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Jean M Whaley,1 Mark Tirmenstein,2 Timothy P Reilly,2 Simon M Poucher,3 JoAnne Saye,4 Shamik Parikh,5 James F List61Bristol-Myers Squibb, Metabolic Disease Discovery Biology, Research and Development, Princeton, NJ, USA; 2Bristol-Myers Squibb, Drug Safety Evaluation, Research and Development, New Brunswick and Princeton, NJ, USA; 3AstraZeneca, Cardiovascular and Gastrointestinal Innovative Medicines Science Unit, Alderley Park, Macclesfield, Cheshire, UK; 4AstraZeneca, Global Safety Assessment, Research and Development, Wilmington, DE, USA; 5AstraZeneca, Cardiovascular, Clinical Development, Wilmington, DE, USA; 6Bristol-Myers Squibb, Global Clinical Development, Research and Development, Princeton, NJ, USAAbstract: Sodium-glucose cotransporter-2 (SGLT2) inhibitors are a novel class of glucuretic, antihyperglycemic drugs that target the process of renal glucose reabsorption and induce glucuresis independently of insulin secretion or action. In patients with type 2 diabetes mellitus, SGLT2 inhibitors have been found to consistently reduce measures of hyperglycemia, including hemoglobin A1c, fasting plasma glucose, and postprandial glucose, throughout the continuum of disease. By inducing the renal excretion of glucose and its associated calories, SGLT2 inhibitors reduce weight and have the potential to be disease modifying by addressing the caloric excess that is believed to be one of the root causes of type 2 diabetes mellitus. Additional benefits, including the possibility for combination with insulin-dependent antihyperglycemic drugs, a low potential for hypoglycemia, and the ability to reduce blood pressure, were anticipated from the novel mechanism of action and have been demonstrated in clinical studies. Mechanism-related risks include an increased incidence of urinary tract and genital infections and the possibility of over-diuresis in volume-sensitive patients. Taken together, the results of Phase III clinical studies generally point to a positive benefit-risk ratio across the continuum of diabetes patients. To date, data on dapagliflozin, a selective SGLT2 inhibitor in development, demonstrate that the kidney is an efficacious and safe target for therapy, and that SGLT2 inhibition may have benefits for patients with type 2 diabetes mellitus beyond glycemic control.Keywords: type 2 diabetes mellitus, SGLT2, dapagliflozin, mechanism of action

Key Points: Meldonium inhibits endogenous carnitine synthesis and tissue uptake, and accelerates urinary carnitine excretion, although the impact of meldonium-mediated muscle carnitine depletion on whole-body fuel selection, and muscle fuel metabolism and its molecular regulation is under-investigated. Ten days of oral meldonium administration did not impact on food or fluid intake, physical activity levels or body weight gain in the rat, whereas it depleted muscle carnitine content (all moieties), increased whole-body carbohydrate oxidation and muscle and liver glycogen utilization, and reduced whole-body fat oxidation. Meldonium reduced carnitine transporter protein expression across muscles of different contractile and metabolic phenotypes. A TaqMan PCR low-density array card approach revealed the abundance of 189 mRNAs regulating fuel selection was altered in soleus muscle by meldonium, highlighting the modulation of discrete cellular functions and metabolic pathways. These novel findings strongly support the premise that muscle carnitine availability is a primary regulator of fuel selection in vivo., Abstract: The body carnitine pool is primarily confined to skeletal muscle, where it regulates carbohydrate (CHO) and fat usage. Meldonium (3-(2,2,2-trimethylhydrazinium)-propionate) inhibits carnitine synthesis and tissue uptake, although the impact of carnitine depletion on whole-body fuel selection, muscle fuel metabolism and its molecular regulation is under-investigated. Male lean Zucker rats received water (control, n = 8) or meldonium-supplemented water (meldonium, n = 8) for 10 days [1.6 g kg -1 body mass (BM) day -1 days 1-2, 0.8 g kg -1  BM day -1 thereafter]. From days 7-10, animals were housed in indirect calorimetry chambers after which soleus muscle and liver were harvested. Food and fluid intake, weight gain and physical activity levels were similar between groups from days 7 to 10. Compared to control, meldonium depleted muscle total carnitine (P < 0.001) and all carnitine esters. Furthermore, whole-body fat oxidation was less (P < 0.001) and CHO oxidation was greater (P < 0.05) compared to the control, whereas soleus and liver glycogen contents were less (P < 0.01 and P < 0.01, respectively). In a second study, male Wistar rats received water (n = 8) or meldonium-supplemented water (n = 8) as above, and kidney, heart and extensor digitorum longus muscle (EDL) and soleus muscles were collected. Compared to control, meldonium depleted total carnitine content (all P < 0.001), reduced carnitine transporter protein and glycogen content, and increased pyruvate dehydrogenase kinase 4 mRNA abundance in the heart, EDL and soleus. In total, 189 mRNAs regulating fuel selection were differentially expressed in soleus in meldonium vs. control, and a number of cellular functions and pathways strongly associated with carnitine depletion were identified. Collectively, these data firmly support the premise that muscle carnitine availability is a primary regulator of fuel selection in vivo., (© 2017 The University of Nottingham. The Journal of Physiology © 2017 The Physiological Society.)

Introduction: Accurate assessment of muscle insulin sensitivity requires measurement of insulin concentration in interstitial fluid (ISF), but has proved difficult. We aimed to optimise measurement of ISF insulin concentrations in rat muscles in vivo using microdialysis., Methods: Factorial experimental design experiments were performed in vitro to determine optimal conditions for insulin recovery with microdialysis probes. These conditions were tested in vivo, adjusted appropriately and used in lean and obese Zucker rats to compare ISF insulin concentrations basally and during hyperinsulinaemic-euglycaemic (HE) clamp., Results: Optimal conditions in vivo were: a 100kDa microdialysis probe inserted in muscle, perfused with 1% BSA, 1.5mM glucose in 0.9% sodium chloride at 1μl/min. Samples were collected into siliconised glass microvials. As a reference for insulin, we established a protocol of inulin infusion, beginning at -80min and reaching equilibrium within 60min. HE clamp, beginning at 0min, increased ISF insulin concentration from 122±56 basally to 429±180pmol/l (P<0.05) in lean rats and from 643±165 to 1087±243pmol/l (P=0.07) in obese rats; ISF insulin concentrations were significantly higher throughout in obese rats. The difference between ISF and plasma insulin concentration (ISF:plasma ratio) was substantially higher in obese rats, but fell to similar values in obese and lean rats during HE clamp., Discussion: Optimising insulin recovery with microdialysis allowed accurate measurement of basal ISF insulin in muscle of lean and obese Zucker rats and indicates insulin transport across capillaries is impaired in obese rats, basally and during hyperinsulinaemia., (Copyright © 2017 Elsevier Inc. All rights reserved.)

In 1997, Congress amended the Individuals with Disabilities Education Act ("IDEA") to include provisions meant to assist school districts in educating students with behavioral needs. These amendments required schools to use functional behavioral assessments ("FBA") and behavior intervention plans ("BIP") under certain circumstances. Congress did not, however, include a definition of or substantive requirements for either system of behavior management. As a result, although BIPs and FBAs are now federally mandated requirements, and it is clear that disregarding behavioral issues is a denial of a free appropriate public education ("FAPE"), the IDEA’s adjudicative standard, there is no clear consensus as to whether a student with behavioral needs must have an FBA or a BIP or what either must include. The IDEA’s lack of guidance has resulted in inconsistent and often contradictory court rulings, and the lack of specific definitions and procedures has allowed schools to develop purportedly legal but substantively deficient behavior evaluations and intervention plans for special needs students. Despite this confusion, some courts have properly looked to the administrative record for guidance on the substantive elements of FBAs and BIPs. Deference to hearing officers, along with other provisions within the IDEA, such as Child Find and inclusion obligations, may assist courts in determining whether a school’s failure to use an FBA and a BIP for a student with behavioral needs falls short of Board of Education v. Rowley’s requirements for FAPE. FBAs and BIPs aim to prevent and correct student misconduct before it escalates and results in drastic disciplinary action. Given the impact punitive school discipline policies have had on students with special needs, the added procedural safeguards these proactive behavior management schemes could provide are imperative to dismantling the school-to-prison pipeline.

Animal models are invaluable tools which allow us to investigate the microbiome-host dialogue. However, experimental design introduces biases in the data that we collect, also potentially leading to biased conclusions. With obesity at pandemic levels animal models of this disease have been developed; we investigated the role of experimental design on one such rodent model. We used 454 pyrosequencing to profile the faecal bacteria of obese (n = 6) and lean (homozygous n = 6; heterozygous n = 6) Zucker rats over a 10 week period, maintained in mixed-genotype cages, to further understand the relationships between the composition of the intestinal bacteria and age, obesity progression, genetic background and cage environment. Phylogenetic and taxon-based univariate and multivariate analyses (non-metric multidimensional scaling, principal component analysis) showed that age was the most significant source of variation in the composition of the faecal microbiota. Second to this, cage environment was found to clearly impact the composition of the faecal microbiota, with samples from animals from within the same cage showing high community structure concordance, but large differences seen between cages. Importantly, the genetically induced obese phenotype was not found to impact the faecal bacterial profiles. These findings demonstrate that the age and local environmental cage variables were driving the composition of the faecal bacteria and were more deterministically important than the host genotype. These findings have major implications for understanding the significance of functional metagenomic data in experimental studies and beg the question; what is being measured in animal experiments in which different strains are housed separately, nature or nurture?

