Juha Rouru

Effects of Intravenously Infused Leptin on Insulin Sensitivity and on the Expression of Uncoupling Proteins in Brown Adipose Tissue

Centrally administered leptin has been shown to increase insulin-stimulated glucose utilization and to favor the expression of uncoupling proteins (UCPs). To study if leptin also has direct peripherally mediated effects on these processes, this hormone (1 mg/day) or its vehicle was infused iv for 4 days to lean rats and insulin-stimulated glucose utilization in skeletal muscle and adipose tissue as well as the expression of UCP messenger RNAs (mRNAs) in brown adipose tissue were measured. Iv leptin administration resulted in decreases in food intake (31%), body weight gain, and plasma insulin levels (45%), in increases in overall (23%) as well as brown adipose tissue and muscle glucose utilization, and in decreases in white adipose tissue glucose uptake. Most of these changes were mimicked, in control rats, by giving them the same amount of food as that consumed by the leptin-infused group (pair-feeding). Iv leptin infusion also favored the expression of UCPs in brown adipose tissue, either by increasing ...

SELECTIVE DEPENDENCE OF INTRACEREBROVENTRICULAR NEUROPEPTIDE Y-ELICITED EFFECTS ON CENTRAL GLUCOCORTICOIDS

It has been reported that hyperphagia and excessive body weight gain of genetically obese rodents were abolished by adrenalectomy. High hypothalamic levels of neuropeptide Y (NPY) were found in obese rodents. A chronic intracerebroventricular (icv) infusion of NPY in normal rats was shown to produce most hormono-metabolic abnormalities of genetically obese animals, and to be inefficient in doing so in adrenalectomized (ADX) rats. The combined presence of NPY and of glucocorticoids thus appeared to be necessary for inducing obesity. This study, therefore, was aimed at determining the consequences of a chronic i.c.v. NPY infusion in ADX rats receiving or not i.c.v. glucocorticoids. It was found that the combined i.c.v. infusion of NPY and dexamethasone in ADX rats increased food intake, body weight, plasma insulin, leptin, and triglyceride levels relative to vehicle-infused ADX controls. The infusion of NPY alone, or of dexamethasone alone in ADX rats failed to produce these effects. In contrast, the icv infusion of NPY alone greatly decreased the expression of brown adipose tissue uncoupling protein-1 and -3. This was not modified by the superimposed infusion of dexamethasone. It is concluded that, although many of centrally elicited NPY effects require the central presence of glucocorticoids, those bearing on the inhibition of uncoupling proteins expression (energy dissipation) do not require central glucocorticoids.

Hypothalamic neuropeptide expression after food restriction in Zucker rats: evidence of persistent neuropeptide Y gene activation.

The Obese Zucker rat is a model of genetic obesity characterized by hyperphagia, hyperinsulinemia and other endocrine abnormalities. In order to elucidate pathogenetic mechanisms contributing to disturbed feeding behavior in these animals, the effect of food restriction on three hypothalamic neuropeptides involved in the control of food intake was studied. Eighteen male obese and 18 lean Zucker rats were randomly divided into two groups: half of the animals were food-restricted for 2 weeks, while the other half served as controls and were fed ad libitum. The levels of preproneuropeptide Y (preproNPY), preprocorticotropin releasing factor (preproCRF) and preprosomatostatin (preproSOM) mRNAs were determined using in situ hybridization technique. In addition, plasma insulin and corticosterone concentrations were analyzed. Food restriction significantly increased the expression of preproNPY mRNA in the arcuate nucleus in both Zucker phenotypes, while the expressions of preproCRF mRNA in the paraventricular nucleus (PVN) and preproSOM mRNA in the periventricular nucleus (PeV) were not altered. The expression of preproNPY mRNA was significantly greater in control obese animals compared to control lean animals. Food restriction lowered plasma insulin levels, but did not change plasma corticosterone levels. It is concluded that food restriction specifically activates NPY gene transcription in the arcuate nucleus the response being similar in both Zucker phenotypes. The results suggest that orexigenic NPY plays a role in the adaptation to altered feeding status.

