Mechanisms of antihyperglycaemic action of efaroxan in mice: time for reappraisal of α2A-adrenergic antagonism in the treatment of type 2 diabetes?

AIMS/HYPOTHESIS: Inspired by recent speculation about the potential utility of α(2A)-antagonism in the treatment of type 2 diabetes, the study examined the contribution of α(2)-antagonism vs other mechanisms to the antihyperglycaemic activity of the imidazoline (±)-efaroxan. METHODS: Effects of the racemate and its pure enantiomers on isolated pancreatic islets and beta cells in vitro, as well as on hyperglycaemia in vivo, were investigated in a comparative manner in mice. RESULTS: In isolated perifused islets, the two enantiomers of efaroxan were equally potent in counteracting inhibition of insulin release by the ATP-dependent K(+) (K(ATP)) channel-opener diazoxide but (+)-efaroxan, the presumptive carrier of α(2)-antagonistic activity, was by far superior in counteracting inhibition of insulin release by the α(2)-agonist UK14,304. In vivo, (+)-efaroxan improved oral glucose tolerance at 100-fold lower doses than (-)-efaroxan and, in parallel with observations made in vitro, was more effective in counteracting UK14,304-induced than diazoxide-induced hyperglycaemia. The antihyperglycaemic activity of much higher doses of (-)-efaroxan was associated with an opposing pattern (i.e. with stronger counteraction of diazoxide-induced than UK14,304-induced hyperglycaemia), which implicates a different mechanism of action. CONCLUSIONS/INTERPRETATION: The antihyperglycaemic potency of (±)-efaroxan in mice is almost entirely due to α(2)-antagonism, but high doses can also lower blood glucose via another mechanism. Our findings call for reappraisal of the possible clinical utility of α(2A)-antagonistic compounds in recently identified subpopulations of patients in which a congenitally higher level of α(2A)-adrenergic activation contributes to the development and pathophysiology of type 2 diabetes.

Survey on paediatric tumour boards in Europe: current situation and results from the ExPo-r-Net project.

BACKGROUND: Under the ExPO-r-NeT project (European Expert Paediatric Oncology Reference Network for Diagnostics and Treatment), we aimed to identify paediatric oncology tumour boards in Europe to investigate the kind of technologies and logistics that are in place in different countries and to explore current differences between regions. METHODS: A 20-question survey regarding several features of tumor boards was designed. Data collected included infrastructure, organization, and clinical decision-making information from the centres. The survey was distributed to the National Paediatric Haematology and Oncology Societies that forwarded the survey to the sites. For comparative analysis, respondents were grouped into four geographical regions. RESULTS: The questionnaire was distributed amongst 30 countries. Response was obtained from 23 (77%) that altogether have 212 paediatric oncology treating centres. A total of 121 institutions answered (57%). Ninety-one percent of the centres hold multidisciplinary boards; however, international second consultations are performed in 36% and only 15% participate on virtual tumor boards. Videoconferencing facilities and standard operational procedures (SOPs) are available in 49 and 43% of the centres, respectively. There were statistically significant differences between European regions concerning meeting infrastructure and organization/logistics: specific room, projecting equipment, access to medical records, videoconferencing facilities, and existence of SOPs. CONCLUSION: Paediatric tumor boards are a common feature in Europe. To reduce inequalities and have equal access to healthcare, a virtual network is needed. Important differences on the functioning and access to technology between regions in Europe have been observed and need to be addressed.

Direct thiazolidinedione action on isolated rat skeletal muscle fuel handling is independent of peroxisome proliferator-activated receptor-gamma-mediated changes in gene expression.

