Hypoglycemia In Mice Research Articles

Potentiation of Glibenclamide Hypoglycaemia in Mice by MK-467, a Peripherally Acting Alpha2-Adrenoceptor Antagonist.

Pharmacological antagonism and genetic depletion of pancreatic α2A-adrenoceptors increase insulin secretion in mice and enhance the insulinotropic action of glibenclamide, a representative of the sulphonylurea class of insulin secretagogues used in the therapy of type 2 diabetes. Antagonism of α2-adrenoceptors in the central nervous system (CNS) causes tachycardia and hypertension, making generalized α2-adrenoceptor blockade unfavourable for clinical use despite its potential to decrease blood glucose levels. The purpose of this study was to test the acute effects of the peripherally acting α2-adrenoceptor antagonist MK-467 alone and in combination with glibenclamide in non-diabetic C57BL/6N mice. Cardiovascular safety was assessed in freely moving mice with radiotelemetry. Dose-dependent decreases in blood glucose and increases in plasma insulin concentrations were seen with the combination of MK-467 and glibenclamide; the combinations were much more potent than glibenclamide or MK-467 alone. Furthermore, MK-467 had no effect on mean arterial pressure or heart rate in freely moving mice and did not prevent the centrally mediated hypotensive effect of the α2-adrenoceptor agonist medetomidine. Thus, peripheral blockade of α2-adrenoceptors does not evoke the same cardiovascular adverse effects as antagonism of CNS α2-adrenoceptors. The current results indicate that the combined use of small doses of a peripherally acting α2-adrenoceptor antagonist with a sulphonylurea drug could provide a novel option for the treatment of type 2 diabetes, especially in patients with increased tonic α2-adrenoceptor-mediated inhibition of insulin secretion.

Deciphering the Hypoglycemic Glucagon Response: Development of a Graded Hyperinsulinemic Hypoglycemic Clamp Technique in Female Mice.

Glucose lowering therapy in type 1 and type 2 diabetes is often associated with hypoglycemic events. To avoid this, glucose lowering therapies need to be developed that support the hypoglycemic defense mechanisms. Such development needs a tool for evaluating counterregulatory mechanisms in vivo. A sustained glucagon release during hypoglycemia is of most importance to hypoglycemic defense mechanisms. We have therefore developed a graded hyperinsulinemic hypoglycemic clamp in mice and used it to evaluate counterregulatory glucagon dynamics. Glucose was clamped at narrow intervals aiming at 2.5, 3.5, 4.5, and 6.0 mmol/L. Glucagon levels were increased during hypoglycemia in a glucose-dependent way with a glucagon counterregulatory threshold between 3.5 and 4.0 mmol/L. Modelling the glucose-glucagon relationship using a hyperbolic curve with the equation: plasma glucagon = -4.20 + 90.79/blood glucose showed high correlation. When comparing this method to the insulin tolerance test as an approach to study glucagon dynamics in vivo, we found that the graded clamp more efficiently evoked a robust, predictable, glucagon response with considerably less variation in blood glucose. In conclusion, we have developed a tool for the study of in vivo glucagon dynamics during hypoglycemia in mice and demonstrated a hyperbolic glucose-counterregulatory glucagon relationship.

55P0110, a Novel Synthetic Compound Developed from a Plant Derived Backbone Structure, Shows Promising Anti-Hyperglycaemic Activity in Mice.

