Vera Schultz

Mechanisms for Increased Glycolysis in the Hypertrophied Rat Heart

Glycolysis increases in hypertrophied hearts but the mechanisms are unknown. We studied the regulation of glycolysis in hearts with pressure-overload LV hypertrophy (LVH), a model that showed marked increases in the rates of glycolysis (by 2-fold) and insulin-independent glucose uptake (by 3-fold). Although the Vmax of the key glycolytic enzymes was unchanged in this model, concentrations of free ADP, free AMP, inorganic phosphate (Pi), and fructose-2,6-bisphosphate (F-2,6-P2), all activators of the rate-limiting enzyme phosphofructokinase (PFK), were increased (up to 10-fold). Concentrations of the inhibitors of PFK, ATP, citrate, and H+ were unaltered in LVH. Thus, our findings show that increased glucose entry and activation of the rate-limiting enzyme PFK both contribute to increased flux through the glycolytic pathway in hypertrophied hearts. Moreover, our results also suggest that these changes can be explained by increased intracellular free [ADP] and [AMP], due to decreased energy reserve in LVH, activating the AMP-activated protein kinase cascade. This, in turn, results in enhanced synthesis of F-2,6-P2 and increased sarcolemma localization of glucose transporters, leading to coordinated increases in glucose transport and activation of PFK.

Phosphofructokinase Isozymes in Pancreatic Islets and Clonal β-Cells (INS-1)

Normal insulin secretion is oscillatory in vivo, and the oscillations are impaired in type II diabetes. We and others have shown oscillations in insulin secretion from isolated perifused islets stimulated with glucose, and in this study we show oscillations in insulin secretion from the glucose-sensitive clonal β-cell line INS-1. We have proposed that the oscillatory insulin secretion may be caused by spontaneous oscillations of glycolysis and the ATP:ADP ratio in the β-cell, analogous to those seen in glycolyzing muscle extracts. The mechanism of the latter involves autocatalytic activation of the key regulatory enzyme, phosphofructokinase (PFK), by its product fructose 1,6-bisphosphate (F16BP). However, of the three PFK subunit isoforms (M-[muscle], L-[liver], and C-type, predominant in fibroblasts), only M-type is activated by micromolar F16BP at near-physiological conditions. We therefore studied PFK isoforms in the β-cell. Western analysis of PFK subunits in isolated rat islets and INS-1 cells showed the presence of M-type, as well as C-type and perhaps lesser amounts of L-type. Kinetic studies of PFK activity in INS-1 cell extracts showed strong activation by micromolar concentrations of F16BP at near-physiological concentrations of ATP (several millimolar) and AMP and fructose 6-phosphate (micromolar), indicative of the M-type isoform. Activation by submicromolar concentrations of fructose 2,6-bisphosphate (F26BP) and potent inhibition by citrate were also observed. The F16BP-stimulatable activity was about one-half of the F26BP-stimulatable activity. These experiments demonstrate that μ-cells contain the M-type isoform of PFK with the requisite regulatory properties for generating glycolytic oscillations that may be the basis of oscillatory insulin secretion.

Respiration in adipocytes is inhibited by reactive oxygen species.

It is a desirable goal to stimulate fuel oxidation in adipocytes and shift the balance toward less fuel storage and more burning. To understand this regulatory process, respiration was measured in primary rat adipocytes, mitochondria, and fat‐fed mice. Maximum O2 consumption, in vitro, was determined with a chemical uncoupler of oxidative phosphorylation (carbonylcyanide p‐trifluoromethoxyphenylhydrazone (FCCP)). The adenosine triphosphate/adenosine diphosphate (ATP/ADP) ratio was measured by luminescence. Mitochondria were localized by confocal microscopy with MitoTracker Green and their membrane potential (ΔψM) measured using tetramethylrhodamine ethyl ester perchlorate (TMRE). The effect of N‐acetylcysteine (NAC) on respiration and body composition in vivo was assessed in mice. Addition of FCCP collapsed ΔψM and decreased the ATP/ADP ratio. However, we demonstrated the same rate of adipocyte O2 consumption in the absence or presence of fuels and FCCP. Respiration was only stimulated when reactive oxygen species (ROS) were scavenged by pyruvate or NAC: other fuels or fuel combinations had little effect. Importantly, the ROS scavenging role of pyruvate was not affected by rotenone, an inhibitor of mitochondrial complex I. In addition, mice that consumed NAC exhibited increased O2 consumption and decreased body fat in vivo. These studies suggest for the first time that adipocyte O2 consumption may be inhibited by ROS, because pyruvate and NAC stimulated respiration. ROS inhibition of O2 consumption may explain the difficulty to identify effective strategies to increase fat burning in adipocytes. Stimulating fuel oxidation in adipocytes by decreasing ROS may provide a novel means to shift the balance from fuel storage to fuel burning.

