Different expression and genetic control for the K99 antigen and its associated adhesin

Cultivation of K99⁺ wild strains and transconjugants in MINCA medium with variable concentrations of glucose, glycerol, alanine, or protein-interfering antibiotics shows that the K99 antigen and its associated adhesin are expressed in different ways. The addition of cAMP to medium containing a K99-repressive concentration of glucose demonstrates that the K99-antigen production is controlled by the cAMP-CRP complex, but this does not occur with the adhesin production. Sub-inhibitory concentrations of protein-interfering antibiotics inhibit the K99-antigen production, but do not correlatively affect the adhesin production. All these experiments suggest that the K99 antigen and the adhesin are different structures whose genes are controlled by different mechanisms.     

Energy on demand: a mechanism for astrocyte–neuron metabolic coupling

A tight link exists between neuronal activity and energy metabolism. This relationship was first proposed by Roy and Sherrington who suggested that brain possesses intrinsic mechanisms to regulate the availability of energy substrates in register with local variations of functional activity. This concept was later confirmed by Sokoloff and colleagues who demonstrated that increased neuronal activity led to increased glucose utilization in almost any areas of the brain tested. Despite wide acceptance of this concept, the cellular and molecular mechanisms that underlie this close relationship between neuronal activity and energy metabolism have remained largely unknown. The extensive analysis carried out by our group will be discussed. Astrocytes appear to be the key cells that operate the coupling between synaptic activity and glucose utilization. Indeed both in vitro and in vivo evidences indicate that astrocytes can detect synaptically released glutamate through sodium‐coupled uptake operated by glutamate transporters. Disruption of sodium homeostasis activates the energy‐demanding Na‐K‐ATPase which promotes glucose uptake and lactate production. Relevance of these findings to functional brain imaging will be discussed.     

Glucose stimulation of hypothalamic MCH neurons involves K(ATP) channels, is modulated by UCP2, and regulates peripheral glucose homeostasis

Blood glucose levels are tightly controlled, a process thought to be orchestrated primarily by peripheral mechanisms (insulin secretion by β cells, and insulin action on muscle, fat, and liver). The brain also plays an important, albeit less well-defined role. Subsets of neurons in the brain are excited by glucose; in many cases this involves ATP-mediated closure of K(ATP) channels. To understand the relevance of this, we are manipulating glucose sensing within glucose-excited neurons. In the present study, we demonstrate that glucose excitation of MCH-expressing neurons in the lateral hypothalamus is mediated by K(ATP) channels and is negatively regulated by UCP2 (a mitochondrial protein that reduces ATP production), and that glucose sensing by MCH neurons plays an important role in regulating glucose homeostasis. Combined, the glucose-excited neurons are likely to play key, previously unexpected roles in regulating blood glucose.     

Acetylation targets the M2 isoform of pyruvate kinase for degradation through chaperone-mediated autophagy and promotes tumor growth

Most tumor cells take up more glucose than normal cells but metabolize glucose via glycolysis even in the presence of normal levels of oxygen, a phenomenon known as the Warburg effect. Tumor cells commonly express the embryonic M2 isoform of pyruvate kinase (PKM2) that may contribute to the metabolism shift from oxidative phosphorylation to aerobic glycolysis and tumorigenesis. Here we show that PKM2 is acetylated on lysine 305 and that this acetylation is stimulated by high glucose concentration. PKM2 K305 acetylation decreases PKM2 enzyme activity and promotes its lysosomal-dependent degradation via chaperone-mediated autophagy (CMA). Acetylation increases PKM2 interaction with HSC70, a chaperone for CMA, and association with lysosomes. Ectopic expression of an acetylation mimetic K305Q mutant accumulates glycolytic intermediates and promotes cell proliferation and tumor growth. These results reveal an acetylation regulation of pyruvate kinase and the link between lysine acetylation and CMA.     