Thiazolidinediones (TZDs) are believed to induce insulin sensitization by modulating gene expression via agonistic stimulation of the nuclear peroxisome proliferator-activated receptor-gamma (PPAR-gamma). We have shown earlier that the TZD troglitazone inhibits mitochondrial fuel oxidation in isolated rat skeletal muscle. In the present study, rat soleus muscle strips were exposed to TZDs to examine whether the inhibition of fuel oxidation is mediated by PPAR-gamma activation. Our findings consistently indicated direct, acute, and PPAR-gamma-independent TZD action on skeletal muscle fuel metabolism. Rapid stimulation of lactate release by 20 micromol/l troglitazone within 30 min suggested that direct TZD action on skeletal muscle in vitro does not rely on changes in gene expression rates (12.6 +/- 0.6 [control] vs. 16.0 +/- 0.8 micromol. g(-1). h(-1) [troglitazone]; P < 0.01). This conclusion was supported by the failure of actinomycin D and cycloheximide to block the effects of troglitazone. Mitochondrial fuel oxidation was consistently inhibited by six different TZDs (percent inhibition of CO(2) production from palmitate after 25 h: troglitazone, -61 +/- 2%; pioglitazone, -43 +/- 7%; rosiglitazone, -22 +/- 6%; BM13.1258, -47 +/- 9%; BM15.2054, -51 +/- 4%; and T-174, -59 +/- 4% [P < 0.005 each]), but not by PPAR-gamma agonistic compounds not belonging to the TZD class (JTT-501, -5 +/- 7% [NS]; prostaglandin J(2), 17 +/- 7% [P < 0.05]), which further argues against dependence on PPAR-gamma activation. In summary, our findings provided good evidence that direct inhibition of mitochondrial fuel oxidation in isolated skeletal muscle is a group-specific effect of TZDs and is independent of PPAR-gamma-mediated gene expression.

Chronic and acute effects of thiazolidinediones BM13.1258 and BM15.2054 on rat skeletal muscle glucose metabolism.

1 New thiazolidinediones BM13.1258 and BM15.2054 were studied with regard to their PPARgamma-agonistic activities and to their acute and chronic effects on glucose metabolism in soleus muscle strips from lean and genetically obese rats. 2 Both BM13.1258 and BM15.2054 revealed to be potent PPARgamma-activators in transient transfection assays in vitro. 3 In insulin-resistant obese rats, but not in lean rats, 10 days of oral treatment with either compound increased the stimulatory effect of insulin on muscle glycogen synthesis to a similar extent (insulin-induced increment in micromol glucose incorporated into glycogen g-1 h-1: control, +1.19+/-0.28; BM13.1258, +2.50+/-0.20; BM15.2054, +2.55+/-0.46; P<0.05 vs control each). 4 In parallel to insulin sensitization, mean glucose oxidation increased insulin-independently in response to BM13.1258 (to 191 and 183% of control in the absence and presence of insulin, respectively; P<0.01 each), which was hardly seen in response to BM15.2054 (to 137 and 124% of control, respectively; ns). 5 Comparable effects on PPARgamma activation and on amelioration of insulin resistance by BM13.1258 and BM15.2054 were therefore opposed by different effects on glucose oxidation. 6 In contrast to chronic oral treatment, acute exposure of muscles to BM13.1258 or BM15.2054 in vitro elicited a distinct catabolic response of glucose metabolism in specimens from both lean and obese rats. 7 The results provide evidence that BM13.1258 and BM15.2054 can affect muscle glucose metabolism via more than one mechanism of action. 8 Further efforts are required to clarify, to what extent other mechanisms besides insulin sensitization via the activation of PPARgamma are involved in the antidiabetic actions of thiazolidinediones.

Survey on paediatric tumour boards in Europe: current situation and results from the ExPo-r-Net project

Background: Under the ExPO-r-NeT project (European Expert Paediatric Oncology Reference Network for Diagnostics and Treatment), we aimed to identify paediatric oncology tumour boards in Europe to investigate the kind of technologies and logistics that are in place in different countries and to explore current differences between regions. Methods: A 20-question survey regarding several features of tumor boards was designed. Data collected included infrastructure, organization, and clinical decision-making information from the centres. The survey was distributed to the National Paediatric Haematology and Oncology Societies that forwarded the survey to the sites. For comparative analysis, respondents were grouped into four geographical regions. Results: The questionnaire was distributed amongst 30 countries. Response was obtained from 23 (77%) that altogether have 212 paediatric oncology treating centres. A total of 121 institutions answered (57%). Ninety-one percent of the centres hold multidisciplinary boards; however, international second consultations are performed in 36% and only 15% participate on virtual tumor boards. Videoconferencing facilities and standard operational procedures (SOPs) are available in 49 and 43% of the centres, respectively. There were statistically significant differences between European regions concerning meeting infrastructure and organization/logistics: specific room, projecting equipment, access to medical records, videoconferencing facilities, and existence of SOPs. Conclusion: Paediatric tumor boards are a common feature in Europe. To reduce inequalities and have equal access to healthcare, a virtual network is needed. Important differences on the functioning and access to technology between regions in Europe have been observed and need to be addressed

No disponible

Peroxisome proliferator-activated receptor-delta, a regulator of oxidative capacity, fuel switching and cholesterol transport.

