inhibitor of protein glycosylation, lowered the number of insulin binding sites.     

Effects of Rapeseed Oil on Activity of Methylmalonyl-CoA Carboxyltransferase in Culture of Streptomyces fradiae

To investigate why more tylosin was produced when Streptomyces fradiae T1558 was cultured in a rapeseed oil medium than in a glucose or starch medium, we measured the activity of methylmalonyl-CoA carboxyltransferase (EC 2.1.3.1) and intracellular propionic acid. The activity of the enzyme, which catalyzes the formation of the precursor of tylosin, protylonolide, was 0.19 U/mg protein in 5 days of culture in rapeseed oil medium, which was 2.5- and 1.3-fold that with the glucose or starch medium, respectively. The intracellular propionic acid concentration was 1.2 g/g of dry weight, which was 4.3- and 2.1-fold that with the glucose or starch medium, respectively. The addition of propionic acid increased tylosin production in batch culture: when 0.2 g/l (final concentration) propionic acid was added to the glucose medium, 3.8 g/l tylosin was produced in 10 days of culture, 4.7-fold the amount without propionic acid. These findings suggest that in glucose medium, intracellular propionic acid is a limiting factor because of the low activity of methylmalonyl-CoA carboxyltransferase of the tylosin biosynthesis pathway.     

Action of β-galactosidase in medium on the Lemna minor (L.) callus polysaccharides

The callus culture of duckweed cultivated on medium containing different concentrations of β-galactosidase was shown to produce the following polysaccharides: pectin lemnan LMC, intracellular AG1, and extracellular AG2 arabinogalactans. The samples of lemnan with 46% galactose residue reduction and 9-46% increased galacturonic acid residue content were obtained at β-galactosidase concentrations of 10⁻³-10⁻¹ mg/mL. The most substantial alterations in the sugar composition of pectin were found to occur in the fraction with a molecular mass of 100-300 kDa. Low concentrations of enzyme failed to influence the sugar composition of intracellular arabinogalactan, whereas high concentrations were shown to decrease the amount of arabinose residues in AG1 and to cause galactan formation. Extracellular galactan was found to be produced on the medium with 10⁻¹ and 1 mg/mL β-galactosidase whereas extracellular arabinogalactan AG2 was shown to be biosynthesized without β-galactosidase or at a β-galactosidase concentration of 10⁻³ mg/mL. Alterations in the sugar composition of polysaccharides were shown to be connected with the increasing activity of α-l-arabinofuranosidase and β-galactosidase, and with the decreasing activity of intracellular polygalacturonase.     

Citric acid accumulation by cycloheximide-sensitive mutant strains of Aspergillus niger

 Mutants having impaired protein synthesis, that is cycloheximide-sensitive mutants of a citric-acid-hyper-accumulating strain, were induced from Aspergillus niger WU-2223L. Selection was on the basis of a presumption that the mutants should be more sensitive to cycloheximide than WU-2223L. In shake culture without methanol as a promotor substance, seven mutants accumulated approximately 1.8–3.5 times as much citric acid as WU-2223L. The best mutant, CHM I-C3, accumulated 69.4 mg citric acid/ml from 120 mg glucose/ml in shake culture without methanol, this amount being 1.1 times the amount accumulated by WU-2223L with methanol. Furthermore, under the conditions without methanol the mutants appeared to be more efficient than WU-2223L in employing the consumed glucose for the accumulation of citric acid. It was also confirmed that CHM I-C3 exhibited a significantly increased level of intracellular NH⁺ ₄ accumulation. The addition of 2% (v/v) methanol or 20 μg cycloheximide/ml to the medium caused a remarkable increase of citric acid accumulation by WU-2223L: about 3.1 and 2.4 times respectively. However, the addition of these substances produced negative effects on citric acid accumulation by the mutants. With 2% (v/v) methanol, WU-2223L showed a remarkably decreased level of protein accumulation but a substantially increased level of intracellular NH⁺ ₄ accumulation. However, these phenomena were also observed in CHM I-C3 without methanol. These results indicate that the intracellular circumstances of the cycloheximide-sensitive mutants without methanol were similar to those of WU-2223L with methanol, and that the impairment of protein synthesis contributed to increased citric acid accumulation by the mutants in the absence of methanol. Received: 21 November 1994 / Received last revision: 10 July 1995 / Accepted: 26 July 1995     