Subchronic treatment with metformin produces anorectic effect and reduces hyperinsulinemia in genetically obese zucker rats

The effect of subchronic metformin treatment on food intake, weight gain and plasma and tissue hormone levels was investigated in genetically obese male Zucker rats and in their lean controls. Metformin hydrochloride (320 mg/kg/day for 14 days in the drinking water) significantly reduced 24 hour food intake both after one and two weeks treatment in obese rats. In contrast, metformin had only a transient effect on food intake in lean animals. The reduced food intake was associated with body weight decrease, particularly in obese rats. Metformin markedly reduced also the hyperinsulinemia of the obese animals without altering their plasma glucose or pancreatic insulin content which may reflect an improved insulin sensitivity after metformin treatment. Metformin did not change plasma corticosterone levels or insulin and somatostatin concentrations in the pancreas. Metformin reduced pyloric region somatostatin content in lean rats. It is concluded that metformin has an anorectic effect and reduces body weight and hyperinsulinemia in genetically obese Zucker rat.

Determination of metformin in plasma by high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of metformin, an oral antidiabetic agent, in plasma is described. Plasma samples containing the internal standard, phenformin, are eluted through Amprep extraction columns before injection into the chromatographic column, packed with microBondapak phenyl. The eluent is monitored at 236 nm. At a mobile phase flow-rate of 1.35 ml/min, the retention times of metformin and phenformin are 2.8 and 5.6 min, respectively. The intra-day coefficients of variation are 1.5 and 4.3% at metformin concentrations of 0.05 and 1 mg/l, respectively.

ANORECTIC EFFECT OF METFORMIN IN OBESE ZUCKER RATS : LACK OF EVIDENCE FOR THE INVOLVEMENT OF NEUROPEPTIDE Y

The hypothalamic neuropeptide Y content and preproneuropeptide Y mRNA expression were studied in metformin-treated (300 mg/kg orally for 12 days), in pair-fed and in ad libitum-fed obese Zucker rats in order to elucidate possible mechanisms involved in the anorectic and body weight reducing effect of chronic metformin treatment in genetically obese Zucker rats. In addition the acute influence of metformin on food intake was studied by comparing its effects after oral and parenteral administration. The concentration of neuropeptide Y in the hypothalamic paraventricular nucleus was significantly higher in the metformin-treated and pair-fed rats when compared to the control animals. The expression of preproneuropeptide Y mRNA in the arcuate nucleus was similar in all three treatment groups. Both chronic metformin treatment and pair-feeding markedly lowered hyperinsulinaemia in these animals. A single subcutaneous dose of metformin (300 mg/kg) reduced food intake only in obese animals, while the same dose of metformin given orally did not affect food intake in either lean or obese animals. It is concluded that the treatment with metformin and pair-feeding, which results in comparable reductions in food intake, body weight gain and hyperinsulinaemia, similarly increase neuropeptide Y concentrations in the paraventricular nucleus while not affecting preproneuropeptide Y mRNA expression in the arcuate nucleus. The increase in hypothalamic neuropeptide Y content may be secondary to the reduction in hyperinsulinaemia during metformin treatment and pair-feeding. Thus, the anorectic effect of chronic metformin treatment cannot be explained by changes in content or expression of hypothalamic neuropeptide Y.

Celiprolol augments the effect of physical exercise on insulin sensitivity and serum lipid levels in chronic heart failure.

Impaired insulin sensitivity has been linked with chronic heart failure (CHF). Exercise has a beneficial effect on insulin sensitivity in healthy subjects. It is used also as an adjunctive therapy in patients with CHF. We studied the effect of randomized treatment with celiprolol, a vasodilating β1‐adrenoceptor antagonist, 200 mg once daily (n=20) or placebo (n=11) on serum lipid levels and insulin sensitivity in patients with CHF. In addition, all subjects participated in a 6‐month exercise training protocol. Thirteen subjects in the celiprolol and eight subjects in the control group were on additional β1‐adrenoceptor antagonist as part of their tailored CHF therapy. Insulin sensitivity was determined using the hyperinsulinemic euglycemic clamp test (diabetic subjects excluded, n=11 for the celiprolol group and n=8 for the placebo group).

Anti-obesity effect of MPV-1743 A III, a novel imidazoline derivative, in genetic obesity

