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).

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.

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.

Cerebrovascular Blood Flow Design and Regulation; Vulnerability in Aging Brain.

Nutrient delivery to the brain presents a unique challenge because the tissue functions as a computer system with in the order of 200,000 neurons/mm3. Penetrating arterioles bud from surface arteries of the brain and penetrate downward through the cortex. Capillary networks spread from penetrating arterioles through the surrounding tissue. Each penetrating arteriole forms a vascular unit, with little sharing of flow among vascular units (collateral flow). Unlike cells in other tissues, neurons have to be operationally isolated, interacting with other neurons through specific electrical connections. Neuronal activation typically involves only a few of the cells within a vascular unit, but the local increase in nutrient consumption is substantial. The metabolic response to activation is transmitted to the feeding arteriole through the endothelium of neighboring capillaries and alters calcium permeability of smooth muscle in the wall resulting in modulation of flow through the entire vascular unit. Many age and trauma related brain pathologies can be traced to vascular malfunction. This includes: 1. Physical damage such as in traumatic injury with imposed shear stress as soft tissue moves relative to the skull. Lack of collateral flow among vascular units results in death of the cells in that vascular unit and loss of brain tissue. 2. Age dependent changes lead to progressive increase in vascular resistance and decrease in tissue levels of oxygen and glucose. Chronic hypoxia/hypoglycemia compromises tissue energy metabolism and related regulatory processes. This alters stem cell proliferation and differentiation, undermines vascular integrity, and suppresses critical repair mechanisms such as oligodendrocyte generation and maturation. Reduced structural integrity results in local regions of acute hypoxia and microbleeds, while failure of oligodendrocytes to fully mature leads to poor axonal myelination and defective neuronal function. Understanding and treating age related pathologies, particularly in brain, requires better knowledge of why and how vasculature changes with age. That knowledge will, hopefully, make possible drugs/methods for protecting vascular function, substantially alleviating the negative health and cognitive deficits associated with growing old.

Ethanol metabolism: The good, the bad, and the ugly.

Throughout the world, ethanol is both an important commercial commodity and a source of major medical and social problems. Ethanol readily passes through biological membranes and distributes throughout the body. It is oxidized, first to acetaldehyde and then to acetate, and finally by the citric acid cycle in virtually all tissues. The oxidation of ethanol is irreversible and unregulated, making the rate dependent only on local concentration and enzyme activity. This unregulated input of reducing equivalents increases reduction of both cytoplasmic and intramitochondrial NAD and, through the latter, cellular energy state {[ATP]/([ADP][Pi])}. In brain, this increase in energy state stimulates dopaminergic neural activity signalling reward and a sense of well being, while suppressing glutamatergic neural activity signalling anxiety and unease. These positive responses to ethanol ingestion are important to social alcohol consumption. Importantly, decreased free [AMP] decreases AMP-dependent protein kinase (AMPK) activity, an important regulator of cellular energy metabolism. Oxidation of substrates used for energy metabolism in the absence of ethanol is down regulated to accommodate the input from ethanol. In liver, chronic ethanol metabolism results in fatty liver and general metabolic dysfunction. In brain, transport of other oxidizable metabolites through the blood-brain barrier and the enzymes for their oxidation are both down regulated. For exposures of short duration, ethanol induced regulatory changes are rapid and reversible, recovering completely when the concentrations of ethanol and acetate fall again. Longer periods of ethanol exposure and associated chronic suppression of AMPK activity activates regulatory mechanisms, including gene expression, that operate over longer time scales, both in onset and reversal. If chronic alcohol consumption is abruptly ended, metabolism is no longer able to respond rapidly enough to compensate. Glutamatergic neural activity adapts to chronic dysregulation of glutamate metabolism and suppression of glutamatergic neural activity by increasing excitatory and decreasing inhibitory amino acid receptors. A point is reached (ethanol dependence) where withdrawal of ethanol results in significant metabolic energy depletion in neurons and other brain cells as well as hyperexcitation of the glutamatergic system. The extent and regional specificity of energy depletion in the brain, combined with hyperactivity of the glutamatergic neuronal system, largely determines the severity of withdrawal symptoms.

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.

Hyperbaric oxygen toxicity in brain: A case of hyperoxia induced hypoglycemic brain syndrome.

