Induction of Salivary Proteins Modifies Measures of Both Orosensory and Postingestive Feedback during Exposure to a Tannic Acid Diet - Figure 4

<p>A–D: White bars represent feeding behaviors measured while animals were consuming the control diet; grey bars represent the same behaviors measured while animals were on the 3% tannic acid diet. The white bar labeled ‘C’ represents a 5-day average of behavioral measures on the control diet prior to exposure to the tannic acid diet. Food intake (A) and meal size (B) were decreased on the first 3 days of exposure to the tannic acid diet but returned to control levels by day 4. Meal number (C) was increased during the first 2 days of exposure to the tannic acid diet but returned to control-levels by day 3. Rate of feeding (D) was decreased throughout the entire exposure to the tannic acid diet buy this effect was most pronounced during the first 3 days.</p

Data depicted in the larger graphs are densitometry units normalized to average control (water replete) protein expressions (which are set to 1).

<p>The first bar (stippled) represents the average water replete expression of protein concentration, while all rats were fed the control diet. The data in the remainder of the bars were collected in the water-deprived condition. The white bar represents the average protein expression of all rats on the day of their first exposure to the brief-access taste test. The white bar with hash marks represents the saliva samples collected the day of the second exposure to the brief-access test by rats that were maintained on the control diet. The gray bar with hash marks represents the saliva samples collected the day of the second exposure to the brief-access test by rats that were maintained on the tannic acid diet between exposures. Statistical analyses were not preformed on these data. Total protein concentration was significantly altered by water deprivation making comparisons between samples unreliable. We have presented them only to illustrate the relative abundance of proteins across treatments. The inset graphs represent the change in densitometry units between the two test sessions (i.e. test 1 protein expression- test 2 protein expression) for the control group (white bar with hash marks) and experimental group (gray bar with hash marks). *Experimental group greater than the control group, P<0.05.</p

The closed circles represent the average licking of the two test groups during their first exposure to the brief-access test (unconditioned licking does not differ between the groups at this time point p>0.05).

<p>The open circles represent the average licking during the second exposure to the brief-access test by rats that were maintained on the control diet. The open triangles represent the average licking during the second exposure to the brief-access test by rats that were maintained on the tannic acid diet between exposures. Lines represent curves fit to the average licking behavior. Rats with an increase in the salivary protein at 19/18.5 and 18 kDa bands show a right-ward shift in the licking response curve demonstrating that they found the tannic acid less aversive in the second exposure than the first exposure, while rats maintained on the control diet did not alter their licking behavior on the second exposure.</p

The sequence of rat protein cystatin S (Uniprot accession P19313).

<p>Bold regions indicate where tryptic peptides detected in proteomic analysis overlay the protein sequence.</p

Data are densitometry units normalized to average control diet protein expressions (set to 1) as well as IgA expression.

<p>White bars represent protein expression measured while the experimental animals were consuming the control diet (average of the final 5 days; depicted by white bars labelled “C”), and grey bars represent expression of the same protein while the experimental animals were consuming the 3% tannic acid diet. The line graphs represent the same protein densitometry measures for animals that were maintained on the control diet for the entire course of the study. *Significant within-subject difference between protein expression on the control and experimental diets (p<0.05, bonferoni corrected for multiple comparisons).</p

Summary of ANOVAs comparing normalized densitometry units of protein bands at each of the listed masses (kDa).

<p>Analyses were conducted with experimental group (control or tannic acid diet) as the between-subjects comparison and time (days) as the within-subjects comparison. *ps<0.05.</p><p>Summary of ANOVAs comparing normalized densitometry units of protein bands at each of the listed masses (kDa).</p

Summary of linear mixed model analyses.

<p>We looked for the predictive value of each of the normalized densitometry units of protein bands at each of the listed masses (kDa) on feeding behaviors. Analyses were restricted to the first four days of exposure to the tannin diet as this represented the dynamic phase over which changes in feeding behavior and salivary protein expression were observed. *ps<0.05.</p><p>Summary of linear mixed model analyses.</p

Representative samples of the glycosylation determination.

<p>The gel contains saliva samples from a single rat after 4 or 12 days exposure to the tannic acid diet. The samples were loaded in duplicate on the same12% SDS/PAGE. The quantity of protein loaded in each lane was varied to allow for maximal visibility of the protein band. After resolving the gel, it was cut into two pieces. Gel A was stained using Pro-Q Emerald 300 glycoprotein detection kit, which detects only glycosylated proteins. Gel B was stained with Coomassie R250 stain, which detects total proteins. Both gels contain both Candy-Cane glycoprotein molecular weight standards and Bio-Rad molecular weight standards.</p