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Inhibition of inosine monophosphate dehydrogenase reduces adipogenesis and diet-induced obesity
Biochemical and Biophysical Research Communications 386 (2009) 351–355
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Inhibition of inosine monophosphate dehydrogenase reduces adipogenesis and diet-induced obesity Hua Su a, Jennifer H. Gunter a, Melissa de Vries b, Tim Connor b, Stephen Wanyonyi b, Felicity S. Newell a, David Segal b, Juan Carlos Molero b, Ofer Reizes c, Johannes B. Prins a, Louise J. Hutley a, Ken Walder b,d, Jonathan P. Whitehead a,* a
Diamantina Institute for Cancer, Immunology and Metabolic Medicine, University of Queensland, Princess Alexandra Hospital, Brisbane, Qld 4102, Australia Metabolic Research Unit, School of Exercise and Nutrition Sciences, Deakin University, Melbourne, Vic., Australia Cleveland Clinic Foundation, Lerner Research Institute, Cleveland, OH, USA d Verva Pharmaceuticals Ltd., Geelong, Vic., Australia b c
a r t i c l e
i n f o
Article history: Received 1 June 2009 Available online 11 June 2009
Keywords: IMPDH Adipocyte Guanine nucleotides Obesity
a b s t r a c t We previously described a putative role for inosine monophosphate dehydrogenase (IMPDH), a rate-limiting enzyme in de novo guanine nucleotide biosynthesis, in lipid accumulation. Here we present data which demonstrate that IMPDH activity is required for differentiation of preadipocytes into mature, lipid-laden adipocytes and maintenance of adipose tissue mass. In 3T3-L1 preadipocytes inhibition of IMPDH with mycophenolic acid (MPA) reduced intracellular GTP levels by 60% (p < 0.05) and blocked adipogenesis (p < 0.05). Co-treatment with guanosine, a substrate in the salvage pathway of nucleotide biosynthesis, restored GTP levels and adipogenesis demonstrating the specificity of these effects. Treatment of diet-induced obese mice with mycophenolate mofetil (MMF), the prodrug of MPA, for 28 days did not affect food intake or lean body mass but reduced body fat content (by 36%, p = 0.002) and adipocyte size (p = 0.03) and number. These data suggest that inhibition of IMPDH may represent a novel strategy to reduce adipose tissue mass. Ó 2009 Elsevier Inc. All rights reserved.
Introduction In vitro systems have been used extensively to dissect the molecular and cellular events involved in adipogenesis, the process which underpins excessive expansion of adipose tissue mass leading to obesity [1]. Adipogenesis is cooperatively modulated by a cascade of transcription factors, including members of the CCAAT/enhancer-binding protein (C/EBP) and the peroxisome proliferator-activated receptor (PPAR) families that are crucial for induction of the adipocyte phenotype [2]. While this knowledge has increased our understanding of the adipogenic program we still have limited information concerning additional regulatory mechanisms important during differentiation. Guanine nucleotides are required for a number of cellular processes, including RNA and DNA synthesis, cell signalling and vesicle trafficking, and are synthesised by de novo and salvage pathways [3]. Inosine monophosphate dehydrogenase (IMPDH) catalyses the ratelimiting step in the de novo pathway, at a branch point in purine biosynthesis, while hypoxanthine phosphoribosyl transferase (HPRT) salvages purine metabolites. IMPDH has been the subject of inten-
* Corresponding author. Fax: +61 (0) 7 3240 5946. E-mail address: [email protected] (J.P. Whitehead). 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.06.040
sive research since it was recognised that IMPDH expression and activity are up-regulated in rapidly proliferating and malignant cells [4,5]. Inhibitors of IMPDH block cell proliferation and induce terminal differentiation in a variety of cell types and are currently used for a number of clinical indications, including immunosuppression following solid-organ transplant [6]. We recently identified a putative role for IMPDH in the regulation of lipid accumulation [7]. Upon treatment of a variety of cell types with insulin or oleic acid IMPDH translocated to lipid droplets and inhibition of this translocation correlated with reduced lipid accumulation. As lipid droplet formation and lipid accretion are defining features of adipogenesis we hypothesized that IMPDH may facilitate efficient lipid accumulation during adipose conversion of preadipocytes. In the current report we have examined this hypothesis in vitro, using the well characterized murine 3T3-L1 cell line, and in vivo, using a mouse model of diet-induced obesity. Materials and methods Reagents and antibodies. Reagents were from Sigma–Aldrich (Castle Hill, Australia) unless otherwise stated. Tissue culture media and foetal calf serum were from Invitrogen (Mount Waverley, Australia) and JRH Biosciences (Brooklyn, Australia). Monoclonal