Ca2+, NAD(P)H and membrane potential changes in pancreatic β-cells by methyl succinate: comparison with glucose

The present study was undertaken to determine the main metabolic secretory signals generated by the mitochondrial substrate MeS (methyl succinate) compared with glucose in mouse and rat islets and to understand the differences. Glycolysis and mitochondrial metabolism both have key roles in the stimulation of insulin secretion by glucose. Both fuels elicited comparable oscillatory patterns of Ca2+ and changes in plasma and mitochondrial membrane potential in rat islet cells and clonal pancreatic beta-cells (INS-1). Saturation of the Ca2+ signal occurred between 5 and 6 mM MeS, while secretion reached its maximum at 15 mM, suggesting operation of a K(ATP)-channel-independent pathway. Additional responses to MeS and glucose included elevated NAD(P)H autofluorescence in INS-1 cells and islets and increases in assayed NADH and NADPH and the ATP/ADP ratio. Increased NADPH and ATP/ADP ratios occurred more rapidly with MeS, although similar levels were reached after 5 min of exposure to each fuel, whereas NADH increased more with MeS than with glucose. Reversal of MeS-induced cell depolarization by Methylene Blue completely inhibited MeS-stimulated secretion, whereas basal secretion and KCl-induced changes in these parameters were not affected. MeS had no effect on secretion or signals in the mouse islets, in contrast with glucose, possibly due to a lack of malic enzyme. The data are consistent with the common intermediates being pyruvate, cytosolic NADPH or both, and suggest that cytosolic NADPH production could account for the more rapid onset of MeS-induced secretion compared with glucose stimulation.

Energy state of bovine cerebral microvessels: Comparison of isolation methods

Isolation procedures employed by various laboratories to obtain cerebral microvessels generally utilize meshes to sieve and collect the microvessels from homogenized brain. This is followed in some cases by further purification using density gradients of Percoll or sucrose, or albumin flotation. We have evaluated microvessels prepared by these methods in terms of ATP content and ATP/ADP ratio, which reflect the cellular energy state, and enrichment of the marker enzymes, alkaline phosphatase and gamma-glutamyltransferase. Albumin flotation generally increased the enrichment of marker enzymes; however, preparations using albumin flotation or a Percoll gradient exhibited considerable variability in ATP content and ATP/ADP ratio with the mean ATP/ADP ratio significantly lower than that observed in microvessels isolated by sieving through meshes. More uniformly high values for both ATP (approximately 1.6 nmole ATP/mg protein) and the ATP/ADP ratio (approximately 2.3) were obtained with meshes alone. Use of a sucrose gradient consistently resulted in preparations with a much lower ATP content and ATP/ADP ratio, compared with preparations obtained with the other methods. Values using the other methods were higher than those previously reported, yet were still lower than the ATP content of about 23 and ATP/ADP ratios of 18 and 7 we found in cultured microvascular endothelium and pericyte, respectively. These low values were not improved by supplying additional fuel to the microvessels during isolation, suggesting they were not the result of fuel deprivations during isolation. Despite the probable damage incurred during isolation, microvessel preparations are a useful in vitro model in which fuel metabolism appears to reflect the prior hormonal/nutritional state of donor animals. However, our data indicate the advisability of measurements of ATP content and ATP/ADP ratio for quality control of preparations used for metabolic studies, especially after Percoll density gradient or albumin flotation steps.

