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, 7 (3), e32511

Reduced Serotonin Reuptake Transporter (SERT) Function Causes Insulin Resistance and Hepatic Steatosis Independent of Food Intake

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Reduced Serotonin Reuptake Transporter (SERT) Function Causes Insulin Resistance and Hepatic Steatosis Independent of Food Intake

Xiaoning Chen et al. PLoS One.

Abstract

Serotonin reuptake transporter (SERT) is a key regulator of serotonin neurotransmission and a major target of antidepressants. Antidepressants, such as selectively serotonin reuptake inhibitors (SSRIs), that block SERT function are known to affect food intake and body weight. Here, we provide genetic evidence that food intake and metabolism are regulated by separable mechanisms of SERT function. SERT-deficient mice ate less during both normal diet and high fat diet feeding. The reduced food intake was accompanied with markedly elevated plasma leptin levels. Despite reduced food intake, SERT-deficient mice exhibited glucose intolerance and insulin resistance, and progressively developed obesity and hepatic steatosis. Several lines of evidence indicate that the metabolic deficits of SERT-deficient mice are attributable to reduced insulin-sensitivity in peripheral tissues. First, SERT-deficient mice exhibited beta-cell hyperplasia and islet-mass expansion. Second, biochemical analyses revealed constitutively elevated JNK activity and diminished insulin-induced AKT activation in the liver of SERT-deficient mice. SERT-deficient mice exhibited hyper-JNK activity and hyperinsulinemia prior to the development of obesity. Third, enhancing AKT signaling by PTEN deficiency corrected glucose tolerance in SERT-deficient mice. These findings have potential implications for designing selective SERT drugs for weight control and the treatment of metabolic syndromes.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. SERT mRNA detected by RT-PCR from WT mice tissues.
Intestine tissue from a SERT−/− mouse is presented as a negative control. 6-month old mice were analyzed.
Figure 2
Figure 2. Characterization of food intake and adiposity of SERT-deficient mice.
A and C. Average daily food intake of 3- and 6-month old mice. SERT mutant mice exhibited significantly reduced food intake during ND and HFD feeding, compared to corresponding WT mice. B and D. Quantification of plasma leptin levels in 16 h fasted mice. SERT mutant mice at the age of 3- and 6-month exhibited higher fasting lepin levels, as compared to corresponding WT controls. E. Body weight at age of 3 months. The differences among WT, SERT−/− and SERT+/− mice were not statistically significant. F. Whole-body fat content at age of 3 months. The differences among WT, SERT−/− and SERT+/− mice were not statistically significant. For example, p = 0.34 for the difference between WT and SERT−/− mice, Student's t-test. G. Body weight at age of 6 months. ND-fed SERT−/− mice exhibited higher body weight compared to ND-fed WT. The difference between SERT+/− and WT was, however, not statistically significant. HFD-fed WT mice exhibited higher body weight compared to ND-fed WT mice. However, the difference between HFD- and ND-fed SERT−/− mice was not statistically significant (p = 0.06, Student's t-test). H. Whole-body fat content at age of 6 months. Fat content was increased in SERT−/− mice fed ND and HFD compared to corresponding WT mice. I. Absolute lean mass at age of 6 months. Lean mass was reduced in HFD-fed SERT−/− mice, as compared to HFD-fed WT mice. Data in panels A–I are presented as means ± SEM. *p<0.05, **p<0.01, ***p<0.001, Student's t-test. The number of animals analyzed is indicated in parentheses. J and K. Lipid content detected by Oil Red O staining of liver sections of WT and SERT−/− mice. Scale bar, 50 µm.
Figure 3
Figure 3. Characterization of glucose homeostasis in SERT-deficient mice.