Hyperbaric oxygen exposure is a recent hazzard for higher animals that originated as humans began underwater construction, exploration, and sports. Exposure can lead to abnormal brain EEG, convulsions, and death, the time to onset of each stage of pathology decreasing with increase in oxygen pressure. We provide evidence that hyperoxia, through oxidative phosphorylation, increases the energy state ([ATP]/[ADP][Pi]) of cells critical to providing glucose to cells behind the blood brain barrier (BBB). Brain cells without an absolute dependence on glucose metabolism; i.e. those having sufficient ATP synthesis using lactate and glutamate as oxidizable substrates, are not themselves very adversely affected by hyperoxia. The increased energy state and decrease in free [AMP], however, suppress glucose transport through the blood brain barrier (BBB) and into cells behind the BBB. Glucose has to pass in sequence through three steps of transport by facilitated diffusion and transporter activity for each step is regulated in part by AMP dependent protein kinase. The physiological role of this regulation is to increase glucose transport in response to hypoxia and/or systemic hypoglycemia. Hyperoxia, however, through unphysiological decrease in free [AMP] suppresses 1) glucose transport through the BBB (endothelial GLUT1 transporters) into cerebrospinal fluid (CSF); 2) glucose transport from CSF into cells behind the BBB (GLUT3 transporters) and (GLUT4 transporters). Cumulative suppression of glucose transport results in local regions of hypoglycemia and induces hypoglycemic failure. It is suggested that failure is initiated at axons and synapses with insufficient mitochondria to meet their energy requirements.

Oxygen dependence of glucose sensing: role in glucose homeostasis and related pathology.

In glucose homeostasis, glucose concentration is sensed by its metabolism through glucokinase (GCK) and oxidative phosphorylation. Because oxidative phosphorylation is an integral part of the sensory system, glucose sensing is necessarily dependent on oxygen pressure. Much of the dependence on oxygen is suppressed by location of glucose sensing cells in tissues with well-regulated blood flow. In healthy individuals the oxygen dependence is primarily observed in response to transient global hypoxia events such as during birth or transition to high altitude. The GCK sensing system is, however, used to control release of both insulin and glucagon, the preeminant hormonal regulators of blood glucose, as well as glucose sensitive neuronal activity. Suppression of oxygen delivery to glucose-sensing cells or interference with regulation of tissue blood flow by either local or systemic causes, stresses the glucose regulatory system. This is true whether the stress is imposed locally, such as by altered oxygen delivery to the pancreas, or globally, as in pulmonary insufficiency or exposure to high altitude. It may be expected that chronic application of this stress predisposes individuals to developing diabetes. Type 2 diabetes is a broad class of diseases characterized by disturbance of glucose homeostasis, i.e., having either hyperglycemia and/or decreased sensitivity to insulin. Given the role of oxidative phosphorylation in glucose sensing, tissue oxygen deprivation may predispose individuals to developing diabetes as well as contributing to the disease itself. This is particularly true in age-related diabetes because the incidence of vascular insufficiency increases markedly with increasing age. NEW & NOTEWORTHY Glucose sensing requires glucose metabolism through glycolysis and oxidative phosphorylation. Dependence of the latter on oxygen concentration imposes an oxygen dependence on glucose sensing. We have used a validated computational model to quantify that dependence. Evidence is presented that tissue oxygenation plays an important role in predisposition of individuals to developing type 2 diabetes and in progression of the disease.

Intra-islet glucagon signaling is critical for maintaining glucose homeostasis.

Glucagon, a hormone released from pancreatic alpha-cells, plays a key role in maintaining proper glucose homeostasis and has been implicated in the pathophysiology of diabetes. In vitro studies suggest that intra-islet glucagon can modulate the function of pancreatic beta-cells. However, because of the lack of suitable experimental tools, the in vivo physiological role of this intra-islet cross-talk has remained elusive. To address this issue, we generated a novel mouse model that selectively expressed an inhibitory designer G protein-coupled receptor (Gi DREADD) in α-cells only. Drug-induced activation of this inhibitory designer receptor almost completely shut off glucagon secretion in vivo, resulting in significantly impaired insulin secretion, hyperglycemia, and glucose intolerance. Additional studies with mouse and human islets indicated that intra-islet glucagon stimulates insulin release primarily by activating ß-cell GLP-1 receptors. These new findings strongly suggest that intra-islet glucagon signaling is essential for maintaining proper glucose homeostasis in vivo. Our work may pave the way toward the development of novel classes of antidiabetic drugs that act by modulating intra-islet cross-talk between α- and ß-cells.