Glucose recruits K(ATP) channels via non-insulin-containing dense-core granules

beta cells rely on adenosine triphosphate-sensitive potassium (K(ATP)) channels to initiate and end glucose-stimulated insulin secretion through changes in membrane potential. These channels may also act as a constituent of the exocytotic machinery to mediate insulin release independent of their electrical function. However, the molecular mechanisms whereby the beta cell plasma membrane maintains an appropriate number of K(ATP) channels are not known. We now show that glucose increases K(ATP) current amplitude by increasing the number of K(ATP) channels in the beta cell plasma membrane. The effect was blocked by inhibition of protein kinase A (PKA) as well as by depletion of extracellular or intracellular Ca(2+). Furthermore, glucose promoted recruitment of the potassium inward rectifier 6.2 to the plasma membrane, and intracellular K(ATP) channels localized in chromogranin-positive/insulin-negative dense-core granules. Our data suggest that glucose can recruit K(ATP) channels to the beta cell plasma membrane via non-insulin-containing dense-core granules in a Ca(2+)- and PKA-dependent manner.     

5-amino-imidazole carboxamide riboside acutely potentiates glucose-stimulated insulin secretion from mouse pancreatic islets by KATP channel-dependent and -independent pathways

AMP-activated protein kinase (AMPK) is an important signaling effector that couples cellular metabolism and function. The effects of AMPK activation on pancreatic beta-cell function remain unresolved. We used 5-amino-imidazole carboxamide riboside (AICAR), an activator of AMPK, to define the signaling mechanisms linking the activation of AMPK with insulin secretion. Application of 300 microM AICAR to mouse islets incubated in 5-14 mM glucose significantly increased AMPK activity and potentiated insulin secretion. AICAR inhibited ATP-sensitive K(+) (K(ATP)) channels and increased the frequency of glucose-induced calcium oscillations in islets incubated in 8-14 mM glucose. At lower glucose concentration (5mM) AICAR did not affect K(ATP) activity or intracellular ([Ca(2+)](i)). AICAR also did not inhibit (86)Rb(+) efflux from islets isolated from Sur1(-/-) mice that lack K(ATP) channels yet significantly potentiated glucose stimulated insulin secretion. Our data suggest that AICAR stimulates insulin secretion by both K(ATP) channel-dependent and -independent pathways.     

Mechanisms of the free fatty acid-induced increase in hepatic glucose production

The associations between obesity, insulin resistance, and type 2 diabetes mellitus are well documented. Free fatty acids (FFA), which are often elevated in obesity, have been implicated as an important link in these associations. Contrary to muscle glucose metabolism, the effects of FFA on hepatic glucose metabolism and the associated mechanisms have not been extensively investigated. It is still controversial whether FFA have substantial effects on hepatic glucose production, and the mechanisms responsible for these putative effects remain unknown. We review recent progress in this area and try to clarify controversial issues regarding the mechanisms responsible for the FFA-induced increase in hepatic glucose production in the postabsorptive state and during hyperinsulinemia.     

Extracellular links in Kir subunits control the unitary conductance of SUR/Kir6.0 ion channels.

Potassium (K+) channels are highly selective for K+ ions but their unitary conductances are quite divergent. Although Kir6.1 and Kir6.2 are highly homologous and both form functional K+ channels with sulfonylurea receptors, their unitary conductances measured with 150 mM extracellular K+ are approximately 35 and 80 pS, respectively. We found that a chain of three amino acid residues N123-V124-R125 of Kir6.1 and S113-I114-H115 of Kir6.2 in the M1-H5 extracellular link and single residues M148 of Kir6.1 and V138 of Kir6.2 in the H5-M2 link accounted for the difference. By using a 3D structure model of Kir6.2, we were able to recognize two independent plausible mechanisms involved in the determination of single channel conductance of the Kir6.0 subunits: (i) steric effects at Kir6.2V138 or Kir6.1M148 in the H5-M2 link influence directly the diffusion of K+ ions; and (ii) structural constraints between Kir6.2S113 or Kir6. 1N123 in the M1-H5 link and Kir6.2R136 or Kir6.1R146 near the H5 region control the conformation of the permeation pathway. These mechanisms represent a novel and possibly general aspect of the control of ion channel permeability.     

