László Drimba

Involvement of cholecystokinin in baseline and post-prandial whole body insulin sensitivity in rats

The objective of the study was to investigate the role of cholecystokinin (CCK) on the food-induced insulin sensitization phenomenon in healthy Long Evans Tokushima Otsuka (LETO) and Otsuka Long Evans Tokushima Fatty (OLETF) rats. Whole body insulin sensitivity determined by hyperinsulinaemic euglycaemic glucose clamping and the rapid insulin sensitivity test served as endpoints. Determinations were done in both fasted and re-fed animals. The involvement of CCK in post-prandial insulin sensitization was assessed by using proglumide, a CCK receptor blocker, by assessment of hypothalamic CCK-1/CCK-2 receptor expression by rt-PCR technique and by plasma insulin immunoreactivity determinations by means of radioimmunoassay as pharmacological, genetic and analytical approaches, respectively. The body weight of the OLETF rats and the amount of food consumed much exceeded those seen with LETO rats. The post-prandial increase in insulin sensitivity was marked in LETO, but not in OLETF rats. Intravenous proglumide attenuated post-prandial insulin sensitivity in LETO rats, with no effect in OLETF rats. Nevertheless, baseline insulin sensitivity was much lower in OLETF than in LETO rats. Treatment with rosiglitazone increased baseline insulin sensitivity of OLETF rats and evoked an increase in CCK-1 receptor gene expression in LETO rats. The results provide evidence for the involvement of CCK receptors in adjustment of both fasting and post-prandial insulin sensitivity. The data obtained with OLETF rats strongly suggest the predominant role of CCK-1 receptors.

Morphological, hemodynamical and hemorheological changes of mature artificial saphenous arterio-venous shunts in the rat model

Artificial femoral arterio‐venous (AV) shunts are widely used in rodent models for studying shunt maturation and to optimize various surgical techniques. However, little is known about complex circulatory, microcirculatory, and hemorheological effects of end‐to‐side saphenous AV shunts. We aimed to study these parameters in mature AV shunts. Studying these questions in CD rats, end‐to‐side anastomoses were made between the left saphenous artery and vein. On the right‐side the nonoperated saphenous vessels served as own control. Furthermore healthy control animals were also investigated. On the 8th to 12th postoperative week microcirculatory and blood flow measurements were performed and blood samples were taken both from the shunts arterial and venous limbs and from the nonoperated side vessels. Hematological parameters, erythrocyte aggregation, and deformability were determined. The entire shunt and the control vessels were removed for histological examinations. The skin microcirculation on shunt side slightly increased on thigh and decreased on paws versus the nonoperated side. Blood flow measurements made directly on the vessels showed that arterial to venous blood flow rate ratio was 1.59 ± 0.29 on nonoperated side and 1.2 ± 0.13 on the shunt side, and 1.49 ± 0.05 in control animals. Erythrocyte aggregation and deformability worsened on the shunt side. Histologically increased number of smooth muscle elements and connective tissue were found in venous limb of the shunts. The artificial AV shunt between the saphenous artery and vein seems to be a suitable model for further functional‐morphological and hemorheological examinations of hemodialysis in various states and diseases.

Investigation of the metabolic effects of chronic clozapine treatment on CCK-1 receptor deficient Otsuka Long Evans Tokushima Fatty (OLETF) rats

Clozapine increases meal size and meal duration, effects similar to the pharmacological blockade or congenital deficiency of CCK-1 receptor. We aimed to investigate the role of CCK-1 receptor in clozapine-induced weight gain and insulin sensitivity in CCK-1 receptor deficient, male Otsuka Long Evans Tokushima Fatty rats (OLETF). Long Evans Tokushima Otsuka (LETO) rats served as healthy control. Animals were orally treated with either clozapine (10mg/kg) or its vehicle over 25 days. Daily metabolic parameters were measured by metabolic cages. The insulin sensitivity was determined by hyperinsulinaemic euglycaemic glucose clamping (HEGC). Adiposity was determined by measuring the perirenal, intraabdominal and epididymal white adipose tissue fat pads. Hypothalamic mRNA expression of CCK-1 and CCK-2 receptor was measured by real-time PCR, plasma insulin by radioimmunoassay. Clozapine failed to increase weight gain or daily food intake, but it increased adiposity, 1st meal size and duration and decreased insulin sensitivity both in OLETF or LETO rats. The glucose infusion rate during the steady state of the HEGC was unaltered, but the metabolic clearance rate of insulin was reduced by the clozapine treatment. Hypothalamic mRNA of CCK-1 and CCK-2 receptor was elevated in LETO rats, but the mRNA of CCK-2 receptor was reduced by clozapine in OLETF rats. Our results suggest that the CCK-1 receptor has no direct role in the clozapine-induced adiposity and insulin resistance. We also demonstrated that atypical antipsychotic treatment can induce insulin resistance in the absence of manifest obesity in male rats.

Identification of PPARγ ligands with One-dimensional Drug Profile Matching.