Starting off with a structure derived from the natural compound multiflorine, a derivatisation program aimed at the discovery and initial characterisation of novel compounds with antidiabetic potential. Design and discovery of the structures was guided by oral bioactivities obtained in oral glucose tolerance tests in mice. 55P0110, one among several new compounds with distinct anti-hyperglycaemic activity, was further examined to characterise its pharmacology and mode of action. Whereas a single oral dose of 55P0110 did not affect basal glycaemia, it markedly improved the glucose tolerance of healthy and diabetic mice (peak blood glucose in glucose tolerance test, mmol/l: healthy mice with 90 mg/kg 55P0110, 17.0±1.2 vs. 10.1±1.1; diabetic mice with 180 mg/kg 55P0110, 23.1±0.9 vs. 11.1±1.4; p<0.001 each). Closer examination argued against retarded glucose resorption from the gut, increased glucose excretion in urine, acute insulin-like or insulin sensitising properties, and direct inhibition of dipeptidyl peptidase-4 as the cause of glucose lowering. Hence, 55P0110 seems to act via a target not exploited by any drug presently approved for the treatment of diabetes mellitus. Whereas the insulinotropic sulfonylurea gliclazide (16 mg/kg) distinctly increased the circulating insulin-per-glucose ratio under basal conditions, 55P0110 (90 mg/kg) lacked such an effect (30 min. after dosing, nmol/mol: vehicle, 2.49±0.27; 55P0110, 2.99±0.35; gliclazide, 8.97±0.49; p<0.001 each vs. gliclazide). Under an exogenous glucose challenge, however, 55P0110 increased this ratio to the same extent as gliclazide (20 min. after glucose feeding: vehicle, 2.53±0.41; 55P0110, 3.80±0.46; gliclazide, 3.99±0.26; p<0.05 each vs. vehicle). By augmenting the glucose stimulated increase in plasma insulin, 55P0110 thus shows distinct anti-hyperglycaemic action in combination with low risk for fasting hypoglycaemia in mice. In summary, we have discovered a novel class of fully synthetic substituted quinazolidines with an attractive pharmacological profile that recommends the structures for further evaluation as candidates for the treatment of diabetes mellitus.

Targeting β-catenin signaling for therapeutic intervention in MEN1-deficient pancreatic neuroendocrine tumours.

Inactivating MEN1 mutations are the most common genetic defects present in sporadic and inherited pancreatic neuroendocrine tumours (PNETs). The lack of interventional therapies prompts us to explore the therapeutic approach of targeting β-catenin signalling in MEN1-mutant PNETs. Here we show the MEN1-encoded scaffold protein menin regulates phosphorylation of β-catenin. β-catenin signalling is activated in MEN1-mutant human and mouse PNETs. Conditional knockout of β-catenin suppresses the tumorigenesis and growth of Men1-deficient PNETs, and significantly prolongs the survival time in mice. Suppression of β-catenin signalling by genetic ablation or a molecular antagonist inhibits the expression of proproliferative genes in menin-null PNETs and potently improves hyperinsulinemia and hypoglycemia in mice. Blockade of β-catenin has no adverse effect on physiological function of pancreatic β-cells. Our data demonstrate that β-catenin signalling is an effective therapeutic target for MEN1-mutant PNETs. Our findings may contribute to individualized and combined medication treatment for PNETs.

Purification and functional characterization of pancreatic insulin from camel (Camelus dromedarius)

Large-scale production of insulin still represents the key step in helping diabetic patients throughout the world. Many species and approaches have been used for the production of insulin. In this study, we purified and characterized for the first time pancreatic insulin from the Arabian camel (Camelus dromedarius) using a modified acid-alcohol extraction method. After extraction insulin was purified using a one-step gel filtration on a Sephadex G-50 column leading to a purification yield of 80mg/kg (20%) of camel pancreas. The purity of camel insulin was assessed by SDS–PAGE and HPLC using insulin from human, bovine and porcine as standards. Molecular weight was determined for purified camel insulin as 5800 Daltons and its amino acid composition is similar to that known for other species. The functional characterization of purified crude camel insulin was demonstrated in vitro by positive competition by radioimmunoassay and in vivo showing camel insulin inducing acute hypoglycaemia in mice. Together, our study reports for the first time the successful purification of functional insulin from camel pancreas with similar properties compared to other insulin species. This is of great interest given that the camel represents considerable economic worth in many countries.

