In the present study, we first demonstrated that trehalose suppressed adipocyte hypertrophy in both WT mice and those with trehalase KO mice. We further showed a negative correlation between the size of mesenteric adipocytes and the proportion of jejunal CLDs. To our knowledge, this is the first study to assess the effects of trehalose on adipocyte hypertrophy and accumulation of jejunal CLDs in trehalase KO mice.
Comparing trehalase KO mice and WT mice fed HFD indicated that trehalose had nearly similar effects in terms of suppression of adipocyte hypertrophy and the jejunal CLD trap rate. Because trehalose is degraded by trehalase in the upper small intestine, the amount of trehalose that reaches the lower intestine is higher in the trehalase KO mice than the WT mice. However, the effects of trehalose on adipocyte size and jejunal lipid droplet trapping were nonetheless equivalent. The similarity in the effects of trehalose on WT and trehalase KO mice suggested that the trehalose-mediated action occurs before trehalose is degraded by trehalase and thus these effects could be induced in sites such as the oral cavity, stomach, duodenum and upper jejunum.
D’Aquila et al. [13] reviewed the significance of jejunal CLDs. Dietary fat consumed as TG is efficiently digested into fatty acids (FA) in the gastrointestinal lumen and is absorbed by enterocytes. The digested products taken up by enterocytes are re-synthesized into TGs and packaged either in chylomicrons (CMs) for secretion or in CLDs for storage. Although the CLDs were thought to be an inactive reservoir of neutral lipids, they are now recognized as dynamic organelles that have functions beyond lipid metabolism [14, 15]. The synthesis of CLDs is thought to buffer enterocytes from FA toxicity and control the rate of synthesis and secretion of CMs [13]. Although all regions of the small intestines can take up and absorb digestive products of TG, the jejunum is the main site for TG metabolite uptake and absorption [16, 17].
When we examined whole intestine tissues histopathologically, the upper jejunum had the strongest intensity of CLD staining and this result was consistent with earlier reports [16, 17]. The finding of substantially more jejunal lipid droplets in the trehalose group compared to the water group was unexpected. Since the accumulation of CLDs suppresses the secretion of CM that migrates to the lymphatic system, according to the review by D’Aquila et al. [13], intestinal CLDs would not necessarily be detrimental. Moreover, this review also reported that the amount CLD increased after meals and decreased upon fasting due to enterocyte turnover such that malabsorption of fat that occurs during steatorrhea did not occur under normal physiological conditions. We also conducted histopathological evaluation of the intestine, liver and pancreas, but observed no abnormal changes associated with trehalose treatment. In particular, there were no differences in the number of intestinal villi and villi length in specific areas between the HFD groups. In contrast, there was a negative correlation between the size of mesenteric adipocytes and the proportion of jejunal CLD content. Trapping lipid droplets in the jejunum could be responsible for the induction of adipocyte hypertrophy suppression.
Xiao et al. [18] performed a single lipid challenge test in which glucose or water was consumed 5 hours after a high fat liquid meal challenge, and a duodenal biopsy was performed 1 hour later to compare the amount of CLDs. Their data indicate that oral glucose mobilizes TGs stored within enterocyte CLDs and provides a substrate for CM synthesis and secretion. At 1 hour after glucose ingestion, the amount of CM-TG was significantly higher and the number of CLDs in the jejunum was significantly lower compared to the water group. This result suggested that glucose suppressed CLD accumulation in the intestine and they were instead rapidly transferred to lymphatic vessels as CM. When glucose was given to mice fed a HFD in our study, we found no suppression of adipocyte hypertrophy [1]. This phenomenon was apparently different from the effect of trehalose on CLDs.
Soriguer et al. [19] reported that plasma levels of glucose, insulin, TG, CM, apoB48, apo A-IV levels and HOMA-IR were all significantly higher in morbidly obese patients with T2DM. But the jejunal wall TG concentration in these patients was markedly lower than in morbidly obese patients without T2DM. In this study, amounts of ApoB48, CM-TG, and HOMA-IR were negatively correlated with jejunal TG. As such, the difference between diabetes and obesity may be due to the suppression of a transition from jejunal lipid droplet traps to CM. These findings were consistent with data for our study.
In terms of the inhibitory effects of trehalose on CM secretion, we measured the amount of APOB48 that passed under the basement membrane of Caco-2 cells. A decrease in the amount of APOB48 was observed in the presence of trehalose. Since APOB48 is a component protein of CM, CM secretion could likely be suppressed by trehalose.
Studies by Debosch et al. [20, 21] indicated that trehalose inhibited glucose transport to induce hepatic autophagy and prevent hepatic steatosis in an SLC2A- and AMPK-dependent manner. The beneficial effects of hepatic AMPK activation in increasing fat oxidation and insulin sensitivity are well documented [22]. In our previous study, trehalose maintained high serum HMW-adiponectin levels in HFD-fed mice compared to the water group [2]. This result suggests that trehalose maintains high levels of HMW-adiponectin that in turn activates AMPK. Meanwhile, Auclair et al. [23] reported that intestinal factors regulate CLDs and showed that increased intestinal AMPK activity reduced lipolysis and decreased secretion of CM from CLDs. We will need to investigate whether there are differences in intestinal AMPK activity with trehalose treatment of mice in future studies.
In recent years, proteins in CLDs have been frequently analyzed to investigate the
relationship between CLDs and systemic lipid metabolism. D’Aquila et al. [24] analyzed the proteome of CLDs isolated from enterocytes harvested from the small intestine of mice following dietary fat challenge. They identified 181 proteins associated with the CLD fraction, 37 of which are related to lipid-related metabolic pathways. Moreover, using confocal and electron microscopy they confirmed that perilipin 3, apolipoprotein A-Ⅳ and acyl-CoA synthetase long-chain family member 5 localized on or around the CLD. However, which proteins actually suppress CM secretion from CLDs remains unclear.
The increase in the rate of jejunum CLDs associated with trehalose treatment compared to the water group may also be due to differences in CLD proteins. Future studies will help clarify what protein(s) is the causative factor of this increase in CLD content. In addition, the effect of lipolysis and lipophagy on CLDs as well as the effect of trehalose on these functions should be investigated.