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pan-IMPDH antibody was described previously [7]. Antibodies for PPARc, C/EBPa, C/EBPb and HPRT were from Santa Cruz (Santa Cruz, CA, USA). Antibodies to b-Tubulin and perilipin were from Upstate (Boronia, Australia) and Research Diagnostics (Concord, MA, USA). Cell culture, assessment of mitotic clonal expansion (SYTO 60 assay), differentiation and triglyceride accumulation. 3T3-L1 preadipocytes were cultured and differentiated as described [8]. The fluorescent nucleic acid stain, SYTO 60 (Invitrogen), was used to measure cell number according to the manufacturers’ instructions [8]. Differentiation was assessed morphologically by observation of lipid accumulation using phase-contrast microscopy. Triglyceride accumulation was determined by quantitation of neutral lipid stained with Oil Red-O [7]. Glycerol-3-phosphate dehydrogenase (G3PDH) activity was measured as described [8]. Assessment of IMPDH activity, Western blotting and HPLC analysis of intracellular nucleotides. IMPDH activity, Western blotting of whole cell lysates (10 lg) and Reverse-Phase HPLC were all performed as described [9]. Murine study with mycophenolate mofetil. Male C57Bl/6J mice were fed ad libitum a high-fat diet (SF04-001, Specialty Feeds, Glen Forrest, WA, Australia) for 10 weeks to induce obesity. The diet was continued for 4 weeks during which animals were treated by twice daily oral gavage with either mycophenolate mofetil (MMF; 60 mg/ kg; n = 8) or vehicle (n = 6). Primary outcome measures were reduction in body weight and fat mass. Food intake and body weight were measured weekly. Body composition was assessed by DEXA on days 0 and 28 using a pDEXA Sabre XL (Norland Stratec, Australia) under anaesthesia. Blood samples were collected from the tail on days 0 and 28 and glucose concentration determined using glucose sticks (AccuChek, Roche Diagnostics, USA). Following centrifugation, plasma was assayed for insulin, leptin and adiponectin (radioimmunoassays; Linco, USA) and total triglycerides and cholesterol (colorimetric assays; Roche Diagnostics, USA). At the completion of the study animals were sacrificed and epididymal and retroperitoneal adipose depots, liver and red gastrocnemius muscles were excised and weighed. Adipose and liver tissues were fixed in formalin, mounted in paraffin blocks, cut into sections and stained with hematoxylin and eosin using standard protocols. Histological examinations (n = 10–12 sections/tissue) were evaluated by two independent operators who were blinded to the treatment groups. Adipocyte cellularity was determined by counting number of cells/field. Average adipocyte volume (lm3) was calculated and total number of adipocytes estimated as described [10]. All experimental protocols were approved by the Deakin University Animal Welfare Committee, and adhered to the guidelines provided by the National Health and Medical Research Council. Statistics. Statistical analysis was performed using SPSS 13.0 (SPSS Inc., Chicago, IL, USA). Data were checked for normality of distribution using a 1-sample Kalmogorov–Smirnov test. For normally distributed data, two-tailed Paired-Samples t-test or independent samples t-test were used to evaluate the significance of the difference in mean values between the different treatments. p < 0.05 was considered statistically significant. Results IMPDH is transiently increased during differentiation of 3T3-L1 cells Upon induction of differentiation, 3T3-L1 preadipocytes execute a well defined program characterized initially (48–72 h) by 1–2 rounds of proliferation known as mitotic clonal expansion (MCE), resulting in a 3–4-fold increase in cell number and concomitant increase in total protein (Fig. 1A) and, subsequently by morphological changes including accumulation of lipid droplets (Fig. 1B) [2].