Abstract A246: Development of an IGF2 super-antagonist by directed evolution of IGF2R.

Abstract Insulin-like growth factor 2 (IGF2) plays an important role both in human and mouse embryonic development and in tumour growth. IGF2 causes IGF1R and insulin receptor isoform A, IR(A), dependent activation of proliferation (MAPK) and survival pathways (AKT-PI3K). IGF2 bioavailability is tightly regulated by six IGF binding proteins and by the IGF2/mannose 6-phosphate receptor (IGF2R). IGF2R is a 15-domain extracellular receptor, frequently mutated in cancer, which clears IGF2 via binding to domain 11 and internalising it for lysosomal degradation. Increased supply of IGF2 occurs in hepatocellular, breast, prostate, colorectal and ovarian tumours. Excessive amounts of IGF2 secreted by some tumours, mainly sarcoma, can lead to activation of IR(A) and cause non-islet cell tumour induced hypoglycaemia. Approaches aiming to counteract the activity of IGF2 by targeting IGF1R often fail due to IGF2-mediated activation of IR(A). A ligand trap that exploits the high specificity and affinity of IGF2R for IGF2 has been designed by the fusion of a mutated form of human domain 11 of IGF2R to a C-terminal human IgG1 Fc domain (Fc-dom11). For the ligand trap to function as a clinically useful IGF2 super-antagonist, the binding affinity of domain 11 to IGF2 was further improved. The binding affinity optimisation strategy consisted of random mutagenesis of domain 11 loops involved in IGF2 binding, followed by yeast surface display in P. Pastoris, FACS and SPR screening, as well as structure-directed mutagenesis, complemented with NMR studies. Several mutants showing enhanced IGF2 binding have been identified, the combination of separately identified mutations improving affinity up to 100 fold. In particular, the mutant domain 113-4D shows an increase in affinity through both on- and off-rate modification attributed to the decrease in flexibility of one of the loops involved in IGF2 binding, as suggested by NMR results. The ability of Fc-dom113-4D to function as an IGF2 antagonist has been tested in vitro, using hepatocellular carcinoma cell lines, and in vivo, where it counteracts IGF2-induced hypoglycaemia in mice. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A246. Citation Format: Susana Frago, Madeleine Strickland, Jennifer Hughes, Christopher Williams, Ryan Nicholls, Lee Garner, Dellel Rezgui, Matthew P. Crump, A. Bassim Hassan. Development of an IGF2 super-antagonist by directed evolution of IGF2R. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A246.

Brain Glucose Transport and Phosphorylation Under Acute Insulin-Induced Hypoglycemia in Mice: An 18F-FDG PET Study

We addressed the questions of how cerebral glucose transport and phosphorylation change under acute hypoglycemia and what the underlying mechanisms of adaptation are. Quantitative (18)F-FDG PET combined with the acquisition of real-time arterial input function was performed on mice. Hypoglycemia was induced and maintained by insulin infusion. PET data were analyzed with the 2-tissue-compartment model for (18)F-FDG, and the results were evaluated with Michaelis-Menten saturation kinetics. Glucose clearance from plasma to brain (K1,glc) and the phosphorylation rate constant increased with decreasing plasma glucose (Gp), in particular at a Gp of less than 2.5 mmol/L. Estimated cerebral glucose extraction ratios taking into account an increased cerebral blood flow (CBF) at a Gp of less than 2 mmol/L were between 0.14 and 0.79. CBF-normalized K1,glc values were in agreement with saturation kinetics. Phosphorylation rate constants indicated intracellular glucose depletion at a Gp of less than 2-3 mmol/L. When brain regions were compared, glucose transport under hypoglycemia was lowest in the hypothalamus. Alterations in glucose transport and phosphorylation, as well as intracellular glucose depletion, under acute hypoglycemia can be modeled by saturation kinetics taking into account an increase in CBF. Distinct transport kinetics in the hypothalamus may be involved in its glucose-sensing function.

