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. Author manuscript; available in PMC: 2021 Jul 1.
Published in final edited form as: Physiol Behav. 2020 Apr 10;221:112910. doi: 10.1016/j.physbeh.2020.112910

HIGH-FAT DIET ALTERS FLUID INTAKE WITHOUT REDUCING SENSITIVITY TO GLUCAGON-LIKE PEPTIDE-1 RECEPTOR AGONIST EFFECTS

K Linnea Volcko 1,2, Quinn E Carroll 1,2, Destiny J Brakey 1,2, Derek Daniels 1,2,3
PMCID: PMC7266080  NIHMSID: NIHMS1585925  PMID: 32283107

Abstract

Rats that are maintained on a high-fat diet (HFD) differ from controls in many ways, but how HFD maintenance affects water intake and drinking behavior has not been well studied. This is unfortunate because diet and obesity may influence fluid balance in humans through a mechanism that is poorly understood. We therefore tested the hypothesis that HFD maintenance affects water intake in rats. Water intake and drinking behavior are, in part, controlled by the actions of glucagon-like peptide-1 (GLP-1), a peptide which is well studied for its hypophagic effects. Previous studies have shown that HFD maintenance impairs the ability of GLP-1 receptor agonists to suppress food intake when the drug is administered peripherally, but not centrally. The effects of GLP-1 on fluid intake are thought to rely more on central receptor activation; therefore, a secondary aim of these experiments was to shed additional light on the location of GLP-1 responsive cells that mediate feeding vs drinking behavior. We maintained male Sprague-Dawley rats on HFD or low-fat diet (LFD) for six weeks and measured body weight, food intake, water intake, and drinking behavior. We then tested the relative contributions of diet and body weight on food intake and water intake after peripheral and central injections of GLP-1 receptor agonist Exendin-4 (Ex4). We found that HFD maintenance reduced the amount of water consumed, when intake was corrected for body weight. Consistent with other reports, rats on HFD showed a smaller suppression of food intake when given Ex4 peripherally, but not centrally. Water intake suppression when given Ex4 did not differ by diet or body weight regardless of injection site, however, adding support to the hypothesis that only central GLP-1 receptors are involved in water intake.

Keywords: high-fat diet, drinking, thirst, GLP-1, Exendin-4, obesity

1. Introduction

Rats that are fed a high-fat diet (HFD) differ from controls on standard chow or on low-fat diets (LFD) in a variety of ways, many of which have been studied extensively. There has been far less research into whether or not HFD affects fluid intake and drinking behavior, although some studies indicate that HFD does indeed affect water intake [1, 2]. The dearth of research on this topic is unfortunate because HFD affects activity of a number of feeding-related peptide systems, and there is ample evidence that many feeding-related peptides have feedingindependent effects on fluid intake. One of the more studied peptides, in this respect, is glucagon-like peptide-1 (GLP-1) [3–5] and previous studies found that GLP-1 effects on food are altered by HFD [6–9]. The current experiments, therefore, examined if water intake and drinking behavior were affected by HFD maintenance, and whether drinking effects of GLP-1 receptor (GLP-1R) activation differs in rats fed HFD compared to controls. Additionally, these experiments provided an opportunity to clarify the site of action for the water-intake suppressive effects of GLP-1.

Although the effects of a HFD on water intake and drinking behavior has not, to our knowledge, been carefully studied in rodents, the human literature hints that diet and obesity may impact drinking. HFD often leads to obesity, and in humans obesity has been correlated with changes in body fluid distribution [10, 11] that are not explained by difference in water content of adipose tissue and fat-free tissues [11], but that may be due to cellular dehydration [12]. People who are under-hydrated are more likely to have a high body mass index (BMI) than those who are more hydrated [13], and drinking a given volume of water is less protective against dehydration in people with obesity than it is in normal-weight individuals [14]. This is perhaps not surprising, because water requirements are higher in people with obesity and water turnover rates correlate with BMI [15]. It is nonetheless striking that despite an increased need due to body size, overweight individuals often fail to consume an adequate amount of water. One reason for this may be diet. Diet can affect fluid balance and weight directly, because low-calorie food often has a higher water content than energy-dense food [13]. For example, children who are properly hydrated eat diets with lower energy content and lower percent energy from fat than do children who are less hydrated [16]. Diet may also affect hydration indirectly by influencing thirst sensation. People with normal BMI who consume diets high in sugar and saturated fat report feeling less thirsty after eating a salty snack, and experience a smaller change in thirst after drinking water, than do those who consume diets lower in sugar and fat [17]. It is therefore possible that there is no causal link between obesity and hypohydration, but that the correlation is driven by the impact of diet on both.