The Central Role of Glucokinase in Glucose Homeostasis: A Perspective 50 Years After Demonstrating the Presence of the Enzyme in Islets of Langerhans.

It is hypothesized that glucokinase (GCK) is the glucose sensor not only for regulation of insulin release by pancreatic ß-cells, but also for the rest of the cells that contribute to glucose homeostasis in mammals. This includes other cells in endocrine pancreas (α- and δ-cells), adrenal gland, glucose sensitive neurons, entero-endocrine cells, and cells in the anterior pituitary. Glucose transport is by facilitated diffusion and is not rate limiting. Once inside, glucose is phosphorylated to glucose-6-phosphate by GCK in a reaction that is dependent on glucose throughout the physiological range of concentrations, is irreversible, and not product inhibited. High glycerol phosphate shuttle, pyruvate dehydrogenase, and pyruvate carboxylase activities, combined with low pentose-P shunt, lactate dehydrogenase, plasma membrane monocarboxylate transport, and glycogen synthase activities constrain glucose-6-phosphate to being metabolized through glycolysis. Under these conditions, glycolysis produces mostly pyruvate and little lactate. Pyruvate either enters the citric acid cycle through pyruvate dehydrogenase or is carboxylated by pyruvate carboxylase. Reducing equivalents from glycolysis enter oxidative phosphorylation through both the glycerol phosphate shuttle and citric acid cycle. Raising glucose concentration increases intramitochondrial [NADH]/[NAD+] and thereby the energy state ([ATP]/[ADP][Pi]), decreasing [Mg2+ADP] and [AMP]. [Mg2+ADP] acts through control of KATP channel conductance, whereas [AMP] acts through regulation of AMP-dependent protein kinase. Specific roles of different cell types are determined by the diverse molecular mechanisms used to couple energy state to cell specific responses. Having a common glucose sensor couples complementary regulatory mechanisms into a tightly regulated and stable glucose homeostatic network.

Metabolic homeostasis: oxidative phosphorylation and the metabolic requirements of higher plants and animals.

A model of oxidative phosphorylation and its regulation is presented, which is consistent with the experimental data on metabolism in higher plants and animals. The variables that provide real-time control of metabolic status are: intramitochondrial [NAD+]/[NADH], energy state ([ATP]/[ADP][Pi]), and local oxygen concentration ([O2]). ATP consumption and respiratory chain enzyme content are tissue specific (liver vs. heart muscle), and the latter is modulated by chronic alterations in ATP consumption (i.e., endurance training etc.). ATP consumption affects the energy state, which increases or decreases as necessary to match synthesis with consumption. [NAD+]/[NADH], local [O2], and respiratory chain content determine the energy state at which match of synthesis and utilization is achieved. Tissues vary widely in their ranges of ATP consumption. Expressed as the turnover of cytochrome c, the rates may change little (7 to 12/s) (liver) or a lot (1 to >300/s) (flight muscle of birds, bats, and insects). Ancillary metabolic pathways, including creatine or arginine kinase, glycerol phosphate shuttle, fatty acid, and citric acid cycle dehydrogenases, are responsible for meeting tissue-specific differences in maximal rate and range in ATP utilization without displacing metabolic homeostasis. Intramitochondrial [NAD+]/[NADH], [ATP], and [Pi] are adjusted to keep [ADP] and [AMP] similar for all tissues despite large differences in ranges in ATP utilization. This is essential because [ADP] and [AMP], particularly the latter, have major roles in regulating the activity of many enzymes and signaling pathways (AMP deaminase, AMP dependent protein kinases, etc.) common to all higher plants and animals.NEW & NOTEWORTHY Oxidative phosphorylation has an intrinsic program that sets and stabilizes cellular energy state ([ATP]/[ADP][Pi]), and thereby metabolic homeostasis. A computational model consistent with regulation of oxidative phosphorylation in higher plants and animals is presented. Focus is on metabolism ancillary to oxidative phosphorylation by which it was integrated into preexisting metabolic regulation and adapted by evolution to develop cells and tissues with differing rates of ATP utilization: i.e., liver versus brain versus muscle.

