Figure 3. GIP overexpression alters hepatic and adipose tissue responses to a high fat diet. A-F. Histological analyses of liver (A, B and C) and epididymal white adipose tissue (eWAT, D, E and F) from Het GIP Tg and WT littermates (18 weeks old) following 13 weeks of LF and HF feedings. Liver sections were stained with H&E (A, scale bar = 100 mm) and the lipid body stain BODIPY 493/503 (B, scale bar = 100 mm). C. BODIPY 493/503 positive signals. BODIPY 493/503 positive signals were quantified as described in Experimental Procedures. Fat sections were stained with H&E (D, scale bar = 100 mm) and GIP Overexpression and Diet-Induced Obesity antibodies against the macrophage antigen MAC-2 (E, scale bar = 25 mm). F. eWAT Crown-Like Structure (CLS) content. MAC-2 positive CLS content was quantified as described in Experimental Procedures. G and H. Gene expression profiles. Total RNA was extracted from eWATs of Het GIP Tg and WT littermates on HF feeding, and RT2 Profiler PCR Arrays were performed to simultaneously quantify mRNA expression levels of multiple genes. I. Altered Insulin-Receptor Substrate 1 & 2 protein expression. Proteins were extracted from eWATs of Het GIP Tg and WT littermates on HF feeding, and Western blot analyses performed using antibodies against IRS-1, IRS-2, and b- actin. All data represent the mean 6 S.E.M. and significance was tested using ANOVA with a Newman-Keuls post hoc test or Student’s t test, where ** represents p,0.05 vs indicated group; NS represents not significant. doi:10.1371/journal.pone.0040156.g003 tissue expansion, including the production of beneficial adipokines such as adiponectin, and the anabolic effects of GIP on the adipocyte may also be physiologically beneficial. In view of the glucose intolerance but resistance to diet-induced obesity observed in GIPR knockout mice [22,33–34], the current study was initiated to test the hypothesis that chronically elevating GIP levels in vivo would increase adipose tissue expansion and exert beneficial effects on glucose homeostasis. In line with earlier studies demonstrating that subcutaneous administration of long- acting GIP analogues resulted in improved b-cell function and glycemic control [10,11,13], GIP Tg mice showed improved insulin sensitivity, glucose tolerance and b-cell function (Figures 1 and 2), as well as reduced steatosis. Despite greatly elevated circulating GIP levels, the improved metabolic profile was not associated with blunted GIP responsiveness (Figure 4). However, greatly elevated levels of GIP in homozygous Tg mice treated with ZnSO4 did induce resistance (Figures 7I and 7J), suggesting that there is a circulating concentration threshold, above which the beneficial effects of GIP on metabolism are lost. In contrast to our initial hypothesis regarding adipose tissue expansion, chronic overexpression of GIP in mice reduced the development of adiposity in response to a HF diet (Figure 1B). Importantly, eWAT from Het GIP Tg mice exhibited substantially decreased macrophage infiltration (Figures 3E and 3F). In order to examine whether this was associated with altered adipose tissue gene expression profiles, eWAT was examined and a number of genes encoding proteins involved in mitochondrial function and inflammatory signaling pathways were found to be down- regulated in Het GIP Tg mice on a HF diet (Figure 3G). This included reductions in expression of the proinflammatory cytokine MCP-1 (Ccl2), plasminogen activator inhibitor type 1 (Serpin1) and interleukin 4 receptor alpha (IL4ra), as well as a component of the NFkB pathway, Ikb kinase b (Ikbkb). Altered expression of enzymes associated with ROS production may also be linked to reduced inflammation. For example, decreased G6pd2 would be expected to deprive the cell of NADPH that is needed for both ROS and NO production [35]. Also of interest was the reduced expression of the angiotensin converting enzyme gene (Ace) since angiotensin II is involved in increasing fat deposition [36], activation of nitric oxide synthase 3 (eNos) and modulating the macrophage phenotype [37]. The increase in angiotensinogen gene (Agt) expression may have been a compensatory response to the decrease in Ace activity. The reduction in Nos3 expression and consequent lowering of NO production could be related to anti- inflammatory effects of GIP and/or altered mitochondrial bio- genesis and function. eNos has been linked to the induction of PGC-1a expression [38–39] and both PGC-1a [40] and Ucp2 [41] are involved in mitochondrial biogenesis, reduction of which may contribute to lowering ROS production. In contrast, the p38 MAPKa gene (Mapk 14) was up-regulated and mitochondrial PLoS ONE | www.plosone.org 6 July 2012 | Volume 7 | Issue 7 | e40156