Sprague-Dawley rats provide an excellent means to tease apart the relative contributions of body weight and diet composition, because just over half of individuals of this strain develop obesity after maintenance on a HFD [18]. Thus, maintaining rats on HFD generates rats that consume HFD but remain lean, and those that consume HFD and become obese. If low-gaining rats on a high-fat diet (HFD-L) behave more similarly to controls than to high-gaining rats on a high-fat diet (HFD-H), then body weight likely explains differences. Conversely, if HFD-L behave more like HFD-H than controls, then diet composition is likely driving differences. By putting Sprague-Dawley rats on HFD we could thus test if water intake and drinking behavior were affected by diet, obesity, or a combination of the two.

In addition to testing the relative contributions of body weight and diet composition on fluid intake, maintaining rats on HFD provides the opportunity to test the hypothesis that GLP-1 effects on drinking require CNS actions of GLP-1. GLP-1R activation suppresses water intake [3–5, 19–21]. Rats maintained on HFD show reduced sensitivity to the hypophagic effect of the GLP-1R agonist exendin-4 (Ex4) after peripheral injection [6–9], but not central injection [6]. The effect of HFD on the fluid intake effect of GLP-1R activation, however, remains an open question. If the effect of HFD on the fluid intake effects of GLP-1 differ from the effect of GLP-1 on food intake, it would suggest distinct populations of GLP-1 responsive neurons that are affected differently by the diet. There is some reason to think this would be the case, because suppression of drinking appears to be more dependent on central receptor activation than suppression of eating. Endogenous GLP-1 reduces food intake by acting on peripheral [22] or central receptors [23–25]. Blocking central GLP-1R reduces the food intake suppression of peripherally administered GLP-1R agonists liraglutide and Ex4, indicating that both peripheral and central receptors mediate the anorexigenic effects of these drugs [26], although recent studies that use a conjugate of Ex4 incapable of crossing the blood-brain barrier have found that without central access, only very high doses have an effect on food intake [27]. Fluid intake, however, appears to involve only central effects of GLP-1. Although GLP-1R agonists delivered intraperitoneally [5, 19], intravenously [20], or centrally [5, 19, 21] suppress fluid intake, changes in circulating GLP-1 were not detected after challenges to fluid homeostasis, but were clear after manipulations of energy homeostasis in the same study [3]. This suggests that challenges to fluid homeostasis engage GLP-1 of central, but not peripheral origin. It remains unclear, however, if altering sensitivity of the peripheral GLP-1 system would cause a change in fluid intake or in the effect of GLP-1 on fluid intake. In this respect, maintenance on HFD provides a means to test the relative importance of peripheral and central GLP-1 systems on fluid intake.

2. Methods

2.1. Animals

Forty-eight male Sprague-Dawley rats were purchased from Envigo at 200 – 225g (n = 30 in cohort 1, n = 18 in cohort 2). Rats were housed in stainless steel wire hanging cages located in a temperature- and humidity-controlled room kept on a 12:12 light:dark cycle. Before diet manipulations, all rats were given ad libitum access to Teklad 2018 rat chow (Envigo, Madison, WI) and tap water. After diet manipulations began, rats were given ad libitum access to their respective diets and water, except where noted for experiments. All experimental protocols were approved by the Institutional Animal Care and Use Committee of the State University of New York at Buffalo.

2.2. Diets and Group Determination

Rats were assigned to one of two diets after an Ex4 responsiveness screening. Approximately 30 min before lights out, rats were given intraperitoneal (ip) injections of 1 μg/kg Ex4 (in sterile 0.9% saline). Water intake, in the absence of food, was measured for 4 hr and compared with the response in each rat to an injection of 1 ml/kg 0.9% saline. The difference in intake between Ex4 and saline was used to rank each individual in terms of its Ex4 responsiveness. The ranked list was used to assign rats to either a HFD or LFD so that groups were matched in Ex4-induced water intake suppression. To allow for subgroup comparisons based on weight gain within the HFD rats, we assigned twice as many rats (n = 20 in cohort 1; n = 12 in cohort 2) to the HFD condition.

A high-fat diet (HFD; 60% kcal from fat; D12492) and its low-fat diet control (LFD; 10% kcal from fat; D12450J) were purchased from Research Diets (New Brunswick, NJ). The sodium content of the HFD and LFD was different: the HFD contained 3.35 g NaCl per 1000 g diet, while the LFD contained 2.45 g NaCl per 1000 g diet. HFD was fully replaced two times per week to prevent spoilage. LFD was changed one time per week.

2.3. Drug Injections and Intake Measures

Exendin-4 (Bachem, King of Prussia, PA) was injected ip at 1 or 3 μg/kg. All rats received a single mock injection, in which a needle was inserted but nothing injected, three days before Ex4 responsiveness screening, and again 3 d before intake experiments. Ex4 was injected icv at a dose of 0.1 μg in 1 μl sterile 0.9% saline. Injections were made through implanted guide cannula with an internal cannula (33 gauge; Plastics One, Roanoke, VA) connected by water-filled PE 50 tubing to a 2 μl Hamilton syringe. Injections took place either immediately before the dark phase (between 45 and 15 min before lights-out) or approximately 2 hr into the light phase, depending on experiment. There were at least 72 hr between injections.

