Reversibility of hyperglycaemia and islet abnormalities in the high fat-fed female ZDF rat model of type 2 diabetes.

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 ß-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, ß-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.

The intravenous glucose tolerance test in cannulated Wistar rats: a robust method for the in vivo assessment of glucose-stimulated insulin secretion.

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.

Glucagon challenge in the rat: a robust method for the in vivo assessment of Glycogen phosphorlyase inhibitor efficacy.

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.

Acetyl-CoA provision and the acetyl group deficit at the onset of contraction in ischemic canine skeletal muscle.

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.

The temporal relationship between glycogen phosphorylase and activation of the pyruvate dehydrogenase complex during adrenaline infusion in resting canine skeletal muscle.

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.

The acetyl group deficit at the onset of contraction in ischaemic canine skeletal muscle.

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.

Bicarbonate-induced alkalosis augments cellular acetyl group availability and isometric force during the rest-to-work transition in canine skeletal muscle.

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.