The measurement of tissue lipid and glycogen contents and the establishment of normal levels of variability are important when assessing changes caused by pathology or treatment. We measured hepatic and skeletal muscle lipid and glycogen levels using (1)H and (13)C MRS at 3 T in groups of subjects with and without type 2 diabetes. Within-visit reproducibility, due to repositioning and instrument errors was determined from repeat measurements made over 1 h. Natural variability was assessed from separate measurements made on three occasions over 1 month. Hepatic lipid content was greater in subjects with diabetes relative to healthy subjects (p = 0.03), whereas levels of hepatic and skeletal muscle glycogen, and of intra- and extra-myocellular lipid, were similar. The single-session reproducibility values (coefficient of variation, CV) for hepatic lipid content were 12% and 7% in groups of subjects with and without diabetes, respectively. The variability of hepatic lipid content over 1 month was greater than the reproducibility, with CV = 22% (p = 0.08) and CV = 44% (p = 0.004) in subjects with and without diabetes, respectively. Similarly, levels of variation in basal hepatic glycogen concentrations (subjects with diabetes, CV = 38%; healthy volunteers, CV = 35%) were significantly larger than single-session reproducibility values (CV = 17%, p = 0.02 and CV = 13%, p = 0.05, respectively), indicating substantial biological changes in basal concentrations over 1 month. There was a decreasing correlation in measurements of both hepatic lipid and glycogen content with increasing time between scans. Levels of variability in intra- and extra-myocellular lipid in the soleus muscle, and glycogen concentrations in the gastrocnemius muscle, tended to be larger than expected from single-session reproducibility, although these did not reach significance., (Copyright © 2013 John Wiley & Sons, Ltd.)

Introduction: Sodium-glucose co-transporter-2 (SGLT2) inhibitors are a novel class of agents for the treatment of type 2 diabetes mellitus (T2DM). By inhibiting SGLT2, they prevent renal glucose reabsorption, resulting in glucosuria., Areas Covered: The rationale for development of SGLT2 inhibitors is reviewed, with particular focus on the nine SGLT2 inhibitors currently in development. The authors compare the potency and SGLT2 selectivity of the agents, as well as the results from both animal and clinical studies, considering the potential implications they may have for clinical use., Expert Opinion: Current evidence suggests that SGLT2 inhibitors have similar efficacy in terms of glycemic control and also demonstrate benefits beyond glycemic reductions, including reductions in body weight and modest reductions in blood pressure. Additionally, they appear to preserve beta-cell function and improve insulin sensitivity. Their mechanism of action allows for combination of SGLT2 inhibitors with other antidiabetic drugs and use across the treatment continuum for T2DM. Potential differences in safety and efficacy based on observed differences in potency and selectivity among the SGLT2 inhibitors, particularly versus SGLT1, remain to be seen. Further long-term data, including post-marketing surveillance, are required to fully determine the safety profile of SGLT2 inhibitors in large patient groups.

Background: Hyperalimentation for 4 weeks is associated with raised liver enzymes and liver fat content (LFC), which are two common features found in individuals with diabetes., Aim: We evaluated the effect of two mixed meal challenges on LFC, liver enzymes and serum bio-markers of liver injury and fibrosis in 16 healthy volunteers (HV) and subjects with type 2 diabetes (T2DM)., Methods: Subjects (HV: 9 male, 7 female, aged 57.9 ± 1.7 years, body mass index (BMI) 27.1 kg/m(2); and T2DM: 11 male, 5 female, aged 62.1 ± 1.3 years, BMI 28.0 ± 0.4 kg/m(2)) consumed two meals at 1 h (884 kcal) and at 6 h (1,096 kcal). LFC determined by (1)H magnetic resonance spectroscopy, serum levels of liver enzymes, hyaluronic acid (HA), procollagen III N-terminal peptide (P3NP) and tissue inhibitor metalloproteinase-1 (TIMP-1) were estimated at time 0 (fasting) and 9 h (postprandial)., Results: Fasting LFC was higher in the T2DM group 7.6 % (4.9, 15.4) [median (inter-quartile range)] than in the HV group 2.3 % (0.8, 5.1) (p < 0.05) while levels of HA, P3NP and TIMP-1 were similar. Following the meal challenge there was no significant change in LFC. Subjects with T2DM had higher post-prandial rise in alanine transaminase (ALT) (p = 0.014), serum HA (p = 0.007) and P3NP (p = 0.015) compared with HV. Fasting LFC correlated with a greater post-prandial increase in P3NP levels in all subjects (Pearson correlation r = 0.53, p = 0.001)., Conclusions: In subjects with T2DM, a mixed meal challenge is associated with a significant elevation in the serum levels of ALT, HA and P3NP without significant changes in LFC. These markers should be performed in the fasted state.

Aims: We examined the effects of the oral dipeptidyl peptidase-4 (DPP-4) inhibitor saxagliptin on glycaemic control and pancreatic β-cell mass and morphology in a mouse model of type 2 diabetes mellitus (T2DM)., Methods: Male C57BL/6 mice (n = 12/group) aged 4 to 6 weeks and weighing >15 g received a high-fat diet throughout this 45-day study. After a 7-day handling period, baseline levels of plasma glucose, plasma insulin and glycated haemoglobin (HbA1c) were assessed. Animals were allocated to one of six groups: compound vehicle control, intraperitoneal streptozotocin (STZ, 50 mg/kg)-treated control and saxagliptin (10 mg/kg) or sitagliptin (10 mg/kg, positive control) initiated 7 days before or 1 day after STZ administration. Endpoints included changes in body weight, food and water consumption, glucose tolerance (approximately 3 weeks post-STZ), fasting glucose and HbA1c and immunohistochemical analyses of the pancreas., Results: Body weight, weight gain and food intake were reduced in STZ versus control mice. DPP-4 inhibitor treatment did not affect these changes, but the increase in water intake observed post-STZ administration was significantly attenuated with DPP-4 inhibitors whether initiated before or after STZ injury. Small but significant improvements in glycaemic control were observed with DPP-4 inhibitors versus the STZ control. Improved β-cell mass and morphology were observed with saxagliptin given pre- or post-STZ and sitagliptin given post-STZ., Conclusions: Saxagliptin mitigated damage to β-cells and improved glycaemic control in this mouse model of T2DM., (© 2012 Blackwell Publishing Ltd.)

Sodium-glucose co-transporter 2 (SGLT2) plays a key role in glucose homeostasis as the key transporter responsible for most renal glucose reabsorption in the proximal tubules of the kidney. Dapagliflozin is a potent, selective, and reversible inhibitor of SGLT2 that lowers blood glucose levels in an insulin-independent fashion. This novel agent has been studied extensively in patients with type 2 diabetes mellitus (T2DM). In these clinical trials, dapagliflozin significantly decreased glycated hemoglobin and fasting plasma glucose levels when administered alone or as add-on treatment in patients who were already receiving metformin, a sulfonylurea (glimepiride), pioglitazone, or insulin. Moreover, dapagliflozin decreased body weight when taken as monotherapy or in combination with metformin, a sulfonylurea, or insulin, and mitigated weight gain in patients receiving pioglitazone. Consistent with preclinical toxicology studies, dapagliflozin has a manageable adverse event profile that is largely predictable from its mechanism of action. While there are no clinically significant negative effects on renal function or electrolytes, dapagliflozin treatment is associated with increased frequencies of urinary tract infections and vulvovaginitis/balanitis. With a mechanism of action that is distinct from and complementary to that of existing antihyperglycemic therapies, dapagliflozin is an effective antihyperglycemic agent that is well tolerated and may enhance weight loss. As such, dapagliflozin promises to become an important adjunctive therapy for comprehensive treatment of T2DM.

Diabetic nephropathy (DN) is a major cause of end-stage renal disease. Yet the pathogenic mechanisms underlying the development of DN are not fully defined, partially due to lack of suitable models that mimic the complex pathogenesis of renal disease in diabetic patients. In this study, we describe early and late renal manifestations of DN and renal responses to long-term treatments with rosiglitazone or high-dose enalapril in ZSF1 rats, a model of metabolic syndrome, diabetes, and chronic renal disease. At 8 weeks of age, obese ZSF1 rats developed metabolic syndrome and diabetes (hyperglycemia, glucosuria, hyperlipidemia, and hypertension) and early signs of renal disease (proteinuria, glomerular collagen IV deposition, tubulointerstitial inflammation, and renal hypertrophy). By 32 weeks of age, animals developed renal histopathology consistent with DN, including mesangial expansion, glomerulosclerosis, tubulointerstitial inflammation and fibrosis, tubular dilation and atrophy, and arteriolar thickening. Rosiglitazone markedly increased body weight but reduced food intake, improved glucose control, and attenuated hyperlipidemia and liver and kidney injury. In contrast, rosiglitazone markedly increased cardiac hypertrophy via a blood pressure-independent mechanism. High-dose enalapril did not improve glucose homeostasis, but normalized blood pressure, and nearly prevented diabetic renal injury. The ZSF1 model thus detects the clinical observations seen with rosiglitazone and enalapril in terms of primary and secondary endpoints of cardiac and renal effects. This and previous reports indicate that the obese ZSF1 rat meets currently accepted criteria for progressive experimental diabetic renal disease in rodents, suggesting that this may be the best available rat model for simulation of human DN.

