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Deletion of PHGDH in adipocytes improves glucose intolerance in diet-induced obese mice
Biochemical and Biophysical Research Communications xxx (2018) 1e6
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Deletion of PHGDH in adipocytes improves glucose intolerance in diet-induced obese mice Keisuke Okabe a, b, Isao Usui b, c, Keisuke Yaku a, Yoshio Hirabayashi d, Kazuyuki Tobe b, Takashi Nakagawa a, e, * a
Department of Metabolism and Nutrition, Graduate School of Medicine and Pharmaceutical Science for Research, University of Toyama, Toyama, 9300194, Japan First Department of Internal Medicine, Graduate School of Medicine and Pharmaceutical Science for Research, University of Toyama, Toyama, 930-0194, Japan c Department of Endocrinology and Metabolism, Dokkyo Medical University, Tochigi, 321-0293, Japan d Neuronal Circuit Mechanisms Research Group, RIKEN Brain Science Institute, Saitama, 351-0198, Japan e Institute of Natural Medicine, University of Toyama, Toyama, 930-0194, Japan b
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Article history: Received 20 August 2018 Accepted 28 August 2018 Available online xxx
Serine is a nonessential amino acid and plays an important role in cellular metabolism. In mammalian serine biosynthesis, 3-phosphoglycerate dehydrogenase (PHGDH) is considered a rate-limiting enzyme and is required for normal development. Although the biological functions of PHGHD in the nervous system have been intensively studied, its function in adipose tissue is unknown. In this study, we found that PHGDH is abundantly expressed in mature adipocytes of white adipose tissue. We generated an adipocyte-specific PHGDH knockout mouse (PHGDH FKO) and used it to investigate the role of serine biosynthesis in adipose tissues. Although PHGDH FKO mice had no apparent defects in adipose tissue development, these mice ameliorated glucose intolerance upon diet-induced obesity. Additionally, we found that the serine levels increase drastically in the adipose tissues of obese wild type mice, whereas no significant rise was observed in PHGDH FKO mice. Furthermore, wild type mice fed a serine-deficient diet also exhibited better glucose tolerance. These results suggest that PHGDH-mediated serine biosynthesis has important roles in adipose tissue glucose metabolism and could be a therapeutic target for diabetes in humans. © 2018 Elsevier Inc. All rights reserved.
Keywords: Serine PHGDH Adipocyte Obesity Glucose intolerance
1. Introduction Serine is a nonessential amino acid that plays an important role in cellular metabolism, including gluconeogenesis, one-carbon metabolism, and phospholipid synthesis [1,2]. Also, it has been shown that serine has diverse biological functions as a signaling molecule. For instance, D-serine, an enantiomer of L-serine, acts as an endogenous agonist of the N-methyl-D-aspartate (NMND) receptor and mediates neuronal excitation [3,4]. L-serine has been shown to be a natural ligand and allosteric activator of pyruvate kinase M2 and supports the proliferation of cancer cells [5]. It has also been reported that serine starvation induces p53-dependent
* Corresponding author. Department of Metabolism and Nutrition, Graduate School of Medicine and Pharmaceutical Science for Research, University of Toyama, 2630 Sugitani, Toyama, Toyama, 930-0194, Japan. E-mail address: [email protected] (T. Nakagawa).
metabolic remodeling in cancer cells and promotes cell survival [6]. In mammals, serine is synthesized from 3-phosphoglycerate through a three-step enzymatic reactions mediated by 3phosphoglycerate dehydrogenase (PHGDH), phosphohydroxythreonine aminotransferase (PSAT1), and phosphoserine phosphatase (PSPH) [1,2]. Serine is also generated by reversible conversion of glycine by serine hydroxymethyltransferase (SHMT) [1,2]. Among these enzymes, PHGDH is considered the rate-limiting enzyme in the serine biosynthesis pathway, and its deficiency in human leads to severe neurological symptoms such as congenital microcephaly, severe psychomotor retardation, and intractable seizures [7]. Accordingly, the systemic deletion of PHGDH in mice results in the developmental abnormalities of the brain and in embryonic lethality [8]. Thus, the biological significance of PHGDH in the nervous system has been elucidated. Although PHGDH is expressed in other tissues, including liver, kidney, and adipose tissue [9e11], the biological importance of serine biosynthesis in
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Please cite this article in press as: K. Okabe, et al., Deletion of PHGDH in adipocytes improves glucose intolerance in diet-induced obese mice, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.08.180
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these tissues is poorly understood. In particular, the biological function of PHGDH in adipose tissue is unknown. Several studies have demonstrated that blood concentrations of branched amino acids, including leucine, isoleucine, valine, tyrosine, and phenylalanine, are tightly associated with future diabetes development in human subjects [12,13]. Another study also indicated that serum levels of glycine are associated with insulin resistance [14]. However, the relationship between serine metabolism and diabetes remains unclear. In this study, we generated an adipocyte-specific PHGDH knockout mouse and used it to investigate the role of serine biosynthesis in adipose tissues.
2. Materials and methods 2.1. Mice Adipocyte-specific PHGDH knockout (FKO) mice were generated by mating PHGDH flox mice [15] with Adiponectin-Cre mice [16]. All mice were maintained under a standard light cycle (12 h light/ dark) and were allowed free access to water and food. For dietinduced obesity experiments, mice were fed a high-fat high-sucrose containing diet (HFHSD) (Research Diets: D12327) for 6 weeks. Serine-deficient diet (Research Diets: A05080213) was fed to 7-weeks old female C57BL/6 mice for 8 weeks. All of the animals care policies and procedures for the experiments were approved by the animal experiment committee at the University of Toyama.
