Food intake regulation

Food intake is controlled by complex physiological mechanisms that are not yet fully understood. Neurotransmitters, nerve pathways and hormones certainly play key roles in this regulation. Different short acting satiety inducing signals, like cholecystokinin or NPY, or orexigenic signals such as orexin, interact with central or peripheral neuronal pathways to influence feed intake. But energy intake and expenditure is also influenced by long term signals. Leptin, a polypeptide produced by fat cells, is such a long-term signal, which induces feed intake reduction and an increase in energy used in the maintenance metabolism. The interaction between long-term and short-term satiety signals is not very well documented yet. By making use of simmondsin as a research tool, we are studying this interaction. Simmondsin (2-cyano methylene-3-hydroxy-4,5-dimethoxycyclohexyl beta-D-glucoside) is an anorexic substance that can be extracted from the seeds of the jojoba plant (Simmondsia chinensis).

1. Simmondsine as a research tool to study the mechanisms of hunger and satiation

It has been shown that simmondsin acts as a satiation inducing substance when mixed in the food of rodents. The effect is short lasting, since the food intake reduction is abolished as soon as simmondsin is withdrawn from the food. The hypothesis that simmondsin exerts its anorexic effect by indirectly acting on the peripheral CCK-A receptors was further explored using two different peripherally CCK-A receptor antagonists; 2-NAP and devazepide. Since 2-NAP and, under certain circumstances, also devazepide do not abolish the food intake reducing activity of simmondsin, the involvement of the CCK-system in the anorexic effect of simmondsin has to be questioned.

By selective breeding, the Otsuka Long Evans Tokushima fatty (OLETF) rat strain was developed in Japanese laboratories out of the LETO strain (Long Evans Tokushima Otsuka rats). It was described that these OLETF rats, lacking an active form of the CCK-A receptor, display hyperphagia and that their food intake could not be reduced by CCK, whereas in LETO rats CCK does reduce food intake. These OLETF rats, lacking the active form of the CCK-A receptor were thus used to further test whether the anorexic activity of simmondsin depends upon intact CCK-A receptors. In short-term experiments, CCK did not, but simmondsin did, induce food intake reduction in the OLETF rats. In long-term experiments, the same effects on food intake were observed. However, the effect of simmondsin on pancreatic and brown adipose tissue (namely a mild hypertrophy and a higher metabolic activity) was not seen in OLETF rats, but present in the LETO rats with intact CCK-A receptors. It might therefore be that the simmondsin-induced anorexia is not CCK-A receptor mediated, but that some physiological effects of simmondsin are CCK-A receptor dependent. Further research, by in vitro experimentation is needed on this topic.

Left: a jojoba plant (Simmondsia chinensis); right: a jojoba nut, from which simmondsin is extracted

In a second series of experiments, the synergistic action of CCK or simmondsin on the one hand, and leptin on the other hand was studied. The synergistic activity of leptin with simmondsin or CCK was tested by simultaneous intraperitoneal administration of leptin with one of the other components. We confirmed an additive, but not always synergistic, effect of CCK and leptin on food intake. This synergism was, according to our results, not strong enough to explain the, in the literature suggested, influence of leptin on the short-term satiety system. We could not determine a synergistic or additive effect between leptin and simmondsin, whereas between CCK and simmondsin an additive effect was observed.

2. Further biological and chemical studies on simmondsin and its derivatives

Besides simmondsin itself, there are several naturally occurring derivatives in the jojoba seeds. However, out of demethylsimmondsin, didemethylsimmondsin and simmondsin ferulate only the simmondsin ferulate has analogous anorexic effects as simmondsin, probably because it is digested to simmondsin in the gut and thus exerts its action. Other synthetically prepared derivatives are also being tested for anorexic effects.
The possible side effects of simmondsin have been studied profoundly in the last three years. We studied the teratological effects of chronic administration of low doses (0.25% mixed in the food) and the haematological effects of chronic administration of higher doses (0.5% mixed in the food) of simmondsin.

Teratological effects of simmondsin treatment (0.25%) in Wistar dams

A good parameter controlling for teratogenicity and foetal development, besides foetal weight, length, survival percentage and litter size, is the ossification pattern of the foetuses. Considering the ossification of foetuses in simmondsin-treated, pair-fed and control dams, together with the other above-described parameters, we concluded that the developmental retardation seen in foetuses from simmondsin treated dams, is merely due to a lower bodyweight gain of the dams themselves. We thus consider the reduced food intake of these animals compared to the control dams to be the major cause of developmental retardation, since the retardation relative to pair-fed animals was insignificant.