Introduction Computational molecular database screening helps to decrease the time and resources needed for drug development. Reintroduction of generic drugs by second medical use patents also contributes to cheaper and faster drug development processes. We screened, in silico, the Food and Drug Administration-approved generic drug database by means of the One-dimensional Drug Profile Matching (oDPM) method in order to find potential peroxisome proliferator-activated receptor gamma (PPARγ) agonists. The PPARγ action of the selected generics was also investigated by in vitro and in vivo experiments. Materials and methods The in silico oDPM method was used to determine the binding potency of 1,255 generics to 149 proteins collected. In vitro PPARγ activation was determined by measuring fatty acid-binding protein 4/adipocyte protein gene expression in a Mono Mac 6 cell line. The in vivo insulin sensitizing effect of the selected compound (nitazoxanide; 50–200 mg/kg/day over 8 days; n = 8) was established in type 2 diabetic rats by hyperinsulinemic euglycemic glucose clamping. Results After examining the closest neighbors of each of the reference set’s members and counting their most abundant neighbors, ten generic drugs were selected with oDPM. Among them, four enhanced fatty acid-binding protein/adipocyte protein gene expression in the Mono Mac 6 cell line, but only bromfenac and nitazoxanide showed dose-dependent actions. Induction by nitazoxanide was higher than by bromfenac. Nitazoxanide lowered fasting blood glucose levels and improved insulin sensitivity in type 2 diabetic rats. Conclusion We demonstrated that the oDPM method can predict previously unknown therapeutic effects of generic drugs. Nitazoxanide can be the prototype chemical structure of the new generation of insulin sensitizers.

Beneficial cardiac effects of cicletanine in conscious rabbits with metabolic syndrome

Background and Purpose: High-fat diet and consequent metabolic syndrome (MS) can lead to elevated risk for cardiac arrhythmias. This preclinical study was to investigate if cicletanine (CIC) could produce cardioprotective effects in conscious rabbits exhibiting the main symptoms of MS. Methods: NZW rabbits that had undergone an 8-week-long cholesterol-enriched diet (1.5%) were instrumented with a pacemaker electrode and randomly assigned into 3 groups according to the oral treatment of either CIC (50 mg·kg−1) or sotalol (25 mg·kg−1) and their placebo b.i.d. over 5 days. Study groups were subjected to either “arrhythmia challenge” by programmed electrical stimulation in the “Arrhythmogenesis” study (N = 54) or global myocardial ischemia by rapid pacing in the “Ventricular Overdrive Pacing-induced Myocardial Ischemia” study (N = 18). The antiarrhythmic effect was evaluated by the establishment of the incidence of programmed electrical stimulation–induced arrhythmias. Proarrhythmia indicators (eg, QTc, Tpeak–Tend) were also measured to assess the cardiac safety profile of CIC. To evaluate the background of antiarrhythmic effect, cardiac cyclic nucleotide (cyclic 3′,5′-guanosine monophosphate [cGMP], cyclic 3′,5′-adenosine monophosphate [cAMP]) and nitric oxide content were determined. The antiischemic effect was characterized by change of intracavital ST segment. Results: Cicletanine treatment significantly decreased the incidence of ventricular arrhythmias, increased cardiac cGMP and nitric oxide content and reduced cardiac cAMP level. Cicletanine did not modify significantly QTc and Tpeak–Tend interval. The ST-segment change in response to rapid pacing was reduced significantly by CIC. (P < 0.05). Conclusions: Cicletanine exerts beneficial cardiac effects in rabbits with symptoms of MS, which may be of influence with regard to the clinical application of the drug.

In Vivo Preclinical Evaluation of a Promising Antiarrhythmic Agent, EGIS‐7229

Preclinical Research

Role of sensory neurons on pancreatic beta cell function and on development of insulin resistance

Methods At the age of 6 weeks, OLETF rats were divided into two groups. The control group was treated with the vehicle for capsaicin, and the capsaicin group was treated with a single subcutaneous dose of 50 mg/kg capsaicin. The next 19 weeks, the metabolic variables (body weight gain, ingested food and water, stool and urine production) were measured by means of metabolic cages. At the end of the treatment period, the glucose-stimulated insulin response was determined by an oral glucose tolerance test (OGTT), whole body insulin sensitivity was determined by means of hyperinsulinaemic euglycaemic glucose clamping, and the hepatic glucose production (HGP) as well as insulin-stimulated peripheral glucose uptake (PGU) were determined by means of [3H]glucose infusion. Fasting plasma insulin levels were determined by RIA and fasting blood glucose values by the glucose oxidase method. Pancreatic beta cell function was characterized by the HOMA-B index based on fasting insulin and glucose levels. Results The body weight of the capsaicin-treated group was significantly lower than that of the control group. There were no changes in the other metabolic parameters. During the OGTT, the control group had a reduced glucose-stimulated response compared to the capsaicin-treated group and the area under the curve values were 1844 ± 124, and 1287 ± 87, respectively (p < 0.5). The whole body insulin sensitivity improved (from 9.4 ± 1.8 to 15.6 ± 2.1 mg/kg/ min) significantly according to the improvement in HGP (from 7.5 ± 1.5 to 12.9 ± 3.1 mg/kg/min) and PGU (from 6.7 ± 1.2 to 2.8 ± 1.1 mg/kg/min). There was no difference in pancreatic beta cell function between the two treatment groups.

The possible link between insulin resistance and increased cardiovascular mortality

Methods Chronically instrumented conscious rabbits were equipped with a right ventricular electrode catheter for pacing and recording the intracavitary electrogram as well as with arterial and venous catheters for blood sampling, blood pressure monitoring and for insulin and glucose infusions, respectively. Hyperinsulinaemia was produced by 2-step hyperinsulinaemic (35.7 ± 7.4 and 103.2 ± 10.5 μU/ml) euglycaemic (5.5 ± 0.5 μU/ml) glucose clamping. Programmed electrical stimulation (PES) was applied for ventricular effective refractory period (VERP) determination and arrhythmia generation.