Food intake was measured by weighing hoppers before and after tests. Spilled food and crumbs were collected below each cage and included in the calculation of remaining food. Water intake measurements consisted of volume, and in some cases drinking microstructure. Volume was calculated by weighing bottles before and after tests (1 gm = 1 ml). Drinking microstructure data were collected using a contact lickometer (designed and constructed by the Psychology Electronics Shop, University of Pennsylvania, Philadelphia, PA) that recorded individual licks. Home cages were affixed with an electrically isolated metal plate with a 3.175-mm-wide opening, through which the rat needed to lick to reach the drinking spout, minimizing the possibility of nontongue contact with the spout.

2.4. Surgical Implantation of Cannulae

Between three and four weeks after the beginning of the diet manipulation, rats in the second cohort were implanted with guide cannula (26 gauge; Plastics One, Roanoke, VA) aimed at the right lateral ventricle. Rats were anesthetized with ketamine (70mg/kg) and xylazine (5mg/kg) and secured to a stereotaxic frame (Kopf, Tujunga, CA). The guide cannula was placed 0.9 mm caudal, and 1.8 mm lateral, to bregma before being lowered through a burr hole in the skull to 2.8 mm below skull surface. Dental cement and bone screws were used to keep the cannula in position. Rats were given a sc injection of carprofen (5 mg/kg) and allowed to recover in shoebox cages 6–7 days before being moved back into the hanging wire cages. Cannula placement was verified by injection of 10 ng angiotensin II (AngII) in 1 μl sterile Tris Buffered Saline. Rats that drank at least 6 ml in 30 min were included in experiments. After the experiments ended accurate cannula placement was confirmed with another injection of 10 ng AngII. Only data from rats that drank at least 6 ml in 30 min were included in subsequent analyses.

2.5. Blood Collection and Osmolality Measures

Blood was collected from rats in cohort 2 as terminal procedure. Approximately 4 hours after lights-on, rats were anesthetized with ketamine (70 mg/kg) and xylazine (5 mg/kg). The chest cavity was opened and 1 ml blood was gently drawn through the right atrium of the heart through a heparinized 20 gauge needle and 1 ml syringe. The blood was transferred to 1.7 ml Eppendorf tubes and immediately centrifuged at 1,500 g for 10 min. Plasma was moved to clean tubes and placed on ice until analysis with a vapor pressure osmometer (Wescor, Logan, UT).

2.6. Experimental Designs

2.6.1. Experiment 1: Effect of Diet on Daily Food Intake, Water Intake, and Plasma Osmolality

Baseline 24-hr food and water intakes were measured on two consecutive days per week for the first five weeks of diet maintenance in the first cohort. Rats were weighed four days each week. Drinking microstructure over a 24-hr period was measured on one occasion during the sixth week after the diet manipulation began.

In the second cohort, overnight intake was measured 7 weeks after start of diet maintenance. Approximately 2 hours after lights-on the next morning, blood samples were collected and plasma osmolality measured.

2.6.2. Experiment 2: Effect of Peripheral Ex4 on Food Intake

Four-hour and overnight food intake was measured after injections of vehicle, 1 μg/kg or 3 μg/kg Ex4 (ip) in a repeated measures design with order of injections counterbalanced. Injections took place 45–15 min before dark phase onset. After the 4-hour measurements, hoppers were returned for the remainder of the dark phase.

2.6.3. Experiment 3: Effect of Peripheral Ex4 on Water Intake

Four-hour water intake, in the absence of food, was measured after injections of vehicle, 1 μg/kg or 3 μg/kg Ex4 (ip) in a repeated measures design with order counterbalanced. Injections took place before dark phase onset (45–15 min before lights-out). Based on previous studies showing that the strongest effect of Ex4 occurs in the first four hours [5], and to minimize the duration of food deprivation, the measures were limited to the first 4 hours after dark onset.

2.6.4. Experiment 4: Effect of Peripheral Ex4 on Water Intake Stimulated by Hypertonic Saline Injections

To test if activating GLP-1 receptors affects drinking behavior differently in rats on a HFD or LFD during times of especially high need, rats were stimulated to drink by subcutaneous (sc) injections of hypertonic saline. Rats were assigned to receive an injection of vehicle or 1 μg/kg Ex4 (ip). Immediately after this injection, they were given 0.1 ml 2% lidocaine followed by 5.5 ml/kg 2 M hypertonic saline (sc), and returned to their home cage. Thirty minutes later, water, but not food, was returned. Water intake and licking behavior were measured for one hour after the return of the bottle.

2.6.5. Experiment 5: Effect of Peripheral Ex4 on Water Intake Stimulated by Overnight Water Deprivation

Water, but not food, was removed from home cages before lights-out. Approximately 2 hr after lights-on, food also was removed. Rats were given an injection of 1 μg/kg Ex4 or vehicle (ip), and returned to their home cage along with a pre-weighed bottle. Drinking behavior was measured for the next three hours.

