Allosteric modulation of ß-cell M3 muscarinic acetylcholine receptors greatly improves glucose homeostasis in lean and obese mice.

Given the global epidemic in type 2 diabetes, novel antidiabetic drugs with increased efficacy and reduced side effects are urgently needed. Previous work has shown that M3 muscarinic acetylcholine (ACh) receptors (M3Rs) expressed by pancreatic ß cells play key roles in stimulating insulin secretion and maintaining physiological blood glucose levels. In the present study, we tested the hypothesis that a positive allosteric modulator (PAM) of M3R function can improve glucose homeostasis in mice by promoting insulin release. One major advantage of this approach is that allosteric agents respect the ACh-dependent spatiotemporal control of M3R activity. In this study, we first demonstrated that VU0119498, a drug known to act as a PAM at M3Rs, significantly augmented ACh-induced insulin release from cultured ß cells and mouse and human pancreatic islets. This stimulatory effect was absent in islets prepared from mice lacking M3Rs, indicative of the involvement of M3Rs. VU0119498 treatment of wild-type mice caused a significant increase in plasma insulin levels, accompanied by a striking improvement in glucose tolerance. These effects were mediated by ß-cell M3Rs, since they were absent in mutant mice selectively lacking M3Rs in ß cells. Moreover, acute VU0119498 treatment of obese, glucose-intolerant mice triggered enhanced insulin release and restored normal glucose tolerance. Interestingly, doses of VU0119498 that led to pronounced improvements in glucose homeostasis did not cause any significant side effects due to activation of M3Rs expressed by other peripheral cell types. Taken together, the data from this proof-of-concept study strongly suggest that M3R PAMs may become clinically useful as novel antidiabetic agents.

Integration of Eukaryotic Energy Metabolism: The Intramitochondrial and Cytosolic Energy States ([ATP]f/[ADP]f[Pi]).

Maintaining a robust, stable source of energy for doing chemical and physical work is essential to all living organisms. In eukaryotes, metabolic energy (ATP) production and consumption occurs in two separate compartments, the mitochondrial matrix and the cytosol. As a result, understanding eukaryotic metabolism requires knowledge of energy metabolism in each compartment and how metabolism in the two compartments is coordinated. Central to energy metabolism is the adenylate energy state ([ATP]/[ADP][Pi]). ATP is synthesized by oxidative phosphorylation (mitochondrial matrix) and glycolysis (cytosol) and each compartment provides the energy to do physical work and to drive energetically unfavorable chemical syntheses. The energy state in the cytoplasmic compartment has been established by analysis of near equilibrium metabolic reactions localized in that compartment. In the present paper, analysis is presented for energy-dependent reactions localized in the mitochondrial matrix using data obtained from both isolated mitochondria and intact tissues. It is concluded that the energy state ([ATP]f/[ADP]f[Pi]) in the mitochondrial matrix, calculated from the free (unbound) concentrations, is not different from the energy state in the cytoplasm. Corollaries are: (1) ADP in both the cytosol and matrix is selectively bound and the free concentrations are much lower than the total measured concentrations; and (2) under physiological conditions, the adenylate energy states in the mitochondrial matrix and cytoplasm are not substantially different.

CK2 acts as a potent negative regulator of receptor-mediated insulin release in vitro and in vivo.

G protein-coupled receptors (GPCRs) regulate virtually all physiological functions including the release of insulin from pancreatic ß-cells. ß-Cell M3 muscarinic receptors (M3Rs) are known to play an essential role in facilitating insulin release and maintaining proper whole-body glucose homeostasis. As is the case with other GPCRs, M3R activity is regulated by phosphorylation by various kinases, including GPCR kinases and casein kinase 2 (CK2). At present, it remains unknown which of these various kinases are physiologically relevant for the regulation of ß-cell activity. In the present study, we demonstrate that inhibition of CK2 in pancreatic ß-cells, knockdown of CK2α expression, or genetic deletion of CK2α in ß-cells of mutant mice selectively augmented M3R-stimulated insulin release in vitro and in vivo. In vitro studies showed that this effect was associated with an M3R-mediated increase in intracellular calcium levels. Treatment of mouse pancreatic islets with CX4945, a highly selective CK2 inhibitor, greatly reduced agonist-induced phosphorylation of ß-cell M3Rs, indicative of CK2-mediated M3R phosphorylation. We also showed that inhibition of CK2 greatly enhanced M3R-stimulated insulin secretion in human islets. Finally, CX4945 treatment protected mice against diet-induced hyperglycemia and glucose intolerance in an M3R-dependent fashion. Our data demonstrate, for the first time to our knowledge, the physiological relevance of CK2 phosphorylation of a GPCR and suggest the novel concept that kinases acting on ß-cell GPCRs may represent novel therapeutic targets.