Hypoglycemia Reduces Vascular Endothelial Growth Factor A Production by Pancreatic Beta Cells as a Regulator of Beta Cell Mass

VEGF-A expression in beta cells is critical for pancreatic development, formation of islet-specific vasculature, and Insulin secretion. However, two key questions remain. First, is VEGF-A release from beta cells coupled to VEGF-A production in beta cells? Second, how is the VEGF-A response by beta cells affected by metabolic signals? Here, we show that VEGF-A secretion, but not gene transcription, in either cultured islets or purified pancreatic beta cells, was significantly reduced early on during low glucose conditions. In vivo, a sustained hypoglycemia in mice was induced with Insulin pellets, resulting in a significant reduction in beta cell mass. This loss of beta cell mass could be significantly rescued with continuous delivery of exogenous VEGF-A, which had no effect on beta cell mass in normoglycemic mice. In addition, an increase in apoptotic endothelial cells during hypoglycemia preceded an increase in apoptotic beta cells. Both endothelial and beta cell apoptosis were prevented by exogenous VEGF-A, suggesting a possible causative relationship between reduced VEGF-A and the loss of islet vasculature and beta cells. Furthermore, in none of these experimental groups did beta cell proliferation and islet vessel density change, suggesting a tightly regulated balance between these two cellular compartments. The average islet size decreased in hypoglycemia, which was also prevented by exogenous VEGF-A. Taken together, our data suggest that VEGF-A release in beta cells is independent of VEGF-A synthesis. Beta cell mass can be regulated through modulated release of VEGF-A from beta cells based on physiological need.

Oral delivery of bioencapsulated exendin‐4 expressed in chloroplasts lowers blood glucose level in mice and stimulates insulin secretion in beta‐TC6 cells

Glucagon-like peptide (GLP-1) increases insulin secretion but is rapidly degraded (half-life: 2min in circulation). GLP-1 analogue, exenatide (Byetta) has a longer half-life (3.3-4h) with potent insulinotropic effects but requires cold storage, daily abdominal injections with short shelf life. Because patients with diabetes take >60000 injections in their life time, alternative delivery methods are highly desired. Exenatide is ideal for oral delivery because insulinotropism is glucose dependent, with reduced risk of hypoglycaemia even at higher doses. Therefore, exendin-4 (EX4) was expressed as a cholera toxin B subunit (CTB)-fusion protein in tobacco chloroplasts to facilitate bioencapsulation within plant cells and transmucosal delivery in the gut via GM1 receptors present in the intestinal epithelium. The transgene integration was confirmed by PCR and Southern blot analysis. Expression level of CTB-EX4 reached up to 14.3% of total leaf protein (TLP). Lyophilization of leaf material increased therapeutic protein concentration by 12- to 24-fold, extended their shelf life up to 15months when stored at room temperature and eliminated microbes present in fresh leaves. The pentameric structure, disulphide bonds and functionality of CTB-EX4 were well preserved in lyophilized materials. Chloroplast-derived CTB-EX4 showed increased insulin secretion similar to the commercial EX4 in beta-TC6, a mouse pancreatic cell line. Even when 5000-fold excess dose of CTB-EX4 was orally delivered, it stimulated insulin secretion similar to the intraperitoneal injection of commercial EX4 but did not cause hypoglycaemia in mice. Oral delivery of the bioencapsulated EX4 should eliminate injections, increase patient compliance/convenience and significantly lower their cost.

