GIP Overexpression and Diet-Induced Obesity Figure 5. Het GIP Tg mice exhibited decreased physical activity but unaltered energy expenditure. After 11–12 weeks of LF or HF diet feeding, WT and Het GIP Tg mice (n = 4,5/group) were placed in individual cages, and energy expenditure and physical activity were assessed during the dark cycle. A. Energy expenditure in LF- and HF diet-fed Het GIP Tg and WT littermates. B. Average home cage activity in Het GIP Tg and WT littermates. C. Average X beam activity in Het GIP Tg and WT littermates. D. Y beam activity in Het GIP Tg and WT littermates. All data represent the mean 6 S.E.M. and significance was tested using ANOVA with a Newman-Keuls post hoc test, where ** represents p,0.05 vs indicated group; NS represents not significant. doi:10.1371/journal.pone.0040156.g005 food intake contributed to the weight loss. Het GIP Tg mice showed significantly decreased physical activity, including homec- age activity and X and Y beam activities during the dark cycle, when compared to WT littermates on both LF and HF diets (Figures 5B, 5C and 5D). As would be anticipated, these changes in locomotor activity were opposite to those reported for GIPRKO mice [22,34]. No significant differences in energy expenditure, determined by indirect calorimetry, were apparent between Het GIP Tg mice and WT littermates on either LF or HF diets (Figure 5A and Figure S1), whereas higher energy expenditure was reported for GIPRKO mice [22]. This apparent discrepancy may be partly due to the fact that energy expenditure was normalized to lean body mass (LBM), as recently recommended [45], since a unit of fat mass has a lower level of oxygen consumption than a unit of lean mass [45]. Overall these results indicated that changes in homecage activity and energy expenditure did not contribute to the reduced weight gain of Het GIP Tg mice and that the metabolic phenotype resulted from increased GIP expression and not blunted GIP responsiveness. Het GIP Tg mice did, however, exhibit a marked reduction in energy intake compared to WT littermates on both LF and HF diets, that was delayed in the light phase but apparent from the onset of the dark phase (Figures 6A and 6B). Reduced adiposity in Het GIP Tg mice was therefore at least partially due to reduced energy intake. The hypothalamus integrates and orchestrates neural, metabolic and humoral signals from the periphery and multiple hypotha- lamic neural pathways are involved in the control of energy intake. A number of effects of GIP on central nervous functions have been demonstrated, including improved cognition, hippocampal syn- aptic plasticity and glucose homeostasis [46], whereas GIPRKO mice demonstrated impairments in learning, synaptic plasticity and neurogenesis [47]. GIP was reported to be highly expressed in the hippocampus and thalamus of adult rat brain, while its expression in rat hypothalamus was relatively low [48], and it was previously shown that GIP expression in the brain was increased in the GIP Tg mouse strain [29,49]. The VMN is a major site for PACAP biosynthesis [50], with mRNA expression regulated by energy status [51]. In the current study, GIP was found to be PLoS ONE | www.plosone.org 8 July 2012 | Volume 7 | Issue 7 | e40156