SERT2/2 mice, compared to their ND-fed controls, with the absolute values of plasma insulin in SERT-deficient mice higher (Figure 4B). To begin to understand the impact of SERT deficiency on pancreatic function, we carried out histological analyses of islets. Consistent with the abundant SERT mRNA in the pancreas (Figure 1), immunohistochemical staining of pancreatic tissues from 6-month old WT mice showed high levels of SERT in non-insulin labeled cells of islets and lower SERT levels in insulin labeled cells (Figure 4D). Histological analysis of hematoxylin and eosin (H&E) stained pancreatic tissue sections of 6-month old mice showed that the overall structure and organization were preserved in SERT2/2 mice (Figure 4E, F). Immunostaining for insulin and glucagon indicated normal distributions of islet alpha- and beta-cells (Figure 4G, H). However, we found that the size and number of islets both were increased in SERT2/2 mice. On average, SERT2/2 mice exhibited 70% more pancreatic islets and a total of a 3-fold increase in beta-cell mass compare to WT mice (Figure 4I, J). To determine the mechanism underlying islet hypertrophy of SERT2/2 mice, we assessed beta-cell proliferation and apoptosis in SERT2/2 and WT mice. Judged by BrdU incorporation of beta cells, there was an approximately 2-fold increase in beta-cell proliferation in SERT2/2 mice (Figure 4K–M). In contrast, SERT2/2 and WT mice showed a similar frequency of apoptosis of the beta cells, as judged by TUNEL staining (Figure S3). Collectively, these results suggest that glucose intolerance of SERT mutant mice is more likely due to reduced response to insulin, rather than reduced pancreatic insulin secretion. Insulin-induced AKT activation is blunted in SERT mutant liver and muscle To investigate the molecular mechanisms underlying the insulin resistance of SERT-deficient mice, we analyzed insulin signaling in the liver of 3- and 6-month old mice. The serine/threonine kinase AKT signaling is critical for PI3K-mediated insulin action on glucose uptake and storage in liver and muscle [29]. While AKT2 deficiency impairs insulin action on liver metabolism [30,31], elevated basal AKT activity contributes to insulin resistance in obese mice induced by HFD [32] and elevated basal PI3K signaling is associated with insulin resistance in skeletal muscle and glucose intolerance in men [33]. Indeed, fasting AKT activity was significantly elevated in the liver of SERT2/2 and SERT+/2 mice compared to WT mice, as judged by the level of phosphorylated AKT (pAKT) using western blot analyses (Figure 5A, B, D, E). Importantly, insulin-induced increase of pAKT was attenuated in SERT2/2 and SERT+/2 mice, although the absolute amounts of pAKT were similar between WT and the SERT mutants following insulin injection (Figure 5B, C, E, F), again, consistent with that seen in glucose intolerant patients [33]. Fasting AKT activity was also elevated while insulin- induced increase of pAKT was attenuated in the muscle of SERT2/2 and SERT+/2 mice (Figure S4). Insulin-receptor substrate (IRS) proteins are cytoplasmic adaptor proteins that couple insulin signals to PI3K activity [29]. Consistent with the elevated basal AKT activity, the basal activity of IRS1 was elevated in the liver of 3- and 6-month old SERT2/2 mice compared to WT mice, as judged by the level of tyrosine phosphorylated IRS1 (pY-IRS1) using western blot analyses (Figure 5G, H, J, K). Further, insulin-induced increase of pY-IRS1 was attenuated in SERT2/2 mice (Figure 5H, I, K, L), parallel to the reduced insulin-induced AKT activation. Collectively, these data suggest that insulin stimulation of PI3K signaling in the liver and muscle is diminished in SERT-deficient mice. 3-month old SERT2/2 mice did not display obesity Distinct SERT Function in Feeding and Obesity (Figure 2E, F) and hepatic steatosis (data not shown), but their IRS1 and AKT responses to insulin stimulation were reduced, further indicating that SERT2/2 mice develop insulin resistance prior to a significant increase in the adiposity. JNK activity was increased in SERT mutants Stress-induced kinases, JNK, S6 kinase (S6K) and p38 MAP kinase, have been shown to inhibit insulin activation of the PI3K pathway [29,34]. The activities of these kinases are regulated by distinct metabolic and physiological stress, and chronic activation of JNK, S6K and p38 is found to promote glucose intolerance and insulin resistance [34,35,36]. To evaluate the activities of those stress kinases prior to and after the development of obesity in SERT deficient mice, we analyzed JNK, S6K, and p38 activities in the liver of 3- and 6-month old mice, using western blot analysis. At 3-month old, WT and SERT2/2 mice exhibited similar fasting S6K and p38 activities, while fasting JNK activity was significantly increased in the SERT2/2 mice, as judged by the levels of phosphorylated S6K (pS6K), p38 (pp38) and JNK (pJNK) (Figure 5M, N). At 6-month old, fasting levels of both pJNK and pp38 were increased in SERT2/2 mice, while pS6K levels were not significantly changed (Figure 5O, P). These data suggest that the basal JNK activity was increased in SERT-deficient mice prior to the development of obesity, while the basal p38 activity was increased in parallel with the increase in the adiposity. JNK activation is thought to inhibit insulin receptor signal transduction by phosphorylating Ser307 of IRS1 [37,38]. To test JNK activity directly, we determined the basal level of pSer307- IRS1 in the liver of SERT2/2 and WT mice. Figure 5M–P show that the basal pSer307-IRS1 levels were significantly elevated in 3- and 6-month old SERT2/2 mice compared to WT controls. These results are consistent with the idea that a constitutive increase in JNK signaling contributes to insulin resistance in SERT deficient mice. Glucose intolerance of SERT-deficient mice can be partially corrected by reducing PTEN activity We next asked whether a genetically induced increase in AKT signaling could restore glucose tolerance of SERT mutant mice. The PTEN phosphatase is a negative regulator of PI3K signaling pathway [39]. PTEN-null mice are lethal but conditional PTEN deletion and PTEN+/2 mice have elevated AKT signaling [39]. We generated SERT2/2; PTEN+/2 and SERT+/2; PTEN+/2 double mutant mice and analyzed their glucose tolerance and insulin sensitivity using GTT and ITT. Consistent with previous reports [40,41], PTEN+/2 mice exhibited greater glucose tolerance and insulin sensitivity than WT mice (Figure 6). Remarkably, the PTEN+/2 mutation fully corrected glucose tolerance during GTT and insulin-induced glucose clearance during ITT in SERT+/2 mice (Figure 6). Glucose tolerance and insulin sensitivity of the SERT2/2; PTEN+/2 double mutants were also improved, compare to SERT2/2 mice (Figure 6). One potential explanation for these observations is that elevated basal AKT and JNK activities desensitize the response to insulin, leading to glucose intolerance, and that reduced PTEN function boosts AKT signaling thus improving glucose handling. However, PTEN+/2 only partially suppressed the metabolic symptoms in SERT2/2 mice, suggesting additional effects of SERT deficiency on metabolic homeostasis. To validate that PTEN+/2 indeed enhanced AKT signaling in SERT2/2 background, we compared pAKT levels in the liver of SERT2/2; PTEN+/2 double mutant mice with that in SERT2/2 mice. As shown in Figure 7A–F, insulin-induced phosphorylation of AKT was increased in the liver of 3- and 6- PLoS ONE | www.plosone.org 6 March 2012 | Volume 7 | Issue 3 | e32511