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Biomedical subjects

F M Matschinsky

Publications and source records attributed to F M Matschinsky.

At least 109 records · Page 6Linked to original sources
PubMed1986

The developmental appearance of a major basilar papilla-specific protein in the chick.

The sensory epithelium of the chick cochlea, the basilar papilla, contains a major protein of approximately 23,000 daltons. This protein was as abundant as actin in the papilla, yet could not be found in significant quantities in any other cochlear tissue. The protein appeared at a time in development when other studies have shown that the chick embryo develops peripheral auditory competence. These observations suggest a role for this protein in cochlear function.

Age Factors↗
PubMed1986

In vitro exhaustion of pancreatic beta-cells.

To learn more about possible limited beta-cell secretory capacity and factors essential for insulin release, a perifusion system was applied that allowed the in vitro study of insulin secretion from isolated pancreatic islets for more than 6 h. Islets isolated from rats were stimulated with various glucose concentrations (7.5, 16.7, and 30 mM), alpha-ketoisocaproate (30 mM), and 30 mM glucose plus 1 mM 3-isobutyl-1-methylxanthine for several hours in Krebs-Ringer-bicarbonate buffer (KRB) or RPMI 1640. Islets showed "exhaustion" with all stimulatory conditions used when KRB was the perifusion medium. This was not prevented by addition of amino acids, phosphate, myo-inositol or arachidonic acid. With RPMI 1640 as the basal medium, exhaustion was not seen at 7.5 mM but was readily approached at higher glucose concentrations. It is possible that the exhaustion phenomenon observed here is due to a depletion of a readily releasable insulin pool.

1-Methyl-3-isobutylxanthine↗
PubMed1986

The glucokinase glucose sensor in human pancreatic islet tissue.

The enzyme glucokinase controls glucose metabolism in islets and is proposed to be the glucose sensor in pancreatic beta-cells. This concept was developed from studies with rodents and it remained to be explored whether it also applies to man. Studies in man were hampered, however, by the difficulty in obtaining well-preserved pancreatic islet tissue and also because the high activity of hexokinase made it difficult to measure glucokinase. To overcome these obstacles, quantitative histochemical sampling techniques were developed allowing precise dissection of pure human islet tissue and a newly designed radiometric microassay was used, avoiding hexokinase interference, and providing the sensitivity necessary to measure the relatively low glucokinase activity in small samples of tissue obtained from brain-dead tissue donors. The present data indicate that glucokinase is present in human pancreatic islet tissue and is not found in the exocrine pancreas. The enzyme's Vmax with D-glucose as substrate was similar to the Vmax for glucose utilization reported previously for intact, isolated human islets and the enzyme's Km for D-glucose was about 5 mM. Since glucokinase was also present in islet tissue of hamster, mouse, and rat, it is suggested that the glucokinase-glucose sensor concept has general applicability and that it could explain many aspects of the physiology and pathology of glucose homeostasis. This well-defined pancreatic islet glucokinase-glucose sensor should, therefore, be incorporated in any comprehensive model of glucose homeostasis.

Adolescent↗
PubMed1986

Identification of glucokinase as an alloxan-sensitive glucose sensor of the pancreatic beta-cell.

Alloxan inactivated glucokinase in intact, isolated pancreatic islets incubated in vitro. Inactivation of glucokinase was antagonized by 30 mM glucose present during incubation of islets with alloxan. Glucokinase partially purified from transplantable insulinomas or rat liver was inactivated by alloxan with a half-maximal effect at 2-4 microM alloxan. Inactivation of purified glucokinase was antagonized by glucose, mannose, and 2-deoxyglucose in order of decreasing potency but not by 3-O-methylglucose. Glucose anomers at 6 and 14 mM were discriminated as protecting agents, with the alpha-anomer more effective than the beta-anomer. Glucokinase was not protected from alloxan inactivation by N-acetylglucosamine, indicating that the reactive site for alloxan is not the active site; therefore, glucose may protect glucokinase by inducing a conformational change. Glucokinase is thought to be the glucose sensor of the pancreatic beta-cell. The finding that glucokinase is inactivated by alloxan and protected by glucose with discrimination of its anomers similar to inhibition of glucose-stimulated insulin secretion by alloxan supports this hypothesis and appears to explain the mechanism for inhibition of hexose-stimulated insulin secretion by this agent and the unique role of glucose and mannose as protecting agents.

