Keith Dickinson

Mitochondrial uncoupling as a target for drug development for the treatment of obesity.

Mitochondrial proton cycling is responsible for a significant proportion of basal or standard metabolic rate, so further uncoupling of mitochondria may be a good way to increase energy expenditure and represents a good pharmacological target for the treatment of obesity. Uncoupling by 2,4‐dinitrophenol has been used in this way in the past with notable success, and some of the effects of thyroid hormone treatment to induce weight loss may also be due to uncoupling. Diet can alter the pattern of phospholipid fatty acyl groups in the mitochondrial membrane, and this may be a route to uncoupling in vivo. Energy expenditure can be increased by stimulating the activity of uncoupling protein 1 (UCP1) in brown adipocytes either directly or through β3‐adrenoceptor agonists. UCP2 in a number of tissues, UCP3 in skeletal muscle and the adenine nucleotide translocase have also been proposed as possible drug targets. Specific uncoupling of muscle or brown adipocyte mitochondria remains an attractive target for the development of antiobesity drugs.

Artifactual uncoupling by uncoupling protein 3 in yeast mitochondria at the concentrations found in mouse and rat skeletal-muscle mitochondria.

Western blots detected uncoupling protein 3 (UCP3) in skeletal-muscle mitochondria from wild-type but not UCP3 knock-out mice. Calibration with purified recombinant UCP3 showed that mouse and rat skeletal muscle contained 0.14 microg of UCP3/mg of mitochondrial protein. This very low UCP3 content is 200-700-fold less than the concentration of UCP1 in brown-adipose-tissue mitochondria from warm-adapted hamster (24-84 microg of UCP1/mg of mitochondrial protein). UCP3 was present in brown-adipose-tissue mitochondria from warm-adapted rats but was undetectable in rat heart mitochondria. We expressed human UCP3 in yeast mitochondria at levels similar to, double and 7-fold those found in rodent skeletal-muscle mitochondria. Yeast mitochondria containing UCP3 were more uncoupled than empty-vector controls, particularly at concentrations that were 7-fold physiological. However, uncoupling by UCP3 was not stimulated by the known activators palmitate and superoxide; neither were they inhibited by GDP, suggesting that the observed uncoupling was a property of non-native protein. As a control, UCP1 was expressed in yeast mitochondria at similar concentrations to that of UCP3 and at up to 50% of the physiological level of UCP1. Low levels of UCP1 gave palmitate-dependent and GDP-sensitive proton conductance but higher levels of UCP1 caused an additional GDP-insensitive uncoupling artifact. We conclude that the uncoupling of yeast mitochondria by high levels of UCP3 expression is entirely an artifact and provides no evidence for any native uncoupling activity of the protein.

AMP decreases the efficiency of skeletal-muscle mitochondria.

Mitochondrial proton leak in rat muscle is responsible for approx. 15% of the standard metabolic rate, so its modulation could be important in regulating metabolic efficiency. We report in the present paper that physiological concentrations of AMP (K(0.5)=80 microM) increase the resting respiration rate and double the proton conductance of rat skeletal-muscle mitochondria. This effect is specific for AMP. AMP also doubles proton conductance in skeletal-muscle mitochondria from an ectotherm (the frog Rana temporaria), suggesting that AMP activation is not primarily for thermogenesis. AMP activation in rat muscle mitochondria is unchanged when uncoupling protein-3 is doubled by starvation, indicating that this protein is not involved in the AMP effect. AMP activation is, however, abolished by inhibitors and substrates of the adenine nucleotide translocase (ANT), suggesting that this carrier (possibly the ANT1 isoform) mediates AMP activation. AMP activation of ANT could be important for physiological regulation of metabolic rate.

Combination of the sodium-glucose cotransporter-2 inhibitor empagliflozin with orlistat or sibutramine further improves the body-weight reduction and glucose homeostasis of obese rats fed a cafeteria diet.

The present study assessed the potential of the sodium glucose-linked transporter (SGLT)-2 inhibitor empagliflozin to decrease body weight when administered alone or in combination with the clinically effective weight-loss agents orlistat and sibutramine in obese rats fed a cafeteria diet. Female Wistar rats were exposed to a cafeteria diet to induce obesity. Empagliflozin was dosed once daily (10, 30, and 60 mg/kg) for 28 days. Combination studies were subsequently performed using a submaximal empagliflozin dose (10 mg/kg) with either sibutramine or orlistat. Body weight, food, and water intake were recorded daily. The effect of drug treatment on glucose tolerance, relevant plasma parameters, and carcass composition was determined. Empagliflozin dose-dependently reduced body weight, plasma leptin, and body fat though increased urinary glucose excretion. The combination of empagliflozin and orlistat significantly reduced body weight compared to animals treated with either drug alone, and significantly improved glucose tolerance, plasma insulin, and leptin compared to vehicle-treated controls. The effect of sibutramine to improve glycemic control in an oral glucose-tolerance test was also significantly increased, with empagliflozin and combination treatment leading to a reduction in carcass fat greater than that observed with either drug alone. These data demonstrate that empagliflozin reduces body weight in cafeteria-fed obese rats. In combination studies, empagliflozin further improved the body-weight or body-fat loss of animals in comparison to orlistat or sibutramine alone. Such studies may indicate improved strategies for the treatment of obese patients with prediabetes or type 2 diabetes.

