Scheduled feeding results in adipogenesis and increased acylated ghrelin.

Ghrelin, known to stimulate adipogenesis, displays an endogenous secretory rhythmicity closely related to meal patterns. Therefore, a chronic imposed feeding schedule might induce modified ghrelin levels and consequently adiposity. Growing Wistar rats were schedule-fed by imposing a particular fixed feeding schedule of 3 meals/day without caloric restriction compared with total daily control intake. After 14 days, their body composition was measured by DEXA and compared with ad libitum-fed controls and to rats daily intraperitoneal injection with ghrelin. Feeding patterns, circadian activity, and pulsatile acylated ghrelin variations were monitored. After 14 days, rats on the imposed feeding schedule displayed, despite an equal daily calorie intake, a slower growth rate compared with ad libitum-fed controls. Moreover, schedule-fed rats exhibiting a feeding pattern with intermittent fasting periods had a higher fat/lean ratio compared with ad libitum-fed controls. Interestingly, ghrelin-treated rats also showed an increase in fat mass, but the fat/lean ratio was not significantly increased compared with controls. In the schedule-fed rats, spontaneous activity and acylated ghrelin levels were increased and associated with the scheduled meals, indicating anticipatory effects. Our results suggest that scheduled feeding, associated with intermittent fasting periods, even without nutrient/calorie restriction on a daily basis, results in adipogenesis. This repartitioning effect is associated with increased endogenous acylated ghrelin levels. This schedule-fed model points out the delicate role of meal frequency in adipogenesis and provides an investigative tool to clarify any effects of endogenous ghrelin without the need for ghrelin administration.

Dose-response effects of PEGylated cholecystokinin on the behavioral satiety sequence.

Cholecystokinin (CCK) is known to have a short biological half-life. In order to prolong the half-life and create a new investigative tool, we previously PEGylated the peptide, yielding PEG-CCK(9), and demonstrated that it had a dose-dependent prolonged anorectic effect. The aim of this study was to investigate whether PEG-CCK(9) reduces food intake by inducing satiation or by abnormal physiological effects, such as pain, malaise, or nausea. An observational study was performed to examine the effects of different doses of PEG-CCK(9) (1, 2, 4, 8, or 16 microg kg(-1)) on feeding and other behaviors. The behavioral sequence associated with satiety (BSS), i.e. the orderly progression from eating, through grooming and activity, to resting, was analyzed. From the lowest dose tested (1 microg kg(-1)), PEG-CCK(9) caused a dose-dependent reduction in food intake due to a dose-related reduction in both the duration and frequency of eating and a dose-dependent increase in duration of rest. A dose-dependent acceleration in the temporal profile of the BSS was observed, while the normal structure of feeding behaviors was well preserved, except at the dose of 16 microg kg(-1) of PEG-CCK(9), at which a decrease in eating rate and grooming behavior was observed, together with the occurrence of a significant number of abdominal cramps. These findings suggest that the hypophagic response to PEG-CCK(9) is mainly induced by natural mechanisms of satiety, although abnormal physiological effects, such as abdominal cramps, might reinforce the food inhibitory effect, especially at high doses of PEG-CCK(9) (>8 microg kg(-1)).

PEGylated cholecystokinin is more potent in inducing anorexia than conditioned taste aversion in rats.

BACKGROUND AND PURPOSE: The physiological involvement of endogenous cholecystokinin (CCK) in the termination of feeding has been challenged by evidence of aversive effects of exogenous CCK8. We previously prolonged the anorectic effect of CCK by conjugation to polyethylene glycol (PEGylation) to produce PEG-CCK9. In this study, we investigated the ability of different doses of PEG-CCK9 to induce conditioned taste aversion (CTA) and satiety and identified the receptors involved in CTA induction. EXPERIMENTAL APPROACH: Induction of CTA, measured by the saccharin preference ratio determined in a two-bottle CTA procedure, and of satiety in adult male Wistar rats after intraperitoneal (i.p.) injection of different doses of PEG-CCK9 (1, 2, 4, 8, 16 or 32 microg kg(-1)) was compared. Devazepide (100 microg kg(-1)) and 2-NAP (3 mg kg(-1)), two selective CCK1-receptor antagonists, were co-administered i.p. with PEG-CCK9 (8 microg kg(-1)) and the CTA effects monitored. KEY RESULTS: PEG-CCK9 dose-dependently induced CTA, with a minimal effective dose of 8 microg kg(-1), whereas the minimal effective dose to induce satiety was 1 microg kg(-1). The CTA effects of PEG-CCK9 were completely abolished by i.p. administration of devazepide prior to PEG-CCK9 treatment and only partially abolished by administration of 2-NAP. CONCLUSIONS AND IMPLICATIONS: Although PEG-CCK9-induced satiety and PEG-CCK9-induced CTA both increased with dose, the conjugate was more potent in inducing satiety, suggesting that the anorexia could not be completely attributed to the aversiveness of the drug. As observed with induction of satiety, PEG-CCK9-induced CTA was mediated by CCK1-receptors.