A. GTT (i.p., 1 g/kg) of 3-month-old mice after 16 h fast. B. GTT (i.p., 1 g/kg) of 6-month-old mice after 16 h fast. C. ITT (i.p., 1 U/kg) of 3-month-old mice after 6 h fast. D. ITT (i.p., 1 U/kg) of 6-month-old mice after 6 h fast. N, number of mice analyzed for each genotype. Data represent mean ± SEM. Student's t-test was used to analyze statistical significance of the difference between the following groups: SERT−/− vs. WT mice on ND feeding, *p<0.05, **p<0.01, ***p<0.001; SERT−/− vs. WT mice on HFD feeding, #p<0.05, ##p<0.01, Student's t-test. At 3 months of age, the differences in GTT of SERT+/− vs. WT mice were not statistically significant; however, there were significant differences between SERT+/− vs. WT mice in ITT. At 6 months of age, both SERT−/− and SERT+/− mice differed significantly from WT mice in GTT and ITT.
Figure 4
Figure 4. Biochemical, histological and immunohistochemical analyses of insulin secretion and pancreatic beta-cell morphology in SERT-deficient mice.
A and B. Quantification of basal levels of plasma insulin in 16 h fasted mice at the age of 3 months (A) and 6 months (B). SERT mutant mice exhibited elevated fasting insulin levels during ND and HFD feeding as compared to corresponding WT control mice, *p<0.05, **p<0.01, ***p<0.001, Student's t-test. The number of mice analyzed is indicated in parentheses. C. Quantification of glucose-induced plasma insulin levels. 6-month old mice fasted for 16 h were injected with glucose (i.p., 1 g/kg). Insulin levels in plasma samples collected before and 15 and 30 min post glucose administration were analyzed. SERT−/− and SERT+/− mice exhibited higher insulin levels at all tested time points, compared to corresponding WT mice. Data represent mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, Student's t-test. N, number of animal analyzed. D. Immunofluorescent staining of pancreata of WT mice. The left panel shows insulin (green), the middle panel shows SERT (red) co-stained with DAPI (blue), and the right panel shows merge of the fluorescence. Noticing SERT staining in both insulin-expressing cells (as pointed by a triangle) and non-insulin cells (as pointed by an arrow) in the islets. Scale bar, 50 µm. E and F. H&E staining of pancreatic tissue sections from WT and SERT−/− mice. Noticing enlarged islet mass in the SERT−/− background. Scale bar, 100 µm. G and H. Triple-staining of pancreas of WT and SERT−/− mice for insulin (green), glucagon (red) and DAPI (blue). Scale bar, 200 µm. I and J. Quantification of islet mass in WT and SERT−/− mice. I. Beta-cell density was determined by calculating the percentage of insulin-labeled area on serial sections of pancreata. J. Islet density was determined by calculating the average number of islets on the pancreatic sections. The value from SERT−/− mice was compared with that of WT mice, *p<0.05, **p<0.01, Student's t-test. The number of mice analyzed is indicated in parentheses. K and L. Representative photomicrographs showing BrdU incorporation (red) in insulin-labeled cells (green) in pancreatic tissue sections of WT and SERT−/− mice. Scale bar, 50 µm. M. Beta-cell proliferation was estimated by calculating the number of beta cells incorporated BrdU relative to the islet area. Data represent mean ± SEM, **P<0.01, Student's t-test. The number of animals analyzes is indicated in parentheses.
Figure 5
Figure 5. Western blot analysis of insulin signaling component activity in SERT-deficient mice.
A–F. AKT activity in the liver of 3- and 6-month old WT and SERT-deficient mice was evaluated by western blot analysis of AKT Ser473 phosphorylation (pAKT) before and after insulin injection. G–L. IRS1 activity in the liver of 3- and 6-month old WT and SERT-deficient mice was evaluated by western blot analysis of tyrosine-phosphorylated IRS1. IRS1 was first immunoprecipitated with anti-IRS1 antibody (IP-IRS1) and then blotted with anti-IRS1 (IB-IRS1) or anti-phosphotyrosine antibodies (IB-pY-IRS1). A, D, G and J show images of representative western blot results. B and E show densitometric quantification of the ratio of