SUMMAR

Glucose is essential for life. It is a major fuel for the body, and under normal circumstances it is the only fuel that the brain uses. Therefore, glucose concentrations in the blood, and in the brain in particular, are kept within narrow ranges. On earth, light and dark are present intermittently, which gives a 24-hour daily rhythm to life. As a result, daily rhythms in activity and feeding occur and energy expenditure and the need for glucose have a rhythmic nature as well. The daily light/dark signal is received by the eyes and transmitted to, amongst others, the biological clock, located in the suprachiasmatic nucleus (SCN) of the hypothalamus. The SCN sends its daily signal to the brain and the rest of the body via neural projections and via daily rhythms in hormones (chapter 1). Energy metabolism is also organized by the SCN. Previous research of our group has revealed a daily rhythm in basal plasma glucose concentrations. Furthermore, glucose tolerance and insulin sensitivity vary over the light/dark (L/D) cycle. The aim of the current thesis was to further elucidate via which pathways the SCN controls peripheral glucose homeostasis. We focused on two processes that influence the plasma glucose concentration: hepatic glucose production and glucose uptake in peripheral tissue.

Plasma glucose concentrations and peripheral glucose disposal are high at the same time of the day, i.e. the onset of the activity period. Therefore it was suggested that glucose production should be high at that moment as well. Hepatic glucose production (HGP) can be stimulated via both neural and hormonal pathways. One of the hormones that stimulate HGP is pancreatic glucagon. We have shown that plasma glucagon concentrations have a daily rhythm that is modulated by the SCN (chapter 2). However, this rhythm did not show its peak at the onset of the activity period, when plasma glucose concentrations are high. Moreover, the daily rhythm in feeding behavior also had a major influence on the plasma glucagon concentrations, whereas the daily rhythm in plasma glucose concentrations is independent of feeding behavior. Therefore, we concluded that glucagon probably is not part of the SCN pathways that modulate the daily rhythm in plasma glucose concentrations. Other hormones that stimulate glucose production, like growth hormone, corticosterone and epinephrine, are, for different reasons, not likely to be involved in the control of the daily rhythm in plasma glucose concentrations. The SCN is therefore more likely to control glucose production via the autonomic nervous system, rather than via hormonal pathways.

Glucagon is a counterregulatory hormone, i.e. it stimulates glucose production to reverse a hypoglycemic event. We have shown that not only basal plasma glucagons concentrations are controlled by the SCN, but that also glucagon responses, due to insulin-induced hypoglycemia, display a daily variation (chapter 3). Besides, other responses to insulin injections and novelty stress, like corticosterone and ACTH but also leptin, showed a daily variation.
Glucose uptake in peripheral tissues is another process that influences the plasma glucose concentration. Therefore, we tested the hypothesis that the SCN modulates the daily rhythm in plasma glucose concentrations by controlling peripheral glucose uptake. Indeed, glucose tolerance, i.e. the speed of glucose disappearance, displays a daily rhythm. Hormones like insulin, but also other nutrients influence glucose uptake. Previous studies have shown that basal plasma insulin concentrations do not show a clear daily rhythm and were shown not to be responsible for the daily rhythm in plasma glucose concentrations. Insulin sensitivity does show a daily rhythm, which correlFates with the daily rhythm in glucose tolerance, but is opposite to the daily rhythm in basal plasma glucose concentrations. However, a daily rhythm in whole body insulin sensitivity cannot explain a differential control of glucose uptake tissues.
Other factors also affect glucose uptake. Free fatty acids, the building bricks of fat, compete with glucose for uptake in cells and oxidation, and thereby also influence plasma glucose concentrations. We hypothesized that plasma FFA concentrations may fluctuate over the L/D cycle and that high FFA concentrations, due to the sleep-induced fasting, may inhibit glucose uptake at the end of the sleep period, thereby causing high plasma glucose concentrations (chapter 4). In spite of other reports about daily fluctuations in lipid metabolism, we did not find a daily variation in plasma FFA in ad libitum fed rats. When rats were fasted, however, we found increased FFA concentrations because fasting increases the rate of lipolysis. In addition, we did find a difference between day and night, with higher night time levels. Rats with SCN lesions, however, already showed increased FFA concentrations during ad libitum condition, but did not respond to fasting with increased FFA concentrations compared to ad libitum fed SCNx rats. We concluded that the SCN modulates daily lipid metabolism by inhibiting lipolysis at certain moments of the L/D cycle. However, the daily rhythm in plasma glucose concentrations is not controlled via the daily variations in lipid metabolism.
Consequently, we aimed to identify the pathway via which the SCN inhibits lipolysis (chapter 4). We hypothesized that the SCN to paraventricular nucleus (PVN) projections might be involved, because of several reasons. First, the SCN sends many inhibitory GABA-ergic projections to the PVN. Second, stimulation of the PVN induces hepatic glucose production, mediated via the sympathetic innervation of the liver. Third, sympathetic stimulation induces lipolysis. Therefore, we stimulated the PVN, but did not find the expected increased plasma FFA concentrations. We concluded that lipid and glucose mobilization are mediated via differential hypothalamic pathways, and that the medial preoptic area (MPO) instead of the PVN is a good candidate for SCN control of lipid metabolism.

As indicated above, glucose tolerance differs throughout the light/dark period. It is unknown, however, which tissues are responsible for the daily change in glucose uptake. In addition, there daily rhythms in glucose uptake might even differ from one tissue to another. Because the autonomic nervous system innervates various tissues that take up glucose, like adipose tissue and skeletal muscle, the SCN possibly regulates the differentiated peripheral glucose uptake via this way. Furthermore, the daily differences in glucose tolerance are possibly caused by a daily variation in insulin-inhibited hepatic glucose production, rather than by a daily variation in glucose uptake. To test these hypotheses, we used radioactively labeled 2-deoxyglucose, a type of glucose that cannot be metabolized (chapter 5). However, this technique proved not to be suitable for our purpose, i.e. to investigate a daily variation in basal glucose uptake. In other studies, this 3H-2DG is used in combination with hyperinsulinemic conditions. Therefore, we were unable to test our hypothesis.

In chapter 2 and 4, we also measured plasma glucagon and FFA concentrations in fasted rats, and found a similarity between these results. Both rhythms had a larger amplitude in the fasted animals, a phenomenon also seen in previous studies in which body temperature in fasted rats was measured. The increased amplitude is mainly due to decreased trough levels during the resting period. The animals seem to save energy by decreasing their energy expenditure during the sleep period. We hypothesize that the SCN controls this process of energy saving via its projections to the MPO (chapter 6).

In conclusion, the current thesis shows that the SCN modulates energy metabolism, and glucose metabolism in particular, in different ways. First, it modulates basal daily rhythms in order to prepare the organism for the daily recurring changes in energy intake and expenditure. Furthermore, responses to acute disturbances of homeostasis are adapted to the time-of-day, because these disturbances may have a different impact at different times of the day. Finally, the importance of the SCN is emphasized in case of energy restriction. When no food is available, energy expenditure should be confined to selective moments of the day, in order to save energy. The animals decrease their energy expenditure during the resting period, but their ability to be active at the onset of the activity period remains intact.