Protein kinase C-dependent phosphorylation and mitochondrial translocation of aldose reductase

Although aldose reductase (AR) is a critical participant in osmoregulation, and the metabolism of glucose and aldehydes derived from lipid peroxidation, post-translational mechanisms regulating its activity have not been identified. In this paper, we report that stimulation of protein kinase C (PKC) in several cell types induces phosphorylation of AR and translocation of the phosphorylated protein to the mitochondria. In vitro, recombinant AR was directly phosphorylated by activated PKC, suggesting that AR may be an in vivo PKC substrate. Together, these observations reveal a novel link between PKC activation and the regulation of glucose and aldehyde metabolism.     

Contribution to glucose tolerance of insulin-independent vs. insulin-dependent mechanisms in mice

To study the contributions of insulin-dependent vs. insulin-independent mechanisms to intravenous glucose tolerance (K(G)), 475 experiments in mice were performed. An intravenous glucose bolus was given either alone or with exogenous insulin or with substances modulating insulin secretion and sensitivity. Seven samples were taken over 50 min. Insulin [suprabasal area under the curve (DeltaAUC(ins))] ranged from 0 to 100 mU. ml(-1). 50 min. After validation against the euglycemic hyperinsulinemic clamp, the minimal model of net glucose disappearance was exploited to analyze glucose and insulin concentrations to measure the action of glucose per se independent of dynamic insulin (S(G)) and the combined effect of insulin sensitivity (S(I)) and secretion. Sensitivity analysis showed that insulin [through disposition index (DI)] contributed to glucose tolerance by 29 +/- 4% in normal conditions. In conditions of elevated hyperinsulinemia, contribution by insulin increased on average to 69%. K(G) correlated with DI but was saturated for DeltaAUC(ins) above 15 mU. ml(-1). 50 min. Insulin sensitivity related to DeltaAUC(ins) in a hyperbolic manner, whereas S(G) did not correlate with the insulin peak in the physiological range. Thus glucose tolerance in vivo is largely mediated by mechanisms unrelated to dynamic insulin and saturates with high insulin.     

Ca2+ influx does not trigger glucose-induced traffic of the insulin granules and alteration of their distribution

This study investigated mechanisms by which glucose increases readily releasable secretory granules via acting on preexocytotic steps, i.e., intracellular granule movement and granule access to the plasma membrane using a pancreatic beta-cell line, MIN6. Glucose-induced activation of the movement occurred at a substimulatory concentration with regard to insulin output. Glucose activation of the movement was inhibited by pretreatment with thapsigargin plus acetylcholine to suppress intracellular Ca2+ mobilization. Inhibitors of calmodulin and myosin light chain kinase also suppressed glucose activation of the movement. Simultaneous addition of glucose with Ca2+ channel blockers or the ATP-sensitive K+ channel opener diazoxide failed to suppress the traffic activation, and addition of these substances on top of glucose stimulation resulted in a further increase. Although stimulatory glucose had minimal changes in the intracellular granule distribution, inhibition of Ca2+ influx revealed increases by glucose of the granules in the cell periphery. In contrast, high K+ depolarization decreased the peripheral granules. Glucose-induced granule margination was abolished when the protein kinase C activity was downregulated. These findings indicate that preexocytotic control of insulin release is regulated by distinct mechanisms from Ca2+ influx, which triggers insulin exocytosis. The nature of the regulation by glucose may explain a part of potentiating effects of the hexose independent of the closure of the ATP-sensitive K+ channel.     