Alloxan↗
PubMed1986

Glucose transport by radiation-induced insulinoma and clonal pancreatic beta-cells.

Sugar uptake was measured in dispersed cells prepared from radiation-induced insulinomas transplantable in NEDH rats and in three clonal beta-cell lines maintained in continuous culture (RIN m5F, RIN 1046, HIT). Uptake of D-glucose and 3-O-methyl-D-glucose by insulinoma cells was rapid so that the intracellular concentration of D-hexoses approximated the concentration in the incubation medium by 15-30 s. L-Glucose was taken up only slowly. 3-O-methyl-D-glucose uptake by RIN m5F, RIN 1046, and HIT cells was slow; with 1 mM 3-O-methylglucose in the medium, equilibrium was attained at 20 min, but with 10 mM 3-O-methylglucose, equilibrium was not attained even at 20 min. In HIT cells incubated with D-glucose for 30 min, the intracellular concentration of glucose was less than the medium glucose concentration, indicating glucose transport is a nonequilibrium reaction in this cell line. These data indicate that radiation-induced insulinoma cells retain the capacity of normal beta-cells to transport sugar at high rates. RIN m5F, RIN 1046, and HIT cells transport sugar slowly, however, and thus differ from normal beta-cells. In RIN m5F, RIN 1046, and HIT cells, unlike in normal beta-cells, glucose transport may be the site regulating glucose metabolism.

3-O-Methylglucose↗
PubMed1985

Inositol 1,4,5-trisphosphate and the endoplasmic reticulum Ca2+ cycle of a rat insulinoma cell line.

Regulation of endoplasmic reticulum (ER) Ca2+ cycling by inositol 1,4,5-trisphosphate (IP3) was studied in saponin-permeabilized RINm5F insulinoma cells. Cells were incubated with mitochondrial inhibitors, and medium Ca2+ concentration established by nonmitochondrial pool(s) (presumably the ER) was monitored with a Ca2+ electrode. IP3 degradation accounted for the transience of the Ca2+ response induced by pulse additions of the molecule. To compensate for degradation, IP3 was infused into the medium. This resulted in elevation of [Ca2+] from about 0.2 microM to a new steady state between 0.3 and 1.0 microM, depending on both the rate of IP3 infusion and the ER Ca2+ content. The elevated steady state represented a bidirectional buffering of [Ca2+] by the ER, as slight displacements in [Ca2+], by small aliquots of Ca2+ or the Ca2+ chelator quin 2, resulted in net uptake or efflux of Ca2+ to restore the previous steady state. When IP3 infusion was stopped, [Ca2+] returned to its original low level. Ninety per cent of the Ca2+ accumulated by the ER was released by IP3 when the total Ca2+ content did not exceed 15 nmol/mg of cell protein. Above this high Ca2+ content, Ca2+ was accumulated in an IP3-insensitive, A23187-releasable pool. The maximal amount of Ca2+ that could be released from the ER by IP3 was 13 nmol/mg of cell protein. The data support the concept that in the physiological range of Ca2+ contents, almost all the ER is an IP3-sensitive Ca2+ store that is capable of finely regulating [Ca2+] through independent influx (Ca2+-ATPase) and efflux (IP3-modulated component) pathways of Ca2+ transport. IP3 may continuously modulate Ca2+ cycling across the ER and play an important role in determining the ER Ca2+ content and in regulating cytosolic Ca2+ under both stimulated and possibly basal conditions.

Adenoma, Islet Cell↗
PubMed1985

Radiometric oil well assay for glucokinase in microscopic structures.