PEGylated cholecystokinin prolongs satiation in rats: dose dependency and receptor involvement.

BACKGROUND AND PURPOSE: Acute intraperitoneal (i.p.) administration of cholecystokinin (CCK) is known to induce a significant, but short-lasting, reduction in food intake, followed by recovery within hours. Therefore, we had covalently coupled CCK to a 10 kDa polyethylene glycol and showed that this conjugate, PEG-CCK(9), produced a significantly longer anorectic effect than unmodified CCK(9). The present study assessed the dose-dependency of this response and the effect of two selective CCK(1) receptor antagonists, with different abilities to cross the blood-brain barrier (BBB), on PEG-CCK(9)-induced anorexia. EXPERIMENTAL APPROACH: Food intake was measured, for up to 23 h, after i.p. administration of different doses (2, 4, 8, 16 and 32 microg kg(-1)) of CCK(9) or PEG-CCK(9) in male Wistar rats. Devazepide (100 microg kg(-1)), which penetrates the BBB or 2-NAP (3 mg kg(-1)), which does not cross the BBB, were coadministered i.p. with PEG-CCK(9) (6 microg kg(-1)) and food intake was monitored. KEY RESULTS: In PEG-CCK(9)-treated rats, a clear dose-dependency was seen for both the duration and initial intensity of the anorexia whereas, for CCK(9), only the initial intensity was dose-dependent. Intraperitoneal administration of devazepide or 2-NAP, injected immediately prior to PEG-CCK(9), completely abolished the anorectic effect of PEG-CCK(9). CONCLUSIONS AND IMPLICATIONS: The duration of the anorexia for PEG-CCK(9) was dose-dependent, suggesting that PEGylation of CCK(9) increases its circulation time. Both devazepide and 2-NAP completely abolished the anorectic effect of i.p. PEG-CCK(9) indicating that its anorectic effect was solely due to stimulation of peripheral CCK(1) receptors.

Mechanism of simmondsin decomposition during sodium hydroxide treatment.

Jojoba seed meal shows appetite-suppressing activity due to the presence of simmondsin. This pharmacological activity disappears with treatment of the meal with sodium hydroxide. To elucidate this mechanism of inactivation, the reaction of simmondsin in 1 N NaOH at 20 degrees C was monitored as a function of time. The end products of the reaction as well as intermediates were isolated and identified. The half-life of simmondsin was approximately 60 min with d-glucose and 2-hydroxy-3-methoxyphenylacetonitrile as reaction end products. The reaction mechanism could be elucidated by the isolation of isosimmondsin and a simmondsin lactone derivative. Those compounds were isolated and purified by a combination of column chromatography and HPLC and identified mainly by HRMS and NMR spectroscopy.

Identification of 4,5-didemethyl-4-O-alpha-D-glucopyranosylsimmondsin and pinitol alpha-D-galactosides in jojoba seed meal (Simmondsia chinensis).

The isolation and identification of two pinitol alpha-D-galactosides from jojoba meal are described. The products were isolated by a combination of preparative HPLC on silica gel and TLC on amino silica gel and were identified by MS, NMR spectroscopy, and chemical derivatization as 5-O-(alpha-D-galactopyranosyl)-3-O-methyl-D-chiro-inositol or 5-alpha-D-galactopyranosyl-D-pinitol and 2-O-(alpha-D-galactopyranosyl)-3-O-methyl-D-chiro-inositol or 2-alpha-D-galactopyranosyl-D-pinitol. The same preparative HPLC method on silica gel allowed a new simmondsin derivative to be isolated and identified as 4,5-didemethyl-4-O-alpha-D-glucopyranosylsimmondsin mainly by NMR spectroscopy and high-resolution mass spectrometry.

Teratological studies in defatted jojoba meal-supplemented rats.