Inflammatory Induction of Human Resistin Causes Insulin Resistance in Endotoxemic Mice

OBJECTIVEAlthough adipocyte-derived murine resistin links insulin resistance to obesity, the role of human resistin, predominantly expressed in mononuclear cells and induced by inflammatory signals, remains unclear. Given the mounting evidence that obesity and type 2 diabetes are inflammatory diseases, we sought to determine the relationship between inflammatory increases in human resistin and insulin resistance.RESEARCH DESIGN AND METHODSTo investigate the role of human resistin on glucose homeostasis in inflammatory states, we generated mice lacking murine resistin but transgenic for a bacterial artificial chromosome containing human resistin (BAC-Retn), whose expression was similar to that in humans. The metabolic and molecular phenotypes of BAC-Retn mice were assessed after acute and chronic endotoxemia (i.e., exposure to inflammatory lipopolysaccharide).RESULTSWe found that BAC-Retn mice have circulating resistin levels within the normal human range, and similar to humans, lipopolysaccharide markedly increased serum resistin levels. Acute endotoxemia caused hypoglycemia in mice lacking murine resistin, and this was attenuated in BAC-Retn mice. In addition, BAC-Retn mice developed severe hepatic insulin resistance under chronic endotoxemia, accompanied by increased inflammatory responses in liver and skeletal muscle.CONCLUSIONSThese results strongly support the role of human resistin in the development of insulin resistance in inflammation. Thus, human resistin may link insulin resistance to inflammatory diseases such as obesity, type 2 diabetes, and atherosclerosis.

Ketogenic Diet Disrupts the Circadian Clock and Increases Hypofibrinolytic Risk by Inducing Expression of Plasminogen Activator Inhibitor-1

Metabolic disorders such as diabetes and obesity are considered risk factors for cardiovascular diseases by increasing levels of blood plasminogen activator inhibitor-1 (PAI-1). Ketogenic diets (KDs) have been used as an approach to weight loss in both obese and nonobese individuals. We examined circadian changes in plasma PAI-1 and its mRNA expression levels in tissues from mice fed with a KD (KD mice), to evaluate its effects on fibrinolytic functions. Two weeks on the kDa increased plasma levels of free fatty acids and ketones accompanied by hypoglycemia in mice. Plasma PAI-1 concentrations were extremely elevated in accordance with mRNA expression levels in the heart and liver, but not in the kidneys of KD mice. Circadian expression of PAI-1 mRNA was phase-advanced for 4.7, 7.9, and 7.8 hours in the heart, kidney, and adipose tissues, respectively, as well as that of circadian genes mPer2 and DBP in KD mice, suggesting that peripheral clocks were phase-advanced by ketosis despite feeding ad libitum under a periodic light-dark cycle. The circadian clock that regulates behavioral activity rhythms was also phase-advanced, and its free-running period was significantly shortened in KD mice. Our findings suggest that ketogenic status increases hypofibrinolytic risk by inducing abnormal circadian expression of PAI-1.

Interleukin‐1β and Insulin Elicit Different Neuroendocrine Responses to Hypoglycemia

Interleukin (IL)-1beta induces a prolonged hypoglycemia in mice that is not caused by a reduction in food intake and is dissociable from insulin effects. There is a peripheral component in the hypoglycemia that the cytokine induces resulting from an increased glucose uptake, an effect that can be exerted in a paracrine fashion at the site where IL-1 is locally produced. However, the maintenance of hypoglycemia is controlled at brain levels because the blockade of IL-1 receptors in the central nervous system inhibits this effect to a large extent. Furthermore, there is evidence that the cytokine interferes with counter regulation to hypoglycemia. Here we report that administration of IL-1 or long-lasting insulin results in different changes in food intake and in neuroendocrine mechanisms 8 h following induction of the same degree of hypoglycemia (40-45% decrease in glucose blood levels). Insulin, but not IL-1, caused an increase in food intake and an endocrine response that tends to reestablish euglycemia. Conversely, a decrease in noradrenergic and an increase in serotonergic activity in the hypothalamus occur in parallel with a reduction of glucose blood levels only in IL-1-treated mice, effects that can contribute to the maintenance of hypoglycemia. These results are compatible with the proposal that IL-1 acting in the brain can reset glucose homeostasis at a lower level. The biologic significance of this effect is discussed.

Acid amyloglucosidase and carbohydrate regulation. 3. The induction of sulphonylurea-stimulated insulin release and its dependence on intracellular monoamines.