2.5. Measurements of serine levels in serum and adipose tissue by GC/MS Serum was mixed with equal volume of methanol, and then centrifuge at 13000g for 10 min at 4 C. Adipose tissues were grinded by Multi Beads Shocker (Yasui Kikai) with methanol and water in the proportion of 1:1 (by volume), and then centrifuge 13000g for 10 min at 4 C. Supernatant was mixed with equal volume of chloroform, and mixture was centrifuge at 13000g for 10 min at 4 C. The upper aqueous phase was taken into a tube and evaporated using Speedvac SPD 1010 (Thermo). Quantification of serine was performed by Agilent 5977 MSD single Quad mass spectrometer coupled to Agilent 7890 Gas Chromatography with selected ion monitoring (SIM) mode. Evaporated samples were derivatized by methoxiamine hydrochloride and N-methyl-N-trimethylsilyltrifluoroacetamide with 1% trimethylchlorosilane (MSTFA þ 1% TMCS, Pierce). Details of GC/MS setting were described in elsewhere [18]. Amounts of metabolite were calculated by integrated sum of area using Mass Hunter Quantitative software (Agilent), and the absolute concentration was calculated using serine standard curve. 2.6. Histological staining After the excision, the adipose tissues were fixed with 4% paraformaldehyde (Wako), and embedded in paraffin. The paraffin sections of 3 mm thickness were subjected to hematoxylin and eosin staining. Sample slides were observed using BX61 microscope (Olympus, Japan).
2.2. Glucose tolerance test (GTT) 2.7. Statistical analysis For the GTT experiments, the mice were fasted for 16 h and then injected with glucose (1 g/Kg body weight) intraperitoneally. The blood glucose concentration was measured using an automatic blood glucose meter (NOVA Biomedical).
Statistical analysis was performed using an unpaired or paired Student's t-test. Data are expressed as the mean ± SD, and significant differences are confirmed statistically when p-value is less than 0.05.
2.3. Western blot analysis
3. Results
Tissues were harvested from PHGDH WT and FKO mice. vWAT, sWAT, and BAT were prepared from gonadal, inguinal, and interscapular region, respectively. Harvested tissues were immediately frozen in liquid nitrogen and preserved in 80 C until utilization. Frozen tissues were grinded by multibeads shocker (Yasui Kikai) with lysis buffer (10 mM Tris HCl, 2 mM EDTA, 0.1% Nonidet P-40, 150 mM NaCl) and centrifuged at 13000g for 10 min at 4 C. Then, supernatants were subjected to western blot analysis. SVF preparation was performed according to the method described elsewhere [17]. Primary antibodies used for western blot analysis were anti-PHGDH (Atlas Antibodies), anti-PSAT1 (Santa Cruz), anti-pan actin (Chemicon), anti-b-Tubulin (Cell Signaling), and anti-b-actin (Cell Signaling).
3.1. PHGDH is abundantly expressed in mature adipocytes of white adipose tissue
2.4. Real-time quantitative PCR Total RNAs were extracted from adipose tissues and 3T3-L1 cells using RNEasy Lipid Tissue Mini Kit (QIAGEN) and TRI Reagent (Molecular Research Center, Inc.), respectively. cDNA was prepared using ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo, Japan) according to the supplier's protocol. Real-time PCR was carried out using THUNDERBIRD SYBR qPCR Mix (Toyobo) on Thermal Cycler Dice Real Time System II (Takara Bio). Quantification was done by Delta Delta Ct method, and Rpl13a was used as a reference gene.
Although mouse ENCODE transcriptome data shows that Phgdh mRNA is ubiquitously expressed in mouse tissues [11], the protein expression of PHGDH in mouse tissues has not yet been determined. Thus, we examined the tissue distribution of PHGDH protein using various mice tissue lysates. Western blotting analysis confirmed that the brain exhibited abundant expression of PHGDH protein. Interestingly, PHGDH was also abundantly expressed in visceral white adipose tissue (vWAT) and subcutaneous white adipose tissue (sWAT), whereas brown adipose tissue (BAT) contained little PHGHD (Fig. 1A). We also quantified mRNA levels of Phgdh, Psat1, and Psph in adipose tissues, and found that vWAT expressed relatively higher levels of these serine synthesis enzyme mRNAs (Fig. 1B). Although the protein expression of PHGDH in WAT was stronger than that in BAT, the protein levels of PSAT1 were almost comparable between vWAT, sWAT, and BAT (Fig. 1C). WAT is divided into the mature adipocyte fraction (MAF) and the stromal vascular fraction (SVF), which includes stromal cells, pericytes, immune cells, and preadipocytes [19]. Therefore, we also determined the localization of PHGDH in WAT using MAF and SVF samples from wild type mice. Most of PHGDH and PSAT1 were expressed in the MAF, suggesting that serine biosynthesis in WAT occurs in mature adipocytes (Fig. 1D). We further examined whether there are any changes in these serine biosynthesis enzymes during adipogenesis
Please cite this article in press as: K. Okabe, et al., Deletion of PHGDH in adipocytes improves glucose intolerance in diet-induced obese mice, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.08.180
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Fig. 1. PHGDH is abundantly expressed in mature adipocytes of white adipose tissue. (A) PHGDH protein expression in various tissues from male C57BL/6 mice. Pan-actin was used as loading control. (B) qPCR analysis of Phgdh, Psat1, Psph in vWAT, sWAT, and BAT from male C57BL/6 mice. Data are presented as mean ± SD (n ¼ 4 for each group). (C) Immunoblot analysis of PHGDH and PSAT1 in vWAT, sWAT, and BAT from male C57BL/6 mice. b-Tubulin was used as loading control. (D) PHGDH protein expression in MAF and SVF derived from vWAT of male C57BL/6 mice. (E) qPCR analysis of Phgdh, Psat1, Psph in 3T3-L1 cells at indicated time points. Data are presented as mean ± SD (n ¼ 3 for each time point). (F) PHGDH and PSAT1 expression during differentiation of 3T3L1 cells. b-actin was used as loading control.
using 3T3-L1 cells, a commonly used model of preadipocyte differentiation. All three genes were downregulated during the induction phase (day 0 to day 2), but expressions increased after differentiation (~day 4; Fig. 1E and F). These data suggest that serine biosynthesis may play some roles in mature adipocytes.