Glucokinase (ATP:D-glucose 6-phosphotransferase, EC 2.7.1.1) plays a pivotal role in hepatic glucose metabolism and serves as the glucose sensor in pancreatic islet beta-cells. Biochemical studies of this enzyme are complicated by the cellular heterogeneity of the liver and the pancreas and because the presence of hexokinases (ATP:D-hexose 6-phosphotransferases, EC 2.7.1.1) seriously interferes with currently available analytical procedures. A radiometric assay was designed to deal with these problems. It is based on the liberation of 3H2O from D-[2-3H(N)]glucose 6-phosphate, the product of the glucokinase reaction, using exogenous phosphoglucose isomerase (D-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9). Interference by hexokinases was largely eliminated by using glucose 6-phosphate as inhibitor and the sensitivity of the assay was greatly increased by using small volumes with the oil well procedure. The assay was sufficiently sensitive to detect about 1 pg of glucokinase. It thus allowed the application of quantitative histochemical procedures to the study of intralobular hepatic glucokinase profiles and the pancreatic beta-cell glucose sensor. The quantitative histochemical procedures were sufficiently sensitive and reliable for measuring important kinetic constants of glucokinase (i.e., the Km and the Hill number) in microscopic samples of tissue.

Animals↗
PubMed1985

Treatment with aldose reductase inhibitor or with myo-inositol arrests deterioration of the electroretinogram of diabetic rats.

Biochemical abnormalities in the retinal pigment epithelium of experimentally diabetic animals include increased sorbitol and decreased myo-inositol. Diabetes also causes a progressive reduction in the amplitude of the c-wave of the electroretinogram of the pigmented rat. The c-wave is generated by the retinal pigmented epithelium. Myo-inositol administration or treatment with sorbinil, an inhibitor of aldose reductase, arrested the decline in the c-wave. Therefore, hyperglycemia-associated defects in myo-inositol or sorbitol metabolism may be involved in the pathogenesis of the electrophysiological deficit of the diabetic retina. The homogeneous cell layer of the pigment epithelium may be a useful tissue model for studying the pathogenesis of the complications of diabetes.

Aldehyde Reductase↗
PubMed1985

Regulatory role of fructose-2,6-bisphosphate in pancreatic islet glucose metabolism remains unsettled.

Fructose-2,6-P2 was measured in perifused, isolated rat pancreatic islets. Fructose-2,6-P2 was present in pancreatic islets at low levels approximately equal to fructose-2,6-P2 content of liver from fasted rats. In islets perifused with glucose at physiologic concentrations, fructose-2,6-P2 was increased from 0.8 microM in the presence of 5.5 mM glucose to 1.0 microM at 10 mM glucose and 1.3 microM at 16.7 mM glucose, but did not increase further at higher glucose concentration. Therefore, only modest increases in the phosphofructokinase-1 activator, fructose-2,6-P2, occur at glucose concentrations stimulating insulin secretion.

Animals↗
PubMed1984

The effect of inositol trisphosphate on Ca2+ fluxes in insulin-secreting tumor cells.

An early event associated with the stimulation of various secretory cells is the breakdown of phosphatidylinositol 4,5-bisphosphate and the mobilization of cellular calcium. Hydrolysis of this inositol lipid by a phosphodiesterase produces inositol trisphosphate (InsP3), a small water-soluble molecule which may serve a messenger function to release Ca2+ from internal stores. In order to assess the role of inositol lipid breakdown in the stimulation of insulin secretion we have examined the effect of InsP3 on Ca2+ fluxes in three different insulin-secreting tumor cells permeabilized by the addition of saponin. A rapid, transient release of Ca2+ from a non-mitochondrial pool occurred upon addition of InsP3 to all three cell types. Half-maximal Ca2+ release from the RIN-1046-38 and RIN-m5F cells was obtained in the concentration range 0.1-0.2 microM. However, the cells obtained from a transplantable tumor of the Syrian hamster were far more sensitive to InsP3 with half-maximal release being observed at 0.025 microM. A partially purified preparation of vesicles was isolated from this tumor which retained its responsiveness to InsP3. Half-maximal Ca2+ release from the vesicles was obtained at 0.2 microM InsP3. Our data are consistent with a role for InsP3 in mediating the increase in cytosolic free Ca2+ which occurs in response to a number of stimuli that promote the secretion of insulin.

