| Caffeine, which has been used for thousands of years, is the most widely consumed bioactive compound and commonly exists in all kinds of beverages, food sources and drugs. The view that the wide consumption of caffeine induces anorectic functions for food and then facilitates people to maintain or to lose weight, is still controversy. Moreover, most of researches for the anorectic functions of caffeine primarily focus on the recogniztion and motivation of animals and human for food sources. However, there are few available reports about the mechanisum, especially the taste perception basis. Taste is the crucial driving and modulating factor for the ingesting behavior of animals. Consequently, on the basis of recent researches including taste perception mechanism, taste plasticity and influencing factors of taste changes, the hypothesis that behavioral changes of sweet taste preference from chronic administrationof caffeine correlate with the characteristic changes of taste peripheral sensor is proposed. We set up a particular model system using ICR mice and perform a series of experiments to explore the correlation between variation of sweet taste preference and changes of fungiform taste buds characters following chronic caffeine exposure. Meanwhile, we also evaluate anatomic alternation of intestinal villus, expression of sweet taste perceptin related proteins, and glucose homeostasis. Finally, the contributions between oral and postoral sensation for the taste sensation variation are estimated by different exposure pattern of caffeine, including free-access stimulator and gavage administration of stimulator. In this study, many techniques are used including behavior method (two-bottle preference test), immunohistochemistry for cofocal laser scanning microscope, Western blot, real-time PCR and enzyme-linked immunosorbent assay (ELISA), etc. The correlations between the potential variation of peripheral sensor of taste and anorectic functions of caffeine are elaborated from several aspects. The primarily results are showed as follows:(1) Variation of taste preference after chronic administrationof caffeineDifferent age mice have distinct taste preference pattern. The preference pattern of periadolescence mice is "indifference-avoidance" and the avoidance threshold is 5.17 mmol/L. However, the preference patterns of adolescence and adult mice are the same "indifference-preference-avoidance" and their preference thresholds are 0.92 mmol/L and 0.09 mmol/L, respectively. Their avoidance thresholds are 5.178 mmol/L and 16.28 mmol/L, respectively. Although after 21 days caffeine exposure, the preference pattern of the mice is not changed, the tolerance of caffeine is enhanced. Compared with control mice, this effect represent the increase of preference ratio in periadolescence and adolescenc caffeine-treated mice and the increase of preference thresholds in adolescence and adult mice with 5.17 and 0.29 mmol/L, respectively.Caffeine administrition have no influences on the preference pattern and the most preference concentration of AK solution, all the mice present "indifference-preference-avoidance". Howevere, the preference threshold and preference ratio are changed and presented different characterization with age. The preference threshold of periadolescence mice is decreased after caffeine exposure and is 0.42 mmol/L, by contrast with 1.33 mmol/L of control mice. The preference ratio and the most preference concentration are not altered. Compared with periadolescence, the preference threshold and preference ratio of adolescence and adult are increased (control to caffeine,0.42 mmol/L/1.33 mmol/L) and decreased, respectively. By contrast with control mice, the preference ratios of adolescence and adult have an average decrease of 6% and 13.83%, respectively. In a word, the caffeine preference patterns of different age mice are reported for the first time, which is significant for other behavior researches using caffeine. The effect of caffeine exposure for sweet perception of mice sufficiently confirms the anorectic function of caffeine for palatable foodstuff.(2) Chronic administration of caffeine alters anatomic characters of fungiform taste budsThe effect of caffeine chronic exposure for total number of taste bud has no dose difference. The total numbers of fungiform taste buds of all age mice, except for adolescence mice of 500 mg/L caffeine-treated, are not affected and located between 100 and 110 per tongue. In view of the irregular traits of fungiform taste buds, we estimate their morphometric changes using the maximal cross-sectional area and longitudinal axis height of fungiform taste buds. Overall, the maximal cross-sectional areas of fungiform taste buds have no significant changes than that of control. But there is a clear decresing trend in adolescence and adult caffeine-treated mice. Moverover, the heights of taste buds and the number of taste cells per taste bud of all age caffeine-treated mice are absolutely decreased. Compared with control mice, the heights of taste buds decrease 11% to 13%(periadolescence),13% to 13%(adolescence), 13% to 13%(adult) and the number of taste cells per taste bud decrease 20% to 14%(adolescence),15% to 8%(adult) between 300mg/L and 500mg/L caffeine exposuring mice. According to forementioned results, chronic administration of caffeine has significant influence on the anatomic characters of fungiform taste buds, including the decrease of taste bud volume from comprehensive action between maximal cross-sectional area, height of taste buds and the number of taste cells per taste bud. These maybe result in the degradation of accepted taste signal information and activity of taste afferent nerve, which lead to the changes of food intake of animal. Furthermore, the larger age of the mice, the biger the effects are.