Tryptophan Fluorescence Yields and Lifetimes as a Probe of Conformational Changes in Human Glucokinase.

Five variants of glucokinase (ATP-D-hexose-6-phosphotransferase, EC 2.7.1.1) including wild type and single Trp mutants with the Trp residue at positions 65, 99, 167 and 257 were prepared. The fluorescence of Trp in all locations studied showed intensity changes when glucose bound, indicating that conformational change occurs globally over the entire protein. While the fluorescence quantum yield changes upon glucose binding, the enzyme's absorption spectra, emission spectra and fluorescence lifetimes change very little. These results are consistent with the existence of a dark complex for excited state Trp. Addition of glycerol, L-glucose, sucrose, or trehalose increases the binding affinity of glucose to the enzyme and increases fluorescence intensity. The effect of these osmolytes is thought to shift the protein conformation to a condensed, high affinity form. Based upon these results, we consider the nature of quenching of the Trp excited state. Amide groups are known to quench indole fluorescence and amides of the polypeptide chain make interact with excited state Trp in the relatively unstructured, glucose-free enzyme. Also, removal of water around the aromatic ring by addition of glucose substrate or osmolyte may reduce the quenching.

Analysis of the co-operative interaction between the allosterically regulated proteins GK and GKRP using tryptophan fluorescence.

Hepatic glucose phosphorylation by GK (glucokinase) is regulated by GKRP (GK regulatory protein). GKRP forms a cytosolic complex with GK followed by nuclear import and storage, leading to inhibition of GK activity. This process is initiated by low glucose, but reversed nutritionally by high glucose and fructose or pharmacologically by GKAs (GK activators) and GKRPIs (GKRP inhibitors). To study the regulation of this process by glucose, fructose-phosphate esters and a GKA, we measured the TF (tryptophan fluorescence) of human WT (wild-type) and GKRP-P446L (a mutation associated with high serum triacylglycerol) in the presence of non-fluorescent GK with its tryptophan residues mutated. Titration of GKRP-WT by GK resulted in a sigmoidal increase in TF, suggesting co-operative PPIs (protein-protein interactions) perhaps due to the hysteretic nature of GK. The affinity of GK for GKRP was decreased and binding co-operativity increased by glucose, fructose 1-phosphate and GKA, reflecting disruption of the GK-GKRP complex. Similar studies with GKRP-P446L showed significantly different results compared with GKRP-WT, suggesting impairment of complex formation and nuclear storage. The results of the present TF-based biophysical analysis of PPIs between GK and GKRP suggest that hepatic glucose metabolism is regulated by a metabolite-sensitive drug-responsive co-operative molecular switch, involving complex formation between these two allosterically regulated proteins.

Regulation of glucagon secretion in normal and diabetic human islets by γ-hydroxybutyrate and glycine.

