Loss of coupling between calcium influx, energy consumption and insulin secretion associated with development of hyperglycaemia in the UCD-T2DM rat model of type 2 diabetes.

AIMS/HYPOTHESIS: Previous studies on isolated islets have demonstrated tight coupling between calcium (Ca(2+)) influx and oxygen consumption rate (OCR) that is correlated with insulin secretion rate (ISR). To explain these observations, we have proposed a mechanism whereby the activation of a highly energetic process (Ca(2+)/metabolic coupling process [CMCP]) by Ca(2+) mediates the stimulation of ISR. The aim of the study was to test whether impairment of the CMCP could play a role in the development of type 2 diabetes. METHODS: Glucose- and Ca(2+)-mediated changes in OCR and ISR in isolated islets were compared with the time course of changes of plasma insulin concentrations observed during the progression to hyperglycaemia in a rat model of type-2 diabetes (the University of California at Davis type 2 diabetes mellitus [UCD-T2DM] rat). Islets were isolated from UCD-T2DM rats before, 1 week, and 3 weeks after the onset of hyperglycaemia. RESULTS: Glucose stimulation of cytosolic Ca(2+) and OCR was similar for islets harvested before and 1 week after the onset of hyperglycaemia. In contrast, a loss of decrement in islet OCR and ISR in response to Ca(2+) channel blockade coincided with decreased fasting plasma insulin concentrations observed in rats 3 weeks after the onset of hyperglycaemia. CONCLUSIONS/INTERPRETATION: These results suggest that phenotypic impairment of diabetic islets in the UCD-T2DM rat is downstream of Ca(2+) influx and involves unregulated stimulation of the CMCP. The continuously elevated levels of CMCP induced by chronic hyperglycaemia in these islets may mediate the loss of islet function.

Endothelial inflammation induced by excess glucose is associated with cytosolic glucose 6-phosphate but not increased mitochondrial respiration.

AIMS/HYPOTHESIS: Exposure of endothelial cells to high glucose levels suppresses responses to insulin, including induction of endothelial nitric oxide synthase activity, through pro-inflammatory signalling via the inhibitor of nuclear factor kappaB (IkappaB)alpha-nuclear factor kappaB (NF-kappaB) pathway. In the current study, we aimed to identify metabolic responses to glucose excess that mediate endothelial cell inflammation and insulin resistance. Since endothelial cells decrease their oxygen consumption rate (OCR) in response to glucose, we hypothesised that increased mitochondrial function would not mediate these cells' response to excess substrate. METHODS: The effects of glycolytic and mitochondrial fuels on metabolic intermediates and end-products of glycolytic and oxidative metabolism, including glucose 6-phosphate (G6P), lactate, CO(2), NAD(P)H and OCR, were measured in cultured human microvascular endothelial cells and correlated with IkappaBalpha phosphorylation. RESULTS: In response to increases in glucose concentration from low to physiological levels (0-5 mmol/l), production of G6P, lactate, NAD(P)H and CO(2) each increased as expected, while OCR was sharply reduced. IkappaBalpha activation was detected at glucose concentrations >5 mmol/l, which was associated with parallel increases of G6P levels, whereas downstream metabolic pathways were insensitive to excess substrate. CONCLUSIONS/INTERPRETATION: Phosphorylation of IkappaBalpha by excess glucose correlates with increased levels of the glycolytic intermediate G6P, but not with lactate generation or OCR, which are inhibited well below saturation levels at physiological glucose concentrations. These findings suggest that oxidative stress due to increased mitochondrial respiration is unlikely to mediate endothelial inflammation induced by excess glucose and suggests instead the involvement of G6P accumulation in the adverse effects of hyperglycaemia on endothelial cells.

Glucose-stimulated increment in oxygen consumption rate as a standardized test of human islet quality.