3.2. Adipocytes-specific PHGDH knockout mice have no obvious developmental defect in adipose tissues To elucidate the physiological significance of serine biosynthesis in adipose tissue, we generated adipocyte-specific PHGDH knockout mice (PHGDH FKO) by crossing PHGDH flox mice with adiponectin-Cre mice [15,16]. PHGDH FKO mice were born and developed with a normal appearance. We confirmed the significant reduction of PHGDH protein in vWAT, sWAT and BAT of PHGDH FKO mice, but the protein levels in the brain was unchanged (Fig. 2A). When PHGDH FKO mice were fed with a normal chow diet, body weight gain and food intake was essentially comparable with those in wild type (WT) controls (Fig. 2B and C). Accordingly, there were no obvious differences in adipose tissue weight between PHGDH FKO and WT mice (Fig. 2D and E). Histological examination also demonstrated that adipose tissues in PHGDH FKO mice exhibited no evident abnormalities compared with controls (Fig. 2F). Together, these data has demonstrates that deletion of PHGDH in adipose tissues has no influence on normal adipose tissue development.
3.3. Deletion of PHGDH in adipocytes improves glucose intolerance in diet-induced obese mice Next, we examined the role of adipose tissue PHGDH in pathological situation. During obesity, adipose tissues expand due to an increase in both adipocyte size and number [20]. Functionally, insulin sensitivity in adipose tissues is impaired, and glucose intolerance is invoked by obesity [21]. To investigate the role of PHGDH in glucose metabolism, we fed PHGDH FKO and WT mice with a high-fat high-sucrose diet (HFHSD). When male PHGDH FKO mice were fed with HFHSD, they exhibited essentially similar weight gain compared with male WT mice (Fig. 3A). In contrast, female PHGDH FKO mice exhibited slightly less weight gain than female WT mice (Fig. 3B), whereas there was no significant difference in the food intake between them (Fig. 3C). To evaluate the glucose metabolism in male and female PHGDH FKO mice, we performed an intraperitoneal glucose tolerance test (ipGTT) after 6 weeks of HFHSD feeding. Male PHGDH FKO mice exhibited similar glucose tolerance to WT mice (Fig. 3D), whereas female PHGDH FKO mice demonstrated significantly improved glucose tolerance compared with WT (Fig. 3E). We also measured serum serine levels to examine the impact of PHGDH deletion on serine metabolism. However, serum serine levels were unaffected in PHGDH FKO mice (Fig. 3F). We then examined the changes of serine levels in WAT of female PHGDH FKO and WT mice. Interestingly, HFHSD feeding significantly increased serine levels in WT mice (Fig. 3G). In contrast, female PHGDH FKO mice demonstrated no significant rise
Please cite this article in press as: K. Okabe, et al., Deletion of PHGDH in adipocytes improves glucose intolerance in diet-induced obese mice, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.08.180
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Fig. 2. Adipocyte-specific PHGDH knockout mice have no obvious developmental defect in adipose tissues. (A) Immunoblot analysis of male PHGDH in vWAT, sWAT, BAT, and brain from PHGDH FKO and WT mice. Pan-actin was used as loading control. (B and C) Body weight changes (B) and food intake (C) of male PHGDH FKO and WT mice fed a normal chow diet. Data are presented as mean ± SD (n ¼ 3 for each group). (D) Representative image of adipose tissues from male PHGDH FKO and WT mice. (E) Quantifications of adipose tissue weight from male PHGDH FKO and WT mice (n ¼ 4 for each group). (F) Hematoxylin-Eosin (HE) staining of vWAT, sWAT, and BAT from male PHGDH FKO and WT mice. Scale bar represents 200 mm.
in WAT serine levels after HFHSD feeding (Fig. 3G). These results imply the pathological importance of increased serine levels in the development of glucose intolerance during diet-induced obesity. 3.4. Serine-deficient diet feeding also exhibits favorable effects on glucose metabolism in mice Results to date suggested that the inhibition of serine synthesis is beneficial for glucose tolerance during diet-induced obesity. We, therefore, investigated the effect of dietary serine restriction on glucose metabolism using female C57BL/6 mice. When C57BL/6 female mice were fed a serine-deficient diet, they exhibited a slightly higher, but not statistically significant, body weight gain compared with control (serine-containing) diet-fed mice (Fig. 4A). We also confirmed that serine restriction had no effect on food intake and serum serine levels (Fig. 4B and C). Importantly, serinedeficient diet-fed mice also exhibited better glucose tolerance (Fig. 4D). These results demonstrate that dietary serine restriction has beneficial effects on glucose metabolism without adverse side effects and could be a promising nutritional intervention for diabetes patients. 4. Discussion In this study, we discovered that PHGDH is abundantly expressed in mature adipocytes of white adipose tissue. We also
observed an increased expression of PHGDH during the differentiation of 3T3L1 pre-adipocytes. Although the deletion of the PHGDH gene in adipose tissue had no obvious impact on normal adipose tissue development, we found that female PHGDH FKO mice exhibited improved glucose tolerance during diet-induced obesity. We also revealed that the WAT serine levels drastically increased after HFHSD feeding in WT mice, whereas no significant rise was observed in PHGDH FKO mice. Furthermore, wild type mice fed a serine-deficient diet also exhibited better glucose tolerance. These results suggest that PHGDH-mediated serine biosynthesis had some important roles on the progression of glucose intolerance during diet induced obesity. It has been reported that PHGDH is overexpressed in several types of human cancer cells, including breast cancers and melanoma [22e25]. In breast cancer cells, serine is necessary for sustaining cellular proliferation, and deletion of PHGDH impaired the cellular proliferations and survival [26,27]. Interestingly, deletion of PHGDH in breast cancer cells has no effect on the intracellular serine levels when they were maintained in serine containing medium. Instead, deletion of PHGDH decreased serine synthesis flux and reduced the PSAT1-mediated production of a-ketoglutarate (a-KG) from glutamate [26]. a-KG is an intermediate of the tricarboxylic acid cycle and also serves as a substrate for histone or DNA demethylase [28]. Thus, it may be possible that PHGDH deletion affects gene expression through a-KG-mediated epigenetic changes and thus influences glucose metabolism pathways.