Paracrine signaling between pancreatic islet ß-cells and α-cells has been proposed to play a role in regulating glucagon responses to elevated glucose and hypoglycemia. To examine this possibility in human islets, we used a metabolomic approach to trace the responses of amino acids and other potential neurotransmitters to stimulation with [U-(13)C]glucose in both normal individuals and type 2 diabetics. Islets from type 2 diabetics uniformly showed decreased glucose stimulation of insulin secretion and respiratory rate but demonstrated two different patterns of glucagon responses to glucose: one group responded normally to suppression of glucagon by glucose, but the second group was non-responsive. The non-responsive group showed evidence of suppressed islet GABA levels and of GABA shunt activity. In further studies with normal human islets, we found that γ-hydroxybutyrate (GHB), a potent inhibitory neurotransmitter, is generated in ß-cells by an extension of the GABA shunt during glucose stimulation and interacts with α-cell GHB receptors, thus mediating the suppressive effect of glucose on glucagon release. We also identified glycine, acting via α-cell glycine receptors, as the predominant amino acid stimulator of glucagon release. The results suggest that glycine and GHB provide a counterbalancing receptor-based mechanism for controlling α-cell secretory responses to metabolic fuels.

GCK-MODY diabetes associated with protein misfolding, cellular self-association and degradation.

GCK-MODY, dominantly inherited mild fasting hyperglycemia, has been associated with >600 different mutations in the glucokinase (GK)-encoding gene (GCK). When expressed as recombinant pancreatic proteins, some mutations result in enzymes with normal/near-normal catalytic properties. The molecular mechanism(s) of GCK-MODY due to these mutations has remained elusive. Here, we aimed to explore the molecular mechanisms for two such catalytically 'normal' GCK mutations (S263P and G264S) in the F260-L270 loop of GK. When stably overexpressed in HEK293 cells and MIN6 ß-cells, the S263P- and G264S-encoded mutations generated misfolded proteins with an increased rate of degradation (S263P>G264S) by the protein quality control machinery, and a propensity to self-associate (G264S>S263P) and form dimers (SDS resistant) and aggregates (partly Triton X-100 insoluble), as determined by pulse-chase experiments and subcellular fractionation. Thus, the GCK-MODY mutations S263P and G264S lead to protein misfolding causing destabilization, cellular dimerization/aggregation and enhanced rate of degradation. In silico predicted conformational changes of the F260-L270 loop structure are considered to mediate the dimerization of both mutant proteins by a domain swapping mechanism. Thus, similar properties may represent the molecular mechanisms for additional unexplained GCK-MODY mutations, and may also contribute to the disease mechanism in other previously characterized GCK-MODY inactivating mutations.

Effects of a glucokinase activator on hepatic intermediary metabolism: study with 13C-isotopomer-based metabolomics.

GKAs (glucokinase activators) are promising agents for the therapy of Type 2 diabetes, but little is known about their effects on hepatic intermediary metabolism. We monitored the fate of (13)C-labelled glucose in both a liver perfusion system and isolated hepatocytes. MS and NMR spectroscopy were deployed to measure isotopic enrichment. The results demonstrate that the stimulation of glycolysis by GKA led to numerous changes in hepatic metabolism: (i) augmented flux through the TCA (tricarboxylic acid) cycle, as evidenced by greater incorporation of (13)C into the cycle (anaplerosis) and increased generation of (13)C isotopomers of citrate, glutamate and aspartate (cataplerosis); (ii) lowering of hepatic [Pi] and elevated [ATP], denoting greater phosphorylation potential and energy state; (iii) stimulation of glycogen synthesis from glucose, but inhibition of glycogen synthesis from 3-carbon precursors; (iv) increased synthesis of N-acetylglutamate and consequently augmented ureagenesis; (v) increased synthesis of glutamine, alanine, serine and glycine; and (vi) increased production and outflow of lactate. The present study provides a deeper insight into the hepatic actions of GKAs and uncovers the potential benefits and risks of GKA for treatment of diabetes. GKA improved hepatic bioenergetics, ureagenesis and glycogenesis, but decreased gluconeogenesis with a potential risk of lactic acidosis and fatty liver.

Foxa2 controls vesicle docking and insulin secretion in mature Beta cells.