Synthetic agonists of peroxisome proliferator-activated receptor (PPAR)-delta have shown a promising pharmacological profile in preclinical models of metabolic and cardiovascular disease. At present, the pharmaceutical development of these drugs exploits the potential to raise plasma HDL-cholesterol in animals and their insulin-sensitising and glucose-lowering properties. PPAR-delta agonists have also proven to be powerful research tools that have provided insights into the role of fatty acid metabolism in human physiology and disease. Activation of PPAR-delta induces the expression of genes important for cellular fatty acid combustion and an associated increase in whole-body lipid dissipation. The predominant target tissue in this regard is skeletal muscle, in which PPAR-delta activation regulates the oxidative capacity of the mitochondrial apparatus, switches fuel preference from glucose to fatty acids, and reduces triacylglycerol storage. These changes counter the characteristic derangements of insulin- resistant skeletal muscle but resemble the metabolic adaptation to regular physical exercise. Apart from effects on fuel turnover, there is evidence for direct antiatherogenic properties, because PPAR-delta activation increases cholesterol export and represses inflammatory gene expression in macrophages and atherosclerotic lesions. Whereas conclusions about the full potential of PPAR-delta as a drug target await the result of large scale clinical testing, ongoing investigation of this nuclear receptor has greatly improved our knowledge of the physiological regulation of whole-body fuel turnover and the interdependence of mitochondrial function and insulin sensitivity.

Progesterone impairs cell respiration and suppresses a compensatory increase in glucose transport in isolated rat skeletal muscle: a non-genomic mechanism contributing to metabolic adaptation to late pregnancy?

AIMS/HYPOTHESIS: The aim of the study was to gain better insight into the mechanisms responsible for impaired glucose metabolism during late pregnancy. We explored the direct effects of progesterone on glucose metabolism of skeletal muscle. METHODS: Specimens of skeletal muscle from untreated rats were incubated with progesterone and rates of substrate fluxes through the various pathways of glucose metabolism were analysed. RESULTS: Progesterone dose-dependently reduced the rates of glucose and pyruvate oxidation (insulin-stimulated rates after 5 h of exposure to 1 and 10 mumol/l progesterone: glucose oxidation, -6 +/- 4%, NS, and -39 +/- 4%, p < 0.001; pyruvate oxidation, -28 +/- 2% and -55 +/- 4%, p < 0.001 each) and increased lactate release (+28 +/- 4% and +58 +/- 9%, p < 0.005 each), which indicated inhibition of mitochondrial respiratory function. Impairment of cell respiration, e.g. by the specific inhibitor rotenone, is known to trigger a compensatory increase in glucose transport, but this response was blunted in the case of progesterone (change of glucose transport in response to 10 mumol/l progesterone vs 60 nmol/l rotenone, both causing a reduction in glucose oxidation by -39%: progesterone, +14 +/- 8% vs rotenone, +84 +/- 23%, p < 0.03). Further experiments dealt with the underlying mechanisms and revealed a rapid mode of action (50 mumol/l progesterone, reduction in insulin-stimulated glucose oxidation after 30 min: -29 +/- 7%, p < 0.01) not affected by blockers of gene expression or the nuclear progesterone receptor. CONCLUSIONS/INTERPRETATION: Progesterone inhibits cell respiration and at the same time suppresses a compensatory increase in glucose transport, causing cellular carbohydrate deficiency in isolated rat skeletal muscle. This effect is mediated by a direct, rapid and non-genomic mechanism and could contribute to pregnancy-associated changes in glucose homeostasis.

Effects of free fatty acids on carbohydrate metabolism and insulin signalling in perfused rat liver.