To look for possible developmental effects in the offspring of jojoba meal-treated Wistar rats, and to distinguish between the effects of reduced food intake and the specific developmental effects of jojoba meal itself, mated female rats were divided into three groups of 20 rats. They received during gestation: (a) normal rodent food (control group); (b) normal rodent food supplemented with 3% defatted jojoba meal (jojoba group); or (c) normal rodent food pair-fed with the jojoba group (pair-fed group). The jojoba meal group showed approximately 30% inhibition of food intake. Ten rats from each group were killed on gestation day 21. Compared to the control group, foetal body weight was reduced in both the jojoba and pair-fed groups, with a greater reduction in the jojoba group. Skeletal ossification was retarded to the same extent in both the jojoba and pair-fed groups. The other 10 rats from each group were left to produce litters. Compared with controls, the body weight of the pups was lower in both the jojoba and pair-fed groups; the reduction was slightly greater in the jojoba group, but this difference disappeared after 1 week. The offspring showed no other abnormalities and reproduced normally. We conclude that, at the dose used, the retardation in foetal skeletal ossification, induced by jojoba meal supplementation during gestation, is due to food intake inhibition. Moreover, the lower birth weight of the young of jojoba-treated dams compared with the pair-fed group is merely due to a lower body weight gain during gestation.

Gas chromatographic analysis of simmondsins and simmondsin ferulates in jojoba meal.

A capillary gas chromatographic method was developed for the simultaneous determination of simmondsins and simmondsin ferulates in jojoba meal, in detoxified jojoba meal, in jojoba meal extracts, and in animal food mixtures.

The vagus nerve is involved in the anorexigenic effect of simmondsin in the rat.

Simmondsin, 2-(cyanomethylene)-3 hydroxy 4,5 dimethoxy cyclohexyl beta-D-glucoside, from jojoba meal reduces food intake in rats. We investigated the mechanism of action simmondsin, by studying the effects of fasting or of vagotomy on the food intake reduction. The food intake reduction was significantly less in fasted rats than in non-fasted rats. The reduction of food intake was also significantly diminished after vagotomy. The results of the present experiments suggest that simmondsin reduces intake of food in rats through the augmentation of satiety, in part vagally mediated.

Effects of simmondsin on food intake, growth, and metabolic variables in lean (+/?) and obese (fa/fa) Zucker rats.

Incorporation of 2.5 g/kg of the anorexigen, simmondsin, in the diet resulted in food intake reduction in both lean and obese Zucker rats; however, the obese rats were much more sensitive to the food intake-reducing activity of simmondsin. In both obese and lean simmondsin-treated Zucker rats, growth was slower than in control rats, but was the same as that in pair-fed animals. The 24 h heat production pattern showed a smaller diurnal variation and a lower mean in obese rats than in lean rats. Food intake reduction, as a result of either simmondsin treatment or pair feeding, caused a decrease in mean heat production. Simmondsin treatment, but not pair feeding, caused a decrease in the diurnal variation of heat production. Plasma total cholesterol levels were increased in both simmondsin-treated and pair-fed obese and lean Zucker rats compared with control animals; this increase was mainly due to an increase in HDL-cholesterol levels. Blood leptin levels in both obese and lean rats decreased with decreased food intake and decreased fat deposition, but in obese rats, simmondsin treatment resulted in an additional decrease in leptin levels. It is concluded that the food intake-reducing effect of simmondsin is more pronounced in obese Zucker rats than in their lean littermates, and except for the simmondsin-specific effects on leptin and total cholesterol values in obese littermates, the effects of simmondsin are related to food intake restriction in obese and lean Zucker rats.

Evaluation of jojoba meal as a potential supplement in the diet of broiler breeder females during laying.

1. This study was undertaken to investigate whether jojoba meal can be used as a food supplement during the laying period of chickens. 2. The size of eggs laid were smaller and the overall production rate was lower compared to control birds on food without jojoba meal supplementation. Furthermore, both ovary and oviduct weights were lower in jojoba fed birds. 3. This lowering of egg size and production rate was caused by factors present in jojoba which interfere with follicle growth, yolk deposition, progesterone production and the follicular maturation processes, resulting in the ovulation of smaller follicles and a lower ovulation rate.

Comparison of the effects of simmondsin and cholecystokinin on metabolism, brown adipose tissue and the pancreas in food-restricted rats.