The induction of sulphonylurea-stimulated insulin release and its dependence on intracellular monoamine levels in the β-cells was studied in mice. Model experiments in vitro on the interaction between monoamines and purified fungal acid amyloglucosidase were performed.1) Glibenclamide-induced hypoglycemia in mice was prolonged by pretreatment with a small dose of purified fungal acid amyloglucosidase, which itself did not influence the blood glucose level.2) Two minutes following the intravenous injection of glibenclamide, the "free" activity of acid amyloglucosidase in islet homogenates prepared in osmotically protected media was increased. This increase was found to be dose dependent.3) Glibenclamide-induced insulin release in adrenalectomized animals was significantly inhibited by pretreatment with the lysosomal stabilizer dexamethasone and significantly enhanced by pretreatment with the lysosomal labilizer progesterone.4) Glibenclamide-induced insulin release in animals pretreated with a small dose of purified fungal acid amyloglucosidase was considerably enhanced. This increase was significantly inhibited following administration of the monoamine precursors L-5-hydroxytryptophan (L-5-HTP) and L-3,4-dihydroxyphenylalanine (L-DOPA) and also after pretreatment with the non-hydrazine monoamine oxidase inhibitor pargyline. L-DOPA was found to be a more potent inhibitor than L-5-HTP. Combined treatment with pargyline and L-DOPA further decreased the response.5) Model experiments in vitro on the interaction between amines and acid amyloglucosidase activity (purified fungal acid amyloglucosidase) showed a pH-dependent inhibitory action of the monoamines dopamine (DA) and 5-hydroxytryptamine (5-HT) on amyloglucosidase activity. DA was more potent than 5-HT.6) From the data presented it is assumed that the induction of sulphonylurea-stimulated insulin release is elicited through the following events in the β-cell: Lysosomal activation - Increase of the "free" activity of the lysosomal acid amyloglucosidase - Hydrolytic glycogenosis through acid amyloglucosidase (reaction product: glucose) - Insulin release. From a functional point of view it is proposed that lysosomes taking part in insulin secreting processes should be designated crinosomes.

Glutamate Is a Positive Autocrine Signal for Glucagon Release

An important feature of glucose homeostasis is the effective release of glucagon from the pancreatic alpha cell. The molecular mechanisms regulating glucagon secretion are still poorly understood. We now demonstrate that human alpha cells express ionotropic glutamate receptors (iGluRs) that are essential for glucagon release. A lowering in glucose concentration results in the release of glutamate from the alpha cell. Glutamate then acts on iGluRs of the AMPA/kainate type, resulting in membrane depolarization, opening of voltage-gated Ca(2+) channels, increase in cytoplasmic free Ca(2+) concentration, and enhanced glucagon release. In vivo blockade of iGluRs reduces glucagon secretion and exacerbates insulin-induced hypoglycemia in mice. Hence, the glutamate autocrine feedback loop endows the alpha cell with the ability to effectively potentiate its own secretory activity. This is a prerequisite to guarantee adequate glucagon release despite relatively modest changes in blood glucose concentration under physiological conditions.

(244) Symptomatic hypoglycemia due to escalating doses of intravenous methadone

Several case reports exist linking propoxyphene, a drug structurally related to methadone, to hypoglycemia. Morphine-induced hypoglycemia in mice has been shown to be inhibited by co-administration of naloxone. Blunted responses to insulin-induced hypoglycemia as well as delayed and inhibited insulin responses to food ingestion have been demonstrated in methadone maintenance patients. However, there are no published reports of methadone causing or contributing to symptomatic hypoglycemia. In this case, a 46 year old non-diabetic man with stage IV rectosigmoid cancer and renal failure that improved after nephrostomy tubes placement presented with severe abdominal pain due to a rapidly growing right pelvic wall mass.

