Martin G. Hulsey

ICV administration of anti-NPY antisense oligonucleotide: effects on feeding behavior, body weight, peptide content and peptide release

Neuropeptide-Y (NPY) is a potent stimulator of feeding, and chronic administration of the peptide has been shown to increase body weight. This study determined the chronic effects of repeated daily injections of an antisense phosphorothioate oligonucleotide complementary to the rat mRNA for NPY (aNPY) on food intake, feeding behavior and body weight change in rats. Five micrograms of the aNPY oligonucleotide in ten microliters of vehicle or a missense control oligonucleotide were administered intracerebroventricularly (ICV) for seven consecutive days. Cumulative food intake, meal size and meal duration were significantly lowered in aNPY-treated animals. Body weight change of aNPY-injected animals was significantly lower than controls, and the effect was reversed after treatments ceased. A two-bottle taste aversion paradigm was employed to determine the behavioral specificity of the anorectic effect, and the phosphorothioate oligonucleotide was found not to be aversive at the dosage used. Following an additional five day injection period, animals were killed and paraventricular nuclei (PVN) were dissected. In vitro release and tissue content of NPY from this brain area were evaluated by heterologous radioimmunoassay. Content of NPY was unchanged in this brain area. Paradoxically, in vitro release of NPY was increased in aNPY-treated animals.

Metabolic Responses to Intracerebroventricular Leptin and Restricted Feeding

Leptin is a hormone secreted by adipocytes, which plays an important role in the control of food intake and metabolic processes. In the current study, a dose-dependent relationship was shown between a bolus intracerebroventricular rat recombinant leptin administration and reductions in food intake and body weight in Sprague-Dawley rats. During the 24 h postinjection period, food intake was decreased by 24, 26, and 52% with 0.625, 2.5, and 10 microg of leptin, respectively. Body weight was reduced by 2, 3, and 5% at 24 h after leptin administration at the doses of 0.156, 2.5, and 10 microg, respectively. Furthermore, indirect calorimetry demonstrated that five daily i.c.v. injections of leptin resulted in an increase in heat production per unit of metabolic body size and fat oxidation by approximately 10 and 48%, respectively. In contrast, food-restricted rats that consumed the equivalent amount of food as leptin-treated rats for 5 days decreased their energy expenditure by 10%. Food restriction was found to decrease respiratory quotient in a similar pattern as the leptin administration. When ad lib feeding was resumed, food-restricted rats quickly recovered their normal food intakes, body weights, and metabolism. Conversely, leptin treatment has prolonged effects on body weight resulting from different metabolic responses than food restriction. Leptin not only suppresses food intake, but also enhances energy expenditure to reduce fat depots.

Intracerebroventricular (i.c.v.) Administration of Mouse Leptin in Rats: Behavioral Specificity and Effects on Meal Patterns

Leptin is a protein that is produced primarily in fat tissue and is thought to be a lipostatic feedback signal for the regulation of body fat stores. The purpose of this study was to determine the behavioral specificity of i.c.v.-administered mouse leptin in rats and to assess the effects on meal patterns. Using a modified two-bottle paradigm we examined the putative aversive response to i.c.v. doses of 1, 5, 7, 10, and 30 microg of mouse leptin. Artificial CSF and intraperitoneal lithium chloride served as negative and positive controls, respectively. Saccharin consumption in all leptin treatments was not significantly different from the negative control. Following a recovery period, rats from the same group were used to assess the effects of a 30-microg i.c.v. dose on cumulative food intake and meal patterns using a computer-based system for acquisition of feeding data. Leptin (i.c.v.) significantly increased intermeal interval and decreased meal size. We, therefore, conclude that mouse leptin, at doses up to 30 microg i.c.v., is not aversive in the rat, and that leptin has a multiphasic effect on meal patterns.

A system for automated recording and analysis of feeding behavior

We have designed and implemented a system that utilizes a network of top-loading balances digitally interfaced to a Macintosh computer. The system simultaneously collects two forms of data which allow the evaluation of the animals biting and/or licking behavior in addition to cumulative food intake and meal patterns. The system is capable of resuming data acquisition following a power failure without user intervention. Plexiglas cages utilized with the system features adjustable tunnel feeders and are appropriate for use with small rodents. Given appropriate caging, the system may be utilized to evaluate the feeding behavior of other species.