Expression of IMPDH protein was also increased, albeit transiently, even after accounting for the increase in cell number/total protein (Fig. 1C). Total IMPDH protein levels and activity were highest around day 3 (Fig. 1C and D). In contrast, HPRT levels increased and remained high in fully differentiated adipocytes (Fig. 1C). As expected the levels of adipogenic markers, including PPARc and perilipin, were increased during differentiation (Fig. 1C). Lipid droplets and perilipin were first observed around day 3, the period where IMPDH expression and activity was highest. Our previous observations implicated a potential role for IMPDH in lipid accumulation, by virtue of its regulated association with lipid droplets [7]. We therefore examined whether IMPDH was associated with lipid droplets during differentiation but found no co-localisation, suggesting IMPDH is unlikely to play a direct role in lipid accretion during adipogenesis (data not shown). However, having established that IMPDH activity increased during 3T3-L1 differentiation we went onto investigate whether this activity was required for efficient adipogenesis. Mycophenolic acid (MPA) inhibits 3T3-L1 differentiation by depleting GTP MPA is a widely used, potent inhibitor of IMPDH [11]. Treatment with MPA for the first 6 days of differentiation inhibited adipogenesis in a dose dependent manner, as determined morphologically and biochemically (Fig. 2A and B). The IC(50) of MPA was around 0.2 lM, while 1 lM MPA was sufficient to completely block adipogenesis. The specificity of these effects was determined in preliminary guanosine titration studies (to restore GTP levels via the HPRT-dependent salvage pathway), and established that 60 lM guanosine was able to prevent the MPA effects (see below). Fig. 2C, showing intracellular nucleotide levels following MPA and/or guanosine treatment, demonstrates that in differentiating cells 1 lM MPA decreased intracellular GTP by 65% and 50% after 1 and 3 days of incubation, respectively. These effects were reversed upon co-treatment with 60 lM guanosine. In comparison, ATP levels were relatively unaffected, with only a modest decrease after 3 days treatment (Fig. 2C). Treatment with MPA in non-differentiating cells was without significant effect. Furthermore, treatment of fully differentiated 3T3-L1 adipocytes with MPA across a broad concentration range (0.1–30 lM) for up to 48 h did not affect insulin-stimulated glucose uptake (data not shown). Guanosine prevents the inhibitory effects of MPA in 3T3-L1 cells In differentiating 3T3-L1 cells, 1 lM MPA blocked MCE and markedly reduced induction of C/EBPa, although C/EBPb and PPARc were relatively unaffected, whilst co-treatment with 60 lM guanosine reversed these effects (Fig. 2D and E). Consistent with several reports in other cell types, IMPDH levels were increased by MPA and reduced by guanosine (Fig. 2E). As before, HPRT expression was induced upon differentiation and this was unaffected by treatment. Moreover, the inhibitory effects of MPA on lipid accumulation and G3PDH activity were ameliorated by co-treatment with guanosine (Fig. 2F and G). In summary, these data demonstrate that IMPDH expression and activity are increased transiently during differentiation of 3T3-L1 cells and inhibition of IMPDH activity blocks differentiation, while co-treatment with guanosine abrogates this inhibition. Mycophenolate mofetil (MMF) induces loss of weight and fat in dietinduced obese mice We next examined the effects of inhibiting IMPDH activity on adipose tissue in vivo, in a mouse model of diet-induced obesity.
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Fig. 1. IMPDH expression and activity increase transiently during 3T3-L1 preadipocyte differentiation. 3T3-L1 preadipocytes were differentiated and harvested at indicated times. (A) MCE and total protein accumulation. Data are expressed as fold increase compared to day 0, and represent the mean + SE of three independent experiments. (B) Photomicrographs showing accumulation of lipid during differentiation (day 0–9). (C) Western blot analysis of cell lysates probed with antibodies as indicated. Data are representative from three independent experiments. (D) Quantitation of IMPDH expression and activity during differentiation. Data are expressed as fold increase of the value at day 0 and are representative of three independent experiments.