Chronic exposure to high glucose decreases myo-inositol in cultured cerebral microvascular pericytes but not in endothelium

SummaryIt has been proposed that the development of diabetic complications may involve a depletion of cellular myo-inositol due to an increase in polyol (sorbitol) formation. We therefore initially examined the effect of diabetes on levels of these metabolites in isolated cerebral microvessels. Compared with microvessels from control rats, microvessels from diabetic animals showed no detectable alteration in myo-inositol levels and a small increase in sorbitol content. To assess whether myo-inositol depletion might occur in only certain microvascular cells, cultured bovine cerebral microvascular pericytes and endothelium were grown for 3 or 18–20 days at 1.1, 5.5, or 22.2 mmol/l glucose. Increased medium glucose concentration resulted in increased sorbitol content in both cell types after both periods of incubation (p<0.05). In contrast, a significant decrease in myo-inositol content (22%, p<0.01) was observed only in pericytes grown for 18–20 days in the high glucose medium. Neither the adenosine 5′-triphosphate content nor the adenosine 5′-triphosphate/adenosine 5′-diphosphate (ATP/ADP) ratio of the pericytes was affected by the medium glucose concentration, indicating that the decrease in myo-inositol was not caused by a deficiency in the cellular energy state affecting the active transport of myo-inositol. These data suggest that myo-inositol depletion occurs selectively in the pericyte, a cell type known to be the site of early morphological changes in diabetes. Furthermore, the depletion apparently requires prolonged exposure to high glucose and is not due to a change in energy state.

Dihydroxyacetone-induced Oscillations in Cytoplasmic Free Ca2+ and the ATP/ADP Ratio in Pancreatic β-Cells at Substimulatory Glucose

Glucose stimulation of pancreatic β-cells causes oscillatory influx of Ca2+, leading to pulsatile insulin secretion. We have proposed that this is due to oscillations of glycolysis and the ATP/ADP ratio, which modulate the activity of ATP-sensitive K+ channels. We show here that dihydroxyacetone, a secretagogue that feeds into glycolysis below the putative oscillator phosphofructokinase, could cause a single initial peak in cytoplasmic free Ca2+ ([Ca2+]i) but did not by itself cause repeated oscillations in [Ca2+]i in mouse pancreatic β-cells. However, in the presence of a substimulatory concentration of glucose (4 mm), dihydroxyacetone induced [Ca2+]i oscillations. Furthermore, these oscillations correlated with oscillations in the ATP/ADP ratio, as seen previously with glucose stimulation. Insulin secretion in response to dihydroxyacetone was transient in the absence of glucose but was considerably enhanced and somewhat prolonged in the presence of a substimulatory concentration of glucose, in accordance with the enhanced [Ca2+]i response. These results are consistent with the hypothesized role of phosphofructokinase as the generator of the oscillations. Dihydroxyacetone may affect phosphofructokinase by raising the free concentration of fructose 1,6-bisphosphate to a critical level at which it activates the enzyme autocatalytically, thereby inducing the pulses of phosphofructokinase activity that cause the metabolic oscillations.

Nutrient Excess and AMPK Downregulation in Incubated Skeletal Muscle and Muscle of Glucose Infused Rats

We have previously shown that incubation for 1h with excess glucose or leucine causes insulin resistance in rat extensor digitorum longus (EDL) muscle by inhibiting AMP-activated protein kinase (AMPK). To examine the events that precede and follow these changes, studies were performed in rat EDL incubated with elevated levels of glucose or leucine for 30min-2h. Incubation in high glucose (25mM) or leucine (100μM) significantly diminished AMPK activity by 50% within 30min, with further decreases occurring at 1 and 2h. The initial decrease in activity at 30min coincided with a significant increase in muscle glycogen. The subsequent decreases at 1h were accompanied by phosphorylation of αAMPK at Ser485/491, and at 2h by decreased SIRT1 expression and increased PP2A activity, all of which have previously been shown to diminish AMPK activity. Glucose infusion in vivo, which caused several fold increases in plasma glucose and insulin, produced similar changes but with different timing. Thus, the initial decrease in AMPK activity observed at 3h was associated with changes in Ser485/491 phosphorylation and SIRT1 expression and increased PP2A activity was a later event. These findings suggest that both ex vivo and in vivo, multiple factors contribute to fuel-induced decreases in AMPK activity in skeletal muscle and the insulin resistance that accompanies it.