The winged-helix transcription factor Foxa2 regulates Pdx1 gene expression and fetal endocrine pancreas development. We show here by inducible gene ablation that Foxa2 inactivation in mature beta cells induces hyperinsulinemic hypoglycemia in Foxa2(loxP/loxP),Pdx1-CreERT2 adult mice. Mutant beta cells exhibited a markedly increased pool of docked insulin granules, some of which were engaged in sequential or compound exocytosis, consistent with increased first-phase glucose-stimulated insulin secretion. Expression of multiple genes involved in vesicular trafficking, membrane targeting, and fuel-secretion pathways is dependent on Foxa2. In addition, impaired cytosolic Ca(2+) oscillations and elevated intracellular cyclic AMP production accompanied this secretory defect and were likely contributors to the sensitization of the exocytotic machinery. Thus, in the absence of Foxa2, alterations in intracellular second-messenger signaling redistribute the insulin granules into the readily releasable pool. We conclude that Foxa2 is required for both fetal pancreas development and the function of mature beta cells.

Green tea polyphenols control dysregulated glutamate dehydrogenase in transgenic mice by hijacking the ADP activation site.

Glutamate dehydrogenase (GDH) catalyzes the oxidative deamination of L-glutamate and, in animals, is extensively regulated by a number of metabolites. Gain of function mutations in GDH that abrogate GTP inhibition cause the hyperinsulinism/hyperammonemia syndrome (HHS), resulting in increased pancreatic ß-cell responsiveness to leucine and susceptibility to hypoglycemia following high protein meals. We have previously shown that two of the polyphenols from green tea (epigallocatechin gallate (EGCG) and epicatechin gallate (ECG)) inhibit GDH in vitro and that EGCG blocks GDH-mediated insulin secretion in wild type rat islets. Using structural and site-directed mutagenesis studies, we demonstrate that ECG binds to the same site as the allosteric regulator, ADP. Perifusion assays using pancreatic islets from transgenic mice expressing a human HHS form of GDH demonstrate that the hyperresponse to glutamine caused by dysregulated GDH is blocked by the addition of EGCG. As observed in HHS patients, these transgenic mice are hypersensitive to amino acid feeding, and this is abrogated by oral administration of EGCG prior to challenge. Finally, the low basal blood glucose level in the HHS mouse model is improved upon chronic administration of EGCG. These results suggest that this common natural product or some derivative thereof may prove useful in controlling this genetic disorder. Of broader clinical implication is that other groups have shown that restriction of glutamine catabolism via these GDH inhibitors can be useful in treating various tumors. This HHS transgenic mouse model offers a highly useful means to test these agents in vivo.

Generation of N-ethyl-N-nitrosourea (ENU) diabetes models in mice demonstrates genotype-specific action of glucokinase activators.

We performed genome-wide mutagenesis in C57BL/6J mice using N-ethyl-N-nitrosourea to identify mutations causing high blood glucose early in life and to produce new animal models of diabetes. Of a total of 13 new lines confirmed by heritability testing, we identified two semi-dominant pedigrees with novel missense mutations (Gck(K140E) and Gck(P417R)) in the gene encoding glucokinase (Gck), the mammalian glucose sensor that is mutated in human maturity onset diabetes of the young type 2 and the target of emerging anti-hyperglycemic agents that function as glucokinase activators (GKAs). Diabetes phenotype corresponded with genotype (mild-to-severe: Gck(+/+) < Gck(P417R/+), Gck(K140E)(/+) < Gck(P417R/P417R), Gck(P417R/K140E), and Gck(K140E/K140E)) and with the level of expression of GCK in liver. Each mutant was produced as the recombinant enzyme in Escherichia coli, and analysis of k(cat) and tryptophan fluorescence (I(320/360)) during thermal shift unfolding revealed a correlation between thermostability and the severity of hyperglycemia in the whole animal. Disruption of the glucokinase regulatory protein-binding site (GCK(K140E)), but not the ATP binding cassette (GCK(P417R)), prevented inhibition of enzyme activity by glucokinase regulatory protein and corresponded with reduced responsiveness to the GKA drug. Surprisingly, extracts from liver of diabetic GCK mutants inhibited activity of the recombinant enzyme, a property that was also observed in liver extracts from mice with streptozotocin-induced diabetes. These results indicate a relationship between genotype, phenotype, and GKA efficacy. The integration of forward genetic screening and biochemical profiling opens a pathway for preclinical development of mechanism-based diabetes therapies.