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Tubulin Fig. 2. MPA blocks differentiation of 3T3-L1 preadipocytes. 3T3-L1 preadipocytes were differentiated in the presence of vehicle (0) or increasing concentrations of MPA. (A) Photomicrographs showing lipid accumulation on day 8. (B) G3PDH activity in cells harvested on day 8. Results are expressed as percentage of control (vehicle) and represent the mean + SE of four independent experiments. (C) Effects of 1 lM MPA, 60 lM guanosine (GUA) or both (M + G) on intracellular GTP and ATP levels in non-differentiated (ND) or differentiated (CON, MPA, GUA and M + G) cells harvested on days 0, 1, 3 and 6. Results are expressed as the mean + SE of three independent experiments. (*p < 0.05 MPA vs. CON). (D and E) 3T3-L1 preadipocytes were untreated (control; CON) or treated with 1 lM MPA, 60 lM guanosine (GUA), or both (M + G) and harvested on day 3 of differentiation. (D) MCE as determined by SYTO 60. Results are presented as increase in cell number compared with non-differentiated post-confluent cells and represent the mean + SE of three independent experiments. (*p < 0.05 MPA vs. CON). (E) Western blot analysis of cell lysates probed with antibodies as indicated. Data are representative from three individual experiments. ND, non-differentiated post-confluent cells. (F and G) Cells were treated as described above with MPA and/or GUA from days 0–6 and harvested on day 8. (F) Lipid accumulation was assessed morphologically (left panel) and by Oil Red-O assay (right panel). Data are presented as percentage of control and represent the mean + SE of six independent experiments. (G) G3PDH activity showing the mean + SE of six (CON; MPA) or three (GUA; M + G) independent experiments. (**p < 0.01 vs. control; ##p < 0.01 vs. MPA).
Twice daily gavage with 60 mg/kg MMF, the pro-drug of MPA, for 28 days was associated with a 6% reduction in weight in obese animals maintained on a high-fat diet (vehicle treated animals 0.75 ± 0.6 g weight reduction, n = 6, p = 0.143; MMF-treated animals 1.6 ± 0.3 g weight reduction, n = 8, p = 0.03). Body composition analysis by DEXA revealed a 36% decrease in total body fat in MMF-treated animals (Fig. 3A), with no change in lean body mass (vehicle control animals: 0.5 ± 1.3 g reduction in lean mass; MMF: 1.5 ± 1.1 g increase; n.s.). Necropsy data confirmed significant MMF treatment-associated loss in epididymal fat and showed a trend toward retroperitoneal fat loss (Fig. 3B). In contrast there was no reduction in brown adipose tissue, liver or skeletal muscle mass (Fig. 3C). Weight loss was not associated with any alteration
in food intake (data not shown). Histology demonstrated a significant reduction in adipocyte size in response to MMF (Fig. 3D) and an estimated 15% reduction in total adipocyte number [10]. Treatment with MMF also decreased the appearance of hepatic steatosis (Fig. 3E). Control (vehicle gavage) animals showed a significant (p < 0.05) increase in fasting insulin over the 4 week study, consistent with a progressive decline in insulin sensitivity (Fig. 4A). This change was not seen in the MMF-treated animals. A similar trend was observed in fasting triglyceride levels (Fig. 4B). Fasting leptin was reduced in the MMF-treated animals, consistent with the observed fat loss (Fig. 4C). Fasting glucose, total cholesterol and adiponectin were not altered by MMF treatment (Fig. 4A–C). F4/80-positive macro-
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Fig. 3. Treatment with MMF reduces white adipose tissue in obese mice. Obese mice were treated twice daily with vehicle or 60 mg/kg MMF by oral gavage for 28 days. (A) Total body fat content as determined by DEXA at day 0 and 28 (*p = 0.02, MMF day 0 cf. 28). (B) Epididymal (Epid) and retroperitoneal (Retro) fat mass at day 28 (*p = 0.036, MMF cf. vehicle). (C) Brown adipose, liver and Quadricep tissue mass at day 28. Tissue weights are shown as % of total body weight. (D) Representative micrographs showing reduced adipocyte size (*p = 0.03, MMF cf. vehicle). (E) Representative micrographs showing reduced hepatic steatosis in MMF-treated animals. (n = 6 vehicle; n = 8 MMF).