BTS 67 582 stimulates insulin secretion from perifused rat pancreatic islets

The novel antidiabetic agent BTS 67 582 (1,1-dimethyl-2-[2-(4-morpholinophenyl)]guanidine monofumarate) demonstrated a concentration-dependent stimulation of insulin release in perifused rat pancreatic islets. EC50 values of 7.7 microM and 6.3 microM were obtained for BTS 67 582 in the presence of 8 mM glucose, after islets were pre-equilibrated with 4 and 8 mM glucose respectively. In contrast, there was little or no stimulation of insulin release at substimulatory (4 mM) or maximal stimulatory (15 mM) glucose concentrations. The plasma EC50 value for the glucose lowering effect of BTS 67 582 in fasted normal rats was 3.9 microM indicating a similar potency in vivo. In islets, BTS 67 582 completely antagonised (EC50 value of 13.2 microM) the actions of the selective ATP-dependent K+ channel opener diazoxide indicating K+ channel blocking activity. BTS 67 582 only weakly reversed the alpha2-adrenoceptor mediated inhibition of insulin release in islets (EC50 of 83 microM). BTS 67 582, like other imidazoline/guanidine insulin releasing agents, appears to promote insulin release via an effect on the islet ATP-dependent K+ channel which is not mediated by binding to the sulphonylurea receptor.

Determination of body composition in conscious adult female Wistar utilising total body electrical conductivity.

Total body electrical conductivity (TOBEC) is a noninvasive method for estimating fat free mass (FFM) in live animals. In this study, we have evaluated the use of the Em-Scan SA-3000, which is claimed by the manufacturers to perform better than earlier analysers. Previous studies in rats using these earlier versions of the TOBEC analyser have always used anaesthesia to minimise movement artefacts. As repeated anaesthesia also has the potential to induce artefacts by disrupting food intake, for example, we have also attempted to determine if this TOBEC analyser can be used to predict body composition in conscious adult weight-stable female Wistar rats. A simplified cafeteria diet was used to produce large variations in body composition (40-350 g fat/carcass) and a full chemical body composition analysis was performed to generate a TOBEC calibration equation. The TOBEC parameter was more strongly correlated to FFM (r(2)=.785) than it was to body weight (r(2)=.669) or other body composition parameters. Using the TOBEC calibration equation to predict fat mass on these data, there was an excellent correlation with the value obtained by chemical analyses (r(2)=.952, slope=0.958). To determine if the TOBEC calibration equation derived from this calibration study would then be useful for the routine estimation of body composition an additional, validation study was performed. This validation study was performed 6 months later, used an independent group of obese female Wistar rats and was undertaken by different TOBEC operators. This validation study, again, showed a good correlation between the TOBEC- and chemical-derived fat mass (r(2)=.918, slope=1.003) indicating stability of the calibration equation with time and independence from operator. We therefore conclude that it is possible to meaningfully estimate body fat changes in conscious rats using this TOBEC analysis system.

A sustained release formulation of novel quininib-hyaluronan microneedles inhibits angiogenesis and retinal vascular permeability in vivo.

Pathologic neovascularisation and ocular permeability are hallmarks of proliferative diabetic retinopathy and age-related macular degeneration. Current pharmacologic interventions targeting VEGF are effective in only 30-60% of patients and require multiple intraocular injections associated with iatrogenic infection. Thus, our goal is to develop novel small molecule drugs that are VEGF-independent are amenable to sustained ocular-release, and which reduce retinal angiogenesis and retinal vascular permeability. Here, the anti-angiogenic drug quininib was formulated into hyaluronan (HA) microneedles whose safety and efficacy was evaluated in vivo. Quininib-HA microneedles were formulated via desolvation from quininib-HA solution and subsequent cross-linking with 4-arm-PEG-amine prior to freeze-drying. Scanning electron microscopy revealed hollow needle-shaped particle ultrastructure, with a zeta potential of -35.5mV determined by electrophoretic light scattering. The incorporation efficiency and pharmacokinetic profile of quininib released in vitro from the microneedles was quantified by HPLC. Quininib incorporation into these microneedles was 90%. In vitro, 20% quininib was released over 4months; or in the presence of increasing concentrations of hyaluronidase, 60% incorporated quininib was released over 4months. Zebrafish hyaloid vasculature assays demonstrated quininib released from these microneedles significantly (p<0.0001) inhibited ocular developmental angiogenesis compared to control. Sustained amelioration of retinal vascular permeability (RVP) was demonstrated using a bespoke cysteinyl leukotriene induced rodent model. Quininib-HA microparticles significantly inhibited RVP in Brown Norway rats one month after administration compared to neat quininib control (p=0.0071). In summary, quininib-HA microneedles allow for sustained release of quininib; are safe in vivo and quininib released from these microneedles effectively inhibits angiogenesis and RVP in vivo.