Glucokinase activation repairs defective bioenergetics of islets of Langerhans isolated from type 2 diabetics.

It was reported previously that isolated human islets from individuals with type 2 diabetes mellitus (T2DM) show reduced glucose-stimulated insulin release. To assess the possibility that impaired bioenergetics may contribute to this defect, glucose-stimulated respiration (Vo(2)), glucose usage and oxidation, intracellular Ca(2+), and insulin secretion (IS) were measured in pancreatic islets isolated from three healthy and three type 2 diabetic organ donors. Isolated mouse and rat islets were studied for comparison. Islets were exposed to a "staircase" glucose stimulus, whereas IR and Vo(2) were measured. Vo(2) of human islets from normals and diabetics increased sigmoidally from equal baselines of 0.25 nmol/100 islets/min as a function of glucose concentration. Maximal Vo(2) of normal islets at 24 mM glucose was 0.40 ± 0.02 nmol·min(-1)·100 islets(-1), and the glucose S(0.5) was 4.39 ± 0.10 mM. The glucose stimulation of respiration of islets from diabetics was lower, V(max) of 0.32 ± 0.01 nmol·min(-1)·100 islets(-1), and the S(0.5) shifted to 5.43 ± 0.13 mM. Glucose-stimulated IS and the rise of intracellular Ca(2+) were also reduced in diabetic islets. A clinically effective glucokinase activator normalized the defective Vo(2), IR, and free calcium responses during glucose stimulation in islets from type 2 diabetics. The body of data shows that there is a clear relationship between the pancreatic islet energy (ATP) production rate and IS. This relationship was similar for normal human, mouse, and rat islets and the data for all species fitted a single sigmoidal curve. The shared threshold rate for IS was ∼13 pmol·min(-1)·islet(-1). Exendin-4, a GLP-1 analog, shifted the ATP production-IS curve to the left and greatly potentiated IS with an ATP production rate threshold of ∼10 pmol·min(-1)·islet(-1). Our data suggest that impaired ß-cell bioenergetics resulting in greatly reduced ATP production is critical in the molecular pathogenesis of type 2 diabetes mellitus.

Repair of diverse diabetic defects of ß-cells in man and mouse by pharmacological glucokinase activation.

Glucokinase activators (GKAs) are being developed and clinically tested for potential antidiabetic therapy. The potential benefits and limitations of this approach continue to be intensively debated. To contribute to the understanding of experimental pharmacology and therapeutics of GKAs, we have tested the efficacy of one of these agents (Piragliatin) in isolated islets from humans with type 2 diabetes mellitus (T2DM), from mice with glucokinase (GK) mutations induced by ethyl-nitroso-urea (ENU) as models of Maturity Onset Diabetes of the Young linked to GK and Permanent Neonatal Diabetes Mellitus linked to GK (PNDM-GK) and finally of islets rendered glucose insensitive by treatment with the sulphonyl urea compound glyburide in organ culture. We found that the GKA repaired the defect in all three instances as manifest in increased glucose-induced insulin release and elevated intracellular calcium responses. The results show the remarkable fact that acute pharmacological activation of GK reverses secretion defects of ß-cells caused by molecular mechanism that differ vastly in nature, including the little understood multifactorial lesion of ß-cells in T2DM of man, the complex GK mutations in mice resembling GK disease and acute sulphonylurea failure of mouse ß-cells in tissue culture. The implications of these results are to be discussed on the theoretical basis underpinning the strategy of developing these drugs and in light of recent results of clinical trials with GKAs that failed for little understood reasons.

Mutational analysis of allosteric activation and inhibition of glucokinase.