The glucose-insulin system is a challenging process to model due to the feedback mechanisms present, hence the implementation of a model-based approach to the system is an on-going and challenging research area. A new approach is proposed here which provides an effective way of characterising glycaemic regulation. The resulting model is built on the premise that there are three phases of insulin secretion, similar to those seen in a proportional-integral-derivative (PID) type controller used in engineering control problems. The model relates these three phases to a biological understanding of the system, as well as the logical premise that the homeostatic mechanisms will maintain very tight control of the system. It includes states for insulin, glucose, insulin action and a state to simulate an integral function of glucose. Structural identifiability analysis was performed on the model to determine whether a unique set of parameter values could be identified from the available observations, which should permit meaningful conclusions to be drawn from parameter estimation. Although two parameters--glucose production rate and the proportional control coefficient--were found to be unidentifiable, the former is not a concern as this is known to be impossible to measure without a tracer experiment, and the latter can be easily estimated from other means. Subsequent parameter estimation using Intravenous Glucose Tolerance Test (IVGTT) and hyperglycaemic clamp data was performed and subsequent model simulations have shown good agreement with respect to these real data., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Introduction: We characterised the development of Type 2 diabetes and associated changes in islet appearance in female ZDF rats and explored its suitability for studies with novel therapeutic agents., Methods: Female ZDF rats were either chow or high fat (60%) fed for up to 36 days and blood glucose and plasma insulin concentration measured. Additionally, we restored two groups of rats back to chow diet after ten and nineteen days of high fat feeding to determine the reversibility. Finally, two other groups of high fat-fed animals were dosed either orally with drug vehicle or had a minipump implanted subcutaneously to determine the effect of dosing method upon the progression of this disease model. The beta cell mass and morphology were assessed by immunohistochemistry for insulin., Results: High fat feeding elevated blood glucose compared to chow-fed controls which peaked by 15 days, and maintained throughout the study. Plasma insulin reached a maximum after 8 days, but declined over the remaining 4 weeks. Assessment of islets revealed marked disruption, dispersion and weaker insulin staining. The area and percentage β-cells were higher in high fat-fed animals. High fat diet treatment reversal when animals were moderately hyperglycaemic, when plasma insulin was still elevated, reversed the hyperglycaemia and maintained islet morphology similar to that of chow-fed animals. In contrast, dietary reversal when plasma insulin was declining, did not prevent continual decline in plasma insulin, β-cell mass or islet disruption. Oral dosing tended to increase blood glucose and decrease plasma insulin whereas administration by minipump lowered blood glucose., Discussion: The obese female ZDF rat offers the opportunity for preclinical evaluation of novel therapies directed towards improving pancreatic function, provided treatment is initiated prior to the precipitous decline in insulin production. Caution should be exercised in comparison of compounds administered by different dosing routes however., (Crown Copyright © 2010. Published by Elsevier Inc. All rights reserved.)

Aims: To investigate whether glucose lowering with the selective sodium glucose transporter 2 (SGLT2) inhibitor dapagliflozin would prevent or reduce the decline of pancreatic function and disruption of normal islet morphology., Methods: Female Zucker diabetic fatty (ZDF) rats, 7-8 weeks old, were placed on high-fat diet. Dapagliflozin (1 mg/kg/day, p.o.) was administered for ∼33 days either from initiation of high-fat diet or when rats were moderately hyperglycaemic. Insulin sensitivity and pancreatic function were evaluated using a hyperglycaemic clamp in anaesthetized animals (n = 5-6); β-cell function was quantified using the disposition index (DI) to account for insulin resistance compensation. Pancreata from a matched subgroup (n = 7-8) were fixed and β-cell mass and islet morphology investigated using immunohistochemical methods., Results: Dapagliflozin, administered from initiation of high-fat feeding, reduced the development of hyperglycaemia; after 24 days, blood glucose was 8.6 ± 0.5 vs. 13.3 ± 1.3 mmol/l (p < 0.005 vs. vehicle) and glycated haemoglobin 3.6 ± 0.1 vs. 4.8 ± 0.26% (p < 0.003 vs. vehicle). Dapagliflozin improved insulin sensitivity index: 0.08 ± 0.01 vs. 0.02 ± 0.01 in obese controls (p < 0.03). DI was improved to the level of lean control rats (dapagliflozin 0.29 ± 0.04; obese control 0.15 ± 0.01; lean 0.28 ± 0.01). In dapagliflozin-treated rats, β-cell mass was less variable and significant improvement in islet morphology was observed compared to vehicle-treated rats, although there was no change in mean β-cell mass with dapagliflozin. Results were similar when dapagliflozin treatment was initiated when animals were already moderately hyperglycaemic., Conclusion: Sustained glucose lowering with dapagliflozin in this model of type 2 diabetes prevented the continued decline in functional adaptation of pancreatic β-cells., (© 2010 AstraZeneca UK Ltd.)

Manganese-enhanced magnetic resonance imaging (MEMRI) is a novel imaging technique capable of monitoring calcium influx, in vivo. Manganese (Mn2+) ions, similar to calcium ions (Ca2+), are taken up by activated cells where their paramagnetic properties afford signal enhancement in T(1)-weighted MRI methodologies. In this study we have assessed Mn2+ distribution in mice using magnetization-prepared rapid gradient echo (MP-RAGE) based MRI, by measuring changes in T(1)-effective relaxation times (T(1)-eff), effective R(1)-relaxation rates (R(1)-eff) and signal intensity (SI) profiles over time. The manganese concentration in the tissue was also determined using inductively coupled plasma atomic emission spectrometry (ICP-AES). Our results show a strong positive correlation between infused dose of MnCl2 and the tissue manganese concentration. Furthermore, we demonstrate a linear relationship between R(1)-eff and tissue manganese concentration and tissue-specific Mn2+ distribution in murine tissues following dose-dependent Mn2+ administration. This data provides an optimized MnCl2 dose regimen for an MP-RAGE based sequence protocol for specific target organs and presents a potential 3D MRI technique for in vivo imaging of Ca2+ entry during Ca2+-dependent processes in a wide range of tissues., (Copyright © 2010 John Wiley & Sons, Ltd.)

The preclinical efficacy and safety of GPi921, a glycogen phosphorylase inhibitor, was assessed following twenty-eight days of administration to Zucker Diabetic Fatty (ZDF) rats. The ZDF rat is an animal model of type 2 diabetes mellitus (TTDM) which develops severe hyperglycemia. Inhibition of glycogen phosphorylase throughout the duration of the study was demonstrated by reductions in twenty-four-hour glucose profiles and glycated hemoglobin levels. In addition, progression towards hyperglycemia was halted in treated but not control animals, which developed hyperglycemia over the twenty-eight days of the study. Biochemical and histopathological analysis revealed large increases in hepatic glycogen, which closely paralleled the development of hepatomegaly and ultimately resulted in increases in hepatic lipids. Furthermore, prolonged glycogen phosphorylase inhibition resulted in an increased incidence and severity of other adverse pathological findings in the liver, such as inflammation, fibrosis, hemorrhage, and necrosis. The observed biochemical and histopathological phenotype of the liver closely resembled that seen in severe cases of human glycogen storage diseases (GSD) and hepatic glycogenosis in poorly controlled diabetes mellitus. These findings revealed that although glycogen phosphorylase inhibitors are efficacious agents for the control of hyperglycemia, prolonged treatment might have the potential to cause significant clinical hepatic complications that resemble those seen in GSD and hepatic glycogenosis.

Introduction: Glucose-stimulated insulin secretion (GSIS) is critical in mammalian fuel homeostasis and is diminished early in the evolution of beta-cell dysfunction, ultimately contributing to the development of Type 2 diabetes. We sought to standardise and validate the intravenous glucose tolerance test (IVGTT), a commonly used technique to assess GSIS, in anaesthetised and conscious cannulated male Han Wistar rats., Methods: Male Han Wistar rats were cannulated via the right jugular vein and left carotid artery. Anaesthetised and chronically cannulated conscious models underwent IVGTT using increasing doses of glucose (0.2, 0.5 and 1.0 g glucose/kg LBM) or following pre-treatment with Exendin-4 (EX-4) before receiving a 0.5 g glucose/kg LBM bolus dose. Blood glucose, plasma insulin and plasma C-peptide were measured at time-points throughout the experiments., Results: Dose-dependent increases in blood glucose, insulin and C-peptide (where measured) were observed following administration of increasing doses of an intravenous glucose bolus in both the anaesthetised and conscious cannulated rats. The 0.5 g glucose/kg LBM bolus resulted in an intermediate response and was used in the second part of the study. EX-4 pre-treatment in combination with glucose resulted in GSIS potentiation, as assessed by plasma insulin measurement alone (anaesthetised model) or insulin and C-peptide measurements (conscious model)., Discussion: The IVGTT was standardised in anaesthetised and conscious cannulated male Han Wistar rats by performing a glucose dose response study and validated by examining GSIS potentiation using EX-4. Based on these results, the 0.5 g glucose/kg LBM bolus dose is recommended as the dose to use to assess GSIS in any standardised screening phase of new compounds with the potential to enhance glucose-sensitive pancreatic function. The experimental conditions described in these studies could be transferred to disease models for more detailed assessment of novel compound efficacy.