Transformation of postingestive glucose responses after deletion of sweet taste receptor subunits or gastric bypass surgery

The glucose-dependent secretion of the insulinotropic hormone glucagon-like peptide-1 (GLP-1) is a critical step in the regulation of glucose homeostasis. Two molecular mechanisms have separately been suggested as the primary mediator of intestinal glucose-stimulated GLP-1 secretion (GSGS): one is a metabotropic mechanism requiring the sweet taste receptor type 2 (T1R2) + type 3 (T1R3) while the second is a metabolic mechanism requiring ATP-sensitive K(+) (K(ATP)) channels. By quantifying sugar-stimulated hormone secretion in receptor knockout mice and in rats receiving Roux-en-Y gastric bypass (RYGB), we found that both of these mechanisms contribute to GSGS; however, the mechanisms exhibit different selectivity, regulation, and localization. T1R3(-/-) mice showed impaired glucose and insulin homeostasis during an oral glucose challenge as well as slowed insulin granule exocytosis from isolated pancreatic islets. Glucose, fructose, and sucralose evoked GLP-1 secretion from T1R3(+/+), but not T1R3(-/-), ileum explants; this secretion was not mimicked by the K(ATP) channel blocker glibenclamide. T1R2(-/-) mice showed normal glycemic control and partial small intestine GSGS, suggesting that T1R3 can mediate GSGS without T1R2. Robust GSGS that was K(ATP) channel-dependent and glucose-specific emerged in the large intestine of T1R3(-/-) mice and RYGB rats in association with elevated fecal carbohydrate throughout the distal gut. Our results demonstrate that the small and large intestines utilize distinct mechanisms for GSGS and suggest novel large intestine targets that could mimic the improved glycemic control seen after RYGB.     

Modeling the Pancreatic α-Cell: Dual Mechanisms of Glucose Suppression of Glucagon Secretion

The mechanism by which glucose induces insulin secretion in β-cells is fairly well understood. Despite years of research, however, the mechanism of glucagon secretion in α-cells is still not well established. It has been proposed that glucose regulates glucagon secretion by decreasing the conductance of either outward ATP-dependent potassium channels (K(ATP)) or an inward store-operated current (SOC). We have developed a mathematical model based on mouse data to test these hypotheses and found that both mechanisms are possible. Glucose metabolism closes K(ATP) channels, which depolarizes the cell but paradoxically reduces calcium influx by inactivating voltage-dependent calcium and sodium channels and decreases secretion. Glucose metabolism also activates SERCA pumps, which fills the endoplasmic reticulum and hyperpolarizes the cells by reducing the inward current through SOC channels and again suppresses glucagon secretion. We find further that the two mechanisms can combine to account for the nonmonotonic dependence of secretion on glucose observed in some studies, an effect that cannot be obtained with either mechanism alone.     

Nutrient-secretion coupling in the pancreatic islet beta-cell: recent advances

Insulin secretion from pancreatic islet beta-cells is a tightly regulated process, under the close control of blood glucose concentrations, and several hormones and neurotransmitters. Defects in glucose-triggered insulin secretion are ultimately responsible for the development of type II diabetes, a condition in which the total beta-cell mass is essentially unaltered, but beta-cells become progressively "glucose blind" and unable to meet the enhanced demand for insulin resulting for peripheral insulin resistance. At present, the mechanisms by which glucose (and other nutrients including certain amino acids) trigger insulin secretion in healthy individuals are understood only in part. It is clear, however, that the metabolism of nutrients, and the generation of intracellular signalling molecules including the products of mitochondrial metabolism, probably play a central role. Closure of ATP-sensitive K+(K(ATP)) channels in the plasma membrane, cell depolarisation, and influx of intracellular Ca2+, then prompt the "first phase" on insulin release. However, recent data indicate that glucose also enhances insulin secretion through mechanisms which do not involve a change in K(ATP) channel activity, and seem likely to underlie the second, sustained phase of glucose-stimulated insulin secretion. In this review, I will discuss recent advances in our understanding of each of these signalling processes.     