GK (glucokinase) is activated by glucose binding to its substrate site, is inhibited by GKRP (GK regulatory protein) and stimulated by GKAs (GK activator drugs). To explore further the mechanisms of these processes we studied pure recombinant human GK (normal enzyme and a selection of 31 mutants) using steady-state kinetics of the enzyme and TF (tryptophan fluorescence). TF studies of the normal binary GK-glucose complex corroborate recent crystallography studies showing that it exists in a closed conformation greatly different from the open conformation of the ligand-free structure, but indistinguishable from the ternary GK-glucose-GKA complex. GKAs did activate and GKRP did inhibit normal GK, whereas its TF was doubled by glucose saturation. However, the enzyme kinetics, GKRP inhibition, TF enhancement by glucose and responsiveness to GKA of the selected mutants varied greatly. Two predominant response patterns were identified accounting for nearly all mutants: (i) GK mutants with a normal or close to normal response to GKA, normally low basal TF (indicating an open conformation), some variability of kinetic parameters (k(cat), glucose S(0.5), h and ATP K(m)), but usually strong GKRP inhibition (13/31); and (ii) GK mutants that are refractory to GKAs, exhibit relatively high basal TF (indicating structural compaction and partial closure), usually show strongly enhanced catalytic activity primarily due to lowering of the glucose S(0.5), but with reduced or no GKRP inhibition in most cases (14/31). These results and those of previous studies are best explained by envisioning a common allosteric regulator region with spatially non-overlapping GKRP- and GKA-binding sites.

Genetic activation of glucokinase in a minority of pancreatic beta cells causes hypoglycemia in mice.

AIMS: Glucokinase (GK) is expressed in the glucose-sensing cells of the islets of Langerhans and plays a critical role in glucose homeostasis. Here, we tested the hypothesis that genetic activation of GK in a small subset of ß-cells is sufficient to change the glucose set-point of the whole islet. MATERIAL AND METHODS: Mouse models of cell-type specific GK deficiency (GKKO) and genetic enzyme activation (GKKI) in a subset of ß-cells were obtained by crossing the αGSU (gonadotropin alpha subunit)-Cre transgene with the appropriate GK mutant alleles. Metabolic analyses consisted of glucose tolerance tests, perifusion of isolated islets and intracellular calcium measurements. KEY FINDINGS: The αGSU-Cre transgene produced genetically mosaic islets, as Cre was active in 15 ± 1.2 % of ß-cells. While mice deficient for GK in a subset of islet cells were normal, unexpectedly, GKKI mice were chronically hypoglycemic, glucose intolerant, and had a lower threshold for glucose stimulated insulin secretion. GKKI mice exhibited an average fasting blood glucose level of 3.5 mM. GKKI islets responded with intracellular calcium signals that spread through the whole islets at 1 mM and secreted insulin at 3 mM glucose. SIGNIFICANCE: Genetic activation of GK in a minority of ß-cells is sufficient to change the glucose threshold for insulin secretion in the entire islet and thereby glucose homeostasis in the whole animal. These data support the model in which ß-cells with higher GK activity function as 'hub' or 'trigger' cells and thus control insulin secretion by the ß-cell collective within the islet.

α Cell dysfunction in islets from nondiabetic, glutamic acid decarboxylase autoantibody-positive individuals.

BACKGROUNDMultiple islet autoantibodies (AAbs) predict the development of type 1 diabetes (T1D) and hyperglycemia within 10 years. By contrast, T1D develops in only approximately 15% of individuals who are positive for single AAbs (generally against glutamic acid decarboxylase [GADA]); hence, the single GADA+ state may represent an early stage of T1D.METHODSHere, we functionally, histologically, and molecularly phenotyped human islets from nondiabetic GADA+ and T1D donors.RESULTSSimilar to the few remaining ß cells in the T1D islets, GADA+ donor islets demonstrated a preserved insulin secretory response. By contrast, α cell glucagon secretion was dysregulated in both GADA+ and T1D islets, with impaired glucose suppression of glucagon secretion. Single-cell RNA-Seq of GADA+ α cells revealed distinct abnormalities in glycolysis and oxidative phosphorylation pathways and a marked downregulation of cAMP-dependent protein kinase inhibitor ß (PKIB), providing a molecular basis for the loss of glucose suppression and the increased effect of 3-isobutyl-1-methylxanthine (IBMX) observed in GADA+ donor islets.CONCLUSIONWe found that α cell dysfunction was present during the early stages of islet autoimmunity at a time when ß cell mass was still normal, raising important questions about the role of early α cell dysfunction in the progression of T1D.FUNDINGThis work was supported by grants from the NIH (3UC4DK112217-01S1, U01DK123594-02, UC4DK112217, UC4DK112232, U01DK123716, and P30 DK019525) and the Vanderbilt Diabetes Research and Training Center (DK20593).

