Mice had continuous access to control diet (control) or 60% high fat diet (diet-induced obese, DIO) from weaning (at 3 weeks of age) until the time of sacrifice at 20 weeks. Two-way ANOVA for body weight revealed a significant interaction (F(2,12)=19.4, p<.002) between diet and time and a main effect for diet, (F(1,12)=14.6, p<.009), with HF fed animals gaining significantly more weight over time, as would be predicted (Supplemental Figure 1
At 20 weeks of age, dopamine-related gene expression was examined within the hypothalamus and reward circuitry. We focused our initial analysis on two genes important to dopamine function that are expressed within both the hypothalamus and the VTA; tyrosine hydroxylase (TH, the rate limiting enzyme in DA synthesis) and dopamine transporter, (DAT, critical for clearing DA from the synapse). In the hypothalamus, both TH and DAT mRNA were significantly increased approximately 3 fold (t(12)=2.2, p=.024, t(12)=3.1, p=.005, respectively), while in the VTA, expression of TH and DAT mRNA was significantly repressed (t(15)=2.5, p=.011, t(13)=2.6, p=.011, respectively) (). Dopamine dysfunction, particularly hypofunction, has been reported in both obese humans and in animal models of obesity, therefore, mRNA expression for additional dopamine-related genes was examined within hypothalamus and the reward circuitry (VTA, NAc and PFC). Levels of D1, D2, DARPP-32 and COMT did not differ in the hypothalamus, however within the reward circuitry, a general decrease in dopaminergic gene expression was observed (). The D1 receptor was decreased in areas that receive significant DA innervation, NAc and PFC (t(10)=8.6, p<.0001, t(12)=4.1, p=.0007, respectively), while D2 levels remained unchanged in these regions. Both receptor subtypes (D1 and D2) were significantly downregulated in the VTA (t(5)=6.9, p=.0005, t(4)=5.9, p=.002, respectively). Further, DARPP-32, which signals downstream of both receptor subtypes, was decreased in all three areas of the reward circuitry, however the difference was only statistically reliable in the PFC (t(11)=3.7, p=.002, VTA and NAc demonstrated a nonsignificant trend (p=.04 and p=.02, respectively, n.s. after Bonferroni correction). COMT, which degrades DA, was not altered in any brain region examined.
Long-term exposure to high-fat (HF) diet alters TH and DAT mRNA expression
Long-term exposure to high-fat (HF) diet affects dopamine-related gene expression in the reward circuitry
In an effort to determine whether chronic HF diet could alter DNA methylation patterns, DNA methylation was interrogated within the promoter region of specific target genes using the MeDIP assay. For both TH (, left) and DAT (, left) promoter methylation was significantly decreased in the hypothalamic samples (p<.05). Interestingly, the opposite pattern was observed in the VTA, such that there was a significant increase in DNA methylation within the promoter regions of both TH and DAT (, right, p<.05 and p<.01, respectively). Levels of methylation within the GAPDH promoter did not differ (data not shown). The direction of the changes in gene expression are consistent with what would be predicted based on the observed patterns of change in DNA methylation (e.g., increased methylation with transcriptional repression).
DNA methylation status of TH and DAT promoters in DIO mice
To evaluate, feeding and related metabolic and reward behaviors, the pattern of food intake was evaluated in control and DIO mice in metabolic chambers. Food intake, as well as locomotor activity and metabolic rate (oxygen consumption) were evaluated every 3 weeks from 8-20 weeks of age, while on their respective diets (controls on control diet, DIO animals on high fat diet, n=4/group). During the 12hr dark period, DIO animals consumed significantly more calories than control animals (main effect group; F(1,24)=85.8, p=.0001, ). This pattern persisted into the light period as well, with DIO animals eating significantly more calories than controls (main effect group; F(1,24)=55.9, p=.0003). When food intake was measured solely by grams consumed rather than kcal consumed, the identical pattern was observed (data not shown). An analysis of meal patterns revealed that DIO animals ate significantly more meals in both the dark and the light (DARK: F(1, 24)= 10.1, p<.02; LIGHT: F(1,24)=7.5, p=.03, ), and meal size was significantly increased during the dark period (F(1, 24)= 5.78, p<.05, ). DIO animals tended to be less active during the dark, however, this difference was not statistically reliable (p=.08, Supplemental Figure 1
), and activity patterns did not differ during the light. With regard to metabolic rate, there was a significant group × time interaction (F(4,24)= 3.8, p=.0156), such that the significant effect of change over time (F(4,24)=10.5, p<.0001) differed between the two groups, while the decrease in metabolic rate over time (F(4,24)=17.8, p<.0001) during the light period was not different between the groups (Supplemental Figure 1
Food intake analysis of DIO mice
We have previously reported that these DIO animals have a reduced preference for sucrose, (Vucetic et al. 2011
). To test whether alterations in response to rewarding stimuli would be observed in other contexts, a separate cohort of mice was tested at late middle age (14 months). In this experiment, 12 hr overnight food intake was evaluated in control and DIO animals two times, once fed the control diet and once fed the HF diet. Normal weight animals ate significantly more food when given access to the HF diet, increasing their intake 47% when given HF diet. In contrast, DIO animals did not increase their intake when fed the HF diet (t(12)