N-terminal kinase (JNK) activity in the liver was constitutively elevated in SERT-deficient mice, whereas insulin-induced serine/ threonine kinase AKT activation in the liver was attenuated. Based on these results, we propose that feeding and metabolism are regulated by separable mechanisms of SERT function and that SERT deficiency impairs the regulation of insulin signaling in peripheral tissues. Results SERT is distributed in multiple central and peripheral sites implicated in the control of metabolism To begin to identify the putative role for SERT function in metabolism, we analyzed the distribution of SERT mRNA in C57BL/6J (WT) mice and mice bearing a targeted deletion in the SERT gene Slc6a4 (SERT2/2) [20] by RT-PCR. Consistent with previous studies [21,22,23], we detected SERT mRNA in the brainstem, hypothalamus, as well as in blood, white adipose tissue, intestine, liver and pancreas (Figure 1). To validate the specificity of the probes, we analyzed SERT2/2 mice. SERT mRNA was not expressed in any of those tissues in SERT2/2 mice (Figure 1). These data support the idea that SERT may contribute to the control of metabolism in multiple central and peripheral tissues. SERT-deficient mice are hypophagic and hyperleptinemic In light of the well-established inhibitory effect of SSRIs on food intake, we monitored daily food consumption by WT and SERT- deficient mice. The amount of food consumed adjusted for body weight by SERT homozygous (SERT2/2) and heterozygous (SERT+/2) mutant mice was reduced compared to age-matched WT mice (Figure 2A, C; Figure S1) during normal diet (ND) feeding. We further analyzed food intake of SERT2/2 mice during high fat diet (HFD) feeding, and found that daily intake of the HFD by SERT2/2 was also reduced (Figure 2C). The levels of plasma triglycerides in SERT2/2 and SERT+/2 mice were indistinguishable from that in WT mice during ND feeding. HFD- fed SERT2/2 mice exhibited lower plasma triglyceride levels compared to HFD-fed WT mice (Figure S2). Because SERT2/2 mice showed a marked increase in extracellular 5-HT in the brain [24], these results could imply that SERT deficiency results in a greater hypothalamic 5-HT signaling leading to suppression of food intake. In addition to 5-HT, the adipocyte-secreted peptide leptin inhibits food intake [25]. To assess the relationship between leptin and reduced food intake in SERT-deficient mice, we measured plasma leptin in SERT-deficient mice fasted for 16 hr. We found that fasting leptin levels were elevated in both SERT2/2 and SERT+/2 mice fed ND (Figure 2B, D). Fasting leptin levels were further increased in SERT2/2 mice fed HFD (Figure 2D). These Figure 1. SERT mRNA detected by RT-PCR from WT mice tissues. Intestine tissue from a SERT2/2 mouse is presented as a negative control. 6-month old mice were analyzed. doi:10.1371/journal.pone.0032511.g001 Distinct SERT Function in Feeding and Obesity data suggest that elevated leptin levels could be a contributing factor for the hypophagia of SERT-deficient mice. SERT-deficient mice progressively develop obesity and liver steatosis but do not substantially increase body weight Contrary to our expectations, we found that SERT-deficient mice developed age-dependent obesity, despite reduced food intake. At 3 months of age, the body weight and body fat content of WT and SERT2/2 mice were similar (Figure 2E, F). At 6- months old, SERT2/2 mice fed ND exhibited higher body weight and higher fat content compared to WT controls (Figure 2G, H). HFD feeding resulted in a further increase in the adiposity (Figure 2H). However, HFD-fed SERT2/2 mice did not substantially further increase body weight compared to ND-fed SERT2/2 mice, but they had reduced lean mass (Figure 2G, I). These data suggest that SERT deficiency promotes nutrient partitioning towards fat rather than simply causing an overall increase in body mass. Previous studies indicated that major depressive and generalized anxiety disorders are over-represented in patients with nonalco- holic hepatic steatosis and such comorbidity is associated with obesity and type II diabetes [26,27]. Consequently, the increased adiposity in SERT2/2 mice prompted us to examine their hepatic fat content. Oil Red O staining of the liver from 3-month old SERT2/2 mice did not show an appreciable increase in fat content (not shown). By contrast, we observed pronounced hepatic steatosis in 6-month old SERT2/2 mice fed ND (Figure 2K). Collectively, these results show that SERT deficiency can lead to obesity and related metabolic disorders, even though it causes a reduction in food intake. SERT mutant mice exhibit glucose intolerance and insulin resistance prior to the development of obesity Since obesity is frequently associated with insulin resistance and type II diabetes, we wished to determine whether the increased adiposity in SERT-deficient mice is associated with altered glucose homeostasis. We found that fasting glucose levels were elevated in SERT2/2 mice at 3- and 6-month of age (Figure 3A, B). Glucose tolerance tests (GTT) indicated that glucose clearance following injection of a bolus of glucose was affected in 3- and 6-month old SERT2/2 mice (Figure 3A, B). Thus, SERT-deficient mice are hyperglycemic and have reduced glucose tolerance. Glucose intolerance could reflect reduced insulin sensitivity of peripheral tissues in SERT-deficient mice. To test this possibility, we performed insulin tolerance tests (ITT). Figure 3C and D show that insulin-induced clearance of blood glucose was compromised in 3- and 6-month old SERT2/2 mice fed ND. SERT+/2 mice also exhibited glucose intolerance and insulin intolerance, albeit the deficits at the age of 3 months were modest (Figure 3), showing that even a partial SERT deficiency may impair glucose homeostasis and insulin responses. To examine the metabolic response to increased dietary fat in SERT-deficient mice, 3-month old mice were fed with a HFD for three months, and their glucose tolerance and insulin sensitivity were analyzed by GTT and ITT, respectively. Both WT and SERT2/2 mice fed the HFD developed significant glucose intolerance (Figure 3B) and insulin resistance (Figure 3D) compared to their ND-fed controls. HFD-fed SERT2/2 mice exhibited higher fasting glucose levels, and higher glucose levels at 60 min and 90 min post-injection of a bolus of glucose during GTT, compared to HFD-fed WT mice (Figure 3B). HFD-fed SERT2/2 mice also exhibited greater insulin resistance than PLoS ONE | www.plosone.org 2 March 2012 | Volume 7 | Issue 3 | e32511