Background and Purpose: Studies in cultured hepatocytes demonstrate glycogen synthase (GS) activation with glycogen phosphorylase (GP) inhibitors. The current study investigated whether these phenomena occurred in vivo using a novel GP inhibitor., Experimental Approach: An allosteric GP inhibitor, GPi688, was evaluated against both glucagon-mediated hyperglycaemia and oral glucose challenge-mediated hyperglycaemia to determine the relative effects against GP and GS in vivo., Key Results: In rat primary hepatocytes, GPi688 inhibited glucagons-mediated glucose output in a concentration dependent manner. Additionally GP activity was reduced and GS activity increased seven-fold. GPi688 inhibited glucagon-mediated hyperglycaemia in both Wistar (65%) & obese Zucker (100%) rats and demonstrated a long duration of action in the Zucker rat. The in vivo efficacy in the glucagon challenge model could be predicted by the equation; % glucagon inhibition=56.9+34.3[log ([free plasma]/rat IC50)], r=0.921). GPi688 also reduced the blood glucose of obese Zucker rats after a 7 h fast by 23%. In an oral glucose tolerance test in Zucker rats, however, GPi688 was less efficacious (7% reduction) than a glycogen synthase kinase-3 (GSK-3) inhibitor (22% reduction), despite also observing activation (by 45%) of GS in vivo., Conclusions and Implications: Although GP inhibition can inhibit hyperglycaemia mediated by increased glucose production, the degree of GS activation induced by allosteric GP inhibitors in vivo, although discernible, is insufficient to increase glucose disposal. The data suggests that GP inhibitors might be more effective clinically against fasting rather than prandial hyperglycaemic control.

Introduction: Glycogen phosphorlyase inhibitors (GPi) act on the glycogenolytic pathway decreasing hepatic glucose output, making them potential candidates for Type 2 diabetes treatment. We established a robust in vivo method to assess GPis efficacy utilising glucagon-stimulated glycogenolysis., Methods: Blood glucose was monitored in both male AP Wistar and AP Zucker rats using tail prick samples pre- and post intraperitoneal or subcutaneous glucagon administration. The effect of glycogen phosphorylase inhibitors GPi296 (6-60 mg kg(-1) po) and DAB (5 mg kg(-1) po) upon glucose response to subcutaneous glucagon were examined in both strains., Results: In the Wistar rat glucagon induced dose related increases in blood glucose, with the maximum increase occurring 20 min post dose (4.0+/-0.88 mmol l(-1), intraperitoneal; and 2.8+/-0.72 mmol l(-1), subcutaneous, ns). Intraperitoneal glucagon administration produced shorter duration blood glucose elevation than observed with the subcutaneous route of administration. In the Zucker rat, no differences were observed between the 10 and 13 week old rats in response to glucagon (3-200 microg kg(-1) subcutaneous). The maximum blood glucose increase was lower in the Wistar rat compared to the Zucker rats (2.9+/-0.20 vs 7.7+/-1.22 mmol l(-1), P<0.0000018). GPi296 and DAB both produced similar inhibition in each strain., Discussion: Subcutaneous glucagon administration induced more sustained increases in blood glucose than intraperitoneal administration. Blood glucose response to glucagon was higher in the Zucker rat compared to the Wistar rat; there was no difference in inhibition mediated by either GPi296 or DAB between the two strains. We believe that subcutaneous glucagon administration produces a robust model for the assessment of GPis in either rat strain.

Introduction: In the assessment of potential new treatments for Type 2 diabetes, robust pharmacological methods are helpful in assessing efficacy, defining dose response, duration of effect and ultimately in deciding whether to progress compounds to the next phase of drug development. Hepatic glucose handling is abnormal in Type 2 diabetes. We evaluated glucagon challenge as a way of assessing effects on the glycogenolytic pathway., Methods: In each of 2 studies healthy subjects received glucagon as an IV bolus of 0.5 mg studied after an overnight fast and plasma glucose was monitored before and for 180 min after glucagon challenge. Study 1 was a double-blind placebo controlled study comparing glucagon administered twice with saline placebo. In study 2, subjects were studied on a single occasion and the glucagon challenge was carried out in the morning and then repeated 7 h later. In study 2, insulin concentrations were also monitored before and after the glucagon challenge., Results: In study 1, glucose rose in a reproducible manner with a peak glucose 20 min after challenge falling to baseline values by 120 min and then fell below values for saline challenge between 120 and 180 min. Analysis of the data showed that the corrected AUC(0-20) min was the most robust variable and could be expected to detect clinically relevant changes in small numbers (<10) of subjects. In study 2, we demonstrated that when glucagon challenge was repeated 7 h after the first challenge, the glucose excursion was highly variable. The plasma insulin response was robust following the initial challenge but variable following the second challenge., Discussion: We have demonstrated that an IV bolus glucagon challenge (0.5 mg) results in a reproducible rise in glucose in healthy volunteers and can be repeated within a week but when repeated on the same day gave a poorly reproducible rise in glucose. Glucagon challenge may be useful in studying novel drugs that affect glycogen handling in the liver.

Background and Purpose: Inhibition of hepatic glycogen phosphorylase is a potential treatment for glycaemic control in type 2 diabetes. Selective inhibition of the liver phosphorylase isoform could minimize adverse effects in other tissues. We investigated the potential selectivity of two indole site phosphorylase inhibitors, GPi688 and GPi819., Experimental Approach: The activity of glycogen phosphorylase was modulated using the allosteric effectors glucose or caffeine to promote the less active T state, and AMP to promote the more active R state. In vitro potency of indole site inhibitors against liver and muscle glycogen phosphorylase a was examined at different effector concentrations using purified recombinant enzymes. The potency of GPi819 was compared with its in vivo efficacy at raising glycogen concentrations in liver and muscle of Zucker (fa/fa) rats., Key Results: In vitro potency of indole site inhibitors depended upon the activity state of phosphorylase a. Both inhibitors showed selectivity for liver phosphorylase a when the isoform specific activities were equal. After 5 days dosing of GPi819 (37.5 micromol kg(-1)), where free compound levels in plasma and tissue were at steady state, glycogen elevation was 1.5-fold greater in soleus muscle than in liver (P < 0.05)., Conclusions and Implications: The in vivo selectivity of GPi819 did not match that seen in vitro when the specific activities of phosphorylase a isoforms are equal. This suggests T state promoters may be important physiological regulators in skeletal muscle. The greater efficacy of indole site inhibitors in skeletal muscle has implications for the overall safety profile of such drugs.

Hepatic insulin resistance in the leptin-receptor defective Zucker fa/fa rat is associated with impaired glycogen synthesis and increased activity of phosphorylase-a. We investigated the coupling between phosphorylase-a and glycogen synthesis in hepatocytes from fa/fa rats by modulating the concentration of phosphorylase-a. Treatment of hepatocytes from fa/fa rats and Fa/? controls with a selective phosphorylase inhibitor caused depletion of phosphorylase-a, activation of glycogen synthase and stimulation of glycogen synthesis. The flux-control coefficient of phosphorylase on glycogen synthesis was glucose dependent and at 10 mm glucose was higher in fa/fa than Fa/? hepatocytes. There was an inverse correlation between the activities of glycogen synthase and phosphorylase-a in both fa/fa and Fa/? hepatocytes. However, fa/fa hepatocytes had a higher activity of phosphorylase-a, for a corresponding activity of glycogen synthase. This defect was, in part, normalized by expression of the glycogen-targeting protein, PTG. Hepatocytes from fa/fa rats had normal expression of the glycogen-targeting proteins G(L) and PTG but markedly reduced expression of R6. Expression of R6 protein was increased in hepatocytes from Wistar rats after incubation with leptin and insulin. Diminished hepatic R6 expression in the leptin-receptor defective fa/fa rat may be a contributing factor to the elevated phosphorylase activity and/or its high control strength on glycogen synthesis.

