Dopamine plays a critical role in the regulation of food intake. DA action in the hypothalamus plays a role in promoting food intake (Meguid et al. 2000), and DA activity in the reward circuitry is associated with the rewarding aspects of food (Volkow et al. 2010) (e.g., fat (Rada et al. 2010) or sucrose (Rada et al. 2005) ingestion result in dopamine release in NAc). Importantly, dopamine dysfunction has been identified in both obese individuals and animals. In the hypothalamus, chronic intake of high fat diet leads to altered dopamine expression and function within the hypothalamus (Lee et al. 2010a, Li et al. 2009, Huang et al. 2005). Further, it has been shown that this altered DA function is a direct response to the increased fat consumption per se, as opposed to the resultant obesity (Li et al. 2009). Additionally, dopamine dysfunction has been noted within the mesocorticolimbic circuitry. Decreased dopamine D2 receptor expression and function in obese rats (Wang et al. 2001, Johnson & Kenny 2010, Stice et al. 2008), and obese humans (Wang et al. 2001, Johnson & Kenny 2010, Stice et al. 2008), decreased extracellular dopamine in the striatum in DIO rats (Geiger et al. 2009) and decreased D1 receptor expression in the NAc of obesity prone rats on a high fat diet (Alsiö et al. 2010) all support the idea of decreased dopamine activity in the reward circuitry in obesity.

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) (Fig 1). 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 (Fig 2). 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.

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 (Fig 3A, left) and DAT (Fig 3B, 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 (Fig 3A, B, 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).

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, Fig 4A). 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, Figure 4B), and meal size was significantly increased during the dark period (F(1, 24)= 5.78, p<.05, Figure 4C). 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).

Importantly, we observed behavioral responses that are consistent with the gene expression changes. Increased dopamine within the hypothalamus in known to promote food intake. In the DIO mice, we observed increased food intake (both as kcal/bw and as grams/bw) and increased meal number, in both the dark and light periods, as well as increased meal size in the dark. It has been shown that increased food intake, particularly within the light period of nocturnal animals like mice, is a primary contributor to increased weight gain (Arble et al. 2009). We also observed a decreased preference for sucrose (reported previously (Vucetic et al. 2011)) and a lack of increase in food consumption when presented with the HF diet, a response that was observed in the normal weight controls. These behaviors are consistent with the gene expression findings that demonstrate an overall decrease in dopaminergic gene expression within the central reward circuitry. Collectively, these behaviors have the potential to promote obesity in two distinct ways; (1) through an increase in food intake and (2) by increasing the drive for palatable food, as the animal with a blunted response to palatable foods may seek and/or consume these food relatively more than a normal animal in order to reach the same rewarding response.

Dopamine dysregulation in obesity has been documented in other animal models. In a model similar to the present experiments, mice were fed a 35% HF diet from age 3-15 weeks, and gene expression in the hypothalamus was investigated (Lee et al. 2010a). Paralleling the present findings, these authors reported upregulation of TH and DAT, with no change in D1 or D2 receptor expression. Another group focused specifically on the ventromedial hypothalamus (as opposed to total hypothalamus) and found decreased TH expression rather than increased (Li et al. 2009), however in that study the HF diet administration started much later (at 12 weeks of age) and was maintained for a shorter length of time (8 weeks as opposed to 17+ weeks), highlighting two key variables that affect how high fat diet can affect dopamine function, the developmental timeperiod and length of administration of the high fat diet. Highlighting this point is the finding that 4 weeks of HF diet administration (relatively short exposure) to 12 week old animals (relatively older animals) was ineffective in altering gene expression levels of TH, DAT, D1 or D2 within hypothalamus (de Leeuw van Weenen et al. 2009).