2.6.6. Experiment 6: Effect of Central Ex4 on Food Intake

Four-hour food intake was measured after injections of vehicle or 0.1 μg Ex4 (icv) in a repeated measures design with order of injections counterbalanced. Injections took place 45–15 min before dark phase onset. Water was available during the test.

2.6.7. Experiment 7: Effect of Central Ex4 on Water Intake

Four-hour water intake, in the absence of food, was measured after injections of vehicle or 0.1 μg Ex4 (icv) in a repeated measures design with order of injections counterbalanced. Injections took place 45–15 min before dark phase onset.

2.7. Data Analysis

Lick analyses were performed using custom software in a MATLAB (MathWorks Inc; Natick, MA) environment before exporting data to Excel (Microsoft Corp, Redmond WA) for additional organization and analyses. Bursts were defined as a series of at least 2 licks with interlick intervals less than 1 sec.

Data were analyzed both by group (HFD-H, HFD-L, and LFD) and diet (HFD and LFD). Mixed-design ANOVAs and one-way ANOVAs (Statistica; StatSoft, Tulsa, OK) were used to test for significant differences between groups. Significant differences (p < 0.05) were further probed by Newman Keuls post-hoc tests. A t-test was used to test for pre-experiment differences in the rats assigned to HFD and LFD groups.

In a few cases, missing data for a time point or dose was estimated using the average change from the previous dose to that dose and adding that to the previous dose. For example, if the 3 μg/kg Ex4 dose was missing for a HFD-H rat, the average change in intake between 1 μg/kg Ex4 and 3 μg/kg Ex4 for all other HFD-H rats was added to the 1 μg/kg Ex4 dose for the rat with the missing data point. This was done in order to not lose data from that individual for the entire test. Data were considered to be outliers, and excluded from an analysis, if greater than two standard deviations above or below the mean.

3. Results

3.1. Body Weight

In the first cohort, rats assigned to HFD and LFD groups based on Ex4 responsiveness did not differ in body weight (t = 0.29, p = 0.77, data not shown) before diet manipulations began. Consistent with previous reports [18] some of the rats on the HFD gained more weight than others. This was used to retrospectively divide the rats on HFD into high- and low-gainers (HFD-H, and HFD-L, respectively). High-gainers all had at least a 70% increase in body weight at the last day of the fifth week, whereas weight gain was less than that for all rats classified as low-gaining. Qualitatively, differences in individuals were noticeable after the first week of the diet (Figure 1A).

Figure 1.

Figure 1.

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Cumulative weight gain (A) and body weight before diet manipulation and during five weeks of maintenance on HFD and LFD (B). High-gaining rats were significantly heavier than low-gaining rats by the beginning of the third week on the diet, and heavier than LFD rats after 5 days on the diet. HFD-L and LFD rats did not differ from one another. * different (p < 0.05) between HFD-H and HFD-L, # different (p < 0.05) between HFD-H and LFD.

Mixed-design ANOVA revealed significant main effects of Time (F32, 864 = 1948.74, p < 0.001) and Group (F2, 27 = 13.22, p < 0.001) as well as a significant Time by Group interaction (F64, 864 = 24.26, p < 0.001). The average weight of rats in the HFD-H was significantly greater than that in the LFD rats after five days on the diet, and also significantly greater than the body weight of HFD-L rats by the beginning of the third week on the diets. HFD-L did not differ in body weight from LFD at any time point (Figure 1B).

In the second cohort, interestingly, only 2 of the 12 rats on HFD had less than a 70% weight gain after 5 weeks of diet maintenance. It is possible that surgery, and the subsequent recovery in shoebox cages, affected their weight trajectory. Consequently, in this cohort the determination of HFD-H vs HFD-L was made more subjectively by examining individual weight gain curves. Four of the 12 rats on HFD appeared to diverge in weight from the rest of the rats on HFD and LFD, and these four were therefore considered the HFD-H group in cohort 2. In the second cohort body weight showed significant main effects of Time (F22, 308 = 1307.65, p < 0.001), Group (F2, 14 = 12.36, p < 0.001), and a significant Time by Group interaction (F44, 308 = 14.00, p < 0.001). Newman-Keuls post-hoc tests indicated that rats assigned to HFD and LFD groups based on Ex4 responsiveness did not differ in body weight before diet manipulations began. HFD-H and HFD-L were significantly different from each other after 10 days on the diet. HFD-L was not significantly different in weight from LFD, but HFD-H differed from LFD after 3 days on the diets.