Potentiation by ouabain of bradykinin-induced catecholamine secretion and calcium influx into cultured bovine adrenal chromaffin cells: Evidence for involvement of Na+ influx associated with the bradykinin B2-receptor

The effect of bradykinin (BK), in the presence of ouabain, an inhibitor of Na⁺-K⁺ ATPase, on catecholamine (CA) secretion was studied in cultured bovine adrenal chromaffin cells, to determine whether Na⁺, as well as Ca²⁺, is involved in BK-receptor mediated CA secretion. BK (10⁻⁸–10⁻⁵M)-induced CA secretion was markedly potentiated by addition of ouabain (10⁻⁵M), was blocked by a BK-B₂ receptor antagonist, and was decreased in Ca²⁺-free medium. BK-induced increase in ⁴⁵Ca²⁺ influx was also potentiated by addition of ouabain. The cultured cells were first incubated with BK for 30 min in Ca²⁺-free medium in the presence or absence of ouabain and then kstimulated for 15 min with Ca²⁺-medium without BK or ouabain. Prior stimulation of the cells, BK induced ²²Na⁺ influx and increased Ca²⁺-induced CA secretion and these stimulatory effects of BK were potentiated by added ouabain. When the cells were stimulated with BK and ouabain in Na⁺-free sucrose medium, the Ca²⁺-induced CA secretion was greatly reduced. These results indicated that activation of the BK-B2 receptor and inhibition of the Na⁺ pump both increase the intracellular Na⁺ level, resulting in increase in Ca²⁺ influx and CA secretion.     

Effect of glucose on Na, K-ATPase activity in cultured bovine aortic endothelial cells.

The effect of high concentrations of glucose on Na, K-ATPase activity and the polyol pathway was studied using cultured bovine aortic endothelial cells. Na, K-ATPase activity was expressed as ouabain-sensitive K+ uptake. A significant decrease in Na, K-ATPase activity with an intracellular accumulation of sorbitol was found in confluent endothelial cells incubated with 400 mg/dl glucose for 96 h. However, there was no significant change in the Na, K-ATPase activity or sorbitol content of the cells incubated with 100 mg/dl glucose plus 300 mg/dl mannitol. The decrease in Na, K-ATPase induced by the high glucose concentration was restored by the simultaneous addition of 10(-4) M ponalrestat (ICI 128,436; Statil), an aldose reductase inhibitor. The addition of this agent also significantly reduced the increase in sorbitol induced by high glucose levels. These results suggest that the decrease in Na, K-ATPase activity induced in cultured aortic endothelial cells by high concentrations of glucose may be caused in part by the accumulation of sorbitol.     

Acid glycohydrolase in Chinese hamster with spontaneous diabetes VII. The lack of short-term glucose-effect in cultured kidney cells

An epithelial cell line, designated CHK-AC_E, was established from the kidney of a spontaneously diabetic Chinese hamster from the highly inbred AC line. CHK-AC_E was separated into two sublines, CHK-AC_E-100 and CHK-AC_E-400, by successive passages in 100 and 400 mg/dl glucose respectively. Extra- and intracellular activities of $\text{N-}\text{acetyl}\text{-β-}\text{D}\text{-}\text{glucosaminidase}$ and β-D-galactosidase were measured in these cultures after exposure to varying concentrations of glucose (100, 200, 300 and 400 mg/dl) for one passage and 10% heated fetal calf serum for 6.5 h before enzyme measurements were taken; no apparent dependence on medium-glucose concentration was found. In serum-free medium, the time-dependent release of both $\text{N-}\text{acetyl}\text{-β-}\text{D}\text{-}\text{glucosaminidase}$ and β-D-galactosidase was sustained for up to 24 h; no significant difference in their activities was found between CHK-AC_E-100 cultures grown in 100 and 400 mg/dl glucose for one passage.     

Phe-met-arg-phe-amide (FMRF-NH2) inhibits insulin and somatostatin secretion and anti-FMRF-NH2 sera detects pancreatic polypeptide cells in the rat islet

FMRF-NH2-like immunoreactivity was localized in the pancreatic polypeptide containing cells of the rat islet. FMRF-NH2 was investigated with regard to its effect on insulin, somatostatin and glucagon secretion from the isolated perfused rat pancreas. FMRF-NH2 (1 microM) significantly inhibited glucose stimulated (300 mg/dl) insulin release (p less than 0.005) and somatostatin release (p less than 0.01) from the isolated perfused pancreas. FMRF-NH2 (1 and 10 microM) was without effect on glucagon secretion, either in low glucose (50 mg/dl), high glucose (300 mg/dl), or during arginine stimulation (5 mM). These findings indicate that these FMRF-NH2 antisera recognize a substance in the pancreatic polypeptide cells of the islet which may be capable of modulating islet beta and D cell activity.     