Mechanism of hyperinsulinism in short-chain 3-hydroxyacyl-CoA dehydrogenase deficiency involves activation of glutamate dehydrogenase.

The mechanism of insulin dysregulation in children with hyperinsulinism associated with inactivating mutations of short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD) was examined in mice with a knock-out of the hadh gene (hadh(-/-)). The hadh(-/-) mice had reduced levels of plasma glucose and elevated plasma insulin levels, similar to children with SCHAD deficiency. hadh(-/-) mice were hypersensitive to oral amino acid with decrease of glucose level and elevation of insulin. Hypersensitivity to oral amino acid in hadh(-/-) mice can be explained by abnormal insulin responses to a physiological mixture of amino acids and increased sensitivity to leucine stimulation in isolated perifused islets. Measurement of cytosolic calcium showed normal basal levels and abnormal responses to amino acids in hadh(-/-) islets. Leucine, glutamine, and alanine are responsible for amino acid hypersensitivity in islets. hadh(-/-) islets have lower intracellular glutamate and aspartate levels, and this decrease can be prevented by high glucose. hadh(-/-) islets also have increased [U-(14)C]glutamine oxidation. In contrast, hadh(-/-) mice have similar glucose tolerance and insulin sensitivity compared with controls. Perifused hadh(-/-) islets showed no differences from controls in response to glucose-stimulated insulin secretion, even with addition of either a medium-chain fatty acid (octanoate) or a long-chain fatty acid (palmitate). Pull-down experiments with SCHAD, anti-SCHAD, or anti-GDH antibodies showed protein-protein interactions between SCHAD and GDH. GDH enzyme kinetics of hadh(-/-) islets showed an increase in GDH affinity for its substrate, α-ketoglutarate. These studies indicate that SCHAD deficiency causes hyperinsulinism by activation of GDH via loss of inhibitory regulation of GDH by SCHAD.

Research and development of glucokinase activators for diabetes therapy: theoretical and practical aspects.

Glucokinase Glucokinase (GK GK ; EC 2.7.1.1.) phosphorylates and regulates glucose metabolism in insulin-producing pancreatic beta-cells, hepatocytes, and certain cells of the endocrine and nervous systems allowing it to play a central role in glucose homeostasis glucose homeostasis . Most importantly, it serves as glucose sensor glucose sensor in pancreatic beta-cells mediating glucose-stimulated insulin biosynthesis and release and it governs the capacity of the liver to convert glucose to glycogen. Activating and inactivating mutations of the glucokinase gene cause autosomal dominant hyperinsulinemic hypoglycemia and hypoinsulinemic hyperglycemia in humans, respectively, illustrating the preeminent role of glucokinase in the regulation of blood glucose and also identifying the enzyme as a potential target for developing antidiabetic drugs antidiabetic drugs . Small molecules called glucokinase activators (GKAs) glucokinase activators (GKAs) which bind to an allosteric activator allosteric activator site of the enzyme have indeed been discovered and hold great promise as new antidiabetic agents. GKAs increase the enzyme's affinity for glucose and also its maximal catalytic rate. Consequently, they stimulate insulin biosynthesis and secretion, enhance hepatic glucose uptake, and augment glucose metabolism and related processes in other glucokinase-expressing cells. Manifestations of these effects, most prominently a lowering of blood glucose, are observed in normal laboratory animals and man but also in animal models of diabetes and patients with type 2 diabetes mellitus (T2DM T2DM ) type 2 diabetes mellitus (T2DM) . These compelling concepts and results sustain a strong R&D effort by many pharmaceutical companies to generate GKAs with characteristics allowing for a novel drug treatment of T2DM.