3.2. Experiment 1: Effect of Diet on Daily Food Intake, Water Intake, and Plasma Osmolality

Rats in the HFD group consumed more calories than did rats in the LFD group. A mixed-design ANOVA revealed a significant main effect of Group (F2, 27 = 23.697, p < 0.001; Figure 2A). Both HFD-H and HFD-L consumed significantly more calories than LFD, and HFD-H consumed more calories than HFD-L. This main effect persisted when corrected for body weight (F2, 27 = 10.32, p < 0.001; Figure 2B), but post-hoc tests on body weight-normalized data did not find differences between HFD-H and HFD-L rats. Post-hoc tests confirmed that both HFD groups ate significantly more as a function of body weight than did rats on the LFD. Mixed-design ANOVA revealed that rats in our diet groups consumed significantly different amounts of NaCl (F2, 27 = 10.32, p < 0.001; data not shown), with post-hoc tests indicating that HFD-H consumed the most sodium and LFD the least.

Figure 2.

Figure 2.

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Food intake in kcal (A) and in kcal normalized by body weight (B). LFD, HFD-H, and HFD-L all consumed significantly different amounts of calories. When this was normalized to body weight, LFD consumed fewer calories than either HFD-H or HFD-L. Bar graph inserts show the main effect of diet.

Water intake, on the other hand, showed a different pattern of results. Rats in all groups drank the same volume of water per day (F2, 27 = 2.667, p = 0.09; Figure 3A), but when normalized to body weight, rats on a LFD drank significantly more than either HFD-H or HFD-L rats (F2, 27 = 5.279, p = 0.01; Figure 3B). Analysis of volume of water consumed in 6-hr bins over a 24-hour period revealed significant main effects of Time (F3, 69 = 282.597, p < 0.001) and Group (F2, 23 = 6.131, p = 0.01), and a significant Time by Group interaction (F6, 69 = 5.321, p < 0.001; Figure 4). Rats on a HFD drank significantly less water during both the first and second halves of the dark phase, with what appeared to be a partial compensation during the light phase, but this difference was not statistically significant. Four outliers were removed in this analysis: one in the first time bin (HFD-H), two in the second time bin (HFD-H and HFD-L), and one in the fourth time bin (LFD). Thus, the analysis above included data from 10 HFD-H, 7 HFD-L, and 9 LFD rats.

Figure 3.

Figure 3.

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Water intake in ml (A) and in ml normalized by body weight (B). Rats in the two diet groups did not drink different amounts of water, but when water consumption was normalized to body weight, those on a LFD drank significantly more than either HFD-H or HFD-L. Bar graph insert shows the main effect of diet.

Figure 4.

Figure 4.

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Water consumption over 24 hours in 6-hour bins. Rats on a HFD drank significantly less in the first two bins, corresponding roughly to the dark phase, than did rats on a LFD.

Analysis of drinking microstructure was used to further probe the differences in drinking observed between HFD and LFD rats. Analysis of 6-hr bins found no differences in burst number (F1, 28 = 0.3599, p = 0.55; Figure 5A), or burst size (F2,24 = 3.1856, p = 0.06) when HFD-H and HFD-L groups were treated separately, but when collapsed by both diet and time, we detected a statistically significant difference in burst size in which HFD rats had smaller average burst size than did rats on LFD (F1, 26 = 5.0529, p = 0.03; Figure 5C). Two outliers were removed from the analysis, both of which were in the HFD group.

Figure 5.

Figure 5.

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Burst number (A) and burst size (B) for the first two 6-hour time bins. Burst size did not differ between diet groups, but there was a significant main effect of diet, with HFD licking fewer times per burst, when the HFD groups were combined for the analysis (C).

Water intake as a function of grams of food consumed did not differ between the diet groups (F2, 27 = 3.142, p = 0.06) when analyzed as three groups, but when HFD groups were combined for analyses, there was a significant main effect of Group (F1, 28 = 6.392 p = 0.02) with rats on a HFD drinking a greater volume of water for each gram of food eaten than rats on a LFD did (Figure 6).

Figure 6.

Figure 6.

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Water intake as a function of food intake. Although there were no statistically significant differences when analyzed as three groups, when the rats on HFD were considered jointly there was a main effect of Diet, with rats on a HFD consuming more water per gram of food eaten than rats on LFD.

Plasma osmolality did not differ when analyzed as three groups (F2, 15 = 2.6, p = 0.11), but when HFD were combined for analyses there was a significant main effect of Group (F1, 16 = 5.5, p = 0.03), with rats on LFD having a significantly higher plasma osmolality than those on a HFD (Figure 7). To test for diet-group differences in the relationship between osmolality and fluid consumed, we used a one-way ANCOVA, and found that diet group had no effect on osmolality when controlling for the amount of fluid consumed during the 20 hr (including the entire dark phase) the day before blood was collected for measures of osmolality (F2,16= 1.8, p = 0.20). This analysis found no evidence for lack of homogeneity in the relationship between intake and osmolality between the diet groups (F2,14 = 1.05, p = 0.38).

Figure 7.

Figure 7.

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Plasma osmolality. Diet did not have a statistically significant effect on plasma osmolality when the data from the three groups of rats were analyzed. When HFD-H and HFD-L were combined, however, osmolality of rats maintained on HFD was slightly lower than rats on LFD (p < 0.05).