BACKGROUND: Elevated circulating free fatty acids (FFAs) induce insulin resistance and play a crucial role in the development of type 2 diabetes, in which fasting hepatic glucose production (HGP) is increased. However, direct effects of FFAs on fasting HGP are still unclear because indirect endocrine and metabolic effects contribute to FFA action. Thus, we aimed to investigate acute direct effects of specific FFAs on fasting HGP, lactate uptake, and insulin signalling. MATERIALS AND METHODS: Isolated livers obtained from 20 h fasted rats were perfused with albumin-bound palmitate or oleate (200 micromol L(-1) each) or vehicle (control) for 180 min (n = 5-7/group). RESULTS: Compared to control, hepatic lactate uptake was increased by palmitate and oleate (~+40%; P < 0.05), while HGP from lactate (~3 mmol L(-1)) and liver glycogen content were similar. Tyrosine phosphorylation (pY) of insulin-receptor-substrate-(IRS)-2 and p70S6-kinase phosphorylation were not affected by FFAs. Palmitate decreased insulin-receptor-beta pY, IRS-1 pY and phosphoinositol-3-kinase expression by 46 +/- 16%, 46 +/- 11% and 20 +/- 9%, respectively (P < 0.03), while oleate reduced Akt phosphorylation by 85 +/- 7% (P < 0.006). CONCLUSIONS: Isolated liver perfusion with saturated or unsaturated FFAs reduced insulin signalling protein phosphorylation at different sites and increased lactate uptake without affecting HGP or glycogen content. These results suggest that at fasting, both saturated and unsaturated FFAs increase hepatic glucose precursor uptake and may, independently of insulin's presence, accelerate protein dephosphorylation of the insulin signalling cascade at different sites.

Insulin does not regulate glucose transport and metabolism in human endothelium.

BACKGROUND: Although endothelial cells express insulin receptors, it is controversially discussed whether the endothelium represents an insulin-responsive tissue. Since available data are primarily restricted to animal endothelial cells, this study tested (i) whether insulin affects glucose metabolism in human endothelium; (ii) whether insulin sensitivity is different in micro- versus macrovascular endothelial cells; and (iii) whether glucose concentration in the incubation medium affects the cells' response to insulin. MATERIALS AND METHODS: Human umbilical vein endothelial cells (HUVECs), human adult saphenous vein endothelial cells (HAVECs), human aortic endothelial cells (HAEC), and human retinal endothelial cells (HRECs) as well as human smooth muscle cells were incubated with/without insulin (0.3 nmol L(-1) or 1 micromol L(-1)). Glucose transport, glycogen synthesis, glycogen content, lactate release, and expression of phospho-Akt, Akt, and endothelial nitric oxide synthase (eNOS) were determined. RESULTS: In HUVECs and HRECs, insulin (1 micromol L(-1)) increased (P < 0.05) eNOS expression by ~70% and doubled Akt phosphorylation, but the latter was by far more pronounced in human smooth muscle cells (+1093 +/- 500%, P < 0.05). In human smooth muscle cells, insulin (1 micromol L(-1)) stimulated glycogen synthesis by 67 +/- 11% (P < 0.01). In human micro- (HRECs) and macrovascular endothelial cells (HUVECs, HAVECs and HAECs), insulin, however, failed to stimulate glucose transport, glycogen synthesis, glycogen content, or lactate release under various conditions, i.e. after glucose deprivation or in medium with normal (5.5 mmol L(-1)) or high glucose (30 mmol L(-1)). CONCLUSIONS: Insulin stimulated glycogen synthesis and Akt phosphorylation in human smooth muscle cells. In human micro- and macrovascular endothelial cells, insulin, however, failed to affect glucose uptake and metabolism under all experimental conditions applied, whereas it increased Akt phosphorylation and eNOS expression.

Activation of PPAR-delta in isolated rat skeletal muscle switches fuel preference from glucose to fatty acids.