Haematological effects of high doses of simmondsin in male Wistar and Sprague Dawley rats

To further characterise the possible toxic effects of simmondsin, a long-term study with high simmondsin doses (0.5%) was started in Sprague Dawley as well as in Wistar rats. During this experiment, blood samples were drawn and analysed, and the results were compared with pair fed and control animals. Autopsies were performed at different time points. From these experiments, we conclude that simmondsin treatment in higher concentrations has a negative influence on the blood composition. These effects are more or less obvious, depending on the rat strain studied. In growing Sprague Dawley rats the simmondsin treatment resulted in a more extreme reduction in food intake than in Wistar rats. The effects of simmondsin, being a reduced hematocrit, augmented red blood cell volume and kidney, pancreatic and liver hypertrophy, were thus also much more expressed in Sprague Dawley rats. Simmondsin-treatment of Sprague Dawley rats even resulted in the death of some animals, while the Wistar rats all survived the simmondsin treatment during the entire experimental period.

This research topîc is no longer continued (dec. 2010)

Contact: Prof. Dr. Marnix Cokelaere

3.  Hydrophobizing cholecystokinin

The well-known effect of CCK on food intake reduction was also studied by a biophysical approach. We hypothesized that the CCK-receptor binding process could be considerably enhanced by first hydrophobizing the hormone, i.e. by linking it to a phospholipid molecule, and subsequently incorporating the phospholipid-CCK complex into the receptor-containing membrane. In this way, it may be expected that less hormone would be required to trigger a given intracellular effect and that the effect will last for a longer time period. However, one major impediment that could hinder the desired messenger-receptor interaction would arise if, for instance, the bioactive region on the receptor is located on an extracellular loop that cannot be reached by the membrane-associated hormone. To overcome this geometrical problem, a flexible spacer arm needs to be included between the hormone and the lipid moiety.

The work we performed is an appraisal of this strategy. In a first step, phosphatidylcholines were converted to their phosphatidylethanolamine (PE)-analogues by a transphosphatidylation reaction, catalyzed by phospholipase D, extracted from Brussels sprouts. Subsequently, the resulting PEs were reacted with the α-biotin,ω-N-hydroxysuccinimidyl ester of poly(ethylene glycol)-carbonate (NHS-PEG-B) in the presence of triethylamine. By carefully checking the various experimental parameters, ultimately we reached a 100% efficiency of the reaction.
Another challenge deals with the application route of this amphiphilic molecule in an organism. Liposomes or vesicles, which - in the past - have been successfully used as biocompatible drug delivery systems, are excellent candidates as vehicles. So-called magnetoliposomes, which we developed a few years ago, can act as a valuable alternative. This new type of vesicle consists of a nanometer-sized iron oxide grain, wrapped in a phospholipid bilayer. A main advantage of these magnetic biocolloids is that they can be relatively easily monitored in vivo by magnetic resonance imaging techniques.

Upon injection in an organism, however, the 'foreign' particles are quickly removed from circulation, mainly by the liver and the spleen. Fortunately, this problem can be avoided by incorporating our PE-PEG complexes at low concentration (5 to 10%) in the (magneto)liposome coat. Since the polymeric chains (molecular weight 3400) are dangling in the surrounding medium, they create a highly flexible and variable surface to the reticulo-endothelial system of the liver. Eventually, this feature leads to the absence of any undesirable immunological response and, as a result, the blood circulation time will be considerably prolonged.

In developing a suitable delivery system, it is of extreme importance to gather very detailed information on the intrinsic binding properties of the PEGylated PE derivative into the different membrane structures. To study this problem, we embarked on a study dealing with the spontaneous transfer behaviour of the PE-PEG-B complexes. In our experimental set-up, small unilamellar vesicles in which the derivatized PE was incorporated (=donor particles) were mixed with magnetoliposomes (=acceptors). At various time intervals, part of the kinetic mixture was separated by high-gradient magnetophoresis and the eluate and retentate fractions were checked for their content of the modified phospholipid. To assess the impact of the PEG-B moiety on the overall transfer event, similar experiments were performed with donors containing the same amount of non-modified PE. As an example, in the case of dipentadecanoylPE no transfer at all was observable, whereas in the case of the derivative with PEG-B a half-time for transfer of only 70 minutes was found. In the very near future, the discrete role of overall membrane characteristics, such as membrane surface charge, degree of fluidity and membrane curvature will be further explored.

Schematic representation for the synthesis of the functionalised phospholipid, used in intermembraneous transfer experiments

Contact: Prof. Dr. Marcel De Cuyper

4. Perspectives

The effects of CCK and simmondsin on leptin production will be further explored. To unravel the anorexic mechanisms of simmondsin, a microstructure analysis of food intake behaviour will be conducted with the aid of sophisticated material. The effects of several satiety inducing hormones such as CCK, leptin, ....and hydrophobised CCK will be compared.