3.3. Experiment 2: Effect of Peripheral Ex4 on Food Intake

Dark-phase food intake was measured after injections of Ex4. The intake measure from one rat after 3 μg/kg dose was lost due to a technical error, and was therefore estimated for the ANOVA by subtracting the average difference between 1 μg/kg and 3 μg/kg for all rats in that diet group from that rat’s 1 μg/kg intake. We saw a main effect of Group (F2, 27 = 4.352, p = 0.02) but there was no main effect of Drug, nor a Group by Drug interaction for intake over the entire dark phase (Figure 8A). Consistent with previous findings [6–9], however, analysis of the first four hours of the dark phase revealed an effect of diet on the suppression of food intake by peripheral Ex4 (Figure 8B). Statistical analysis of these data did not find a significant main effect of Group, but did detect a significant main effect of Drug (F2, 54 = 41.092, p < 0.001) and a significant Group by Drug interaction (F4, 54 = 2.632, p = 0.04). Post-hoc test revealed that kilocalories consumed as a function of body weight was lower after injections of 3 μg/kg Ex4 in LFD than in HFD-L (p = 0.04). HFD-H did not differ from either LFD. Combining HFD-H and HFD-L for the analyses showed a significant Group by Drug interaction (F2, 56 = 4.84, p = 0.01) with post-hoc tests indicating that kilocalories per kilograms body weight consumed after 3 μg/kg Ex4 was significantly different in rats on HFD and on LFD.

Figure 8.

Figure 8.

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Food intake in kcal, normalized to body weight, after vehicle, 1 μg/kg Ex4, or 3 μg/kg Ex4 is shown for the entire dark phase (A) or the first 4 hours after injection (B). At 4 hours, there was a Group by Drug interaction, with Newman Keuls post hoc tests indicating that the 3 μg/kg dose of Ex4 produced a larger suppression of intake relative to body weight in rats fed a LFD than low-gaining rats fed a HFD. The effect was not statistically significant when data from the entire dark phase were analyzed.

3.4. Experiment 3: Effect of Peripheral Ex4 on Water Intake

To isolate the effects of GLP-1R activation on water intake, we removed food hoppers before beginning the experiment. Ex4 treatment was associated with less water intake relative to body weight in the first four hours of the dark phase (F2, 54 = 16.004, p < 0.001), but there was no main effect of Group (F2, 27 = 2.223, p = 0.13), and, unlike our observations of food intake, there was no Drug by Group interaction (F4, 54 = 0.1113, p = 0.98; Figure 9). The intake for the 1 μg/kg dose was estimated for one HFD-H and one LFD rat, by subtracting the average difference between vehicle and 1 μg/kg for all rats in that diet group from that rat’s 1 μg/kg intake. Combining HFD-H and HFD-L did not change the outcome of the analysis.

Figure 9.

Figure 9.

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Water intake as a function of body weight after ip injections of vehicle, 1 μg/kg, or 3 μg/kg of Ex4. Ex4 reduced 4-hour intake, but there was no main effect of Group nor a Drug by Group interaction.

3.5. Experiment 4: Effect of Peripheral Ex4 on Water Intake Stimulated by Hypertonic Saline Injections

Rats in HFD and LFD groups were given injections of hypertonic saline and used to test the effect of peripheral Ex4 on water intake. There was a main effect of Drug, indicating that Ex4 (1 μg/kg) suppressed drinking in all three groups (F1, 24 = 5.3371, p = 0.03) without a main effect of Group observed (F2, 24 = 1.2759, p = 0.30), nor was there a Group by Drug interaction (F2, 24 = 2.1643, p = 0.14) (Figure 10A). A subjective analysis of the graph raises the possibily of an undetected interaction in which the HFD-H group is more sensitive to Ex4 than any other group. Alhough not borne out by the statistical analysis, the direction of the effect is, nevertheless, the opposite of what would be expected based on previous findings and the prediction that HFD leads to a less sensitive GLP-1 system. Combining HFD-H and HFD-L did not change the outcome of the analysis.

Figure 10.

Figure 10.

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Water intake normalized to body weight after ip injections of vehicle or 1 μg/kg of Ex4 after sc 5.5 ml/kg 2 M hypertonic saline (A) or 15 hours of water deprivation (B). Ex4 reduced body weight-normalized intake, but there was no main effect of Group nor a Drug by Group interaction.

3.6. Experiment 5: Effect of Peripheral Ex4 on Water Intake Stimulated by Overnight Water Deprivation

Similar to what was observed in the previous experiments, 1 μg/kg Ex4 had a suppressive effect on water intake (F1, 23 = 21.684, p < 0.001). We did not detect differences in the magnitude of suppression as a function of body weight between diet groups (F2, 23 = 0.432, p = 0.65), nor did we find a Group by Drug interaction (F2, 23 = 0.144, p = 0.87; Figure 10B). Combining HFD-H and HFD-L did not change the outcome of the analysis.