MPV-1743 A III ((+/-)-4-(5-fluoro-2,3-dihydro-1H-inden-2-yl)-1H-imidazole) is a novel imidazoline derivative. In this study, it was shown to bind with high affinity to alpha2-adrenoceptor subtypes alpha2A (IC50) = 0.66 +/- 0.06 nM), alpha2B (IC50) = 3.8 +/- 0.53 nM), alpha2C (IC50) = 3.1 +/- 0.61 nM) in the recombinant S115 cells and to alpha2D (IC50 = 0.94 +/- 0.10 nM) in the rat submandibular gland. MPV-1743 A III also showed remarkably high affinity to alpha1-adrenoceptors (IC50 = 150 +/- 12 nM) in the rat cerebral cortex and to imidazoline I2b-binding sites (IC50) = 150 +/- 5.0 nM) in the rat liver. The functional alpha2-adrenoceptor antagonistic effect of MPV-1743 A III was demonstrated by studying the ability of orally administered MPV-1743 A III to reverse and prevent the alpha2-adrenoceptor agonist detomidine-induced mydriasis in rat. The anti-obesity effect of MPV-1743 A III was investigated in genetically obese (fa/fa) Zucker rats in two different phases of obesity. Chronic treatment with MPV-1743 A III (0.3 3 mg/kg per day p.o. for 3 weeks) dose dependently decreased weight gain in early-phase obesity. In fully established obesity, GDP binding to mitochondria and expression of uncoupling protein mRNA were increased in brown adipose tissue by MPV-1743 A III indicating an activation of non-shivering thermogenesis. The present study shows that MPV- 1743 A III has a modest anti-obesity effect in the genetic rodent model of obesity. The relative importance of alpha2- and alpha1-adrenoceptors and imidazoline I2b-binding sites in mediating the effects of MPV-1743 A III needs further evaluation.

Hypothalamic neurochemistry and feeding behavioral responses to clonidine, an alpha-2-agonist, and to trifluoromethylphenylpiperazine, a putative 5-hydroxytryptamine-1B agonist, in genetically obese Zucker rats.

Genetically obese Zucker rats are hyperphagic, hyperinsulinemic and hyperlipemic. In order to investigate pathophysiological mechanisms underlying hyperphagia in these animals, monoamine metabolism and turnover were studied in discrete hypothalamic nuclei known to participate in the control of feeding behavior. Neurochemical studies in genetically obese Zucker rats and in their lean littermate controls were complemented by investigating feeding behavioral responses to the acute administration of clonidine (15 and 30 micrograms/kg i.p.), an alpha 2-adrenoceptor agonist, and to trifluoromethylphenylpiperazine (TFMPP; 1, 2 and 5 mg/kg s.c.), a putative serotonergic 5-hydroxytryptamine-1B receptor agonist. Obese Zucker rats had significantly lower concentrations of 5-hydroxyindoleacetic acid, the main deaminated metabolite of 5-hydroxytryptamine, in the nucleus paraventricularis (PVN) and in the nucleus ventromedialis (VMN), when compared to their lean littermate controls. The rate of accumulation of 5-hydroxytryptophan after decarboxylase inhibition was reduced in the PVN, nucleus supraopticus, nucleus periventricularis and nucleus suprachiasmaticus of the obese rats. No differences were observed in basal concentrations of norepinephrine, dopamine or 3,4-dihydroxyphenylacetic acid between obese and lean Zucker rats in the brain areas studied. However, the rate of accumulation of 3,4-dihydroxyphenylalanine was lower in the VMN and in the median eminence of the obese rats. The feeding behavioral tests showed significantly augmented hyperphagic responses to clonidine in obese Zucker rats. The anorexic effect of TFMPP was similar in both phenotypes. It is concluded that serotonergic activity is reduced in obese Zucker rats, particularly in the PVN, which plays a key role in the control of feeding behavior. The reduced serotonergic activity may be associated with enhanced alpha 2-adrenoceptor-mediated feeding responses in obese Zucker rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative determination of free and total dopamine in human plasma by LC–MS/MS: the importance of sample preparation

BACKGROUND Two methods have been developed and validated for the determination of free and total dopamine in human plasma. They are based on solid-phase extraction of the analyte from the matrix by covalent complexation with phenylboronic acid, followed by derivatization with ethylchloroformate. The derivative is quantified by reversed-phase liquid chromatography on a C18 column and positive electrospray ionization MS/MS. RESULTS The high selectivity obtained, in combination with the stable and relatively non-polar nature of the derivatized analyte, enables the reliable quantification of dopamine in the range 0.05 to 20 ng/ml in a 5 min run time, using only 100 µl of sample. Total dopamine concentrations are determined (range 1 to 400 ng/ml) by including an acidic hydrolysis step, which converts the sulphate and glucuronide conjugates to free dopamine prior to extraction. The method was applied to quantify free and total dopamine levels in human plasma after dosing with the anti-Parkinsons drug combination L-dopa/carbidopa with and without entacapone. CONCLUSION A sensitive and selective LC-MS/MS method has been developed and validated for the determination of free and total dopamine in human plasma. This article demonstrates how essential careful optimization of the sample preparation procedures was for developing a successful method.