Standardized assessment of islet quality is imperative for clinical islet transplantation. We have previously shown that the increment in oxygen consumption rate stimulated by glucose (DeltaOCR(glc)) can predict in vivo efficacy of islet transplantation in mice. To further evaluate the approach, we studied three factors: islet specificity, islet composition and agreement between results obtained by different groups. Equivalent perifusion systems were set up at the City of Hope and the University of Washington and the values of DeltaOCR(glc) obtained at both institutions were compared. Islet specificity was determined by comparing DeltaOCR(glc) in islet and nonislet tissue. The DeltaOCR(glc) ranged from 0.01 to 0.19 nmol/min/100 islets (n = 14), a wide range in islet quality, but the values obtained by the two centers were similar. The contribution from nonislet impurities was negligible (DeltaOCR(glc) was 0.12 nmol/min/100 islets vs. 0.007 nmol/min/100 nonislet clusters). The DeltaOCR(glc) was statistically independent of percent beta cells, demonstrating that DeltaOCR(glc) is governed more by islet quality than by islet composition. The DeltaOCR(glc), but not the absolute level of OCR, was predictive of reversal of hyperglycemia in diabetic mice. These demonstrations lay the foundation for testing DeltaOCR(glc) as a measurement of islet quality for human islet transplantation.

Identification and characterization of a novel isoform of the vesicular gamma-aminobutyric acid transporter with glucose-regulated expression in rat islets.

Pancreatic islets are unique outside the nervous system in that they contain high levels of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), synthesized by the enzyme glutamic acid decarboxylase (GAD). Since the role that GABA plays in the islet and the mechanisms whereby the two major GAD isoforms (GAD65 and GAD67) function as diabetes-associated autoantigens are unknown, continued characterization of the islet GAD-GABA system is important. We previously demonstrated that the GABA and glycine transporter vesicular inhibitory amino acid transporter (VIAAT also known as VGAT) is present in rat islets. Here we identify a novel 52 kDa variant of VIAAT in rat islets: VIAAT-52 (V52). V52 is an amino-terminally truncated form of VIAAT (V57) that likely results from utilization of a downstream start site of translation. V57 and V52 display different patterns of post-translational modification and cellular expression. Our results have indicated that islet content of V52, but not V57, is responsive to changes in glucose concentration and other extracellular conditions. VIAAT is expressed in the islet alpha cells, but there have been conflicting findings regarding the presence of VIAAT in the beta cells. Here we have also provided additional evidence for the presence of VIAAT in islet beta cells and show that the beta cell line INS-1 expresses V57. V52 may be better adapted than V57 to the unique rat alpha cell GAD-GABA system, which lacks GAD65 and in which VIAAT traffics to secretory granules rather than just to synaptic microvesicles.

Quantitative evaluation of a monoclonal antibody and its fragment as potential markers for pancreatic beta cell mass.

Antibodies, due to their high specificities and retention, represent potential beta cell imaging agents, however their slow clearance from the blood may preclude their use. Antibody fragments (Fabs) have much higher clearance and if they can be made with similar binding characteristics, would be more efficacious agents. An existing beta cell specific antibody (K14D10) and its Fab were evaluated with a previously developed screening assay. The Fab and the intact immunoglobulin (IgG) had similar affinities (6 - 20 nM), binding sites (300 000 - 700 000 sites/cell), and binding kinetics (t (1/2) = 8 - 18 minutes) for beta cells. However, the cellular specificity was far below the estimated requisite values needed to overcome the very low beta cell mass in the pancreas. The Fab cleared the blood twice as fast as the IgG, but did not preferentially accumulate into pancreas. Thus, generation of Fabs from IgGs with high beta cell binding and blood clearance appears feasible, but in order for molecules to be useful for tracking beta cell mass, antibodies of greater cellular specificity will have to be used.

Mutants of glucokinase cause hypoglycaemia- and hyperglycaemia syndromes and their analysis illuminates fundamental quantitative concepts of glucose homeostasis.