Dopamine dysregulation within reward circuitry has been observed as well, and most findings report a decreased level of gene/protein expression and/or function of the dopamine system within the reward circuitry. D2 downregulation has been documented in response to chronic high fat diet and obesity (Johnson & Kenny 2010), coupled with a decrease in brain reward threshold that persisted for 14 days after the removal of the HF diet. In a recent notable publication, rats were fed a high fat/high sucrose diet for 5 weeks beginning at 8 weeks of age, and were classified as obesity prone or resistant depending on weight gain (Alsiö et al. 2010). Obesity prone rats showed a decrease in D1 receptor in the NAc (similar to our findings). This decrease persisted after 18 days of withdrawal from the palatable diet. The decrease in D1 expression was linked to the consumption of the palatable diet, rather than weight gain per se, because a group that was pair fed restricted amounts of the palatable diet (consumed the diet but did not gain weight) also showed a decrease in D1 expression. A convergent finding comes from rats fed a restricted amount of HF diet that showed reduced DA turnover in the NAc in response to HF diet consumption in the absence of significant weight gain (Davis et al. 2008). In mice, DAT binding in the NAc was decreased after 20 days on HF diet, and persisted for at least 7 days after removal of the high fat diet (South & Huang 2008). Examination of obesity prone and obesity resistant rats consuming a control diet and therefore not different in weight found that obesity prone rats showed decreased levels of dopamine in the NAc, suggesting that the reduction in dopamine did not require the animal to be obese (Rada et al. 2010). However, these rats had been fed a HF diet for 5 days to classify them as obesity prone or resistant, so the dopamine dysfunction may have been linked to a differential response to the HF diet. Additionally, basal and amphetamine-induced dopamine release in striatum are reduced in obese rats fed a chronic high fat diet (Geiger et al. 2009). Importantly, similar phenomenon have also been observed in humans, as obese women when compared to normal weight women show reduced activation in the caudate in response to consumption of a palatable solution (Stice et al. 2008).

An important consideration in all of these reports is the relative importance of the consumption of the HF diet versus the resultant obesity. In our study (and others (Johnson & Kenny 2010, Geiger et al. 2009)), it is not possible to distinguish which of these stimuli may drive the changes in dopamine related gene expression. However, in other studies described above ((Alsiö et al. 2010, Davis et al. 2008, Rada et al. 2010)) dopaminergic changes are observed in animals fed a HF diet in the absence of obesity, suggesting that consumption of the HF diet, rather than the obesity and associated physiological changes, is sufficient to alter dopamine system function.

Given that there is support that these changes in dopamine-related gene expression persist after the removal of the diet (detailed in previous paragraph (Alsiö et al. 2010, South & Huang 2008, Johnson & Kenny 2010)), we examined whether epigenetic modifications may contribute to changes in gene expression. DNA methylation in particular may represent an epigenetic modification that is relatively stable over time. We focused our detailed analyses on TH and DAT, given the opposite pattern of responses that was observed in the reward circuitry and the hypothalamus. As predicted, we found that DNA methylation levels in the promoter regions of TH and DAT, paralleled the gene expression changes (increased methylation related to decreased expression). These data identify differential DNA methylation as a mechanistic link between consumption of high fat diet and/or obesity and the resultant changes in dopamine-related gene expression. Differential DNA methylation in the brain in response to diverse early life challenges has been reported (Coupé et al. 2010, Murgatroyd et al. 2009, Vucetic et al. 2010a, Vucetic et al. 2010b, Weaver et al. 2004, Plagemann et al. 2009, Niculescu & Lupu 2009, Niculescu et al. 2006), however, similar findings in the postnatal brain are more limited. BDNF and Fkbp5 are differentially methylated during memory consolidation (Lubin et al. 2008) and in response to chronic corticosterone exposure (Lee et al. 2010b), respectively. In DIO mice, hypomethylation of melanocortin 4 receptor has been reported in whole brain (Widiker et al. 2010), while we have recently reported differential DNA metylation within the μ-opioid receptor promoter in reward circuitry regions. Given reports of the persistence of dopaminergic gene alteration in the absence of the high fat diet (Alsiö et al. 2010, South & Huang 2008, Johnson & Kenny 2010), the question of whether differential DNA methylation will persist with removal of the diet or normalization of body weight and whether DNA methylation is responsible for persistent changes in gene expression represents important next steps in this line of investigation.

Supp Fig S1