We examined the effects of increasing acetylcarnitine and acetyl-CoA availability at rest, independent of pyruvate dehydrogenase complex (PDC) activation, on energy production and tension development during the rest-to-work transition in canine skeletal muscle. We aimed to elucidate whether the lag in PDC-derived acetyl-CoA delivery toward the TCA cycle at the onset of exercise can be overcome by increasing acetyl group availability independently of PDC activation or is intimately dependent on PDC-derived acetyl-CoA. Gracilis muscle pretreated with saline or sodium acetate (360 mg/kg body mass) (both n = 6) was sampled repeatedly during 5 min of ischemic contraction. Acetate increased acetylcarnitine and acetyl-CoA availability (both P < 0.01) above control at rest and throughout contraction (P < 0.05), independently of differences in resting PDC activation between treatments. Acetate reduced oxygen-independent ATP resynthesis approximately 40% (P < 0.05) during the first minute of contraction. No difference in oxygen-independent ATP resynthesis existed between treatments from 1 to 3 min of contraction; however, energy production via this route increased approximately 25% (P < 0.05) above control in the acetate-treated group during the final 2 min of contraction. Tension development was approximately 20% greater after 5-min contraction after acetate treatment than in control (P < 0.05). In conclusion, at the immediate onset of contraction, when PDC was largely inactive, increasing cellular acetyl group availability overcame inertia in mitochondrial ATP regeneration. However, after the first minute, when PDC was near maximally activated in both groups, it appears that PDC-derived acetyl-CoA, rather than increased cellular acetyl group availability per se, dictated mitochondrial ATP resynthesis.

We previously established that activation of the pyruvate dehydrogenase complex (PDC) using dichloroacetate (DCA) reduced the reliance on substrate-level phosphorylation (SLP) at the onset of exercise, with normal and reduced blood flow. PDC activation also reduced fatigue development during contraction with reduced blood flow. Since these observations, several studies have re-evaluated our observations. One study demonstrated a performance benefit without a reduction in SLP, raising a question mark over PDC's role in the regulation of ATP regeneration and our interpretation of fatigue mechanisms. Using a model of muscle contraction similar to the conflicting study (i.e. tetanic rather than twitch stimulation), we re-examined this question. Using canine skeletal muscle, one group was infused with saline while the other was pretreated with 300 mg (kg body mass)(-1) DCA. Muscle biopsies were taken at rest, peak tension (1 min) and after 6 min of tetanic electrical stimulation (75 ms on-925 ms off per second) and blood flow was limited to 25% of normal values observed during contraction. DCA reduced phosphocreatine (PCr) degradation by 40% during the first minute of contraction, but did not prevent the almost complete depletion of PCr stores at 6 min, while muscle fatigue did not differ between the two groups. During intermittent tetanic stimulation PCr degradation was 75% greater than with our previous 3 Hz twitch contraction protocol, despite a similar rate of oxygen consumption at 6 min. Thus, in the present study enhanced acetyl group availability altered the time course of PCr utilization but did not prevent the decline towards depletion. Consistent with our earlier conclusions, DCA pretreatment reduces muscle fatigue only when SLP is attenuated. The present study and our met-analysis indicates that enhanced acetyl group availability results in a readily measurable reduction in SLP when the initial rate of PCr utilization is approximately 1 mmol (kg dry mass)(-1) s(-1) or less (depending on intrinsic mitochondrial capacity). When measured early during an uninterrupted period of muscle contraction, acetyl group availability is likely to influence SLP under any condition where mitochondria are responsible for a significant proportion of ATP regeneration.

Expression of the glycogen-targeting protein PTG promotes glycogen synthase activation and glycogen storage in various cell types. In this study, we tested the contribution of phosphorylase inactivation to the glycogenic action of PTG in hepatocytes by using a selective inhibitor of phosphorylase (CP-91149) that causes dephosphorylation of phosphorylase a and sequential activation of glycogen synthase. Similar to CP-91194, graded expression of PTG caused a concentration-dependent inactivation of phosphorylase and activation of glycogen synthase. The latter was partially counter-acted by the expression of muscle phosphorylase and was not additive with the activation by CP-91149, indicating that it is in part secondary to the inactivation of phosphorylase. PTG expression caused greater stimulation of glycogen synthesis and translocation of glycogen synthase than CP-91149, and the translocation of synthase could not be explained by accumulation of glycogen, supporting an additional role for glycogen synthase translocation in the glycogenic action of PTG. The effects of PTG expression on glycogen synthase and glycogen synthesis were additive with the effects of glucokinase expression, confirming the complementary roles of depletion of phosphorylase a (a negative modulator) and elevated glucose 6-phosphate (a positive modulator) in potentiating the activation of glycogen synthase. PTG expression mimicked the inactivation of phosphorylase caused by high glucose and counteracted the activation caused by glucagon. The latter suggests a possible additional role for PTG on phosphorylase kinase inactivation.

Peripheral vascular disease (PVD) is generally accepted to result in the failure of skeletal muscle blood flow to increase adequately at the onset of muscular work. There are currently no routine pharmacological interventions towards the treatment of PVD, however, recent Phase III trials in the USA have demonstrated the clinical potential of the phosphodiesterase III inhibitor Cilostazol for pain-free and maximal walking distances in patients with intermittent claudication. PVD is characterized by a marked reliance on oxygen-independent routes of ATP regeneration (phosphocreatine hydrolysis and glycolysis) in skeletal muscle during contraction and the rapid onset of muscular pain and fatigue. The accumulation of metabolic by-products of oxygen-independent ATP production (hydrogen and lactate ions and inorganic phosphate) has long been associated with an inhibition in contractile function in both healthy volunteers and PVD patients. Therefore, any strategy that could reduce the reliance upon ATP re-synthesis from oxygen-independent routes, and increase the contribution of oxygen-dependent (mitochondrial) ATP re-synthesis, particularly at the onset of exercise, might be expected to improve functional capacity and be of considerable therapeutic value. Historically, the increased contribution of oxygen-independent ATP re-synthesis to total ATP generation at the onset of exercise has been attributed to a lag in muscle blood flow limiting oxygen delivery during this period. However, recent evidence suggests that limited inertia is present at the level of oxygen delivery, whilst considerable inertia exists at the level of mitochondrial enzyme activation and substrate supply. In support of this latter hypothesis, we have reported on a number of occasions that activation of the pyruvate dehydrogenase complex, using pharmacological interventions, can markedly reduce the dependence on ATP re-synthesis from oxygen-independent routes at the onset of muscle contraction. This review will focus on these findings and will highlight the pyruvate dehydrogenase complex as a novel therapeutic target towards the treatment of peripheral vascular disease, or any other disease state where premature muscular fatigue is prevalent due to metabolite accumulation.

Fibroblast growth factor 2 (FGF2) is expressed ubiquitously in mesodermal and neuroectodermal cells. Human FGF2 occurs in isoforms translated from a common mRNA by alternative use of AUG (low-molecular weight isoforms) and CUG (high-molecular weight isoforms) start codons. Whereas the high-molecular weight isoforms function in an intracrine manner, the low-molecular weight isoform functions as autocrine, paracrine, and intracrine ligands. FGF2's signals are mediated by a family of high- and low-affinity receptors. The nuclear localization of FGF2 appears to be essential for its mitogenic effects with different isoforms localizing in different nuclear substructures. By regulating the transcription or activity of multiple other genes, FGF2 plays an important role in proliferation, differentiation, and survival of cells of almost all organ systems. Its potent angiogenic effects observed in tissue culture models and healthy animals have prompted clinical trials testing effects of FGF2 protein or genetic material on ischemic tissues. Unfortunately, FGF2-mediated therapeutic angiogenesis has yielded inconclusive results. One possible reason is that single-gene therapy is not sufficient to support the numerous effectors required to generate mature vessels assembled by multiple cells, including pericytes, smooth muscle cells, and endothelial cell subtypes. Another possible reason is that potentially negative effects of dyslipidemia, a common finding in patients with macro- and microvascular disease, on gene therapy have not been taken into account. We have demonstrated that electronegative low-density lipoprotein (LDL) from hypercholesterolemic human plasma downregulates FGF2 by both transcriptional and posttranscriptional mechanisms. In our models, FGF2 downregulation results in angiostasis and endothelial cell apoptosis. Deprivation of endogenous FGF2 may lead to dysregulation of the activities of other survival and angiogenesis-related genes. Delineation of the molecular mechanisms modulating the expression and actions of FGF2 may provide the basis for novel therapeutic interventions.

The present study examined the effect of adrenaline infusion on the activation status of glycogen phosphorylase and the pyruvate dehydrogenase complex (PDC) and on the accumulation of glucose-6-phosphate (G-6-P) and acetylcarnitine in resting canine skeletal muscle. The study was performed in an effort to gain some insight into the temporal relationship between glycogen phosphorylase and PDC activation in vivo in skeletal muscle, which is currently unresolved. Multiple muscle samples were obtained from canine brachial muscle (n = 10) before and during (1, 3, 7 and 15 min) adrenaline infusion (0.14 microg (kg body mass)(-1) min(-1), I.V.). Adrenaline infusion increased glycogen phosphorylase "a" by > 2-fold above basal levels after 3 min (pre-infusion = 9.2 +/- 1.1 vs. 3 min = 22.3 +/- 4.0 mmol glucosyl units (kg dry muscle)(-1) min(-1), P < 0.05). The concentration of G-6-P increased transiently from its basal concentration at 1 min (pre-infusion = 1.5 +/- 0.2 vs. 1 min = 4.4 +/- 0.9 mmol kg dry muscle)(-1), P < 0.01), declined to its pre-infusion concentration at 3 min (P < 0.05), and then increased again after 7 min of infusion (P < 0.05). The PDC was activated following 7 min of adrenaline infusion (pre-infusion = 0.22 +/- 0.04 vs. 7 min = 1.04 +/- 0.15 mmol acetyl-CoA (kg wet muscle)(-1) min(-1), P < 0.01), and this degree of activation was maintained for the duration of infusion. During the first 3 min of infusion, the concentration of acetylcarnitine declined (pre-infusion = 3.8 +/- 0.3 vs. 3 min = 1.6 +/- 0.2 mmol (kg dry muscle)(-1), P < 0.05), before transiently increasing at 7 min above the 3 min concentration (3 min = 1.6 +/- 0.2 vs. 7 min = 5.1 +/- 1.0 mmol (kg dry muscle)(-1), P < 0.01). This is the first study to demonstrate that adrenaline can indirectly activate the PDC in skeletal muscle in vivo at rest. The results demonstrate that adrenaline increased glycogen phosphorylase activation and glycolytic flux within 3 min of infusion, but took several more minutes to activate the PDC. This temporal relationship, combined with a probable adrenaline-induced increase in metabolic rate (and thereby resting ATP demand), resulted in the biphasic changes in G-6-P and acetylcarnitine with infusion time.