AIMS/HYPOTHESIS: Mutations of the glucokinase gene cause hyperglycaemia or hypoglycaemia. A quantitative understanding of these defects of glucose homeostasis linked to the glucokinase gene was lacking. Therefore a database of kinetic variables of wild-type and 20 missense mutants of glucokinase was developed and used in mathematical modelling to predict the thresholds for glucose-stimulated insulin release. METHODS: Recombinant human glucokinase was generated in E. coli. The k(cat), glucose S(0.5), ATP K(m), and Hill number of glucokinase were determined. Inhibition by Stearoyl CoA and glucokinase regulatory protein and thermal stability were assayed for all mutants kinetically similar to wild-type glucokinase. A mathematical model predicting the threshold for glucose-stimulated insulin release was constructed. This model is based on the two substrate kinetics of glucokinase and the kinetic variables of the database. It is assumed that both glucokinase gene alleles are equally expressed in beta-cells and that induction of glucokinase occurs as a function of basal blood glucose. RESULTS: Large changes, varying greatly between mutants were found in nearly all variables. Glucokinase flux at threshold for glucose-stimulated insulin release was about 25 % of total phosphorylating potential in the normal beta-cell and this was used to predict thresholds for the mutant heterozygotes. Clinical data for maturity onset diabetes of the young type linked to the glucokinase gene and familial hyperinsulinaemic hypoglycaemia linked to the glucokinase gene and the glucokinase kinetic data of this study were used to test the model. The model predicts fasting blood glucose between 3 and 7 mmol/l in these cases. CONCLUSION/INTERPRETATION: A kinetics database of wild-type and 20 mutants of glucokinase was developed. Many kinetic differences were found for the mutants. The mathematical model to calculate the threshold for glucose-stimulated insulin release predicts fasting blood glucose between 3 and 7 mmol/l in subjects with glucokinase gene mutations. [Diabetologia 42: 1175-1186]

Measurement and modeling of glucose-6-phosphatase in pancreatic islets.

In the beta-cells of the pancreas, glucose phosphorylation carried out by glucokinase is the rate-controlling step in glycolysis, and the kinetic characteristics of glucokinase govern to a high degree the dose-response relationship between glucose and insulin release. Because glucose-6-phosphatase (G-6-Pase) opposes the action of glucokinase, it may have a regulatory role in the release of insulin in response to glucose if the enzyme is present in the beta-cells. A number of researchers have reported finding high levels of G-6-Pase in islets, but quantitation of its activity remains controversial, mainly because of difficulties in solubilizing a particulate enzyme. Therefore a method developed to measure functional glucose phosphorylation activity in intact brain was applied (Chi, M. M.-Y., M. E. Pusateri, J. G. Carter, B. J. Norris, D. B. McDougal, Jr., and O. H. Lowry. Anal. Biochem. 161: 508-513, 1987), and the rates of accumulation and disappearance of 2-deoxyglucose 6-phosphate (DG-6-P) in freshly harvested islets were determined as a measure of glucose cycling. Islets were incubated in the presence of 30 mM 2-deoxyglucose (DG) for 60 min, and subsequently the incubation medium was replaced with medium containing no DG, but instead high levels of mannoheptulose as a blocker of phosphorylation. The content of DG-6-P in the islets was measured at strategic times during the protocol. As predicted by a mathematical model, DG-6-P accumulated in the presence of DG and decayed after its washout. Both of these results are consistent with islets containing dephosphorylation activity for this substrate. The kinetic curves were fit using a mathematical model, and the maximal G-6-Pase activity was estimated to be 0.13 +/- 0.005 micromol x g(-1) x min(-1). However, when the physiological effect of this amount of G-6-Pase activity was assessed by use of a model of glycolysis, it was found that the impact on glucose cycling and usage was insignificant. It was concluded that normal islets do contain measurable activity for dephosphorylating glucose 6-phosphate but that this enzymatic reaction does not play a role in glucose metabolism and sensing by the normal beta-cell.

Structural instability of mutant beta-cell glucokinase: implications for the molecular pathogenesis of maturity-onset diabetes of the young (type-2).