In this study, we investigated the analogies between the physiological effects of simmondsin, a satiety-inducing glycoside extracted from jojoba seeds, and the gastro-intestinal satiation peptide, cholecystokinin. The effects of intraperitoneal injection of the biological active CCK-octapeptide on the pancreas, interscapular brown adipose tissue, growth performance and energy metabolism in normal-fed, severely food intake-restricted (50 % of normal food intake) or moderately food intake-restricted (65 % of normal food intake) growing rats were compared to the effects of 0.25 % simmondsin mixed in the food, inducing moderate food intake reduction (65 % of normal) in rats. Cholecystokinin induced pancreatic hypertrophy. In normal fed rats, cholecystokinin had no effect on brown adipose tissue or growth, while, in severely food intake-restricted rats, it caused brown adipose tissue hypertrophy and reduced growth. In moderately food intake-restricted rats, both cholecystokinin and simmondsin induced pancreatic hypertrophy, increased brown adipose weight and metabolism and caused a slight decrease in growth. We conclude that cholecystokinin may decrease growth performance in fast growing severely food intake-restricted rats by stimulating brown adipose tissue metabolism, probably because of protein shortage induced by pancreatic hyperstimulation. Simmondsin has similar effects. These results support the hypothesis that endogenous cholecystokinin is involved in the effects of simmondsin in rats.

Reproductive performance of rats treated with defatted jojoba meal or simmondsin before or during gestation.

The effects on food intake, growth and reproductive performance parameters of defatted jojoba meal and pure simmondsin, an extract from jojoba meal, were compared in female Wistar rats. Rats fed 0.15% simmondsin or 3% defatted jojoba meal (equivalent to 0.15% simmondsin) for 8 weeks before conception showed a similar reduction in food intake (about 20%) and a similar growth retardation compared with controls. Both treatments induced a reduction in the number of corpora lutea on gestation day 16: this effect could be ascribed to the lower food intake before conception because it was also observed in rats pair-fed to the treated ones. Rats given feed containing 0.15% simmondsin or 3% defatted jojoba meal during days 1-16 of gestation showed a similar reduction in food intake relative to controls. Foetal and placental weights were reduced, relative to controls, to a similar extent in both groups, and the reductions were slightly greater than in the corresponding pair-fed groups. We conclude that the effects on food intake, growth and reproductive performance that were seen after feeding rats defatted jojoba meal were due to the simmondsin content of the meal. The simmondsin induced reduction in food intake and probably also a relative protein shortage.

Unidentified Factors in Jojoba Meal Prevent Oviduct Development in Broiler Breeder Females.

Supplementation of feed with jojoba meal, as a means for autonomous feed restriction, was successful in depressing feed intake and controlling body weight of broiler breeder pullets to the extent recommended by the breeder company. However, these broiler breeders never produced eggs. At the level of ovary, normal follicle development and maturation did occur. A considerable number of ovulations occurred which were not followed by oviposition. After ovulation, the ova could not be captured by the oviduct, because of the small size of the oviduct, resulting in "internal laying". The virtual absence of oviduct development cannot be explained presently but it must be due to some yet unidentified factor(s) in jojoba meal which prevent(s) the normal development of the oviduct. These factors may be acting by abnormally increasing plasma progesterone or triiodothyronin levels and/or directly by themselves interfering with oviduct development. The nature of these factors requires further investigations.

Influence of feeding pattern on thyroid hormones in long-term food-restricted rats.

The effect of a long-term 50% food restriction on plasma thyroid hormone levels was studied in adult male rats, which received their reduced daily amount of food either as one meal or 5 different meals. Compared to controls, a long-term food restriction reduced total 24 h-mean plasma T4, T3 and TSH concentrations. The total T4 level was more reduced in rats fed their reduced ration in one meal than in rats given food in several meals. This resulted in a higher T3/T4 ratio in the former group of rats than in the other groups (free access to food; food in several meals). In the control group and in the food-restricted group receiving their food in one meal, significant diurnal variations in total T4, T3 and TSH levels were seen. The T3 rhythm was abolished in food-restricted rats receiving their food spread over several meals, but T4 and TSH rhythms were not. We conclude that total peripheral thyroid hormone levels infood-restricted rats are influenced, not only by the amount of food but also by the feeding pattern.

Devazepide reverses the anorexic effect of simmondsin in the rat.