Please cite this article in press as: K. Okabe, et al., Deletion of PHGDH in adipocytes improves glucose intolerance in diet-induced obese mice, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.08.180
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Fig. 3. Deletion of PHGDH in adipocytes improves glucose intolerance in diet-induced obese mice. (A and B) Body weight changes of male (A) or female (B) PHGDH FKO and WT mice fed with HFHSD. Data are presented as mean ± SD (n ¼ 10 for each group). (C) Food intake of female PHGDH FKO and WT mice fed with HFHSD. Data are presented as mean ± SD (n ¼ 10 for each group). (D and E) Intraperitoneal glucose tolerance test (ipGTT) of male (D) or female (E) PHGDH FKO and WT mice fed with HFHSD. Data are presented as mean ± SD (n ¼ 10 for each group). (F) Plasma serine concentration in female PHGDH FKO and WT mice fed with HFHSD. Data are presented as mean ± SD (n ¼ 20 for each group). (G) Relative serine levels in female PHGDH FKO and WT mice fed with HFHSD. Data are presented as mean ± SD (n ¼ 7e12 for each group). Asterisk (*) indicated the statistical significance when p-value is less than 0.05.
Fig. 4. Serine-deficient diet feeding also exhibits favorable effects on glucose metabolism in mice. (AeD) Body weight changes (A), food intake (B), Plasma serine concentration(C), and ipGTT (D) of female C57BL/6 mice fed with serine-deficient diet or control diet. Data are presented as mean ± SD (n ¼ 5e6 for each group). Asterisk (*) indicates the statistical significance when p-value is less than 0.05, and N.S. means not significant.
Serine is also a major source for one-carbon metabolism, which contributes to the production of folic acid and S-adenosine methionine (SAM) [1]. In particular, SAM is an important donor for histone and DNA methylation [28]. Currently, the effect of PHGDH deletion on SAM levels has not been determined. However, it is possible that serine-derived SAM contributes to altering methylation patterns and affects the expression of genes related to glucose metabolism. From this viewpoint, serine biosynthesis may regulate both methylation and demethylation, and the gene regulation mechanisms affected by serine metabolism may be more complex than currently appreciated. It has been reported that hepatic PSAT1 regulates insulin sensitivity during diet-induced obesity [29]. During genetic- and diet-induced obesity, PSAT1 is downregulated in the liver, and the suppression of PSAT1 induces the gene expression of tribbles homolog 3 (TRB3). Furthermore, an adenovirus-mediated increase of PSAT1 in the liver improves glucose intolerance in obese mice through the suppression of the TRB3 pathway [29]. These results
indicate that the activation of the serine synthesis pathway in the liver is beneficial to preserving insulin sensitivity during obesity. In general, the liver is an organ that produces glucose from glycogenic amino acids, including serine, whereas adipose tissues are organs that consume glucose. Thus, their metabolic activities are often opposed during various nutritional changes, including fasting and obesity [29]. It will, therefore, be important to generate a liverspecific PHGDH KO mouse to clarify the role of serine biosynthesis during obesity. In this study, we confirmed favorable effects only in female PHGHD KO mice. It well known that gender differences affects to the obesity and glucose intolerance possibly through sex hormones [30]. Of note, estrogen receptor status is related to PHGDH expression levels in breast cancer cells [26]. Thus, it is possible that sex hormone may influence serine biosynthesis and its related metabolic pathways in obese mice. Although serine deficiency in humans causes severe neuronal and mental disorders [7], PHGDH deletion in adipocytes and
Please cite this article in press as: K. Okabe, et al., Deletion of PHGDH in adipocytes improves glucose intolerance in diet-induced obese mice, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.08.180
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dietary serine restriction exhibits favorable effects on glucose handling in mice. Symptoms in human serine deficiency probably attributes to decreased serine levels in the brain [8], and the results from brain-specific PHGDH knockout mice also support this idea [15]. Thus, it is important to specifically decrease adipose tissue serine levels to achieve only favorable metabolic effects. In the dietary serine restriction experiment, we only depleted serine, and not glycine, which is reversibly converted to serine by SMTH. Additionally, the serum serine levels and body weight were unaffected by dietary serine restriction. Thus, these results suggest that dietary serine restriction is a safe nutritional intervention, and may be applicable to the treatment of human diabetic patients. Acknowledgement We are grateful to Ms. Tomomi Kubo for the care and husbandry of PHGDH FKO mice. This work was supported by the Research Grant from Banyu Life Science Foundation International and the University of Toyama President Grant to TN. JSPS KAKENHI (Grant Number 18K16193) to KO also supported this study. Transparency document
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Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.08.180. [20]
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Deletion of PHGDH in adipocytes improves glucose intolerance in diet-induced obese mice Keisuke Okabe a, b, Isao Usui b, c, Keisuke Yaku a, Yoshio Hirabayashi d, Kazuyuki Tobe b, Takashi Nakagawa a, e, * a