Adenoma, Islet Cell↗
PubMed1984

An enzymatic fluorimetric assay for myo-inositol.

An enzymatic assay for myo-inositol (MI) is described. The method is based on the oxidation of MI by NAD+-dependent myo-inositol dehydrogenase, coupled to reoxidation of NADH with oxalacetate and malate dehydrogenase. The resultant malate is measured fluorimetrically. Several variations of the assay are described for measuring MI in serum and in tissues in amounts ranging from 0.2 pmol to 8 nmol. Highest sensitivity is achieved by applying an oil-well technique for handling small droplets, and by using the principle of enzymatic cycling. The potential of the technique is illustrated by MI measurements in several tissues of normal and diabetic rats and Chinese hamsters.

Animals↗
PubMed1984

Metabolic adaption of pancreatic islet tissue in aging rats.

A previous report indicating reduced glucose metabolism in pancreatic islets isolated from old as compared with young rats was reinvestigated. With a modified islet isolation procedure it was found that islets from 12- to 18-month-old rats had increased glucose use, elevated glucokinase, phosphofructokinase and glucose-6-phosphate dehydrogenase when compared with islets from 2-month-old controls. Glucose-induced insulin release in vitro of islets from the older rats was also improved by the more careful method of islet isolation but did not achieve rates observed with islets from young rats. The data suggest an age-related activation of pancreatic islet cell metabolism, possibly in response to overstimulation by increased peripheral insulin resistance, characteristic of older obese rats.

Aging↗
PubMed1984

New perspectives on pancreatic islet glucokinase.

Control of blood sugar involves the complex interaction of the pancreatic glucose-sensing beta-cells with the liver, which serves as the primary site of glucose disposal after a meal. Glucokinase occupies an important role in controlling glucose phosphorylation and metabolism both in the liver and in pancreatic islets. In the beta-cells, glucokinase functions as pacemaker of glycolysis at physiological glucose levels. It determines the unique characteristics of islet hexose usage, that is, the rate, affinity, cooperativity, and anomeric discrimination of glucose metabolism. Because glycolysis controls hexose-induced insulin release, glucokinase is considered the best-qualified candidate for the elusive glucose sensor of beta-cells. A deficiency of glucokinase would disturb glucose homeostasis. Decreased islet glucokinase would diminish islet glycolysis and would result in a higher set point of beta-cells for glucose-induced insulin release. Decreased liver glucokinase would cause less efficient hepatic glucose disposal. Human maturity-onset diabetes (type II diabetes) has these characteristics. It is thus conceivable that certain forms of type II diabetes are due to a glucokinase deficiency.

Animals↗
PubMed1984

Computer modeling identifies glucokinase as glucose sensor of pancreatic beta-cells.

Previous work from this laboratory indicates that glucokinase serves as the glucose sensor of pancreatic islets. Here we show by nonlinear computer optimization that the kinetic properties of glucokinase (together with hexokinase, known to be present in islets) account for the observed glycolytic rates in islets as a function of glucose level. Alternative enzymes that have been suggested to perform the same function as glucokinase, N-acetyl-D-glucosamine kinase and glucose-6-phosphatase, are shown to have incompatible properties, including a poor fit, different curve shapes, and unreasonable parameter values resulting from optimization. Their activities in islets are shown to be too low to account for observed glucose usage rates. This work endorses our previous proposal that glucokinase acts as the glucose sensor in pancreatic islet cells.

Blood Glucose↗
PubMed1984

Pharmacological modifications of insulin release in vitro from fuel-responsive transplantable insulinomas.