Metabolic Homeostasis in Life as We Know It: Its Origin and Thermodynamic Basis.

Living organisms require continuous input of energy for their existence. As a result, life as we know it is based on metabolic processes that extract energy from the environment and make it available to support life (energy metabolism). This metabolism is based on, and regulated by, the underlying thermodynamics. This is important because thermodynamic parameters are stable whereas kinetic parameters are highly variable. Thermodynamic control of metabolism is exerted through near equilibrium reactions that determine. (1) the concentrations of metabolic substrates for enzymes that catalyze irreversible steps and (2) the concentrations of small molecules (AMP, ADP, etc.) that regulate the activity of irreversible reactions in metabolic pathways. The result is a robust homeostatic set point (-ΔGATP) with long term (virtually unlimited) stability. The rest of metabolism and its regulation is constrained to maintain this set point. Thermodynamic control is illustrated using the ATP producing part of glycolysis, glyceraldehyde-3-phosphate oxidation to pyruvate. Flux through the irreversible reaction, pyruvate kinase (PK), is primarily determined by the rate of ATP consumption. Change in the rate of ATP consumption causes mismatch between use and production of ATP. The resulting change in [ATP]/[ADP][Pi], through near equilibrium of the reactions preceding PK, alters the concentrations of ADP and phosphoenolpyruvate (PEP), the substrates for PK. The changes in ADP and PEP alter flux through PK appropriately for restoring equality of ATP production and consumption. These reactions appeared in the very earliest lifeforms and are hypothesized to have established the set point for energy metabolism. As evolution included more metabolic functions, additional layers of control were needed to integrate new functions into existing metabolism without changing the homeostatic set point. Addition of gluconeogenesis, for example, resulted in added regulation to PK activity to prevent futile cycling; PK needs to be turned off during gluconeogenesis because flux through the enzyme would waste energy (ATP), subtracting from net glucose synthesis and decreasing overall efficiency.

Palmitic acid acutely inhibits acetylcholine- but not GLP-1-stimulated insulin secretion in mouse pancreatic islets.

Fatty acids, acetylcholine, and GLP-1 enhance insulin secretion in a glucose-dependent manner. However, the interplay between glucose, fatty acids, and the neuroendocrine regulators of insulin secretion is not well understood. Therefore, we studied the acute effects of PA (alone or in combination with glucose, acetylcholine, or GLP-1) on isolated cultured mouse islets. Two different sets of experiments were designed. In one, a fixed concentration of 0.5 mM of PA bound to 0.15 mM BSA was used; in the other, a PA ramp from 0 to 0.5 mM was applied at a fixed albumin concentration of 0.15 mM so that the molar PA/BSA ratio changed within the physiological range. At a fixed concentration of 0.5 mM, PA markedly inhibited acetylcholine-stimulated insulin release, the rise of intracellular Ca(2+), and enhancement of cAMP production but did not influence the effects of GLP-1 on these parameters of islet cell function. 2-ADB, an IP(3) receptor inhibitor, reduced the effect of acetylcholine on insulin secretion and reversed the effect of PA on acetylcholine-stimulated insulin release. Islet perfusion for 35-40 min with 0.5 mM PA significantly reduced the calcium storage capacity of ER measured by the thapsigargin-induced Ca(2+) release. Oxygen consumption due to low but not high glucose was reduced by PA. When a PA ramp from 0 to 0.5 mM was applied in the presence of 8 mM glucose, PA at concentrations as low as 50 microM significantly augmented glucose-stimulated insulin release and markedly reduced acetylcholine's effects on hormone secretion. We thus demonstrate that PA acutely reduces the total oxygen consumption response to glucose, glucose-dependent acetylcholine stimulation of insulin release, Ca(2+), and cAMP metabolism, whereas GLP-1's actions on these parameters remain unaffected or potentiated. We speculate that acute emptying of the ER calcium by PA results in decreased glucose stimulation of respiration and acetylcholine potentiation of insulin secretion.