ICV administration of anti-corticotropin-releasing factor antisense oligonucleotide: effects on feeding behavior and body weight

Corticotropin-releasing factor (CRF) has been reported to reduce food intake and body weight, and numerous studies suggest a role for CRF in putative mechanisms for the regulation of body energy. This study investigated the effects of ICV-administered antisense phosphorothioate oligonucleotides, directed against the CRF mRNA, on feeding behavior and body weight in rats. Sixteen male HSD rats were cannulated in the lateral ventricle, and given ad libitum access to tap water and a ground chow diet. Feeding behavior was recorded by computer, and meal patterns were assessed. Rats were given 3 micrograms each of two anti-CRF oligonucleotides (aCRF) or two control oligos in the hour before the onset of the nocturnal cycle for ten consecutive days. Cumulative food intake was assessed at 3, 6, 12 and 24 h after each injection, as well as over the 10-day injection period. Compared to missense controls, rats receiving the antisense oligonucleotides ate significantly more at 6 h (P = 0.01), but not at 3, 12, 24 h, or during the entire 10-day injection period (P > 0.05). There was no effect on body weight change, meal size, or meal interval (P > 0.05). These data indicate that daily administration of anti-CRF oligonucleotides has a significant short-term stimulatory effect on feeding behavior, but does not have a long-term effect on feeding or body weight gain.

An anorectic agent from adipose tissue of overfed rats : effects on feeding behavior

Parabiosis and blood-transfer studies with rodents suggest the existence of humoral factors capable of affecting energy balance. The nature and origin of these factors is undetermined. Aqueous extracts of adipose tissue from overfed rats significantly reduce food intake when administered intraperitoneally (IP) or intracerebroventricularly (ICV). We term the agent(s) responsible for this effect adipose satiety factor (ASF). A single IP dose of ASF, equivalent to 44 mg crude protein, suppresses cumulative food intake for over 12 h. ASF, prepared using a combination of adipose tissue from obese Zucker rats and overfed rats, is more potent per unit of protein than ASF prepared exclusively using adipose tissue from overfed rats. A single ICV dose of this hybrid preparation, equivalent to 14.6 micrograms of crude protein, suppresses cumulative food intake by 40% for up to 48 h. By ultrafiltration, the molecular weight associated with maximal ASF activity is between 30 and 100 kilodaltons (kDa). The behavioral specificity of ASF-induced anorexia is demonstrated using meal pattern, taste aversion, and differential starvation paradigms.

Effects of Intracerebroventricularly Administered Leptin on Protein Selection in the Rat

The effect of centrally administered rat leptin on selection of 5 and 30% protein diets was investigated in male Sprague-Dawley rats with indwelling i.c.v. cannulas. Leptin (0 vs 2.5 microg/day) was administered for 4 consecutive days, followed by an 8-day withdrawal period. Total intake was reduced to approximately 50% of that in the vehicle injected group during each day following leptin administration. Intake of both the 5 and 30% diets was reduced. Vehicle-treated rats selected a 13-15% CP diet. Diet selection in leptin-treated rats was not different during the first day, but on Days 2-4, leptin-treated rats selected a 10% CP diet. Intake began to normalize within 24-48 h after the last treatment, and was not different by Day 3 of the withdrawal period. Body weight was reduced by leptin treatment, and despite the normalization of food intake, did not recover during the withdrawal period. Rats were sacrificed at the end of the 8-day withdrawal period. Despite the reduction in body and carcass weights, liver, kidney, heart, and soleus muscle weights were not different between control and leptin-treated groups when expressed on an absolute or relative basis. However, epididymal and retroperitoneal fat pad weights were still reduced 56 and 78%, respectively, in rats that had been previously treated with leptin for 4 days and then not treated for 8 days. In addition, circulating T3 levels remained elevated in rats that had been treated with leptin. Centrally administered leptin has little effect on muscle mass, but had potent effects on intake of nonobese rats and a sustained effect on adipose tissue mass, thyroid hormone status, and body weight after withdrawal. Results from rats selecting between diets varying in protein content suggest that leptin may cause avoidance of protein.

The Role of Animals In Nutrition Research

To suggest the futility of animal research, antivivisectionists often point to preventive health measures such as diet. Ironically, animal experimentation has played and will continue to play a pivotal role in nutrition research.

11 - Computer Program for Kinetic Modeling of Gene Expression in Neurons

Publisher Summary This chapter describes a computer program that can be used to compare experimental data with theoretical predictions regarding the induction of gene products. The purpose in writing the program was to integrate quantitative data concerning rates of synthesis and decay for gene products. These values may be obtained using run-on assays for nuclear transcription with appropriate corrections, quantitative assays for mRNA by solution hybridization, and immunological or enzymatic assays for peptides or proteins. As semiquantitative data can be used, the technique could be applied to studies using in situ hybridization or immunological detection by light or electron microscopy. The third use is didactic, because the program integrates data from molecular and cellular biology, indicating the enormous amplification involved in mammalian gene expression, the relative concentrations of mRNA and protein, and the predicted lag between induction of mRNA and protein. The kinetic model described in the chapter predicts that the time required for induction of an mRNA or protein to 50% of a new steady-state level cannot be shorter than the respective half-lives of these molecules. Many neuronal genes that are affected by transsynaptic stimulation probably never attain a steady state, because the time course of adaptation by this mechanism is much longer than the periods over which neuronal activity fluctuates. The program represents an initial step toward making computer modeling of gene expression available to any laboratory that possesses a microcomputer.