The catalytic function and thermal stability of wild-type and mutant recombinant human pancreatic beta-cell glucokinase was investigated. The mutants E70K and E300K, which are thought to be the cause of impaired insulin production by the pancreatic beta-cell and decreased glucose uptake by the liver of patients with maturity-onset diabetes of the young, were found to be functionally indistinguishable from the wild-type, i.e. their kcat.S0.5, inflection point and h were normal. However, these two mutants showed markedly reduced stability under a variety of test conditions. Glucokinase instability, not low enzyme catalytic activity, may be the cause of diabetes mellitus with E70K and E300K mutants.

Effect of a glucokinase inhibitor on energy production and insulin release in pancreatic islets.

Glucokinase has exclusively high control strength on glucose usage in the pancreatic beta-cell. However, glucokinase also has extraordinarily high control strength on insulin secretion, which is linked to the phosphate potential, [ATP]/([ADP][Pi]) (F.M. Matschinsky, Y.Liang, P. Kesavan, L. Wang, P. Froguel, G. Velho, D. Cohen, M.A. Permutt, Y. Tanizawa, T.L. Jetton, K. Niswender, and M.A. Magnuson. J. Clin. Invest. 92: 2092-2098, 1993). We propose that the ATP produced via the tricarboxylic acid cycle is approximately constant, irrespective of the glucose level. Furthermore, the component of ATP production that is derived from glycolysis and glycolytically derived NADH, which is shuttled into the mitochondria, is a critical signal controlling the ionic events leading to insulin secretion, as suggested previously (M. J. MacDonald. Diabetes 39: 1461-1466, 1990 and I.D. Dukes, M.S. McIntyre, R.J. Mertz, L.H. Philipson, M.W. Roe, B. Spencer, and J.F. Worley III. J. Biol. Chem. 269: 10979-10982, 1994). To test this hypothesis, glucose usage, oxidation, and insulin secretion were measured in cultured rat islets over a wide range of concentrations of glucose and mannoheptulose, an inhibitor of glucokinase. These data were fit to a mathematical model that predicts that glucokinase will govern the rate of glucose usage and ATP production and will also have a strong, but not complete, control over the rate of glucose oxidation, the phosphate potential, and insulin release. Mannoheptulose caused an inhibition of all three fluxes. The estimates of the mechanistic parameters of the model [maximal velocity (Vmax) and Michaelis constant for glucokinase, Vmax for hexokinase and glucose transport, and the inhibition constant of mannoheptulose to glucokinase] were similar to those obtained in vitro. Thus the data are consistent with a model in which the primary importance of glycolysis in transducing the glucose signal into changes of the phosphate potential imparts to glucokinase a high control strength on glucose-induced insulin secretion.

Effect of glucose on uptake of radiolabeled glucose, 2-DG, and 3-O-MG by the perfused rat liver.

In the transition from the fasting to the fed state, plasma glucose levels rise, and the liver converts from an organ producing glucose to one of storage. To determine the effect of glucose on hepatic glucose uptake, radiolabeled glucose, 2-deoxyglucose, and 3-O-methylglucose were injected into perfused rat livers during different nontracer glucose levels, and the concentrations in the outflow were measured. A mathematical model was developed that described the behavior of the injected compounds as they traveled through the liver and was used to simulate and fit the experimental results. The rates of membrane transport, glucokinase, glucose-6-phosphatase, and the consumption of glucose 6-phosphate were estimated. Membrane transport for all of the tracers decreased as nontracer glucose increased, demonstrating competitive inhibition of the glucose transporter. In contrast, the consumption of injected [2-14C]glucose increased when glucose was elevated, demonstrating that glucose caused an activation of enzyme activity that overcame the competitive inhibition of transport and phosphorylation. When glucose was elevated, the rate coefficient of glucokinase did not decrease, indicating that glucokinase was stimulated by glucose. Both changes would lead to the increased glycogen synthesis and decreased glucose production rate observed in vivo during the fasted-to-fed transition.

Mathematical model of beta-cell glucose metabolism and insulin release. I. Glucokinase as glucosensor hypothesis.