Convergent and Diastereoselective Synthesis of the Polycyclic Pyran Core of Saudin

The natural product saudin was found to induce hypoglycemia in mice and, therefore, could be an appealing lead structure for the development of new agents to treat diabetes. A diastereoselective tandem Stille-oxa-electrocyclization reaction has been developed which provides access to the core structure of saudin in a rapid and convergent manner. This new reaction has been extended to the convergent preparation of a series of polycyclic pyran systems. Progress has been made on the advancement of these complex pyran systems toward the natural product. A complete account of these synthetic efforts is presented.

Counterregulatory deficits occur within 24 h of a single hypoglycemic episode in conscious, unrestrained, chronically cannulated mice.

Hypoglycemia-induced counterregulatory failure is a dangerous complication of insulin use in diabetes mellitus. Controlled hypoglycemia studies in gene knockout models, which require the use of mice, would aid in identifying causes of defective counterregulation. Because stress can influence counterregulatory hormones and glucose homeostasis, we developed glucose clamps with remote blood sampling in conscious, unrestrained mice. Male C57BL/6 mice implanted with indwelling carotid artery and jugular vein catheters were subjected to 2 h of hyperinsulinemic glucose clamps 24 h apart, with a 6-h fast before each clamp. On day 1, blood glucose was maintained (euglycemia, 178 +/- 4 mg/dl) or decreased to 62 +/- 1 mg/dl (hypoglycemia) by insulin (20 mU x kg(-1) x min(-1)) and variable glucose infusion. Donor blood was continuously infused to replace blood sample volume. Baseline plasma epinephrine (32 +/- 8 pg/ml), corticosterone (16.1 +/- 1.8 microg/dl), and glucagon (35 +/- 3 pg/ml) were unchanged during euglycemia but increased significantly during hypoglycemia, with a glycemic threshold of approximately 80 mg/dl. On day 2, all mice underwent a hypoglycemic clamp (blood glucose, 64 +/- 1 mg/dl). Compared with mice that were euglycemic on day 1, previously hypoglycemic mice had significantly higher glucose requirements and significantly lower plasma glucagon and corticosterone (n = 6/group) on day 2. Epinephrine tended to decrease, although not significantly, in repeatedly hypoglycemic mice. Pre- and post-clamp insulin levels were similar between groups. We conclude that counterregulatory responses to acute and repeated hypoglycemia in unrestrained, chronically cannulated mice reproduce aspects of counterregulation in humans, and that repeated hypoglycemia in mice is a useful model of counterregulatory failure.

Increased glucose tolerance and reduced adiposity in the absence of fasting hypoglycemia in mice with liver-specific Gsα deficiency

The G protein G(s)alpha is essential for hormone-stimulated cAMP generation and is an important metabolic regulator. We investigated the role of liver G(s)-signaling pathways by developing mice with liver-specific G(s)alpha deficiency (LGsKO mice). LGsKO mice had increased liver weight and glycogen content and reduced adiposity, whereas survival, body weight, food intake, and metabolic rates at ambient temperature were unaffected. LGsKO mice had increased glucose tolerance with both increased glucose-stimulated insulin secretion and increased insulin sensitivity in liver and muscle. Fed LGsKO mice were hypoglycemic and hypoinsulinemic, with low expression of hepatic gluconeogenic enzymes and PPARgamma coactivator-1. However, LGsKO mice maintained normal fasting glucose and insulin levels, probably due to prolonged breakdown of glycogen stores and possibly increased extrahepatic gluconeogenesis. Lipid metabolism was unaffected in fed LGsKO mice, but fasted LGsKO mice had increased lipogenic and reduced lipid oxidation gene expression in liver and increased serum triglyceride and FFA levels. LGsKO mice had very high serum glucagon and glucagon-like peptide-1 levels and pancreatic alpha cell hyperplasia, probably secondary to hepatic glucagon resistance and/or chronic hypoglycemia. Our results define novel roles for hepatic G(s)-signaling pathways in glucose and lipid regulation, which may prove useful in designing new therapeutic targets for diabetes and obesity.