Department of Metabolism and Nutrition, Graduate School of Medicine and Pharmaceutical Science for Research, University of Toyama, Toyama, 9300194, Japan First Department of Internal Medicine, Graduate School of Medicine and Pharmaceutical Science for Research, University of Toyama, Toyama, 930-0194, Japan c Department of Endocrinology and Metabolism, Dokkyo Medical University, Tochigi, 321-0293, Japan d Neuronal Circuit Mechanisms Research Group, RIKEN Brain Science Institute, Saitama, 351-0198, Japan e Institute of Natural Medicine, University of Toyama, Toyama, 930-0194, Japan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 August 2018 Accepted 28 August 2018 Available online xxx
Serine is a nonessential amino acid and plays an important role in cellular metabolism. In mammalian serine biosynthesis, 3-phosphoglycerate dehydrogenase (PHGDH) is considered a rate-limiting enzyme and is required for normal development. Although the biological functions of PHGHD in the nervous system have been intensively studied, its function in adipose tissue is unknown. In this study, we found that PHGDH is abundantly expressed in mature adipocytes of white adipose tissue. We generated an adipocyte-specific PHGDH knockout mouse (PHGDH FKO) and used it to investigate the role of serine biosynthesis in adipose tissues. Although PHGDH FKO mice had no apparent defects in adipose tissue development, these mice ameliorated glucose intolerance upon diet-induced obesity. Additionally, we found that the serine levels increase drastically in the adipose tissues of obese wild type mice, whereas no significant rise was observed in PHGDH FKO mice. Furthermore, wild type mice fed a serine-deficient diet also exhibited better glucose tolerance. These results suggest that PHGDH-mediated serine biosynthesis has important roles in adipose tissue glucose metabolism and could be a therapeutic target for diabetes in humans. © 2018 Elsevier Inc. All rights reserved.
Keywords: Serine PHGDH Adipocyte Obesity Glucose intolerance
1. Introduction Serine is a nonessential amino acid that plays an important role in cellular metabolism, including gluconeogenesis, one-carbon metabolism, and phospholipid synthesis [1,2]. Also, it has been shown that serine has diverse biological functions as a signaling molecule. For instance, D-serine, an enantiomer of L-serine, acts as an endogenous agonist of the N-methyl-D-aspartate (NMND) receptor and mediates neuronal excitation [3,4]. L-serine has been shown to be a natural ligand and allosteric activator of pyruvate kinase M2 and supports the proliferation of cancer cells [5]. It has also been reported that serine starvation induces p53-dependent
* Corresponding author. Department of Metabolism and Nutrition, Graduate School of Medicine and Pharmaceutical Science for Research, University of Toyama, 2630 Sugitani, Toyama, Toyama, 930-0194, Japan. E-mail address: [email protected] (T. Nakagawa).
metabolic remodeling in cancer cells and promotes cell survival [6]. In mammals, serine is synthesized from 3-phosphoglycerate through a three-step enzymatic reactions mediated by 3phosphoglycerate dehydrogenase (PHGDH), phosphohydroxythreonine aminotransferase (PSAT1), and phosphoserine phosphatase (PSPH) [1,2]. Serine is also generated by reversible conversion of glycine by serine hydroxymethyltransferase (SHMT) [1,2]. Among these enzymes, PHGDH is considered the rate-limiting enzyme in the serine biosynthesis pathway, and its deficiency in human leads to severe neurological symptoms such as congenital microcephaly, severe psychomotor retardation, and intractable seizures [7]. Accordingly, the systemic deletion of PHGDH in mice results in the developmental abnormalities of the brain and in embryonic lethality [8]. Thus, the biological significance of PHGDH in the nervous system has been elucidated. Although PHGDH is expressed in other tissues, including liver, kidney, and adipose tissue [9e11], the biological importance of serine biosynthesis in
https://doi.org/10.1016/j.bbrc.2018.08.180 0006-291X/© 2018 Elsevier Inc. All rights reserved.
Please cite this article in press as: K. Okabe, et al., Deletion of PHGDH in adipocytes improves glucose intolerance in diet-induced obese mice, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.08.180
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these tissues is poorly understood. In particular, the biological function of PHGDH in adipose tissue is unknown. Several studies have demonstrated that blood concentrations of branched amino acids, including leucine, isoleucine, valine, tyrosine, and phenylalanine, are tightly associated with future diabetes development in human subjects [12,13]. Another study also indicated that serum levels of glycine are associated with insulin resistance [14]. However, the relationship between serine metabolism and diabetes remains unclear. In this study, we generated an adipocyte-specific PHGDH knockout mouse and used it to investigate the role of serine biosynthesis in adipose tissues.
2. Materials and methods 2.1. Mice Adipocyte-specific PHGDH knockout (FKO) mice were generated by mating PHGDH flox mice [15] with Adiponectin-Cre mice [16]. All mice were maintained under a standard light cycle (12 h light/ dark) and were allowed free access to water and food. For dietinduced obesity experiments, mice were fed a high-fat high-sucrose containing diet (HFHSD) (Research Diets: D12327) for 6 weeks. Serine-deficient diet (Research Diets: A05080213) was fed to 7-weeks old female C57BL/6 mice for 8 weeks. All of the animals care policies and procedures for the experiments were approved by the animal experiment committee at the University of Toyama.
