We deprived flies of sleep using two methods: a mechanical stimulus and a light stimulus. The mechanical stimulus produced sleep loss when applied at night, but not during the day. When chronically sleep-restricted during the night, flies only partly compensated for the sleep loss, consistent with studies in both rodents and humans 
. When exposed to the same mechanical stimulation during the day, flies slept more than controls. Yet energy stores in both groups of flies displayed a pattern that was consistent across lines and sexes: glycogen levels decreased, while triglycerides increased. These data suggest that the changes in energy stores that we observed are not due to chronic partial sleep loss. Supporting evidence is provided by our findings for flies deprived of sleep using the light stimulus. While not effective at chronically depriving males of sleep, the additional light produced greater sleep loss in females than the mechanical stimulus. However, the pattern of decreased glycogen and increased triglycerides seen after mechanical stimulation was not seen in females stimulated by light. Instead, triglycerides were not significantly altered as compared to the controls, and glycogen was significantly reduced in only one line (w1118
). Taken together, the data indicate that chronic partial sleep loss per se does not impact energy stores. Furthermore, our circadian clock shift experiment was not intended to deprive flies of sleep, but they did lose sleep. Despite the sleep loss, the clock shift experiment had little effect on triglycerides, reinforcing the conclusion that changes in triglycerides were a result of factors other than sleep loss. The effect of the clock shift on glycogen varied among sexes and lines. Since the clock shift was not de-coupled from the sleep loss, two conclusions are possible: alterations in the molecular circadian clock affected glycogen stores, or the sleep loss in combination with changes in the molecular circadian clock affected glycogen. Recent evidence that the fly circadian clock controls feeding behavior argues for the former conclusion. Peripheral clocks in the fat body (analogous to the mammalian liver) inhibited nighttime feeding 
and disruption of this peripheral clock reduced glycogen levels in the fat body, an effect which was opposed by disrupting the circadian clock in neuronal cells 
. Although the current study examined whole-body glycogen, these findings nevertheless imply that the circadian clock can affect glycogen stores in the fly.
It is not clear why the mechanically stimulated flies exhibited increases in triglycerides while having a reduction in glycogen stores. To keep the flies awake, the mechanical stimulus physically perturbs the flies, which may result in an increase in their activity. For example, loss of glycogen has been observed in both the heads and bodies of female flies after sleep deprivation by hand tapping, whether the flies were stimulated during their normal sleep period or during their active period 
. One possibility is that the large reduction in whole-body glycogen that we observed could be due to increases in locomotor activity elicited by the mechanical stimulus. However, if changes glycogen or triglycerides were solely mediated by changes in total activity counts resulting from altered sleep, then one would always observe the same relationship between these nutrients and sleep time. The observations herein suggest that the total amount of activity is not the sole determinant of glycogen or triglyceride levels.
Furthermore, we did not observe a consistent pattern between changes in energy stores and changes in waking activity. Nor was waking activity uniformly increased with the application of the mechanical stimulus, underscoring the previously observed lack of correlation between sleep time and waking activity 
We suggest that the increase in triglycerides we observed after mechanical stimulation may be induced as part of the stress response. A number of stress response pathways are conserved between mammals and flies 
. Recent studies have shown that stress pathway molecules such as c-Jun N-terminal kinase, which is conserved in flies, influence insulin signaling and fat storage 
. Flies deprived of sleep using the mechanical stimulus have increased expression of genes involved in stress response pathways, including genes involved in the inflammatory, oxidative stress, and unfolded protein responses 
, which may account for the increase in triglycerides we observed. Thus, stress is more likely the stronger influence on triglyceride level when flies are mechanically stimulated. How stress might alter triglyceride level, whether through changes in physiology or behaviors such as feeding, remains to be determined.
When human sleep was restricted to four hours for six days, impaired carbohydrate metabolism and endocrine function were observed 
, leading the authors to suggest that long-term sleep loss might produce metabolic changes increasing the likelihood of obesity and type 2 diabetes 
. In this experiment, we did not observe changes in glycogen or triglyceride stores that would suggest similar metabolic changes take place with chronic partial sleep loss in flies. However, in a parallel study, we observed a genetic link between endogenous sleep and energy stores 
. Based upon the metabolic effects of stress discussed above, we suggest that stress may contribute to the effects of short sleep on metabolism.