(3) Effect of chronic administration of caffeine for the expression of sweet taste perception related protein of mice fungiform taste budsThe results of immunofluorescence show that the a-gustducin immunopositive taste cells per taste bud of caffeine-treated mice are lower than that of control mice. Compared with control mice, the number of immunopositive taste cells per taste bud decrease 22% to 29% (periadolescence),15% to 33% (adolescence),23% to 37% (adult) between 500 mg/L and 300 mg/L caffeine exposuring mice. The lower dose caffeine solution (300 mg/L) have larger effect than the higher dose (500 mg/L) (P<0.05). Furthermore, caffeine exposure has more evidently influence on the number of immunopositive taste cells per taste bud of male mice than that of female mice (P<0.05). Compared with control mice, the number of immunopositive taste cells per taste bud decrease 22% to 15%(periadolescence),15% to 9%(adolescence),23% to 16% (adult) between male and female caffeine-treated mice. Consequently, male mice and 300 mg/L caffeine solution are used in following experiments. The quantitative changes of a-gustducin of fungifrom taste bud are further demonstrated by Western blot results. Compared with control mice, there are 1.43-fold (periadolescence),1.29-fold (adolescence),1.28-fold (adult) dereases in a-gustducin protein abundance of caffeine-treated mice. The quantitative changes of PLCβ2 are also analysed and also take on a decreasing trend. Compared with control mice, there are 1.35-fold (periadolescence),1.41-fold (adolescence),1.08-fold (adult) dereases in protein abundance. By contrast, sweet receptor protein T1R2 and T1R3 have an inverse changing trend. Compared with control mice, there are 3.28/1.81-fold (periadolescence),3.29/1.94-fold (adolescence),2.55/2.05-fold increases in T1R2/T1R3 protein abundance. However, there is no available regular and significant changes for SNAP25, which a key protein to modulate neurotransmitter releasing. The specific function of caffeine exposure for taste perception related protein abundance is determined by the results of four different kinds of tastant including caffeine, quinine, AK and sucrose.In short, according to forementioned three parts results, it is possible that the changes of characters of peripheral taste organ (taste buds) contribute anorectic function of caffeine for palatable food comsumption。These characters mainly include the maximal cross-sectional area and the height of taste buds, total taste cells per taste bud and taste perception related proteins.(4) Effect of chronic administration of caffeine for sweet taste perception related proteins expression abundance of different intestine and intestinal glucose homeostasisCaffeine exposure significantly increases the height of intestinal villus and the ratio of villus/crypt. Compare with control mice, there are 8%,6%,3% increases of height of intestinal villus and 7%,3%,7% decreases of depth of crypt in duodenum, jejunum and ileum, respectively. Therefore, the ratios of villus/crypt are considerably increased and have a 9%,9% and 10% increase, respectively. Immuno-fluorescence co-localizatin results show that sweet taste perception related proteins present through the whole intestine and co-express in each other, which provide foundation for intestinal sweet taste recognization. The Western blot results demonstrate that caffeine exposure significantly increases the sweet receptor proteins T1R2 and T1R3 abundance. Compared with control mice, there are 2.38/1.74,1.61/2.17 and 1.71/2.30-fold increase of sweet receptor protein T1R2/T1R3 in duodenum, jejunum and ileum respectively. Except for a-gustducin of duodenum, there are no clearly changes in a-gustducin and SNAP25 abundance between control and caffeine-treated mice. According to previous references, which demonstrate sweet receptor protein T1R2/T1R3 and gustdcuin play an important role in intestinal hormones release and glucose homeostasis, as well as our experimental results, we evaluate the release of intestinal hormones including GLP-1 and insulin of caffeine-treated mice by gavage administration of 2 g/kg for fasting mice. The results show that caffeine-treated mice have stronger response to glucose loading from intestine and mainly present the level of GLP-1 and insulin rapid increasing. Meanwhile caffeine treatments also significantly enhance the glucose tolerance of mice. In conclusion, forementioned experimental results denmonstrate that chronic caffeine exposure has significant influence on anatomic characters of intestinal villus, sweet receptor proteins abundances, intestinal hormones release (GLP-1 and insulin) and glucose homeostasis. However, the correlations between them need number of experiments to be elaborated.(5) Contribution of postoral function of caffeine for the characteristic changes of peripheral taste sensorWe estimated the postoral function of caffeine for the characteristic changes of peripheral taste sensor using different exposure pattern including free-access and gavage administration of caffeine solution. The results show that in any case, chronic administration of caffeine exerts significantly influence on the characters of fungiform taste buds including deareases of longitudinal axis, cross-sectional area, taste cells number, a-gustducin immunopositive cells, a-gustducin expression abundance of taste bud and increase of sweet receptor protein T1R2/T1R3 expression abundance. However, gavage administration has smaller influences on the characters of taste buds than free-access caffeine solution, which indicates that postoral changes of caffeine in part determince the characteristic variation of taste buds. |
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