2.5. Measurements of serine levels in serum and adipose tissue by GC/MS Serum was mixed with equal volume of methanol, and then centrifuge at 13000g for 10 min at 4 C. Adipose tissues were grinded by Multi Beads Shocker (Yasui Kikai) with methanol and water in the proportion of 1:1 (by volume), and then centrifuge 13000g for 10 min at 4 C. Supernatant was mixed with equal volume of chloroform, and mixture was centrifuge at 13000g for 10 min at 4 C. The upper aqueous phase was taken into a tube and evaporated using Speedvac SPD 1010 (Thermo). Quantification of serine was performed by Agilent 5977 MSD single Quad mass spectrometer coupled to Agilent 7890 Gas Chromatography with selected ion monitoring (SIM) mode. Evaporated samples were derivatized by methoxiamine hydrochloride and N-methyl-N-trimethylsilyltrifluoroacetamide with 1% trimethylchlorosilane (MSTFA þ 1% TMCS, Pierce). Details of GC/MS setting were described in elsewhere [18]. Amounts of metabolite were calculated by integrated sum of area using Mass Hunter Quantitative software (Agilent), and the absolute concentration was calculated using serine standard curve. 2.6. Histological staining After the excision, the adipose tissues were fixed with 4% paraformaldehyde (Wako), and embedded in paraffin. The paraffin sections of 3 mm thickness were subjected to hematoxylin and eosin staining. Sample slides were observed using BX61 microscope (Olympus, Japan).
2.2. Glucose tolerance test (GTT) 2.7. Statistical analysis For the GTT experiments, the mice were fasted for 16 h and then injected with glucose (1 g/Kg body weight) intraperitoneally. The blood glucose concentration was measured using an automatic blood glucose meter (NOVA Biomedical).
Statistical analysis was performed using an unpaired or paired Student's t-test. Data are expressed as the mean ± SD, and significant differences are confirmed statistically when p-value is less than 0.05.
2.3. Western blot analysis
3. Results
Tissues were harvested from PHGDH WT and FKO mice. vWAT, sWAT, and BAT were prepared from gonadal, inguinal, and interscapular region, respectively. Harvested tissues were immediately frozen in liquid nitrogen and preserved in 80 C until utilization. Frozen tissues were grinded by multibeads shocker (Yasui Kikai) with lysis buffer (10 mM Tris HCl, 2 mM EDTA, 0.1% Nonidet P-40, 150 mM NaCl) and centrifuged at 13000g for 10 min at 4 C. Then, supernatants were subjected to western blot analysis. SVF preparation was performed according to the method described elsewhere [17]. Primary antibodies used for western blot analysis were anti-PHGDH (Atlas Antibodies), anti-PSAT1 (Santa Cruz), anti-pan actin (Chemicon), anti-b-Tubulin (Cell Signaling), and anti-b-actin (Cell Signaling).
3.1. PHGDH is abundantly expressed in mature adipocytes of white adipose tissue
2.4. Real-time quantitative PCR Total RNAs were extracted from adipose tissues and 3T3-L1 cells using RNEasy Lipid Tissue Mini Kit (QIAGEN) and TRI Reagent (Molecular Research Center, Inc.), respectively. cDNA was prepared using ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo, Japan) according to the supplier's protocol. Real-time PCR was carried out using THUNDERBIRD SYBR qPCR Mix (Toyobo) on Thermal Cycler Dice Real Time System II (Takara Bio). Quantification was done by Delta Delta Ct method, and Rpl13a was used as a reference gene.
Although mouse ENCODE transcriptome data shows that Phgdh mRNA is ubiquitously expressed in mouse tissues [11], the protein expression of PHGDH in mouse tissues has not yet been determined. Thus, we examined the tissue distribution of PHGDH protein using various mice tissue lysates. Western blotting analysis confirmed that the brain exhibited abundant expression of PHGDH protein. Interestingly, PHGDH was also abundantly expressed in visceral white adipose tissue (vWAT) and subcutaneous white adipose tissue (sWAT), whereas brown adipose tissue (BAT) contained little PHGHD (Fig. 1A). We also quantified mRNA levels of Phgdh, Psat1, and Psph in adipose tissues, and found that vWAT expressed relatively higher levels of these serine synthesis enzyme mRNAs (Fig. 1B). Although the protein expression of PHGDH in WAT was stronger than that in BAT, the protein levels of PSAT1 were almost comparable between vWAT, sWAT, and BAT (Fig. 1C). WAT is divided into the mature adipocyte fraction (MAF) and the stromal vascular fraction (SVF), which includes stromal cells, pericytes, immune cells, and preadipocytes [19]. Therefore, we also determined the localization of PHGDH in WAT using MAF and SVF samples from wild type mice. Most of PHGDH and PSAT1 were expressed in the MAF, suggesting that serine biosynthesis in WAT occurs in mature adipocytes (Fig. 1D). We further examined whether there are any changes in these serine biosynthesis enzymes during adipogenesis
Please cite this article in press as: K. Okabe, et al., Deletion of PHGDH in adipocytes improves glucose intolerance in diet-induced obese mice, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.08.180
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Fig. 1. PHGDH is abundantly expressed in mature adipocytes of white adipose tissue. (A) PHGDH protein expression in various tissues from male C57BL/6 mice. Pan-actin was used as loading control. (B) qPCR analysis of Phgdh, Psat1, Psph in vWAT, sWAT, and BAT from male C57BL/6 mice. Data are presented as mean ± SD (n ¼ 4 for each group). (C) Immunoblot analysis of PHGDH and PSAT1 in vWAT, sWAT, and BAT from male C57BL/6 mice. b-Tubulin was used as loading control. (D) PHGDH protein expression in MAF and SVF derived from vWAT of male C57BL/6 mice. (E) qPCR analysis of Phgdh, Psat1, Psph in 3T3-L1 cells at indicated time points. Data are presented as mean ± SD (n ¼ 3 for each time point). (F) PHGDH and PSAT1 expression during differentiation of 3T3L1 cells. b-actin was used as loading control.
using 3T3-L1 cells, a commonly used model of preadipocyte differentiation. All three genes were downregulated during the induction phase (day 0 to day 2), but expressions increased after differentiation (~day 4; Fig. 1E and F). These data suggest that serine biosynthesis may play some roles in mature adipocytes.