Considerable debate surrounds the identity of the precise cellular site(s) of inertia that limit the contribution of mitochondrial ATP resynthesis towards a step increase in workload at the onset of muscular contraction. By detailing the relationship between canine gracilis muscle energy metabolism and contractile function during constant-flow ischaemia, in the absence (control) and presence of pyruvate dehydrogenase complex activation by dichloroacetate, the present study examined whether there is a period at the onset of contraction when acetyl-coenzyme A (acetyl-CoA) availability limits mitochondrial ATP resynthesis, i.e. whether a limitation in mitochondrial acetyl group provision exists. Secondly, assuming it does exist, we also aimed to identify the mechanism by which dichloroacetate overcomes this "acetyl group deficit". No increase in pyruvate dehydrogenase complex activation or acetyl group availability occurred during the first 20 s of contraction in the control condition, with strong trends for both acetyl-CoA and acetylcarnitine to actually decline (indicating the existence of an acetyl group deficit). Dichloroacetate increased resting pyruvate dehydrogenase complex activation, acetyl-CoA and acetylcarnitine by approximately 20-fold (P < 0.01), approximately 3-fold (P < 0.01) and approximately 4-fold (P < 0.01), respectively, and overcame the acetyl group deficit at the onset of contraction. As a consequence, the reliance upon non-oxidative ATP resynthesis was reduced by approximately 40 % (P < 0.01) and tension development was increased by approximately 20 % (P < 0.05) following 5 min of contraction. The present study has demonstrated, for the first time, the existence of an acetyl group deficit at the onset of contraction and has confirmed the metabolic and functional benefits to be gained from overcoming this inertia.

Increasing blood bicarbonate content has long been cited as a potential mechanism to improve contractile function. We investigated whether sodium bicarbonate-induced metabolic alkalosis could positively affect force development during the rest-to-work transition in ischaemic skeletal muscle. Secondly, assuming it could, we investigated whether bicarbonate could augment acetyl group availability through the same equilibrium reaction as sodium acetate pre-treatment and whether this underpins, at least in part, its ergogenic effect. Multiple biopsy samples were obtained from the canine gracilis muscle during 5 min of electrically evoked ischaemic contraction, which enabled the determination of the time course of acetyl group accumulation, substrate utilisation, pyruvate dehydrogenase complex activation and tension development in animals treated with saline (control; n = 6) or sodium bicarbonate (n = 5). Treatment with bicarbonate elevated acetylcarnitine content above the control level at rest (P < 0.05), but at no time point during subsequent contraction. The pyruvate dehydrogenase complex was activated following 40 s of contraction in both groups, with no differences existing between treatments at any time point. The requirement for ATP re-synthesis from non-oxygen-dependent routes was no different between groups at any time point during contraction. No difference in peak twitch force production existed between groups. However, at 3 min of stimulation, tension development was better maintained in the bicarbonate group (P < 0.05), being approximately 20% greater than control following 5 min of contraction (P < 0.05). The results demonstrate, for the first time, that bicarbonate can augment acetyl group availability prior to contraction, independent of pyruvate dehydrogenase complex activation, but cannot influence the requirement for non-oxidative ATP re-synthesis during subsequent contraction. It would appear, therefore, that the bicarbonate-induced improvement in muscle tension development was probably mediated through the metabolic alkalosis and not via the increased availability of acetyl groups within the cell.

The oxygen deficit at the onset of submaximal exercise represents a period when the energy demand of contraction cannot be met solely by mitochondrial ATP generation, and as a consequence there is an acceleration of ATP re-synthesis from oxygen-independent routes (phosphocreatine hydrolysis and glycolysis). Historically, the origin of the oxygen deficit has been attributed to a lag in muscle blood flow and oxygen availability at the onset of exercise which limits mitochondrial respiration. However, more recent evidence suggests that considerable inertia exists at the level of mitochondrial enzyme activation and substrate supply. In support of this latter hypothesis, we have reported on a number of occasions that pharmacological activation of the pyruvate dehydrogenase complex (and consequent stockpiling of acetyl groups), using dichloroacetate or exercise interventions, can markedly reduce the degree of ATP re-synthesis from oxygen-independent routes during the rest-to-work transition period. This review will focus on these findings, and will offer the hypothesis that acetyl group delivery to the tricarboxylic acid cycle limits mitochondrial flux at the onset of exercise--the so-called acetyl group deficit.

The current study describes a model in the anesthetized cat which may be used to investigate pharmacological interventions for the treatment of vascular "steal" during muscle contraction. An artificial stenosis was placed around both the descending abdominal aorta and the left external iliac artery. Arterial blood flow was measured in the right and left external iliac and right femoral arteries. Contraction of the left hindquarters, induced by electrical stimulation of the left sciatic and femoral nerves, resulted in an increase of left iliac artery blood flow from 14.7+/-2.0 to 30.5+/-2.8 ml min-1 (p<0.001). A simultaneous reduction of blood flow measured at the level of the right femoral artery (6.7+/-0.1 to 4.5+/-0.7 ml min(-1), p<0.01) was observed in the noncontracting hindlimb (y = 7.76 - 0.10x, correlation coefficient = -0.865). The magnitude of each response was reproducible within the same animal. Administration of 8-phenyltheophylline, a nonselective antagonist of adenosine receptors, reduced the functional hyperemia response within the left external iliac artery by 32% and reduced the "steal" from the vascular bed perfused by the right femoral artery by 21%.

1. Male cats were anaesthetized with alphaxalone-alphadolone and breathed spontaneously following tracheotomy, Using coloured microspheres, muscle blood flow was measured at rest and during two periods of contraction elicited by simultaneous stimulation of the left sciatic and femoral nerves at 3 Hz for 10 min. In one group, the hindlimb blood flow was allowed to increase during muscle contraction and in another group the perfusion of the hindlimb was limited by a stenosis on the left external iliac artery. 2. In the absence of flow restriction, soleus muscle blood flow increased from 18.9 +/- 3.8 to 30.4 +/- 3.3 ml min-1 (100 g tissue)-1 (n = 6; P < 0.02) and gastrocnemius muscle blood flow increased from 24.8 +/- 5.9 to 61.6 +/- 12.8 ml min-1 (100 g tissue)-1 (n = 6; P < 0.01) during contraction. The bradykinin (BK) antagonists HOE 140 (1 mg kg-1, i.v.) did not affect the response in either the soleus (37.7 +/- 7.3 ml min-1 (100 g tissue)-1; n = 6; n.s.) or the gastrocnemius (62.9 +/- 7.9 ml min-1 (100 g tissue)-1; n = 6; n.s.) muscles. 3. In the stenosis group, soleus muscle blood flow increased from 9.8 +/- 2.3 to 22.9 +/- 4.9 ml min-1 (100 g tissue)-1 (n = 6; P < 0.01) and gastrocnemius muscle blood flow increased from 15.8 +/ 3.4 to 36.4 +/- 5.5 ml min-1 (100 g tissue)-1 (n = 6; P < 0.01) during contraction. Following administration of HOE 140, functional hyperaemia in the soleus muscle was unaffected (blood flow, 17.8 +/- 2.2 ml min-1 (100 g tissue)-1, n = 6; n.s.) white blood flow in the gastrocnemius muscle was reduced to 21.8 +/- 6.0 ml min-1 (100 g tissue)-1 (n = 6; P < 0.05). 4. The results show that BK does not contribute ot functional hyperaemia associated with twitch contraction at 3 Hz when blood flow is unrestricted, but may contribute up to 40% of the vasodilation of predominantly glycolytic muscle groups such as the gastrocnemius when flow is restricted. BK plays no role in hyperaemia associated with twitch contraction of oxidative muscle groups such as the soleus.