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Fig. 4. Treatment with MMF reduces plasma insulin and leptin in obese mice. Obese mice were treated twice daily with vehicle or 60 mg/kg MMF by oral gavage for 28 days. Fasting biochemical parameters were measured by standard assay at day 0 and day 28. (A) Insulin and glucose. (B) Triglycerides and Cholesterol. (C) Leptin and adiponectin. (*p < 0.05, day 0 cf. day 28 within treatment; #p < 0.05, day 28 MMF cf. vehicle; n = 6 vehicle; n = 8 MMF).
phages were observed in adipose tissue of vehicle and MMF-treated mice, however, no difference was observed in the number of macrophages present following MMF treatment (data not shown). There were no clinical, biochemical or histological signs of toxicity in MMF-treated animals. Discussion The hypothesis that prompted these studies was based on observations implicating a role for IMPDH in lipid accumulation by virtue of its regulated association with lipid droplets [7]. In the current report we characterised key features of guanine metabolism during differentiation of 3T3-L1 cells and investigated the effects of IMPDH inhibition on adipose tissue in vivo. While we observed no association of IMPDH with lipid droplets during adi-
pogenesis our results demonstrated that inhibition of IMPDH blocked differentiation of 3T3-L1 cells and this effect could be prevented by guanosine supplementation. Consistent with this, treatment of diet-induced obese mice with the IMPDH inhibitor MMF reduced adipose tissue mass. Nucleotide biosynthesis is essential for a variety of cellular processes [3]. Dependence on the de novo and salvage guanine nucleotide biosynthesis pathways varies between cell types and contributes to the differential sensitivity to IMPDH inhibitors [3,11]. The relative importance of these pathways in adipogenesis had not been examined. We found that IMPDH expression and activity increased transiently during differentiation of the 3T3-L1 cells. In contrast, HPRT expression increased and was maintained upon adipocyte maturation suggesting the balance between the de novo and salvage pathways may change. We investigated the requirement for IMPDH activity during adipogenesis by employing the widely used inhibitor MPA. A major consequence of inhibition of IMPDH with MPA is a reduction in DNA synthesis, which leads to a block in proliferation, promoting terminal differentiation in a range of neoplastic cell types [6]. We found that inhibition of IMPDH by MPA (1 lM) blocked DNA synthesis and terminal differentiation of the 3T3-L1 cells, highlighting the importance of MCE in this system [12,13]. Consistent with this, MPA treatment was most effective at reducing GTP levels during the period of MCE. Indeed, treatment of differentiating 3T3-L1 cells with MPA for 3 days during the period of MCE (days 0–2) reduced differentiation by 60–70% compared with only a 10–20% reduction when cells were treated with MPA post-MCE (days 3–5) (H.S. and J.P.W. – unpublished observations). Recent work from Arner and colleagues strengthens a model whereby acquisition of new adipocytes contributes to the dynamics of fat cell turnover, with increased rates of adipogenesis reported in obese subjects [1], providing further support for therapeutic strategies that target such processes [14]. Although inhibitors of IMPDH, including MMF and mizoribine, have been used extensively as immunosuppressive agents for a number of years we have been unable to find data describing their effects, or lack thereof, on adipose tissue mass in humans. Limited information comes from studies in rodents. Three reports suggest inhibiting IMPDH may reduce body mass although specific measurement of fat and lean body mass was not reported [15–17]. To investigate the potential effects of inhibiting IMPDH on adipose tissue in vivo we performed a study in a mouse model of diet-induced obesity treated with MMF. Treatment with MMF induced significant weight loss and this was largely due to a reduc-
H. Su et al. / Biochemical and Biophysical Research Communications 386 (2009) 351–355