To quantitatively test the theory that glucokinase controls the rate of glucose metabolism and therefore the rate of insulin secretion, a minimal mathematical model of glycolysis in the pancreatic beta-cell was developed. The model represents our current hypothesis of how the normal beta-cell transduces the glucose signal. In this report, the model was used to address questions regarding the control strength of transport, hexokinase, glucose-6-phosphatase, and phosphofructokinase in the metabolism of glucose. The hypothesis that fructose 6-phosphate and a protein regulator modulate glucokinase activity was evaluated by simulation analysis, as was the possibility that glucose-6-phosphatase, working in concert with phosphofructokinase, can modulate the glucose-sensing system. It was found that, in the absence of glucose-6-phosphatase, transport, hexokinase, and phosphofructokinase do not greatly influence the rate of glucose metabolism unless their activities are dramatically altered from the measured values. Glucose metabolism was profoundly affected by the activity of glucokinase. However, in the presence of glucose-6-phosphatase, the ratio of glucose-6-phosphatase to phosphofructokinase activities was a very important parameter, and this potential control mechanism deserves more attention. The results further support the notion that glucokinase is indeed the glucosensor of the beta-cell and that modeling the system in toto provides quantitative evaluation needed to interpret the experimental tests of hypotheses.

Comprehensive model of transport and metabolism of adenosine and S-adenosylhomocysteine in the guinea pig heart.

Regulation of blood flow and mitochondrial respiration in the heart would be clarified by improved knowledge of interstitial concentrations and cellular production rates of adenosine; however, these variables cannot be measured directly. To interpret indexes that are available, a comprehensive mathematical model was developed, based on a large body of experimental data. The model describes most of the important pathways of capillary-tissue transport and cellular metabolism of adenosine in the guinea pig heart. It includes capillary flow, solute transport between tissue regions, nonlinear enzyme kinetics for adenosine kinase and adenosine deaminase, and reversible biunireactant kinetics for S-adenosylhomocysteine hydrolase in cardiomyocytes and endothelial cells, intracellular production of adenosine via AMP hydrolysis and transmethylation, and extracellular production of adenosine. A single set of parameter values for the model was obtained in the first stage of the analysis by taking certain values directly from published sources, other values were subject to specific constraints, and other values were determined by parameter optimization. The effects of flow and endothelial metabolism on the relation between interstitial and venous adenosine concentrations were determined. The relation between myocardial adenosine production rate and S-adenosylhomocysteine accumulation in the presence of excess homocysteine was estimated. In the second stage of the analysis, the model was used to investigate the mechanism of myocardial adenosine production, without changing the parameter values. Cellular adenosine production rates were estimated by fitting measurements of venous adenosine release obtained during altered energetic conditions in experiments by different investigators. The original results showed a dissociation between measurements of cytosolic AMP concentrations and venous adenosine release. It is concluded that 1) it is essential to account for the effect of flow on interstitial and venous adenosine concentrations, since decreased flow may produce effects outwardly resembling inhibition of the enzyme 5'-nucleotidase, 2) adenosine concentrations in epicardial transudate are not in equilibrium with interstitial fluid, and 3) the rate of cellular adenosine production increases monotonically with free cytosolic concentrations of AMP during a variety of alterations in energy balance of the guinea pig heart.

Insulin binding to individual rat skeletal muscles.