Susceptibility of the developing brain to acute hypoglycemia involving A1adenosine receptor activation

It has been suggested that the developing brain is less vulnerable to the adverse effects of hypoglycemia than the mature brain; however, this issue remains controversial. We also do not know the magnitude or duration of hypoglycemia needed to trigger hypoglycemic brain injury during development. To address this issue a series of in vivo and in vitro studies were performed. First, we established an acute model of insulin-induced hypoglycemia in mice by administering 3 U/kg of neutral-protamine Hagadorn insulin subcutaneously. When we examined degenerating neurons in hippocampus and striatum by TUNEL labeling, injury was observed after 4 h of hypoglycemia in postnatal day (P)7 mice, and we observed more cell injury in animals rendered hypoglycemic at P7 than at P21. Studies of hippocampal slice cultures revealed that reduction in glucose concentration induced more neuronal injury in slices prepared from P3 and P7 than from P14 and P21 mice. Treatment of slices with an adenosine A(1) receptor (A(1)AR) antagonist reduced the hypoglycemic damage, whereas agonists increased damage, particularly in slices prepared from very young pups. This suggests a critically important role for A(1)ARs, which was further demonstrated by the reduction of hypoglycemic damage in hippocampal slices prepared from A(1)AR(-/-) mice. Furthermore, insulin-induced hypoglycemia in P7 A(1)AR(-/-) mice did not increase TUNEL-positive cells, but a major increase was seen in A(1)AR(+/-) mice. These observations show that the developing nervous system is indeed sensitive to acute hypoglycemic injury and that A(1)AR activation contributes to damage induced by hypoglycemia, particularly in immature mouse brain.

Combined corticotropin-releasing hormone and glucocorticoid deficiency does not enhance counterregulatory responses after recurrent hypoglycemia in mice

Glucocorticoids and corticotropin-releasing hormone (CRH) have been proposed to inhibit counterregulatory responses to recurrent hypoglycemia. We used the CRH knockout (CRH KO) mouse to test the hypothesis that combined CRH and glucocorticoid deficiency would prevent development of counterregulatory deficits after repeated hypoglycemia. To develop a mouse model of recurrent hypoglycemia, we first tested the effects of daily lente insulin injection on counterregulatory responses to acute hypoglycemia in male C57BL/6 mice. Treatment with up to 250 U/kg per day lente insulin resulted in significantly greater decreases in plasma glucose, suggestive of impaired counterregulation, after hypoglycemia induced by acute insulin injection. Plasma catecholamine responses to hypoglycemia in repeatedly hypoglycemic C57BL/6 mice were unexpectedly higher than in naive mice, which we interpreted as a compensatory response to the greater decreases in plasma glucose. Lente insulin doses had to be reduced (50-75 U/kg per day) for CRH KO mice to survive repeated hypoglycemia. Wild-type (WT) mice treated with 50 to 75 U/kg per day lente insulin exhibited enhanced sympathetic activity after hypoglycemia, resembling the compensatory responses associated with impaired glucose homeostasis in C57BL/6 mice treated with 250 U/kg per day lente insulin. During acute hypoglycemia, CRH KO mice maintained higher plasma glucose levels that correlated with higher plasma norepinephrine and greater glycogen mobilization. Recurrent hypoglycemia did not enhance liver glycogen depletion or the markedly impaired glucocorticoid and epinephrine responses to hypoglycemia in CRH KO mice. Previously hypoglycemic CRH KO mice also did not display the further increases in sympathetic activity that in WT mice were suggestive of compensation for impaired counterregulation. Despite the apparent resistance of CRH KO mice to the counterregulatory effects of repeated hypoglycemia, their greater mortality after hypoglycemia tolerated by WT mice indicates that combined CRH and glucocorticoid deficiency does not significantly improve counterregulation after repeated hypoglycemia.