pAKT vs. total AKT, and H and K show densitometric quantification of the ratio of pY-IRS1 vs. total IRS1. Insulin (−), basal activity determined from the tissues collected from16 h fasted mice. Insulin (+), insulin-induced activity determined from the tissues collected from mice 20 min post insulin injection. The value of WT treated with insulin and mutants with and without insulin treatment is normalized to that of WT without insulin treatment. C and F show the ratio of pAKT before and after insulin injection for each genotype. I and L show the ratio of pY-IRS1 before and after insulin injection. The basal pAKT and pY-IRS1 were elevated, but the net increase in pAKT and pY-IRS1 following insulin injection was attenuated in SERT mutant mice. M–P. Western blot analysis of phospho-Ser307-IRS1, phospho-JNK (pJNK), phospho-S6K (pS6K), and phospho-p38 MAPK (pp38) in the liver of 16 h fasted 3- and 6-month old SERT mutant mice. M and O show images of representative western blot results. N and P show densitometric quantification of pSer307-IRS1, pJNK, pS6K and pp38. The amounts of phosphorylated proteins are normalized to that of total corresponding proteins. Shown are the relative values of SERT−/− mice to WT, with the average value from WT mice defined as 1. Data represent mean ± SEM, *p<0.05, **p<0.01, Student's t-test. The number of mice analyzed is indicated in parentheses.
Figure 6
Figure 6. Reduced PTEN function corrected glucose tolerance in SERT mutants.
A and B show GTT (i.p., 1 g/kg), and C and D show ITT (i.p., 1 U/kg). The SERT−/−; PTEN+/− mice exhibited improved insulin sensitivity at 3 months of age and improved glucose tolerance at 6 months of age compared to age-matched SERT−/− mice, *p<0.05, ***p<0.001. The SERT+/−; PTEN+/− mice exhibited significantly improved glucose tolerance and insulin sensitivity at the age of 3- and 6-months, compared to age-matched SERT+/−, #p<0.05, ##p<0.01, ###p<0.001, Student's t-test. N, number of mice analyzed.
Figure 7
Figure 7. Western blot analysis of insulin signaling component activity in SERT−/−;PTEN+/− mice.
A–F. AKT activity in the liver of 3- and 6-month old SERT−/−; PTEN+/− and SERT−/− mice was evaluated by AKT Ser473 phosphorylation (pAKT) before and after insulin injection. G–J. IRS1 activities in the liver of 3- and 6-month old SERT−/−;PTEN+/− and SERT−/− mice were evaluated by tyrosine-phosphorylated IRS1. IRS1 was first immunoprecipitated with anti-IRS1 antibody (IP-IRS1) and then blotted with anti-IRS1 (IB-IRS1) or anti-phosphotyrosine antibodies (IB-pY-IRS1). A, D, G and I show images of representative western blot results. B and E show densitometric quantification of the ratio of pAKT vs. total AKT, and H and J show densitometric quantification of the ratio of pY-IRS1 vs. total IRS1. Insulin (−), basal activity determined from the tissues collected from16 h fasted mice. Insulin (+), insulin-induced activity determined from the tissues collected from mice 20 min post insulin injection. The value of WT treated with insulin and mutants with and without insulin treatment is normalized to that of WT without insulin treatment. C and F show the ratio of pAKT before and after insulin injection for each genotype. The net increase in pAKT amounts following insulin injection was elevated in SERT−/−; PTEN+/− mice relative to SERT−/− mice, *p<0.05, Student's t-test. K–N. Western blot analysis of phospho-JNK (pJNK) and phospho-Ser307-IRS1 in the liver of 16 h fasted 3- and 6-month old SERT−/−; PTEN+/− and SERT−/− mice. K and M show images of representative western blot results. L and N show densitometric quantification of pJNK and pSer307-IRS1. The amounts of phosphorylated proteins were normalized to that of total corresponding proteins. Shown are the relative values of SERT−/−; PTEN+/− mice to SERT−/− mice, with the average value from SERT−/− mice defined as 1. Data represent mean ± SEM. The number of mice analyzed is indicated in parentheses.

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