tion in white adipose tissue, with no change in brown adipose tissue, liver and muscle weight. In epididymal adipose tissue, where the effects of MMF were most marked, treatment with MMF reduced adipocyte size and number. Recent evidence indicates that adipose tissue in mice exhibits dynamic remodelling when animals are maintained on a high-fat diet, with marked changes in adipocyte cell number, cell death and cell size occurring after 8–20 weeks of high fat feeding [18]. Future studies will be required to determine which of these parameters are altered by MMF treatment. Circulating leptin levels were reduced in MMF-treated animals, consistent with the reduction in white adipose tissue. We also observed a relative reduction in fasting insulin in response to MMF, with no concomitant change in glucose levels, suggesting an improvement in insulin sensitivity. Other indicators of metabolic benefit induced by MMF included reduction in hepatic steatosis and a trend towards reduced triglyceride levels. MMF-treated animals showed no toxicity and food intake was similar to controls. Thus, the fat loss observed may relate to increased fuel oxidation/metabolic rate and/or a reduction in adipogenesis. Unlike leptin, total adiponectin levels were unchanged. Taking into account the reduction in adipose tissue this suggests that total adiponectin levels (adjusted for adipose tissue mass) were increased. Future studies will examine the mechanism underpinning the MMF-induced fat loss in more detail. The dose of MMF used in this study (total of 120 mg/kg/day) is similar to that used to achieve immunosuppression in murine studies [19,20]. Subsequent studies will also aim to determine if the dose relationship to fat loss and immunosuppression differ. Whilst obesity-related adipose tissue inflammation and insulin resistance is characterised by increased infiltration of macrophages [21,22], there was no difference in macrophage number in adipose tissue or liver of MMF-treated animals compared with control animals, suggesting MMF treatment does not affect inflammation in these tissues over the study period. Nevertheless, reduced adipocyte size and number correlate to metabolic improvements in obesity and recent evidence suggests that pro-inflammatory cytokines from the adipocyte can affect peripheral insulin sensitivity in the absence of changes to adipose tissue macrophages [23]. This provides a potential mechanism for the improved metabolic parameters. In summary, we have demonstrated that IMPDH inhibition blocks adipogenesis of the murine 3T3-L1 cell line and that MMF induces significant fat loss in diet-induced obese mice. These data suggest that inhibition of IMPDH may represent a novel therapeutic strategy to reduce the increase in fat mass which underpins obesity associated weight gain. Acknowledgments This work was funded by the Australian National Health and Medical Research Council, Diabetes Australia, the Lions Medical Research Foundation, the Princess Alexandra Foundation and the University of Queensland.
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Inhibition of inosine monophosphate dehydrogenase reduces adipogenesis and diet-induced obesity Hua Su a, Jennifer H. Gunter a, Melissa de Vries b, Tim Connor b, Stephen Wanyonyi b, Felicity S. Newell a, David Segal b, Juan Carlos Molero b, Ofer Reizes c, Johannes B. Prins a, Louise J. Hutley a, Ken Walder b,d, Jonathan P. Whitehead a,* a
Diamantina Institute for Cancer, Immunology and Metabolic Medicine, University of Queensland, Princess Alexandra Hospital, Brisbane, Qld 4102, Australia Metabolic Research Unit, School of Exercise and Nutrition Sciences, Deakin University, Melbourne, Vic., Australia Cleveland Clinic Foundation, Lerner Research Institute, Cleveland, OH, USA d Verva Pharmaceuticals Ltd., Geelong, Vic., Australia b c
a r t i c l e
i n f o
Article history: Received 1 June 2009 Available online 11 June 2009
Keywords: IMPDH Adipocyte Guanine nucleotides Obesity
a b s t r a c t We previously described a putative role for inosine monophosphate dehydrogenase (IMPDH), a rate-limiting enzyme in de novo guanine nucleotide biosynthesis, in lipid accumulation. Here we present data which demonstrate that IMPDH activity is required for differentiation of preadipocytes into mature, lipid-laden adipocytes and maintenance of adipose tissue mass. In 3T3-L1 preadipocytes inhibition of IMPDH with mycophenolic acid (MPA) reduced intracellular GTP levels by 60% (p < 0.05) and blocked adipogenesis (p < 0.05). Co-treatment with guanosine, a substrate in the salvage pathway of nucleotide biosynthesis, restored GTP levels and adipogenesis demonstrating the specificity of these effects. Treatment of diet-induced obese mice with mycophenolate mofetil (MMF), the prodrug of MPA, for 28 days did not affect food intake or lean body mass but reduced body fat content (by 36%, p = 0.002) and adipocyte size (p = 0.03) and number. These data suggest that inhibition of IMPDH may represent a novel strategy to reduce adipose tissue mass. Ó 2009 Elsevier Inc. All rights reserved.