Pyruvate dehydrogenase complex (PDC) activation status has been described as being central in the regulation of tissue substrate oxidation as outlined by the glucose fatty-acid cycle. In the present study we examined the effects of reduced lipolysis, with use of nicotinate, and increased PDC activation, with use of dichloroacetate (DCA), on substrate utilization during 20 min of submaximal steady-state contraction (approximately 80% of maximal O2 uptake) in canine gracilis skeletal muscle. At rest, PDC activation was unchanged by nicotinate but was approximately 2.5-fold higher in the DCA group than in the control group (P < 0.05). During contraction, PDC activation status increased to 3.5 mmol acetyl-CoA.min-1.kg-1 at 37 degrees C in the control group, remained at 4.5 mmol acetyl-CoA.min-1.kg-1 at 37 degrees C in the DCA group, but only increased to 2.2 mmol acetyl-CoA.min-1.kg-1 at 37 degrees C in the nicotinate group (P < 0.05). However, the estimated amount of carbohydrate oxidized during the 20-min contraction was similar across groups and did not follow the degree of PDC activation (81.2 +/- 22.9, 95.9 +/- 11.7, and 89.3 +/- 18.9 mmol glucosyl units/kg dry muscle for control, nicotinate, and DCA, respectively). Thus it would appear that, during steady-state contraction, PDC activation status does not determine the rate of carbohydrate oxidation in skeletal muscle.

The role of beta- and alpha-adrenoceptors in the total vascular capacitance responses to changing pressure in vascularly isolated carotid sinuses of anesthetized and atropinized dogs was investigated. A change in vascular capacitance was determined by measuring the shift of blood in and out of a reservoir that was connected to the aorta and maintained at a constant pressure. Changes in carotid sinus pressure from 135 to 57 mmHg and back to 137 mmHg resulted in a rapid vascular capacitance response of approximately 30 ml in the absence of adrenoceptor antagonists. Administration of a beta2-adrenoceptor antagonist (ICI-118551) caused a significant enhancement of the capacitance responses to similar decreases and increases in carotid sinus pressure (approximately 130%). Administration of a beta1-adrenoceptor antagonist (CGP-20712A) did not cause any further enhancement of the responses. However, an alpha-blocker (phentolamine) reduced the responses by 75%. The results suggest that in the presence of a beta2-adrenoceptor antagonist vascular capacitance responses to loading and unloading of baroreceptors are greatly enhanced and that patients suffering from orthostatic syncope may benefit from this kind of drug.

Skeletal muscle contraction during ischemia, such as that experienced by peripheral vascular disease patients, is characterized by rapid fatigue. Using a canine gracilis model, we tested the hypothesis that a critical factor determining force production during ischemia is the metabolic response during the transition from rest to steady state. Dichloroacetate (DCA) administration before gracilis muscle contraction increased pyruvate dehydrogenase complex activation and resulted in acetylation of 80% of the free carnitine pool to acetylcarnitine. After 1 min of contraction, phosphocreatine (PCr) degradation in the DCA group was approximately 50% lower than in the control group (P < 0.05) during conditions of identical force production. After 6 min of contraction, steady-state force production was approximately 30% higher in the DCA group (P < 0.05), and muscle ATP, PCr, and glycogen degradation and lactate accumulation were lower (P < 0.05 in all cases). It appears, therefore, that an important determinant of contractile function during ischemia is the mechanisms by which ATP regeneration occurs during the period of rest to steady-state transition.

1. The present study investigated the contribution of the A2A adenosine receptor subtype to the functional hyperaemia response evoked by muscle contraction in anaesthetized cats when muscle blood flow was limited. 2. Application of a stenosis reduced the hindlimb blood flow at rest from 9.67 +/- 1.80 to 5.53 +/- 0.91 ml min(-1) (kg body mass)(-1) and during muscle contraction from 36.80 +/- 2.55 to 11.11 +/- 1.19 ml min(-1) (kg body mass)(-1) (P < 0.001). The force produced by the extensor digitorum longus and tibialis anterior (EDL-TA) muscle groups was also reduced, from 9.66 +/- 0.56 to 4.10 +/- 0.4 N (kg muscle mass)(-1) (P < 0.01). 3. The selective A2A adenosine receptor antagonist ZM241385 (3 mg kg(-1), I.V.) had no effect upon the hindlimb vascular conductance or muscle contraction responses in the presence of the flow-limiting stenosis. 4. In contrast, in the absence of the flow restriction the vascular conductance response was reduced by 27.5 +/- 5.0% (P < 0.05), whilst the isometric force produced by the EDL-TA muscle group was unaffected (pre- vs. post-contraction, 5.8 +/- 0.8 vs. 4.6 +/- 1.0 N (kg muscle mass)(-1) contraction). Oxygen consumption by the contracting hindlimb muscles was maintained (1.71 +/- 0.25 vs. 1.69 +/- 0.26 ml min(-1) (kg body mass)(-1)) by an increase in the oxygen extraction (51.9 +/- 4.9 vs. 66.2 +/- 6.1%; P< 0.01). 5. These results confirm previous data showing that adenosine, acting at the A2A receptor subtype, can contribute up to 30% of the functional hyperaemia response in the hindlimb of anaesthetized cats under free flow conditions. However, when blood flow is limited by a stenosis, antagonism of the A2A adenosine receptor does not affect functional hyperaemia.

4-(2-[7-Amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5] triazin-5-ylamino]ethyl)phenol (ZM 241385) is currently the most selective for the A2a adenosine receptor antagonist. This paper describes the in-vivo activity of ZM 241385 after administration by both oral and intraduodenal routes. In conscious spontaneously hypertensive rats, ZM 241385 (1-10 mg kg-1) selectively attenuated the mean arterial blood pressure response produced by exogenous adenosine (1 mg kg-1 min-1, i.v.) by up to 45% after oral administration. Activity of ZM 241385 was maintained for at least 6 h after 3 and 10 mg kg-1 (p.o.). In conscious normotensive cats, ZM 241385 attenuated the blood pressure responses to adenosine (0.6-1.0 mg kg-1, i.v.) by 94% after 10 mg kg-1 (p.o.) and by up to 74% after 0.3 mg kg-1 (i.v.). Duration of action of ZM 241385 up to 12 h (36% inhibition) was observed after 3 mg kg-1 (p.o.). In anaesthetized dogs and cats, ZM 241385, after intraduodenal administration (1-10 mg kg-1), produced a rapid (dose ratio 100-fold 15 min after administration of 10 mg kg-1 in the cat) and prolonged (dose ratio of 14 at 6 h after administration of 10 mg kg-1) attenuation of the vasodilatation responses to adenosine receptor stimulation. When administered by this route ZM 241385 was six times more potent than theophylline in the cat and at least twice as potent as theophylline in the dog. In conclusion, ZM 241385 is a potent, selective A2a adenosine receptor antagonist which is orally active, with a good duration of action by the enteric route in cat, rat and dog. It could therefore be used to evaluate the role of adenosine A2a receptors in the action of adenosine in-vivo.

The purpose of the present study was to investigate the functional consequences of KATP modulation in the normal and the metabolically inhibited guinea-pig isolated diaphragm using the K+ channel openers cromakalim, pinacidil, RP49356 (N-methyl-2-(3-pyridil)-tetrahydrothiopyran-2-carbothiami de-1-oxide) and ZM260384 (2-(2,2-bis(difluoromethyl)-6-nitro-3,4-dihydro-2H-1,4-benzoxazine -4-yl)pyridine-N-oxide) and the K+ channel inhibitors glibenclamide, phentolamine and ciclazindol. All K+ channel openers accelerated the decline in function induced by intermittent tetanic contractions following metabolic inhibition and delayed the development of contracture. Cromakalim also improved the recovery of twitch tension following 10 min intermittent tetanic stimulation in the hypoxic guinea-pig diaphragm preparation. Of the K+ channel inhibitors tested, only ciclazindol, at the highest concentration tested (10 microM), significantly delayed the decline in tetanic tension following metabolic inhibition in the guinea-pig isolated diaphragm. None of the inhibitors significantly accelerated the development of contracture. All inhibitors however, antagonised the actions of the K+ channel opener, cromakalim. The results indicate that opening of KATP can accelerate the decline in function following metabolic inhibition in the guinea-pig isolated diaphragm. In the absence of K+ channel openers however, KATP does not appear to contribute to this decline under the conditions of the present study.

The in vivo characterisation of ZM 241385 (4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-+ ++ylamino] ethyl)phenol), a novel, non-xanthine, selective adenosine A2A antagonist is described. In anaesthetised dogs ZM 241385 (i.v.) was 140-fold more potent in attenuating vasodilator responses to exogenous adenosine in the constant flow perfused hind limb than the bradycardic effects. In pithed rats in which blood pressure was supported by angiotensin II infusion, ZM 241385 (10 mg kg-1, i.v.) did not inhibit the hypotensive or bradycardic effects of the A3/A1 receptor agonist N(6)-2-(4-amino-3-iodophenyl)ethyladenosine (APNEA). In conscious spontaneously hypertensive rats, ZM 241385 (3-10 mg kg-1, p.o.) selectively attenuated the mean arterial blood pressure response produced by exogenous adenosine. No inhibition of the bradycardic effects of adenosine was observed following these doses of ZM 241385. The results indicate that ZM 241385 can be used to evaluate the role of adenosine A2A receptors in the action of adenosine in vivo.