3.2. Adipocytes-specific PHGDH knockout mice have no obvious developmental defect in adipose tissues To elucidate the physiological significance of serine biosynthesis in adipose tissue, we generated adipocyte-specific PHGDH knockout mice (PHGDH FKO) by crossing PHGDH flox mice with adiponectin-Cre mice [15,16]. PHGDH FKO mice were born and developed with a normal appearance. We confirmed the significant reduction of PHGDH protein in vWAT, sWAT and BAT of PHGDH FKO mice, but the protein levels in the brain was unchanged (Fig. 2A). When PHGDH FKO mice were fed with a normal chow diet, body weight gain and food intake was essentially comparable with those in wild type (WT) controls (Fig. 2B and C). Accordingly, there were no obvious differences in adipose tissue weight between PHGDH FKO and WT mice (Fig. 2D and E). Histological examination also demonstrated that adipose tissues in PHGDH FKO mice exhibited no evident abnormalities compared with controls (Fig. 2F). Together, these data has demonstrates that deletion of PHGDH in adipose tissues has no influence on normal adipose tissue development.
3.3. Deletion of PHGDH in adipocytes improves glucose intolerance in diet-induced obese mice Next, we examined the role of adipose tissue PHGDH in pathological situation. During obesity, adipose tissues expand due to an increase in both adipocyte size and number [20]. Functionally, insulin sensitivity in adipose tissues is impaired, and glucose intolerance is invoked by obesity [21]. To investigate the role of PHGDH in glucose metabolism, we fed PHGDH FKO and WT mice with a high-fat high-sucrose diet (HFHSD). When male PHGDH FKO mice were fed with HFHSD, they exhibited essentially similar weight gain compared with male WT mice (Fig. 3A). In contrast, female PHGDH FKO mice exhibited slightly less weight gain than female WT mice (Fig. 3B), whereas there was no significant difference in the food intake between them (Fig. 3C). To evaluate the glucose metabolism in male and female PHGDH FKO mice, we performed an intraperitoneal glucose tolerance test (ipGTT) after 6 weeks of HFHSD feeding. Male PHGDH FKO mice exhibited similar glucose tolerance to WT mice (Fig. 3D), whereas female PHGDH FKO mice demonstrated significantly improved glucose tolerance compared with WT (Fig. 3E). We also measured serum serine levels to examine the impact of PHGDH deletion on serine metabolism. However, serum serine levels were unaffected in PHGDH FKO mice (Fig. 3F). We then examined the changes of serine levels in WAT of female PHGDH FKO and WT mice. Interestingly, HFHSD feeding significantly increased serine levels in WT mice (Fig. 3G). In contrast, female PHGDH FKO mice demonstrated no significant rise
Please cite this article in press as: K. Okabe, et al., Deletion of PHGDH in adipocytes improves glucose intolerance in diet-induced obese mice, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.08.180
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Fig. 2. Adipocyte-specific PHGDH knockout mice have no obvious developmental defect in adipose tissues. (A) Immunoblot analysis of male PHGDH in vWAT, sWAT, BAT, and brain from PHGDH FKO and WT mice. Pan-actin was used as loading control. (B and C) Body weight changes (B) and food intake (C) of male PHGDH FKO and WT mice fed a normal chow diet. Data are presented as mean ± SD (n ¼ 3 for each group). (D) Representative image of adipose tissues from male PHGDH FKO and WT mice. (E) Quantifications of adipose tissue weight from male PHGDH FKO and WT mice (n ¼ 4 for each group). (F) Hematoxylin-Eosin (HE) staining of vWAT, sWAT, and BAT from male PHGDH FKO and WT mice. Scale bar represents 200 mm.
in WAT serine levels after HFHSD feeding (Fig. 3G). These results imply the pathological importance of increased serine levels in the development of glucose intolerance during diet-induced obesity. 3.4. Serine-deficient diet feeding also exhibits favorable effects on glucose metabolism in mice Results to date suggested that the inhibition of serine synthesis is beneficial for glucose tolerance during diet-induced obesity. We, therefore, investigated the effect of dietary serine restriction on glucose metabolism using female C57BL/6 mice. When C57BL/6 female mice were fed a serine-deficient diet, they exhibited a slightly higher, but not statistically significant, body weight gain compared with control (serine-containing) diet-fed mice (Fig. 4A). We also confirmed that serine restriction had no effect on food intake and serum serine levels (Fig. 4B and C). Importantly, serinedeficient diet-fed mice also exhibited better glucose tolerance (Fig. 4D). These results demonstrate that dietary serine restriction has beneficial effects on glucose metabolism without adverse side effects and could be a promising nutritional intervention for diabetes patients. 4. Discussion In this study, we discovered that PHGDH is abundantly expressed in mature adipocytes of white adipose tissue. We also
observed an increased expression of PHGDH during the differentiation of 3T3L1 pre-adipocytes. Although the deletion of the PHGDH gene in adipose tissue had no obvious impact on normal adipose tissue development, we found that female PHGDH FKO mice exhibited improved glucose tolerance during diet-induced obesity. We also revealed that the WAT serine levels drastically increased after HFHSD feeding in WT mice, whereas no significant rise was observed in PHGDH FKO mice. Furthermore, wild type mice fed a serine-deficient diet also exhibited better glucose tolerance. These results suggest that PHGDH-mediated serine biosynthesis had some important roles on the progression of glucose intolerance during diet induced obesity. It has been reported that PHGDH is overexpressed in several types of human cancer cells, including breast cancers and melanoma [22e25]. In breast cancer cells, serine is necessary for sustaining cellular proliferation, and deletion of PHGDH impaired the cellular proliferations and survival [26,27]. Interestingly, deletion of PHGDH in breast cancer cells has no effect on the intracellular serine levels when they were maintained in serine containing medium. Instead, deletion of PHGDH decreased serine synthesis flux and reduced the PSAT1-mediated production of a-ketoglutarate (a-KG) from glutamate [26]. a-KG is an intermediate of the tricarboxylic acid cycle and also serves as a substrate for histone or DNA demethylase [28]. Thus, it may be possible that PHGDH deletion affects gene expression through a-KG-mediated epigenetic changes and thus influences glucose metabolism pathways.
