Measurement of nitric oxide synthase and its mRNA in genetically obese (ob/ob) mice

doi:10.1016/0024-3205(95)02089-2

Life Sciences

Volume 57, Issue 14, 25 August 1995, Pages 1327-1331

https://doi.org/10.1016/0024-3205(95)02089-2 Get rights and content

Abstract

Recent studies have suggested a role for nitric oxide (NO) in the regulation of food intake. The obese (ob/ob) mouse is a genetic model of obesity. Previously, it has been demonstrated that ob/ob mice show a marked weight reduction when treated with a nitric oxide synthase inhibitor. In the studies reported here, we demonstrate increased levels of nitric oxide synthase (NOS) and its mRNA in the hypothalamus of genetically obese (ob/ob) mice compared to their lean littermate controls (ob/c). NOS levels were 0.016 ±0.001 nmol/mg/min in ob/ob compared to 0.009 ± 0.001 in ob/c (p < 0.01) and NOS mRNA was 32.0 ± 5.0 pg NOS mRNA/mg total RNA in ob/ob compared to 12.4 ± 4.0 in ob/c (p < 0.05). These studies further support the possibility of a role for nitric oxide in the regulation of food intake.

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NPY can also carry out its orexigenic effect by stimulating nitric oxide (NO) in PVN neurons (Morley et al., 1999, 2011; Sadler and Wilding, 2004). It has been reported that NO can have an important role in the regulation of food intake (Morley et al., 1995; Ueta et al., 1995). Although most studies have been carried out during the prenatal period (Koski et al., 1986; Kozak et al., 2000), studies performed during the postnatal period have also shown that nutritional status causes impairments in the structure of organs whose critical developmental windows occur during weaning (Hausman et al., 1991; Lanoue and Koski, 1994).
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The gut hormone and neuropeptide ghrelin was initially identified in the periphery as a compound released in the bloodstream in response to a negative energetic status. In the central nervous system (CNS), ghrelin mainly acts on the hypothalamus and the limbic system, with its best-known biological role being the regulation of appetitive functions. Recent research has shown that ghrelin is not an indispensable factor in the regulation of food intake. However, it plays a key role in the metabolic changes of lipids, mainly those involving hypothalamic NOS, AMPK, CaMKK2, CPT1 and UCP2 proteins. Ghrelin participates in the regulation of memory processes and the feeling of pleasure resulting from eating, both of which are metabolism-dependent and may be essential for the successful achievement of adaptive appetitive behavior. Ghrelin exerts its biological effect through a complicated network of neuroendocrine links, including the melanocortin and endocannabinoid systems. The activity of ghrelin is connected with circadian and annual fluctuations, which depend on seasons and food availability.
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In recent years, there have been a large number of neuropeptides discovered that regulate food intake. Many of these peptides regulate food intake by increasing or decreasing nitric oxide (NO). In the current study, we compared the effect of the food modulators ghrelin, NPY and CCK in NOS KO mice. Satiated homozygous and heterozygous NOS KO mice and their wild type controls were administered ghrelin ICV. Food intake was measured for 2 h post injection. Ghrelin did not increase food intake in the homozygous NOS KO mice compared to vehicle treated NOS KO mice, whereas food intake was increased in the wild type controls compared to vehicle treated wild type controls. NPY was administered ICV and food intake measured for 2 h. Homozygous NOS KO mice showed no increase in food intake after NPY administration, whereas the wild type controls did. In our final study, we administered CCK intraperitoneally to homozygous and heterozygous NOS KO mice and their wild type controls after overnight food deprivation. Food intake was measured for 1 h after injection. CCK inhibited food intake in wild type mice after overnight food deprivation, however, CCK failed to inhibit food intake in the NOS KO mice. The heterozygous mice showed partial food inhibition after the CCK. The current results add further support to the theory that NO is a central mediator in food intake.
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Nitric oxide antagonism reverses the orexigenic effect of NPY. Genetically obese mice (ob/ob) have elevated levels of nitric oxide synthase (NOS) and its messenger RNA [25]. Long-term administration of a NOS antagonist causes weight loss in obese (ob/ob) and diabetic mice [26].
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Included are the nervous, cardiovascular, renal, pulmonary, endocrine, and immune systems, where NO acts as a second messenger [9,10]. Evidence is emerging that NO is an important regulator of food intake [11]. Nitric oxide has been reported to interact with many neuropeptides and affect feeding.
Leptin, which is produced in proportion to adiposity, has been reported to regulate feeding behaviors. Previous researchers reported that inhibition of nitric oxide (NO) synthase (NOS) decreased food intake, while L-arginine attenuated this effect. Recently, studies showed that NO plays an important role as a mediator of feeding behavior induced by a variety of neuropeptides. We investigated whether the anorectic effect of leptin is mediated by nitric oxide in broilers and Leghorns. In the first experiment, leptin was intracerebroventricularly (ICV) administered into the right lateral ventricle of broilers and food intake monitored at 15-min intervals through 180 min postinjection. L-arginine attenuated the decrease in food intake induced by leptin. In the second experiment, leptin was coinjected ICV with NG-nitro-arginine methyl ester HC1 (L-NNA), a NOS inhibitor. In the following study, we investigated whether the decreased feeding induced by leptin (10 μg/l0 μl) is mediated by nitric oxide in chickens. Three week old chickens were administered two levels of leptin (A = aCSF, B = 10 μg/l0 μl) into the right lateral ventricle, and nitrate and nitrite (nitric oxide metabolites) were monitored 30-min postinjection. The results showed leptin decreased NO formation significantly compared with the control group. These results suggest that NO interacts with leptin in the central nervous system to modulate feeding behavior in the chicken.
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Measurement of nitric oxide synthase and its mRNA in genetically obese (ob/ob) mice

Abstract

Anal. Biochem.

Europ. J. Pharmacol.

Life Sci.

Life Sci.

Neuropharmacology

Nature Lond.

Diabetologia