The response of insulinoma tissue to glucose, alpha-ketoisocaproate, and the modifiers of insulin release, tolbutamide, isoproterenol, and acetylcholine, was studied. Tumor tissue was transplanted under the kidney capsule of 14 rats, and the tumor-bearing kidneys were perfused in vitro about 8 weeks later. The plasma glucose concentration of these animals was 85.0 +/- 7.0 mg/dl, while the plasma insulin concentration was 13.8 +/- 1.5 ng/ml (normal, 180.5 +/- 7.0 mg/dl and 2.6 +/- 0.5 ng/ml, respectively; n = 26). Glucose (30 mM) evoked a 3- to 5-fold increase in insulin secretion, similar to the increase seen when either 100 micrograms/ml tolbutamide or 0.5 micrograms/ml isoproterenol were added to the perfusion medium containing 5 mM glucose. Propranolol at 50 micrograms/ml, but not at 20 micrograms/ml, inhibited insulin release stimulated by isoproterenol. Acetylcholine (10 or 100 microM) did not stimulate insulin secretion. alpha-Ketoisocaproate caused the highest insulin release of all stimuli studied. Glucagon or somatostatin release was not seen in any of the experiments. These results show that the tumor tissue transplanted under the kidney capsule responds not only to model fuels, but also to the sulfonylurea class of drugs and to adrenergic agents.

Acetylcholine↗
PubMed1984

Fuel-induced insulin release in vitro from insulinomas transplanted into the rat kidney.

We studied the release of insulin, glucagon, and somatostatin in response to glucose, glyceraldehyde (GA), and alpha-ketoisocaproate (KIC) from rat kidneys containing transplanted insulinomas. Kidneys were perfused about 11 wk after transplantation when the plasma glucose concentration of the fed animals had decreased from 180 +/- 7 to 95.1 +/- 9.9 mg/dl and plasma insulin concentrations had increased from 2.6 +/- 0.5 to 14.2 +/- 2.0 ng/ml. The insulin content of the tumor-containing kidney ranged from 40 to 679 micrograms; the glucagon and somatostatin concentrations ranged from undetectable levels to 3.7 micrograms and 248 ng, respectively. The average response to 30 mM glucose and 10 mM GA was a four- to fivefold increase in insulin secretion, whereas 30 mM KIC caused a 16- to 28-fold increase. In vitro stimulation of the insulinoma with 30 mM glucose primed the beta-cell response to a second stimulus following a short rest period. Cytochalasin B did not enhance this primed glucose response. Diazoxide inhibited glucose, GA, and KIC-stimulated insulin release. Glucose, GA, and KIC stimulated glucagon release in 2 of 17 insulinomas studied here. Somatostatin release was not seen in any of the experiments. These findings show that this islet cell tumor transplanted under the kidney capsule releases insulin in response to physiologic and model fuel substances. Thus, this particular transplantable tumor offers an opportunity to study the biochemistry and biophysics that underlie fuel-stimulated insulin release.

Adenoma, Islet Cell↗
PubMed1983

Discrimination of glucose anomers by glucokinase from liver and transplantable insulinoma.

Phosphorylation of alpha- and beta-D-glucose by glucokinase from rat liver or a radiation-induced, transplantable insulinoma was investigated. Glucokinase partially purified by ion exchange chromatography on DEAE-Cibacron blue F3GA agarose was incubated for brief periods (1 or 3 min) with glucose anomers. Glucokinase from both liver and insulinoma tissue had a higher affinity for alpha-D-glucose (S0.5 = 6-7 mM) than beta-D-glucose (S0.5 = 12-14 mM). The maximum velocity was 15-20% lower for alpha-D-glucose than beta-D-glucose. Cooperative rate dependence with respect to glucose concentration was observed with both anomers (nH = 1.4). These kinetic data imply that both anomers of glucose are phosphorylated by glucokinase, however, at the physiological range of glucose concentrations below 15 mM, the higher affinity of alpha-D-glucose results in higher rates than with beta-D-glucose. At clearly pathological glucose concentrations exceeding 20 mM, the observed velocities are slightly higher with beta- than alpha-D-glucose. Glucokinase is thought to be the glucose sensor of pancreatic beta cells. The present data indicating a preferential phosphorylation of alpha-D-glucose compared to beta-D-glucose by glucokinase, supports the glucokinase-glucose sensor hypothesis, because it parallels the well established greater potency of alpha-D-glucose as a stimulant of insulin release.

Adenoma, Islet Cell↗