AIMS/HYPOTHESIS: GW501516, an agonist of peroxisome proliferator-activated receptor-delta (PPAR-delta), increases lipid combustion and exerts antidiabetic action in animals, effects which are attributed mainly to direct effects on skeletal muscle. We explored such actions further in isolated rat skeletal muscle. MATERIALS AND METHODS: Specimens of rat skeletal muscle were pretreated with GW501516 (0.01-30 mumol/l) for 0.5, 4 or 24 h and rates of fuel metabolism were then measured. In addition, effects on mitochondrial function were determined in isolated rat liver mitochondria. RESULTS: At concentrations between 0.01 and 1 mumol/l, GW501516 dose-dependently increased fatty acid oxidation but reduced glucose utilisation in isolated muscle. Thus after 24 h of preincubation with 1 mumol/l GW501516, palmitate oxidation increased by +46+/-10%, and the following decreased as specified: glucose oxidation -46+/-8%, glycogen synthesis -42+/-6%, lactate release -20+/-2%, glucose transport -15+/-6% (all p<0.05). Reduction of glucose utilisation persisted independently of insulin stimulation or muscle fibre type, but depended on fatty acid availability (the effect on glucose transport in the absence of fatty acids was an increase of 30+/-9%, p<0.01), suggesting a role for the glucose-fatty acid cycle. At higher concentrations, GW501516 uncoupled oxidative phosphorylation by direct action on isolated mitochondria. CONCLUSIONS/INTERPRETATION: GW501516-induced activation of PPAR-delta reduces glucose utilisation by skeletal muscle through a switch in mitochondrial substrate preference from carbohydrate to lipid. High concentrations of GW501516 induce mitochondrial uncoupling independently of PPAR-delta.

Troglitazone directly inhibits CO(2) production from glucose and palmitate in isolated rat skeletal muscle.

Troglitazone is a nuclear peroxisome proliferator-activated receptor-gamma agonist with insulin-sensitizing properties that has been introduced for the treatment of type 2 diabetes. To further elucidate its mechanism of action, this study examined direct troglitazone effects on glucose and palmitate utilization in isolated rat soleus muscle. Exposure of muscle specimens for 25 h to 5 micromol/liter troglitazone resulted in the distinct inhibition of insulin-stimulated mitochondrial fuel oxidation as indicated by decreased rates of CO(2) produced from glucose (glucose converted to CO(2), nanomoles per gram per hour: control, 1461 +/- 192 versus troglitazone, 753 +/- 80, P <.0001) and palmitate (palmitate converted to CO(2), nanomoles per gram per hour: control, 75 +/- 5 versus troglitazone, 20 +/- 2, P <.0001). Blunted fuel oxidation was accompanied by increased rates of anaerobic glycolysis (lactate release, micromoles per gram per hour: control, 17.3 +/- 1.0 versus troglitazone, 49.2 +/- 2.7, P <.0001) and glucose transport ([(3)H]2-deoxyglucose transport, cpm per milligram per hour: control, 540 +/- 46 versus troglitazone, 791 +/- 61, P <.0001), as well as by decreased rates of glycogen synthesis (glucose incorporation into glycogen, micromoles per gram per hour: control, 2.00 +/- 0.26 versus troglitazone, 1.02 +/- 0.13, P <.001). Such shift toward anaerobic glucose utilization also was seen in the absence of insulin and with short-term troglitazone exposure for 90 min, indicating an underlying mechanism that is rapid and independent of concomitant insulin stimulation. The results demonstrate direct and acute inhibition of fuel oxidation to CO(2) by troglitazone in rat skeletal muscle in vitro.

Expression of uncoupling protein-3 mRNA in rat skeletal muscle is acutely stimulated by thiazolidinediones: an exercise-like effect?

AIMS/HYPOTHESIS: We examined whether thiazolidinediones (TZDs) acutely affect uncoupling protein-3 ( UCP-3) expression in skeletal muscle and plasma NEFA in Sprague-Dawley rats. METHODS: Expression of UCP-3 mRNA in hindlimb muscles and plasma NEFA were measured after a single intraperitoneal injection of TZDs in healthy male rats. RESULTS: Independent of which TZD was injected (50 micromol/kg), UCP-3 expression in gastrocnemius muscle was distinctly increased after 6 h (increase vs vehicle-injected control: pioglitazone, 10.3+/-3.2-fold, p=0.03; rosiglitazone, 8.7+/-1.2-fold, p=0.001; RWJ241947, 9.5+/-2.7-fold, p=0.03). This was accompanied by elevated plasma NEFA (control 158+/-13 micromol/l; pioglitazone, 281+/-40 micromol/l, p=0.03; rosiglitazone, 276+/-27 micromol/l, p=0.005; RWJ241947, 398+/-51 micromol/l, p=0.004). The increase in plasma NEFA could in part have mediated TZD-induced UCP-3 expression, but increased UCP-3 mRNA was also found in isolated muscle after 2 h of TZD exposure in vitro (25 micromol/l pioglitazone, 1.7+/-0.3-fold, p=0.046), suggesting that TZDs act directly and independently of NEFA on skeletal muscle. CONCLUSIONS/INTERPRETATION: In healthy rats, a single dose of TZDs rapidly increases UCP-3 mRNA in skeletal muscle and plasma NEFA. This effect resembles the acute response to a bout of exercise.