Studies of insulin binding to skeletal muscle, performed using sarcolemmal membrane preparations or whole muscle incubations of mixed muscle or typical red (soleus, psoas) or white [extensor digitorum longus (EDL), gastrocnemius] muscle, have suggested that red muscle binds more insulin than white muscle. We have evaluated this hypothesis using cryostat sections of unfixed tissue to measure insulin binding in a broad range of skeletal muscles; many were of similar fiber-type profiles. Insulin binding per square millimeter of skeletal muscle slice was measured by autoradiography and computer-assisted densitometry. We found a 4.5-fold range in specific insulin tracer binding, with heart and predominantly slow-twitch oxidative muscles (SO) at the high end and the predominantly fast-twitch glycolytic (FG) muscles at the low end of the range. This pattern reflects insulin sensitivity. Evaluation of displacement curves for insulin binding yielded linear Scatchard plots. The dissociation constants varied over a ninefold range (0.26-2.06 nM). Binding capacity varied from 12.2 to 82.7 fmol/mm2. Neither binding parameter was correlated with fiber type or insulin sensitivity; e.g., among three muscles of similar fiber-type profile, the EDL had high numbers of low-affinity binding sites, whereas the quadriceps had low numbers of high-affinity sites. In summary, considerable heterogeneity in insulin binding was found among hindlimb muscles of the rat, which can be attributed to heterogeneity in binding affinities and the numbers of binding sites. It can be concluded that a given fiber type is not uniquely associated with a set of insulin binding parameters that result in high or low binding.

Rapid reduction and return of surface insulin receptors after exposure to brief pulses of insulin in perifused rat hepatocytes.

Hepatic receptors are normally exposed to discrete pulses of insulin and glucagon at intervals of 8 to 16 min. Using a multicolumn system for perifusing hepatocytes, we investigated the effect of this pattern on the normal processing of the insulin receptor. Surface-receptor binding was measured in acid-washed cells harvested from individual columns. The number of high-affinity surface receptors fell to a nadir 1 min after the end of a 3-min square-wave pulse of insulin. The maximum reduction reached 45% of baseline at amplitudes of 1000 microU/ml or above. The number of surface binding sites returned to baseline 15 min after the end of the pulse, but the affinity constant of the high-affinity receptor was unchanged. The reduction of surface binding was dose dependent, with an ED50 of 251 +/- 34 microU/ml. Prolonging the pulse to 60 min did not affect the nadir or the rate of restoration of the surface-receptor population. The change in surface binding was reduced at 15 degrees C and abolished at 4 degrees C. After a pulse, the pattern of change was a period of rapid decline to a nadir (t1/2 less than or equal to 1 min) that persisted for 3-5 min, followed by restoration of surface binding that reached baseline in 10-15 min. This same pattern was present after six ED95 pulses delivered at intervals of 15 min. These data indicate that the internalization of hepatocyte surface receptors and their recycling and reinsertion into the plasma membrane can be entrained to pulses at the physiologic pulse frequency.

A kinetic analysis of hepatocyte responses to a glucagon pulse: mechanism and metabolic consequences of differences in response decay times.

Pulsatile administration of glucagon to perifused rat hepatocytes stimulates hepatocyte glucose production (HGP) more effectively than continuous administration. Having established that this effect was due to delayed relaxation of glucagon-stimulated HGP (t1/2 for decay = 3.54 +/- 0.60 min) we wished to examine the mechanism of response termination. Delayed dissociation of glucagon from its receptor was excluded by the brisk washout of [125I]glucagon from perifusion columns (t1/2 = 1.00 +/- 0.13) and the rapid decay in glucagon-stimulated cAMP released into the perifusion medium (t1/2 = 1.14 +/- 0.12). The relaxation of the HGP response to a pulse of administered cAMP was comparable to the decay in glucagon-stimulated HGP (t1/2 = 3.28 +/- 0.22). Furthermore, the phosphodiesterase inhibitor isobutyl-methylxanthine did not alter the decay of the HGP response to glucagon despite increasing the amplitude of the response (t1/2 = 3.04 +/- 0.36). These data place the rate-limiting step for HGP relaxation distal to cAMP generation and degradation. The decay of the beta-hydroxybutyrate response to a glucagon pulse was not different from the cAMP response (t1/2 = 1.14 +/- 0.23), whereas the decay of gluconeogenesis from lactate was not significantly different from HGP relaxation (t1/2 = 1.94 +/- 0.08). We conclude that rate-limiting events for HGP relaxation occur distal to the second messenger cascade; however, ketogenesis is more closely coupled to the kinetics of cAMP. These results may help to explain the absence of excessive ketosis during fasting in normal humans, who secrete glucagon episodically at 10- to 14-min intervals.