Simmondsin, a glycoside extracted from jojoba meal (Simmondsia chinensis), causes a reduction in food intake after oral administration. To investigate the mechanism by which simmondsin reduces food intake, fasted and free-feeding rats were given simmondsin-supplemented food and simultaneously injected with devazepide, a specific antagonist of peripheral-type cholecystokinin receptors (CCKA receptors). In free-feeding rats, supplementation of food with 0.5% simmondsin caused a reduction in food intake of +/- 40% in the period of 4 h following food presentation. Intraperitoneal injection of 100 micrograms devazepide/kg body weight prevented this effect. In rats fasted for 20 h, the food intake in the 30 min after presentation of food supplemented with 0.15% or 0.50% simmondsin was reduced in a dose-related manner; this was also inhibited by simultaneous application of devazepide. It is suggested that peripheral CCKA receptors are involved in the effect of simmondsin on food intake. However, a direct effect of simmondsin on CCKA receptors has been excluded, since simmondsin was unable to cause contraction of the guinea-pig gallbladder in vitro.

Partial food restriction increases hepatic inner ring deiodinating activity in the chicken and the rat.

The effects of a long term partial food restriction were studied in chickens and rats. In chickens the treatment resulted in increased plasma T4 levels while T4 levels in rats remained unchanged. Plasma T3 decreased in both species. In vitro hepatic outer ring deiodinating type I (ORD-I) activity was not influenced by the food restriction, suggesting that the amount of ORD-I enzyme present in the liver remained unchanged. In vitro hepatic inner ring deiodinating type III activity, on the contrary, was greatly increased in both species. This increase may contribute to the decreased circulating T3 levels by increasing the degradation of T3 and diverting the deiodination of T4 to rT3 instead of to T3.

Influence of long-term simmondsin administration on thyroid hormone levels in adult rats.

Treatment of adult rats for 28 days with 0.50% of simmondsin induced a reduction of food intake of about 40%. The degree of emaciation was the same in simmondsin treated rats (SM) and pair-fed controls (PF). Diurnal variation of plasma thyroid hormone concentrations was determined. The reduction in food intake induced a decrease of mean 24 h plasma T3, T4 and TSH concentrations. Plasma T4 concentrations were decreased more in SM than in PF at almost all times of the day. No difference was seen between SM and PF for plasma T3 and TSH concentrations. Liver outer ring deiodinating activity (type I) remained unchanged compared to controls in PF and SM, but liver inner ring deiodinating activity (type III) was increased 4 fold in both SM and PF. It was concluded that the food intake reduction induced by simmondsin produced a decrease in plasma TSH concentrations which in turn reduced plasma T4 concentrations. Decreased plasma T4 concentrations together with an increased degradation of plasma T3 resulted in lower plasma T3 concentrations.

Extraction and liquid chromatographic method for the determination of simmondsin in plasma.

A solid-phase method for the extraction of simmondsin from plasma was developed along with a liquid chromatographic method for the quantitative measurement of this compound. The extraction of simmondsin is realised with activated carbon. Chromatography is performed on a 25 x 0.46 cm I.D. RP-18, 5-micron HPLC column with a water-methanol (85:15, v/v) mobile phase and ultraviolet absorbance detection at 217 nm. The limit of detection is 100 ng (using 1 ml of plasma). The linear quantitation range is 0.1-200 micrograms/ml.

Jojoba meal (Simmondsia chinensis) in the diet of broiler breeder pullets: physiological and endocrinological effects.

The present studies evaluated the ability of jojoba meal (JO) to inhibit feed intake of broiler breeder pullets to limit body weight gain as recommended by the breeder company. A first experiment, using graded levels of JO supplementation (0 to 12%), was conducted to establish appropriate JO supplementation. Adequate reduction of growth rate was obtained with 4% JO supplementation. However, notwithstanding their similar growth rate, 4% JO chickens consumed considerably more feed compared with feed-restricted chickens. The dose-dependent impairment of feed intake with increasing levels of JO supplementation was also associated with increased plasma growth hormone and thyroxine and with decreased plasma insulin-like growth factor-I and triiodothyronine concentrations compared with 0% JO chickens. A second experiment included a pair-fed group. Notwithstanding their similar feed intake, 4% JO chickens gained significantly less body weight compared with their pair-fed counterparts. The 4% JO chickens also had a longer feed transit time per kilogram body weight. Again, circulating levels of the somatotrophic and thyrotrophic hormones were altered according to the dietary treatment. From all these observations, it was concluded that the growth retardation caused by JO supplementation was provoked by an inhibition of appetite linked with the simmondsin content of JO as well as by other antinutritional compounds affecting digestibility.