1. The present study was designed to investigate the contribution of the A(2A) adenosine receptor subtype in the functional hyperaemia response during muscle contraction. 2. In cats anaesthetized with sodium pentobarbitone and breathing spontaneously following tracheotomy, the left sciatic and femoral nerves were electrically stimulated at 3 Hz for 20 min to induce muscle contraction, and hindlimb blood flow was measured with a flow probe. The contribution of the A(2A) adenosine receptor subtype was assessed using ZM 241385, a potent and selective A(2A) adenosine receptor antagonist. 3. In a control group, the muscle isometric tension measured in the extensor digitorum longus-tibialis anterior muscle group was 6.64 +/- 0.66 kg (100 g muscle mass)(-1) and hindlimb vascular conductance was 0.22 +/- 0.03 ml mmHg(-1)(kg body mass)(-1) at 20 min of contraction. Administration of vehicle did not affect these parameters upon a second contraction period: 6.31 +/- 0.61 kg (100 g muscle mass)(-1) and 0.23 +/- 0.03 ml mmHg(-1) (kg body mass)(-1), respectively. Total hindlimb conductance during contraction was unaffected (5.5 +/- 3.7% decrease). 4. ZM 241385 (1.0 mg kg(-1)) did not alter the amount of force produced by the muscle at 20 min of contraction. Hindlimb conductance response was reduced by 27.1 +/- 4.8% following the A(2A) selective adenosine receptor antagonist, similar to that observed with the non-selective antagonist 8-phenyltheophylline. 5. These results show that adenosine acting at the A(2A) subtype receptor can contribute up to 30% of the functional hyperaemia response in the hindlimb of anaesthetized cats.

The metabolic effects of partial ischemia on canine skeletal muscle were examined during 20 min of isometric contraction. A reduction in blood flow of approximately 75% resulted in an approximate 40% reduction in contractile function. Muscle lactate accumulation and phosphocreatine (PCr) hydrolysis were greater during ischemia, indicating a greater reliance on anaerobic ATP regeneration. Pyruvate dehydrogenase transformation to its active form (PDCa) during contraction was not affected by ischemia, such that PDCa did not appear to be a determinant of skeletal muscle fatigue. Acetylcarnitine concentration was greater during ischemic contraction and inversely correlated with PCr concentration (r = -0.79, P<0.01). Furthermore, acetylcarnitine accumulation and PCr degradation correlated with the degree of skeletal muscle fatigue (r = 0.56, P<0.05 and r = 0.70, P<0.01, respectively). Thus the greater the acetyl group oxidation, the lesser the contribution from anaerobic ATP provision and, subsequently, the smaller the degree of muscle fatigue observed. The metabolic characteristics of this model of ischemic muscle contraction are indistinguishable from the normal metabolic responses observed with increasing contractile intensity.

Skeletal muscle contractile function is impaired during acute ischemia such as that experienced by peripheral vascular disease patients. We therefore, examined the effects of dichloroacetate, which can alter resting metabolism, on canine gracilis muscle contractile function during constant flow ischemia. Pretreatment with dichloroacetate increased resting pyruvate dehydrogenase complex activity and resting acetylcarnitine concentration by approximately 4- and approximately 10-fold, respectively. After 20-min contraction the control group had demonstrated an approximately 40% reduction in isomeric tension whereas the dichloroacetate group had fatigued by approximately 25% (P < 0.05). Dichloroacetate resulted in less lactate accumulation (10.3 +/- 3.0 vs 58.9 +/- 10.5 mmol.kg-1 dry muscle [dm], P < 0.05) and phosphocreatine hydrolysis (15.6 +/- 6.3 vs 33.8 +/- 9.0 mmol.kg-1 dm, P < 0.05) during contraction. Acetylcarnitine concentration fell during contraction by 5.4 +/- 1.8 mmol.kg-1 dm in the dichloroacetate group but increased by 10.0 +/- 1.9 mmol.kg-1 dm in the control group. In conclusion, dichloroacetate enhanced contractile function during ischemia, independently of blood flow, such that it appears oxidative ATP regeneration is limited by pyruvate dehydrogenase complex activity and acetyl group availability.

The A2a adenosine receptor (AR) mediates several important physiological effects of adenosine, including vasodilation and inhibition of platelet aggregation. Until recently, no antagonist radioligand of sufficient selectivity or affinity was available. We describe the synthesis and characterization by radioligand binding of 125I-4-(2-[7-amino-2-{2-furyl}-{1,2,4}triazolo{2,3-a}- {1,3,5}triazin-5-yl-amino]ethyl)phenol (125I-ZM241385) in membranes from two cell types that express A2a ARs. Membranes from Chinese hamster ovary (CHO) cells expressing a recombinant canine A2a AR bound 125I-ZM241385 with high affinity, and agonist competition experiments with 2-(p-carboxyethyl)-phenylamino-5'-N-carboxamidoadenosine, 5'-N-ethylcarboxamidoadenosine, and (-)-N6[(R)-phenylisopropyl]adenosine revealed a potency order characteristic of an A2a AR binding site. Membranes from bovine striatum, which contain a native A2a AR, also bound 125I-ZM214385 with similarly high affinity and also displayed a pharmacological profile for displacement of radioligand binding that was consistent with that of an A2a AR. Also, under conditions in which 125I-ZM241385 bound with high affinity to a recombinant rat A2a AR expressed in CHO cells, no specific binding was detectable in membranes from CHO cells expressing functional rat A1, A2b, or A3 ARs, indicating that over the range of concentrations used in radioligand binding assays, 125I-ZM214385 is a highly selective antagonist radioligand for study of A2a ARs within a given species.

1. This paper describes the in vitro pharmacology of ZM 241385 (4-(2-[7-amino-2-(2-furyl) [1,2,4]-triazolo[2,3-a][1,3,5]triazin- 5-yl amino]ethyl) phenol), a novel non-xanthine adenosine receptor antagonist with selectivity for the A2a receptor subtype. 2. ZM 241385 had high affinity for A2a receptors. In rat phaeochromocytoma cell membranes, ZM 241385 displaced binding of tritiated 5'-N-ethylcarboxamidoadenosine (NECA) with a pIC50 of 9.52, (95% confidence limits, c.l., 9.02-10.02). In guinea-pig isolated Langendorff hearts, ZM 241385 antagonized vasodilatation of the coronary bed produced by 2-chloroadenosine (2-CADO) and 2-[p-(2-carboxyethyl) phenethylamino]-5'-N-ethylcarboxamidoadenosine (CGS21680) with pA2 values of 8.57 (c.l., 8.45-8.68) and 9.02 (c.l., 8.79-9.24) respectively. 3. ZM 241385 had low potency at A2b receptors and antagonized the relaxant effects of adenosine in the guinea-pig aorta with a pA2 of 7.06, (c.l., 6.92-7.19). 4. ZM 241385 had a low affinity at A1 receptors. In rat cerebral cortex membranes it displaced tritiated R-phenylisopropyladenosine (R-PIA) with a pIC50 of 5.69 (c.l., 5.57-5.81). ZM 241385 antagonized the bradycardic action of 2-CADO in guinea-pig atria with a pA2 of 5.95 (c.l., 5.72-6.18). 5. ZM 241385 had low affinity for A3 receptors. At cloned rat A3 receptors expressed in chinese hamster ovary cells, it displaced iodinated aminobenzyl-5'-N-methylcarboxamido adenosine (AB-MECA) with a pIC50 of 3.82 (c.l., 3.67-4.06). 6. ZM 241385 had no significant additional pharmacological effects on the isolated tissues used in these studies at concentrations three orders of magnitude greater than those which block A2a receptors. At 10 microM it displayed only minor inhibition of the bradycardic effects in guinea-pig atria to some concentrations of carbachol. At 10 microM, ZM 241385 had a small inhibitory effect on relaxant effects of isoprenaline in guinea-pig aortae but no effect on sodium nitrite-induced relaxation. ZM 241385(100 microM) was without effect on phenylephrine-induced tone in guinea-pig aortae.7. ZM 241385 (10 microM) had no inhibitory effect on rat hepatocyte phosphodiesterase types I, II, III and IV but caused a small inhibition of the calcium calmodulin-activated type I enzyme.8. ZM 241385 is the most selective adenosine A2a receptor antagonist yet described and is therefore a useful tool for characterization of responses mediated by A2 adenosine receptors.

The potential cytoprotective effect of the purine transport inhibitor S-(p-nitrobenzyl)-6-thioinosine (NBTI) in a model of myocardial ischaemia and reperfusion was investigated in the anaesthetised ferret. The left anterior descending coronary artery (LAD) was occluded for 90 min, producing ischaemia in 53 +/- 3% of the left ventricular free wall, followed by 240 min reperfusion. NBTI (0.5 mg kg-1, i.v.) was given prior to ischaemia or prior to reperfusion. In addition the effect of purine transport inhibition was investigated in animals subjected to ischaemia without reperfusion. NBTI reduced infarct size from 84.0 +/- 1.7 to 71.4 +/- 3.7% of the area at risk (P < 0.05) when given prior to occlusion of the LAD. NBTI was ineffective however when given 15 min prior to reperfusion. NBTI had no effect upon infarct size produced by ischaemia without reperfusion. The effect of NBTI was independent of significant changes in myocardial blood flow during ischaemia and reperfusion or upon neutrophil infiltration.