3.7. Experiment 6: Effect of Central Ex4 on Food Intake

A 0.1 μg Ex4 injection into the lateral ventricle suppressed 4-hour caloric intake as a function of body weight (F1, 13 = 21.36, p < 0.001; Figure 11A). Intake was not affected by Group (F2, 13 = 0.41, p = 0.67), nor did we find a Group by Drug interaction (F2, 13 = 0.44, p = 0.65). This was also true when HFD-H and HFD-L were combined.

Figure 11.

Figure 11.

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Intake of food (A) and water (B) normalized to body weight after icv injections of vehicle or 0.1 μg Ex4. Four hours after injection, Ex4 reduced body weight-normalized intake of food and water, but there was no main effect of Group nor a Drug by Group interaction.

3.8. Experiment 7: Effect of Central Ex4 on Water Intake

A 0.1 μg Ex4 injection into the lateral ventricle suppressed 4-hour water intake as a function of body weight, in the absence of food (F1, 13 = 21.46, p < 0.001; Figure 10B). Although there appeared to an attenuation of the effect of Ex4 in the HFD-H group, objective statistical analysis of these data found that intake was not affected by Group (F2, 13 = 0.32, p = 0.73), nor did we find a Group by Drug interaction (F2, 13 = 1.59, p = 0.24; Figure 11B). This was also true when HFD-H and HFD-L groups were combined.

4. Discussion

The present results show that rats maintained on a diet that is high in fat drink less than rats on a standard chow diet. This is in agreement with previous studies [1, 2], and we have added to this by showing that the reason rats on HFD drink less than controls is because of changes in burst size. Diet-induced difference in drinking behavior are potentially interesting because of the high rates of dehydration, and rising rates of obesity, in many Western countries. Diets that emphasize lower carbohydrate consumption over low fat consumption continue to gain popularity [28], making the intersection of diet and drinking behavior especially relevant. Studies using human participants indicate that a high BMI is associated with changes in body fluid distribution [10, 11] and hydration [13, 14]. Diet can influence not only body weight and fluid distribution, but also can affect fluid intake, through higher water content of lower-calorie foods [13] and by changing thirst perception [17]. HFD may, therefore, mediate the relationship between body weight and fluid balance, and our results provide some support for this idea.

We found that when rats were maintained on a 60% HFD, they drank less than controls maintained on a LFD, when corrected for body weight. An analysis of subgroups within rats given HFD indicates that the difference in drinking was a function of the diet composition, but not body weight. Specifically, when rats in the HFD group were divided based on high and low weight gain, the weight-corrected daily water intake of these subgroups did not differ, and water intake by both groups was significantly lower than intake by rats in the LFD group. Further analysis found that differences in dark-phase intake, when drinking is normally highest, accounted for the group differences, and this difference was driven largely by differences in burst size. Indeed, rats maintained on HFD licked fewer times per drinking burst than did controls. Based on previous studies of drinking microstructure [29, 30] this suggests that the diet altered orosensory feedback of water while the postingestive feedback remained unchanged. Although we are unaware of differences in water content of the diets, any presumed higher water content in the HFD or perceived dryness of the LFD remain unlikely to explain the difference in intake because rats on HFD drank more, not less, per gram of food eaten than did controls. In other words, if a lower water content or greater perceived dryness of the LFD were driving the differences, the expectation would be for rats on LFD to drink more per grams of food in order to compensate for the lower water content. We observed the opposite of this prediction. Moreover, rats on HFD consumed significantly more grams of salt than rats on LFD did. Although studies in humans have found that increasing salt consumption can reduce water intake [31] we think it is most likely that the difference in normalized water intake between rats on HFD and LFD is more directly related to differences in fat content of the diets. Although a better understanding of fat detection is emerging [32, 33], the means by which this interacts with circuits controlling fluid intake remains unexplored.

Although rats on HFD drank less water than did LFD controls, they did not show signs of dehydration, as measured by plasma osmolality. This is in line with what others have shown: some studies found no differences in plasma osmolality between rats on HFD and LFD [34, 35], while others have found that HFD significantly reduces plasma osmolality [36]. It remains possible, however, that HFD do not differ much from controls in osmolality, but do show an alteration in total volume. This does not appear to be the case, however, because rodent studies have found either no diet-related differences in total body water [37], or found that obese rats had less, not more, intracellular and extracellular water [38]. There are several reasons why rats maintained on HFD could drink less without showing differences in plasma osmolality. One reason may be that metabolic water can be produced by oxidizing adipose tissue and dietary fat [39]. Some species increase body fat catabolism when they are water deprived [40], so it is possible that having a fat reserve and eating a HFD protects the organism from dehydration. Nonetheless, while children who produce more metabolic water are more likely to eat a diet high in fat, they are also more likely to have low hydration [16]. Furthermore, fat is not prioritized as fuel [41] and lower rates of fat oxidation are associated with obesity in both rats [42] and humans [43]. It is also possible that kidney function is altered by HFD, leading to differences in water and solute retention and excretion. In support of this, one study found that rats on HFD drank less water than controls, and that the rats on HFD compensated for this by excreting more concentrated urine [2]. In short, there are several possible explanations that could account for why drinking less water does not affect plasma osmolality in rats maintained on HFD. A more complete explanation of the mechanism responsible for the difference requires further study.