Introduction In vitro systems have been used extensively to dissect the molecular and cellular events involved in adipogenesis, the process which underpins excessive expansion of adipose tissue mass leading to obesity [1]. Adipogenesis is cooperatively modulated by a cascade of transcription factors, including members of the CCAAT/enhancer-binding protein (C/EBP) and the peroxisome proliferator-activated receptor (PPAR) families that are crucial for induction of the adipocyte phenotype [2]. While this knowledge has increased our understanding of the adipogenic program we still have limited information concerning additional regulatory mechanisms important during differentiation. Guanine nucleotides are required for a number of cellular processes, including RNA and DNA synthesis, cell signalling and vesicle trafficking, and are synthesised by de novo and salvage pathways [3]. Inosine monophosphate dehydrogenase (IMPDH) catalyses the ratelimiting step in the de novo pathway, at a branch point in purine biosynthesis, while hypoxanthine phosphoribosyl transferase (HPRT) salvages purine metabolites. IMPDH has been the subject of inten-
* Corresponding author. Fax: +61 (0) 7 3240 5946. E-mail address: [email protected] (J.P. Whitehead). 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.06.040
sive research since it was recognised that IMPDH expression and activity are up-regulated in rapidly proliferating and malignant cells [4,5]. Inhibitors of IMPDH block cell proliferation and induce terminal differentiation in a variety of cell types and are currently used for a number of clinical indications, including immunosuppression following solid-organ transplant [6]. We recently identified a putative role for IMPDH in the regulation of lipid accumulation [7]. Upon treatment of a variety of cell types with insulin or oleic acid IMPDH translocated to lipid droplets and inhibition of this translocation correlated with reduced lipid accumulation. As lipid droplet formation and lipid accretion are defining features of adipogenesis we hypothesized that IMPDH may facilitate efficient lipid accumulation during adipose conversion of preadipocytes. In the current report we have examined this hypothesis in vitro, using the well characterized murine 3T3-L1 cell line, and in vivo, using a mouse model of diet-induced obesity. Materials and methods Reagents and antibodies. Reagents were from Sigma–Aldrich (Castle Hill, Australia) unless otherwise stated. Tissue culture media and foetal calf serum were from Invitrogen (Mount Waverley, Australia) and JRH Biosciences (Brooklyn, Australia). Monoclonal
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pan-IMPDH antibody was described previously [7]. Antibodies for PPARc, C/EBPa, C/EBPb and HPRT were from Santa Cruz (Santa Cruz, CA, USA). Antibodies to b-Tubulin and perilipin were from Upstate (Boronia, Australia) and Research Diagnostics (Concord, MA, USA). Cell culture, assessment of mitotic clonal expansion (SYTO 60 assay), differentiation and triglyceride accumulation. 3T3-L1 preadipocytes were cultured and differentiated as described [8]. The fluorescent nucleic acid stain, SYTO 60 (Invitrogen), was used to measure cell number according to the manufacturers’ instructions [8]. Differentiation was assessed morphologically by observation of lipid accumulation using phase-contrast microscopy. Triglyceride accumulation was determined by quantitation of neutral lipid stained with Oil Red-O [7]. Glycerol-3-phosphate dehydrogenase (G3PDH) activity was measured as described [8]. Assessment of IMPDH activity, Western blotting and HPLC analysis of intracellular nucleotides. IMPDH activity, Western blotting of whole cell lysates (10 lg) and Reverse-Phase HPLC were all performed as described [9]. Murine study with mycophenolate mofetil. Male C57Bl/6J mice were fed ad libitum a high-fat diet (SF04-001, Specialty Feeds, Glen Forrest, WA, Australia) for 10 weeks to induce obesity. The diet was continued for 4 weeks during which animals were treated by twice daily oral gavage with either mycophenolate mofetil (MMF; 60 mg/ kg; n = 8) or vehicle (n = 6). Primary outcome measures were reduction in body weight and fat mass. Food intake and body weight were measured weekly. Body composition was assessed by DEXA on days 0 and 28 using a pDEXA Sabre XL (Norland Stratec, Australia) under anaesthesia. Blood samples were collected from the tail on days 0 and 28 and glucose