Please cite this article in press as: K. Okabe, et al., Deletion of PHGDH in adipocytes improves glucose intolerance in diet-induced obese mice, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.08.180
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Fig. 3. Deletion of PHGDH in adipocytes improves glucose intolerance in diet-induced obese mice. (A and B) Body weight changes of male (A) or female (B) PHGDH FKO and WT mice fed with HFHSD. Data are presented as mean ± SD (n ¼ 10 for each group). (C) Food intake of female PHGDH FKO and WT mice fed with HFHSD. Data are presented as mean ± SD (n ¼ 10 for each group). (D and E) Intraperitoneal glucose tolerance test (ipGTT) of male (D) or female (E) PHGDH FKO and WT mice fed with HFHSD. Data are presented as mean ± SD (n ¼ 10 for each group). (F) Plasma serine concentration in female PHGDH FKO and WT mice fed with HFHSD. Data are presented as mean ± SD (n ¼ 20 for each group). (G) Relative serine levels in female PHGDH FKO and WT mice fed with HFHSD. Data are presented as mean ± SD (n ¼ 7e12 for each group). Asterisk (*) indicated the statistical significance when p-value is less than 0.05.
Fig. 4. Serine-deficient diet feeding also exhibits favorable effects on glucose metabolism in mice. (AeD) Body weight changes (A), food intake (B), Plasma serine concentration(C), and ipGTT (D) of female C57BL/6 mice fed with serine-deficient diet or control diet. Data are presented as mean ± SD (n ¼ 5e6 for each group). Asterisk (*) indicates the statistical significance when p-value is less than 0.05, and N.S. means not significant.
Serine is also a major source for one-carbon metabolism, which contributes to the production of folic acid and S-adenosine methionine (SAM) [1]. In particular, SAM is an important donor for histone and DNA methylation [28]. Currently, the effect of PHGDH deletion on SAM levels has not been determined. However, it is possible that serine-derived SAM contributes to altering methylation patterns and affects the expression of genes related to glucose metabolism. From this viewpoint, serine biosynthesis may regulate both methylation and demethylation, and the gene regulation mechanisms affected by serine metabolism may be more complex than currently appreciated. It has been reported that hepatic PSAT1 regulates insulin sensitivity during diet-induced obesity [29]. During genetic- and diet-induced obesity, PSAT1 is downregulated in the liver, and the suppression of PSAT1 induces the gene expression of tribbles homolog 3 (TRB3). Furthermore, an adenovirus-mediated increase of PSAT1 in the liver improves glucose intolerance in obese mice through the suppression of the TRB3 pathway [29]. These results
indicate that the activation of the serine synthesis pathway in the liver is beneficial to preserving insulin sensitivity during obesity. In general, the liver is an organ that produces glucose from glycogenic amino acids, including serine, whereas adipose tissues are organs that consume glucose. Thus, their metabolic activities are often opposed during various nutritional changes, including fasting and obesity [29]. It will, therefore, be important to generate a liverspecific PHGDH KO mouse to clarify the role of serine biosynthesis during obesity. In this study, we confirmed favorable effects only in female PHGHD KO mice. It well known that gender differences affects to the obesity and glucose intolerance possibly through sex hormones [30]. Of note, estrogen receptor status is related to PHGDH expression levels in breast cancer cells [26]. Thus, it is possible that sex hormone may influence serine biosynthesis and its related metabolic pathways in obese mice. Although serine deficiency in humans causes severe neuronal and mental disorders [7], PHGDH deletion in adipocytes and
Please cite this article in press as: K. Okabe, et al., Deletion of PHGDH in adipocytes improves glucose intolerance in diet-induced obese mice, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.08.180
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dietary serine restriction exhibits favorable effects on glucose handling in mice. Symptoms in human serine deficiency probably attributes to decreased serine levels in the brain [8], and the results from brain-specific PHGDH knockout mice also support this idea [15]. Thus, it is important to specifically decrease adipose tissue serine levels to achieve only favorable metabolic effects. In the dietary serine restriction experiment, we only depleted serine, and not glycine, which is reversibly converted to serine by SMTH. Additionally, the serum serine levels and body weight were unaffected by dietary serine restriction. Thus, these results suggest that dietary serine restriction is a safe nutritional intervention, and may be applicable to the treatment of human diabetic patients. Acknowledgement We are grateful to Ms. Tomomi Kubo for the care and husbandry of PHGDH FKO mice. This work was supported by the Research Grant from Banyu Life Science Foundation International and the University of Toyama President Grant to TN. JSPS KAKENHI (Grant Number 18K16193) to KO also supported this study. Transparency document
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Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.08.180. [20]
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Please cite this article in press as: K. Okabe, et al., Deletion of PHGDH in adipocytes improves glucose intolerance in diet-induced obese mice, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.08.180