Glutamate dehydrogenase: role in regulating metabolism and insulin release in pancreatic ß-cells.

Regulation of insulin release and glucose homeostasis by pancreatic ß-cells is dependent on the metabolism of glucose by glucokinase (GK) and the influence of that activity on oxidative phosphorylation. Genetic alterations that result in hyperactivity of mitochondrial glutamate dehydrogenase (GDH-1) can cause hypoglycemia-hyperammonemia following high protein meals, but the role of GDH-1 remains poorly understood. GDH-1 activity is strongly inhibited by GTP, to near zero in the absence of ADP, and cooperatively activated ( n = 2.3) by ADP. The dissociation constant for ADP is near 200 µM in vivo, but leucine and its nonmetabolized analog 2-amino-2-norbornane-carboxylic acid (BCH) can activate GDH-1 by increasing the affinity for ADP. Under physiological conditions, as [ADP] increases GDH-1 activity remains very low until ~35 µM (threshold) and then increases rapidly. A model for GDH-1 and its regulation has been combined with a previously published model for glucose sensing that coupled GK activity and oxidative phosphorylation. The combined model (GK-GDH-core) shows that GK activity determines the energy state ([ATP]/[ADP][Pi]) in ß-cells for glucose concentrations > 5 mM ([ADP] < 35 µM). As glucose falls < 5 mM the [ADP]-dependent increase in GDH-1 activity prevents [ADP] from rising above ~70 µM. Thus, GDH-1 dynamically buffers ß-cell energy metabolism during hypoglycemia, maintaining the energy state and the basal rate of insulin release. GDH-1 hyperactivity suppresses the normal increase in [ADP] in hypoglycemia. This leads to hypoglycemia following a high protein meal by increasing the basal rate of insulin release (ß-cells) and decreasing glucagon release (α-cells). NEW & NOTEWORTHY A model of ß-cell metabolism and regulation of insulin release is presented. The model integrates regulation of oxidative phosphorylation, glucokinase (GK), and glutamate dehydrogenase (GDH-1). GDH-1 is near equilibrium under physiological conditions, but the activity is inhibited by GTP. In hypoglycemia, however, GK activity is low and [ADP], a potent activator of GDH-1, increases. Reducing equivalents from GDH dynamically buffers the intramitochondrial [NADH]/[NAD+], and thereby the energy state, preventing hypoglycemia-induced substrate deprivation.

Inhibition of cholinergic potentiation of insulin secretion from pancreatic islets by chronic elevation of glucose and fatty acids: Protection by casein kinase 2 inhibitor.

OBJECTIVES: Chronic hyperlipidemia and hyperglycemia are characteristic features of type 2 diabetes (T2DM) that are thought to cause or contribute to ß-cell dysfunction by "glucolipotoxicity." Previously we have shown that acute treatment of pancreatic islets with fatty acids (FA) decreases acetylcholine-potentiated insulin secretion. This acetylcholine response is mediated by M3 muscarinic receptors, which play a key role in regulating ß-cell function. Here we examine whether chronic FA exposure also inhibits acetylcholine-potentiated insulin secretion using mouse and human islets. METHODS: Islets were cultured for 3 or 4 days at different glucose concentration with 0.5 mM palmitic acid (PA) or a 2:1 mixture of PA and oleic acid (OA) at 1% albumin (PA/BSA molar ratio 3.3). Afterwards, the response to glucose and acetylcholine were studied in perifusion experiments. RESULTS: FA-induced impairment of insulin secretion and Ca2+ signaling depended strongly on the glucose concentrations of the culture medium. PA and OA in combination reduced acetylcholine potentiation of insulin secretion more than PA alone, both in mouse and human islets, with no evidence of a protective role of OA. In contrast, lipotoxicity was not observed with islets cultured for 3 days in medium containing less than 1 mM glucose and a mixture of glutamine and leucine (7 mM each). High glucose and FAs reduced endoplasmic reticulum (ER) Ca2+ storage capacity; however, preserving ER Ca2+ by blocking the IP3 receptor with xestospongin C did not protect islets from glucolipotoxic effects on insulin secretion. In contrast, an inhibitor of casein kinase 2 (CK2) protected the glucose dependent acetylcholine potentiation of insulin secretion in mouse and human islets against glucolipotoxicity. CONCLUSIONS: These results show that chronic FA treatment decreases acetylcholine potentiation of insulin secretion and that this effect is strictly glucose dependent and might involve CK2 phosphorylation of ß-cell M3 muscarinic receptors.