HFD reduces the suppressive effects of peripherally administered GLP-1R agonist Ex4 [6–9]. The present experiments extend this finding to male Sprague-Dawley rats that consumed HFD but remained lean. This is inconsistent, however, with a previous report that suggested a lack of differences in Ex4 responses in Sprague-Dawley rats as a function of HFD [44]. This inconsistency could be due to differences in the experimental design or in the lack of a control group tested at the same time in the previous study, or because the previous study did not subdivide HFD rats into those that gained weight and those that remained lean. Nevertheless, the smaller suppression of food intake after GLP-1R activation in a subset of rats in the HFD group suggests that HFD reduces the sensitivity of the effect of the GLP-1 system on food intake, at least under some circumstances. The fact that it was the HFD-L, and not HFD-H, that showed a smaller suppression of intake implies that diet has a larger effect on changing the sensitivity than body weight does, and, additionally, that obesity combined with a HFD may actually counteract the blunting of GLP-R sensitivity. On the whole, these studies are consistent with our present finding that in the absence of pharmacological manipulation of the GLP-1 system, rats in the HFD group consumed significantly more kilocalories than did rats on a LFD, as would be expected if the intake suppressive effects of the endogenous GLP-1 system were somehow attenuated.

The findings related to food intake were, however, the opposite of what we observed with respect to water intake, in which HFD maintenance was associated with less intake, not more intake, when normalized to body weight. We also did not detect statistical differences between diet groups in the efficacy of water intake suppression caused by peripheral Ex4 injections, whether intake was stimulated by time of day, hypertonic saline injection, or water deprivation. Accordingly, it seems reasonable to conclude that any effect HFD has on the GLP-1 system is more selective to the feeding-related aspects of the system, demonstrating the separability of the feeding and drinking effects of GLP-1.

Although peripheral Ex4 injections produce smaller reductions in food intake in rats on HFD than in controls [6–9], this is not the case when Ex4 is given icv [6]. This implies that HFD alters peripheral, but not central GLP-1 receptors. Indeed, endogenous GLP-1 appears to act on both peripheral [22] and central [23–25] receptor populations to induce hypophagia. The GLP-1R agonist Ex4 also binds to both populations of receptors [26], though it appears that without central access, only very high doses have an effect on food intake [27]. Nonetheless, a HFDrelated decrease in sensitivity to the hypophagic effect of Ex4 after peripheral injection, leaves open the possibility that simultaneous peripheral and central GLP-1R activation synergistically reduces intake. GLP-1 effects on water intake, on the other hand, are thought to involve only central, and not peripheral, GLP-1R activation [3]. The present experiments provide further support for that hypothesis. Although peripheral Ex4 reliably suppressed less water intake, we did not detect differences in this effect between rats on HFD and LFD, regardless of the intake stimulus. Accordingly, the present results support the previous suggestion that the effect of GLP-1 on water intake primarily involves CNS substrates. Moreover, the results provide additional evidence that the feeding and drinking effects of GLP-1 involve separable substrates.

High-fat diets have a multitude of effects on organisms. We have shown here that HFD influences water intake and drinking behavior, adding detail to this entry of the list of functions affected by diet. More specifically, our data show that HFD maintenance causes male rats to drink less as a function of body weight. This is largely a function of reduced drinking during the dark phase and appears to be a function of altered orosensory feedback, rather than a change in post-ingestive feedback. These data are especially interesting in light of the current high rates of dehydration in human populations, and the tendency to consume diets with high percentages of fat. The findings also build upon the existing literature on GLP-1 and drinking behavior by showing that, unlike with food, HFD does not desensitize the response to peripherally administered Ex4. Neither food nor water intake shows diet-related changes in suppression of intake by centrally administered Ex4. This, in turn, provides further support for the hypothesis that GLP-1 mediated water intake occurs only through central receptor activation and demonstrates the need for additional research on the separable elements of the GLP-1 system and how they diverge to effect feeding and drinking.

Highlights.

  • High-fat diet was associated with less drinking

  • Drinking effects of a central or peripheral GLP-1 agonist were unaffected by diet

  • Hypophagia caused by a peripheral injection of GLP-1 agonist was reduced in HFD rats

  • Hypophagia caused by a central injection of GLP-1 agonist was unaffected by diet

Acknowledgements

Dr. Ann-Marie Torregrossa provided valuable feedback and helpful interpretation of the results. Alex Schick, Savitoj Kaur, and Ana Suarez-Venot provided excellent technical support. Financial support was provided by National Institutes of Health award DK107500 (DD).

Footnotes

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