A STUDY OF THE METABOLISM AND RELEASE OF DOPAMINE AND AMINO ACIDS FROM NERVE ENDINGS ISOLATED FROM SHEEP CORPUS STRIATUM

Abstract— Synaptosomes prepared from sheep corpus striatum showed a linear rate of respiration over a 90 min period of incubation in Krebs-bicarbonate medium containing glucose (10 mm ) and the rate of respiration was stimulated by electrical pulses. Dopamine was released from synaptosome beds to the medium by either electrical pulses or 56mm -K⁺ (10min), increasing 108% and 76% respectively above control levels of release. The presence of d- or 1-amphetamine (0.12mm ) in the incubation medium (40 min) increased the accumulation of dopamine in the medium by 310 and 275% respectively and 56mm -K⁺ also caused a significant increase in the release of glutamate, GABA and aspartate. Radioactively labelled dopamine was synthesized by the synaptosomes from l -[¹⁴C]tyrosine, l -DOPA or dl -DOPA, and electrical pulses caused a 35% increase in the rate of dopamine production from [U-¹⁴C] tyrosine. No increased release of [¹⁴C]dopamine in response to depolarizing stimuli was found to occur when synaptosome beds were transferred from medium containing radioactive precursors to fresh medium for further incubation (20 min). In the presence of 1- and d-amphetamine, accumulation of ¹⁴C-labelled doparnine in the incubation media was increased 129% and 380% respectively, the latter was partially depressed by absence of calcium from the medium. Three radioactively labelled metabolites formed by synaptosomes during incubation in dl -[2-¹⁴C]DOPA were detected; the major ones were dihydroxyphenylacetic acid and homovanillic acid and the third was unidentified. When the synaptosome beds were transferred to medium containing no radioactive precursors, it was found that labelled dihydroxyphenylacetic acid was 7 times more abundant than labelled dopamine in the incubation medium (20 min) and one-third as abundant in the synaptosomes. The dihydroxyphenylacetic acid n Ci/dopamine n Ci ratio was greatly affected by K⁺ stimulation, decreasing 52% and 34% in the incubation medium and synaptosomes respectively. A pathway of dihydroxyphenylacetic acid degradation was shown to occur through decarboxylation. These results are discussed in terms of the compartmentation of dopamine and its metabolism. It is proposed that one pool of dopamine is released by depolarizing agents and during the period of incubation it is replaced by synthesis from the endogenous tyrosine (19.5 nmol/100 mg protein) and not by the labelled dopamine in the synaptosome. The synaptosomal pool of dopamine which is radioactively labelled after pulse labelling with dl -[2-¹⁴C]DOPA appears to be prone to oxidation to DOPAC and homovanillic acid which are preferentially released from the synaptosomes.     

Unimpaired formation of hormone-stimulated inositol triphosphate in human mesangial cells under hyperglycemic conditions

The relationship between bulk cellular myo-inositol content and phosphatidylinositol metabolism was evaluated in a human mesangial cell line under euglycemic and hyperglycemic conditions. Mesangial cells maintained in high glucose medium displayed a concentration-dependent fall in myo-inositol as measured by gas-liquid chromatography. Measurements of phosphatidylinositol, phosphatidylinositol 4-monophosphate and phosphatidylinositol 4,5-biphosphate mass revealed slight but statistically insignificant increases in cells exposed to high glucose containing medium. CDP-diacylglycerol: myo-inositol 3-phosphatidylinositol transferase activity, measured in plasma membranes from mesangial cells grwon under control and hyperglycemic conditions, was kinetically similar with Michaelis constants (K_m values) for myo-inositol of 2.9 and 2.1 mM, respectively. Finally hormone-stimulated intracellular calcium mobilization and myo-inositol 1,4,5-triphosphate mass was measured from mesangial cells grown under normal and hyperglycemic conditions. Both intracellular calcium and inositol triphosphate formation were unchanged in cells previously exposed to high glucose conditions (400 mg/dl) compared to cells grown under normal glucose concentration (100 mg/dl). These data indicate that bulk changes in myo-inositol induced by hyperglycemia are neither associated with alterations in basal levels of inositol containing glycerolipids nor with changes in hormone-stimulated calcium mobilization and inositol trisphosphate formation under conditions of short term changes in extracellular glucose.     