Signaling through the Phosphatidylinositol 3-Kinase (PI3K)/Mammalian Target of Rapamycin (mTOR) Axis Is Responsible for Aerobic Glycolysis mediated by Glucose Transporter in Epidermal Growth Factor Receptor (EGFR)-mutated Lung Adenocarcinoma

Oncogenic epidermal growth factor receptor (EGFR) signaling plays an important role in regulating global metabolic pathways, including aerobic glycolysis, the pentose phosphate pathway (PPP), and pyrimidine biosynthesis. However, the molecular mechanism by which EGFR signaling regulates cancer cell metabolism is still unclear. To elucidate how EGFR signaling is linked to metabolic activity, we investigated the involvement of the RAS/MEK/ERK and PI3K/AKT/mammalian target of rapamycin (mTOR) pathways on metabolic alteration in lung adenocarcinoma (LAD) cell lines with activating EGFR mutations. Although MEK inhibition did not alter lactate production and the extracellular acidification rate, PI3K/mTOR inhibitors significantly suppressed glycolysis in EGFR-mutant LAD cells. Moreover, a comprehensive metabolomics analysis revealed that the levels of glucose 6-phosphate and 6-phosphogluconate as early metabolites in glycolysis and PPP were decreased after inhibition of the PI3K/AKT/mTOR pathway, suggesting a link between PI3K signaling and the proper function of glucose transporters or hexokinases in glycolysis. Indeed, PI3K/mTOR inhibition effectively suppressed membrane localization of facilitative glucose transporter 1 (GLUT1), which, instead, accumulated in the cytoplasm. Finally, aerobic glycolysis and cell proliferation were down-regulated when GLUT1 gene expression was suppressed by RNAi. Taken together, these results suggest that PI3K/AKT/mTOR signaling is indispensable for the regulation of aerobic glycolysis in EGFR-mutated LAD cells.     

Programmed autophagy in the Drosophila fat body is induced by ecdysone through regulation of the PI3K pathway

Eukaryotic cells catabolize their own cytoplasm by autophagy in response to amino acid starvation and inductive signals during programmed tissue remodeling and cell death. The Tor and PI3K signaling pathways have been shown to negatively control autophagy in eukaryotes, but the mechanisms that link these effectors to overall animal development and nutritional status in multicellular organisms remain poorly understood. Here, we reveal a complex regulation of programmed and starvation-induced autophagy in the Drosophila fat body. Gain-of-function genetic analysis indicated that ecdysone receptor signaling induces programmed autophagy whereas PI3K signaling represses programmed autophagy. Genetic interaction studies showed that ecdysone signaling downregulates PI3K signaling and that this represents the effector mechanism for induction of programmed autophagy. Hence, these studies link hormonal induction of autophagy to the regulatory function of the PI3K signaling pathway in vivo.     

The death effector domain-containing DEDD forms a complex with Akt and Hsp90, and supports their stability

Insulin secretion and glucose transport are the major mechanisms to balance glucose homeostasis. Recently, we found that the death effector domain-containing DEDD inhibits cyclin-dependent kinase-1 (Cdk1) function, thereby preventing Cdk1-dependent inhibitory phosphorylation of S6 kinase-1 (S6K1), downstream of phosphatidylinositol 3-kinase (PI3K), which overall results in maintenance of S6K1 activity. Here we newly show that DEDD forms a complex with Akt and heat-shock protein 90 (Hsp90), and supports the stability of both proteins. Hence, in DEDD^−/− mice, Akt protein levels are diminished in skeletal muscles and adipose tissues, which interferes with the translocation of glucose-transporter 4 (GLUT4) upon insulin stimulation, leading to inefficient incorporation of glucose in these organs. Interestingly, as for the activation of S6K1, suppression of Cdk1 is involved in the stabilization of Akt protein by DEDD, since diminishment of Cdk1 in DEDD^−/− cells via siRNA expression or treatment with a Cdk1-inhibitor, increases both Akt and Hsp90 protein levels. Such multifaceted involvement of DEDD in glucose homeostasis by supporting both insulin secretion (via maintenance of S6K1 activity) and glucose uptake (via stabilizing Akt protein), may suggest an association of DEDD-deficiency with the pathogenesis of type 2 diabetes mellitus.     