Failure of leptin to affect basal and insulin-stimulated glucose metabolism of rat skeletal muscle in vitro.

Studies on different isolated tissues have provided evidence that leptin may directly modulate cellular glucose handling. The present study was performed to elucidate leptin's action on basal and insulin-stimulated glucose metabolism in native muscle tissue, which under physiological circumstances is the quantitatively most important target tissue of insulin. Isolated rat soleus muscle strips were incubated for 1 h in the absence or presence of leptin (0, 1, 10, or 100 nmol/l) under basal or insulin-stimulated conditions (10 nmol/l). No effects of leptin were found on the rates of 3H-2-deoxy-glucose transport (basal: control, 314+/-14; 1 nmol/l leptin, 320+/-17; 10 nmol/l leptin, 314+/-13; 100 nmol/l leptin, 322+/-16; insulin-stimulated: control, 690+/-33; 1 nmol/l leptin, 691+/-29; 10 nmol/l leptin, 665+/-26; 100 nmol/l leptin, 664+/-27; cpm x mg(-1) x h(-1); NS vs respective control) and on net glucose incorporation into glycogen (basal: control, 1.75+/-0.18; 1 nmol/l leptin, 2.01+/-0.13; 10 nmol/l leptin, 1.92+/-0.11; 100 nmol/l leptin, 1.81+/-0.13; insulin-stimulated: control, 5.98+/-0.40; 1 nmol/l leptin, 5.93+/-0.30; 10 nmol/l leptin, 5.46+/-0.25; 100 nmol/l leptin, 5.85+/-0.30; micromol x g(-1) x h(-1); NS vs respective control). In parallel, leptin failed to affect rates of aerobic and anaerobic glycolysis as well as muscle glycogen content. Further experiments revealed that the inability of leptin to directly affect muscle glucose handling prevailed independently of muscle fiber type (soleus and epitrochlearis muscle), of ambient insulin concentrations (0-30 nmol/l), and of leptin exposure time (1 h or 6 h). Thus, our findings fail to support speculations about a physiological role of direct insulin-mimetic or insulin-desensitizing effects of leptin on skeletal muscle tissue.

Metabolic response to anisoosmolarity of rat skeletal muscle in vitro.

Isolated rat hepatocytes exhibit an insulin-like anabolic response to hypoosmotic incubation and a glucagon-like catabolic response to hyperosmotic incubation. Recently, a distinct glycogenic response to hypoosmotic treatment was likewise reported for cultured rat myotubes. The present study examines the effects of anisoosmolar exposure on glucose metabolism in freshly isolated rat soleus muscle strips. Under the same experimental conditions as used for cultured myotubes, hypoosmolarity reduced net glycogen synthesis to 52%, while hyperosmolarity stimulated glycogen storage to 231% of isoosmolar control (nmol glucose incorporated into glycogen g(-1) x h(-1): hypoosmolar, 34+/-3; isoosmolar, 65+/-8; hyperosmolar, 150+/-11; p<0.01 each vs. isoosmolar). The responses of native skeletal muscle to anisoosmolarity are therefore in opposition to what has been described for hepatocytes and cultured myotubes. Further experiments on rat skeletal muscle revealed that the observed lack of a glycogenic response to hypoosmolarity persisted independent of medium composition, specifically with regard to prevailing glucose and K+ concentrations. In conclusion, hypoosmotic exposure inhibits glycogen synthesis in isolated rat soleus muscle, which clearly argues against the hypothesis that osmotic changes and cell swelling may be physiologically relevant stimulators of muscle glycogen synthesis.