Glucose monitoring at the thenar: evaluation of upper dermal blood glucose kinetics during rapid systemic blood glucose changes.

OBJECTIVE: We compared blood glucose measurements at the thenar with those at the fingertip during glucose increase and decrease that was rapid enough to induce glucose differences between the forearm and the fingertip. METHODS: A rapid glucose increase was induced by oral glucose; subsequently, a rapid glucose decrease was induced by intravenous insulin in 16 insulin-treated patients with diabetes. Capillary samples were taken in parallel from the thenar and fingertip. Different glucose monitors (FreeStyle, OneTouch Ultra, Soft-Sense) were used. Additional samples were taken from the forearm (n = 10 patients) in order to demonstrate that the blood glucose change achieved was rapid enough to principally induce glucose differences at alternative sites. RESULTS: Neither blood glucose at baseline (135 +/- 34 vs. 136 +/- 41 mg/dl, p = 0.86) nor glucose amplitude during increase (190 +/- 35 vs. 188 +/- 41 mg/dl, p = 0.65) or decrease (255 +/- 32 vs. 257 +/- 45 mg/dl, p = 0.83) differed significantly between the fingertip and the thenar. Intra-individual average thenar-fingertip glucose difference was - 2 +/- 12 (p = 1.00) and + 5 +/- 9 mg/dl (p = 0.11). In the subgroup, intra-individual average forearm-finger difference was - 50 +/- 19 (p < 0.01) and + 45 +/- 11 mg/dl (p < 0.01) during glucose-increase and decrease, respectively. There were no obvious device-specific differences. CONCLUSIONS: Blood glucose measurements at the thenar are a safe alternative to measurements at the fingertip at steady state as well as during blood glucose change that is sufficiently rapid to induce clinically relevant differences between forearm and fingertip.     

The role of glutathione in rat adipocyte pentose phosphate cycle activity

An assay for reduced and oxidized glutathione was adapted to isolated rat epididymal adipocytes in order to correlate pentose phosphate cycle activity and glutathione metabolism. In collagenase-digested adipocytes the [GSH/GSSG] molar ratio was in excess of 100. Cells incubated for 1 hr with low glucose concentrations (0.28–0.55 mm) had higher GSH contents (3.2 μg/10⁶ cells) than in the absence of glucose (2.3 μg/10⁶ cells). The glutathione oxidant diamide caused a dose-related decrease in intracellular GSH, an increase in GSSG released into the medium, but no detectable change in the low intracellular GSSG content. The intracellular content of GSH and amount of GSSG released into the medium were therefore taken to reflect the glutathione status of the adipocytes most closely. Addition of H₂O₂ to a concentration of 60 μm to adipocytes caused to decline within 5 min in GSH content, which was less severe and more rapid to recover in the presence of 1.1 mm glucose, suggesting that the concomitant stimulation of glucose C-1 oxidation induced by the peroxide in the presence of glucose provided NADPH for regeneration of GSH. Further evidence for tight coupling between adipocyte [GSH/GSSG] ratios and pentose phosphate cycle activity was that (i) lowering intracellular GSH to 35–60% of control values by agents as diverse in action as t-butyl hydroperoxide, diamide, or the sulfhydryl blocker N-ethylmaleimide resulted in optimal stimulation of glucose C-1 oxidation and fractional pentose phosphate cycle activity, and (ii) incubating adipocytes directly with 2.5 mm GSSG resulted in a slight increase in glucose C-1 oxidation and when 0.5 mm NADP⁺ was also added a synergistic effect on pentose phosphate cycle activity was found. On the other hand, electron acceptors such as methylene blue did not lower cellular GSH content, but did stimulate the pentose phosphate cycle, confirming a site of action independent of glutathione metabolism. The results show that (i) glucose metabolism by the pentose phosphate cycle contributes to regeneration of GSH and that (ii) glutathione metabolism either directly or via coupled changes in [NADPH/NADP⁺] ratios may play a significant role in short-term control of the pentose phosphate cycle.