Mathematical Modeling of Interacting Glucose-Sensing Mechanisms and Electrical Activity Underlying Glucagon-Like Peptide 1 Secretion

Intestinal L-cells sense glucose and other nutrients, and in response release glucagon-like peptide 1 (GLP-1), peptide YY and other hormones with anti-diabetic and weight-reducing effects. The stimulus-secretion pathway in L-cells is still poorly understood, although it is known that GLP-1 secreting cells use sodium-glucose co-transporters (SGLT) and ATP-sensitive K⁺-channels (K(ATP)-channels) to sense intestinal glucose levels. Electrical activity then transduces glucose sensing to Ca²⁺-stimulated exocytosis. This particular glucose-sensing arrangement with glucose triggering both a depolarizing SGLT current as well as leading to closure of the hyperpolarizing K(ATP) current is of more general interest for our understanding of glucose-sensing cells. To dissect the interactions of these two glucose-sensing mechanisms, we build a mathematical model of electrical activity underlying GLP-1 secretion. Two sets of model parameters are presented: one set represents primary mouse colonic L-cells; the other set is based on data from the GLP-1 secreting GLUTag cell line. The model is then used to obtain insight into the differences in glucose-sensing between primary L-cells and GLUTag cells. Our results illuminate how the two glucose-sensing mechanisms interact, and suggest that the depolarizing effect of SGLT currents is modulated by K(ATP)-channel activity. Based on our simulations, we propose that primary L-cells encode the glucose signal as changes in action potential amplitude, whereas GLUTag cells rely mainly on frequency modulation. The model should be useful for further basic, pharmacological and theoretical investigations of the cellular signals underlying endogenous GLP-1 and peptide YY release.     

High levels of glucose induce “metabolic memory” in cardiomyocyte via epigenetic histone H3 lysine 9 methylation

Diabetic patients continue to develop inflammation and cardiovascular complication even after achieving glycemic control, suggesting a "metabolic memory". Metabolic memory is a major challenge in the treatment of diabetic complication, and the mechanisms underlying metabolic memory are not clear. Recent studies suggest a link between chromatin histone methylation and metabolic memory. In this study, we tested whether histone 3 lysine-9 tri-methylation (H3K9me3), a key epigenetic chromatin marker, was involved in high glucose (HG)-induced inflammation and metabolic memory. Incubating cardiomyocyte cells in HG resulted in increased levels of inflammatory cytokine IL-6 mRNA when compared with myocytes incubated in normal culture media, whereas mannitol (osmotic control) has no effect. Chromatin immunoprecipitation (ChIP) assays showed that H3K9me3 levels were significantly decreased at the promoters of IL-6. Immunoblotting demonstrated that protein levels of the H3K9me3 methyltransferase, Suv39h1, were also reduced after HG treatment. HG-induced apoptosis, mitochondrial dysfunction and cytochrome-c release were reversible. However, the effects of HG on the expression of IL-6 and the levels of H3K9me3 were irreversible after the removal of HG from the culture. These results suggest that HG-induced sustained inflammatory phenotype and epigenetic histone modification, rather than HG-induced mitochondrial dysfunction and apoptosis, are main mechanisms responsible for metabolic memory. In conclusion, our data demonstrate that HG increases expression of inflammatory cytokine and decreases the levels of histone-3 methylation at the cytokine promoter, and suggest that modulating histone 3 methylation and inflammatory cytokine expression may be a useful strategy to prevent metabolic memory and cardiomyopathy in diabetic patients.