The thermodynamic basis of glucose-stimulated insulin release: a model of the core mechanism.

A model for glucose sensing by pancreatic ß-cells is developed and compared with the available experimental data. The model brings together mathematical representations for the activities of the glucose sensor, glucokinase, and oxidative phosphorylation. Glucokinase produces glucose 6-phosphate (G-6-P) in an irreversible reaction that determines glycolytic flux. The primary products of glycolysis are NADH and pyruvate. The NADH is reoxidized and the reducing equivalents transferred to oxidative phosphorylation by the glycerol phosphate shuttle, and some of the pyruvate is oxidized by pyruvate dehydrogenase and enters the citric acid cycle. These reactions are irreversible and result in a glucose concentration-dependent reduction of the intramitochondrial NAD pool. This increases the electrochemical energy coupled to ATP synthesis and thereby the cellular energy state ([ATP]/[ADP][Pi]). ATP and Pi are 10-100 times greater than ADP, so the increase in energy state is primarily through decrease in ADP The decrease in ADP is considered responsible for altering ion channel conductance and releasing insulin. Applied to the reported glucose concentration-dependent release of insulin by perifused islet preparations (Doliba et al. 2012), the model predicts that the dependence of insulin release on ADP is strongly cooperative with a threshold of about 30 µmol/L and a negative Hill coefficient near -5.5. The predicted cellular energy state, ADP, creatine phosphate/creatine ratio, and cytochrome c reduction, including their dependence on glucose concentration, are consistent with experimental data. The ability of the model to predict behavior consistent with experiment is an invaluable resource for understanding glucose sensing and planning experiments.

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.

Functional and Metabolomic Consequences of KATP Channel Inactivation in Human Islets.

Loss-of-function mutations of ß-cell KATP channels cause the most severe form of congenital hyperinsulinism (KATPHI). KATPHI is characterized by fasting and protein-induced hypoglycemia that is unresponsive to medical therapy. For a better understanding of the pathophysiology of KATPHI, we examined cytosolic calcium ([Ca2+] i ), insulin secretion, oxygen consumption, and [U-13C]glucose metabolism in islets isolated from the pancreases of children with KATPHI who required pancreatectomy. Basal [Ca2+] i and insulin secretion were higher in KATPHI islets compared with controls. Unlike controls, insulin secretion in KATPHI islets increased in response to amino acids but not to glucose. KATPHI islets have an increased basal rate of oxygen consumption and mitochondrial mass. [U-13C]glucose metabolism showed a twofold increase in alanine levels and sixfold increase in 13C enrichment of alanine in KATPHI islets, suggesting increased rates of glycolysis. KATPHI islets also exhibited increased serine/glycine and glutamine biosynthesis. In contrast, KATPHI islets had low γ-aminobutyric acid (GABA) levels and lacked 13C incorporation into GABA in response to glucose stimulation. The expression of key genes involved in these metabolic pathways was significantly different in KATPHI ß-cells compared with control, providing a mechanism for the observed changes. These findings demonstrate that the pathophysiology of KATPHI is complex, and they provide a framework for the identification of new potential therapeutic targets for this devastating condition.

ß-arrestin-2 is an essential regulator of pancreatic ß-cell function under physiological and pathophysiological conditions.

ß-arrestins are critical signalling molecules that regulate many fundamental physiological functions including the maintenance of euglycemia and peripheral insulin sensitivity. Here we show that inactivation of the ß-arrestin-2 gene, barr2, in ß-cells of adult mice greatly impairs insulin release and glucose tolerance in mice fed with a calorie-rich diet. Both glucose and KCl-induced insulin secretion and calcium responses were profoundly reduced in ß-arrestin-2 (barr2) deficient ß-cells. In human ß-cells, barr2 knockdown abolished glucose-induced insulin secretion. We also show that the presence of barr2 is essential for proper CAMKII function in ß-cells. Importantly, overexpression of barr2 in ß-cells greatly ameliorates the metabolic deficits displayed by mice consuming a high-fat diet. Thus, our data identify barr2 as an important regulator of ß-cell function, which may serve as a new target to improve ß-cell function.

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.