concentration determined using glucose sticks (AccuChek, Roche Diagnostics, USA). Following centrifugation, plasma was assayed for insulin, leptin and adiponectin (radioimmunoassays; Linco, USA) and total triglycerides and cholesterol (colorimetric assays; Roche Diagnostics, USA). At the completion of the study animals were sacrificed and epididymal and retroperitoneal adipose depots, liver and red gastrocnemius muscles were excised and weighed. Adipose and liver tissues were fixed in formalin, mounted in paraffin blocks, cut into sections and stained with hematoxylin and eosin using standard protocols. Histological examinations (n = 10–12 sections/tissue) were evaluated by two independent operators who were blinded to the treatment groups. Adipocyte cellularity was determined by counting number of cells/field. Average adipocyte volume (lm3) was calculated and total number of adipocytes estimated as described [10]. All experimental protocols were approved by the Deakin University Animal Welfare Committee, and adhered to the guidelines provided by the National Health and Medical Research Council. Statistics. Statistical analysis was performed using SPSS 13.0 (SPSS Inc., Chicago, IL, USA). Data were checked for normality of distribution using a 1-sample Kalmogorov–Smirnov test. For normally distributed data, two-tailed Paired-Samples t-test or independent samples t-test were used to evaluate the significance of the difference in mean values between the different treatments. p < 0.05 was considered statistically significant. Results IMPDH is transiently increased during differentiation of 3T3-L1 cells Upon induction of differentiation, 3T3-L1 preadipocytes execute a well defined program characterized initially (48–72 h) by 1–2 rounds of proliferation known as mitotic clonal expansion (MCE), resulting in a 3–4-fold increase in cell number and concomitant increase in total protein (Fig. 1A) and, subsequently by morphological changes including accumulation of lipid droplets (Fig. 1B) [2].
Expression of IMPDH protein was also increased, albeit transiently, even after accounting for the increase in cell number/total protein (Fig. 1C). Total IMPDH protein levels and activity were highest around day 3 (Fig. 1C and D). In contrast, HPRT levels increased and remained high in fully differentiated adipocytes (Fig. 1C). As expected the levels of adipogenic markers, including PPARc and perilipin, were increased during differentiation (Fig. 1C). Lipid droplets and perilipin were first observed around day 3, the period where IMPDH expression and activity was highest. Our previous observations implicated a potential role for IMPDH in lipid accumulation, by virtue of its regulated association with lipid droplets [7]. We therefore examined whether IMPDH was associated with lipid droplets during differentiation but found no co-localisation, suggesting IMPDH is unlikely to play a direct role in lipid accretion during adipogenesis (data not shown). However, having established that IMPDH activity increased during 3T3-L1 differentiation we went onto investigate whether this activity was required for efficient adipogenesis. Mycophenolic acid (MPA) inhibits 3T3-L1 differentiation by depleting GTP MPA is a widely used, potent inhibitor of IMPDH [11]. Treatment with MPA for the first 6 days of differentiation inhibited adipogenesis in a dose dependent manner, as determined morphologically and biochemically (Fig. 2A and B). The IC(50) of MPA was around 0.2 lM, while 1 lM MPA was sufficient to completely block adipogenesis. The specificity of these effects was determined in preliminary guanosine titration studies (to restore GTP levels via the HPRT-dependent salvage pathway), and established that 60 lM guanosine was able to prevent the MPA effects (see below). Fig. 2C, showing intracellular nucleotide levels following MPA and/or guanosine treatment, demonstrates that in differentiating cells 1 lM MPA decreased intracellular GTP by 65% and 50% after 1 and 3 days of incubation, respectively. These effects were reversed upon co-treatment with 60 lM guanosine. In comparison, ATP levels were relatively unaffected, with only a modest decrease after 3 days treatment (Fig. 2C). Treatment with MPA in non-differentiating cells was without significant effect. Furthermore, treatment of fully differentiated 3T3-L1 adipocytes with MPA across a broad concentration range (0.1–30 lM) for up to 48 h did not affect insulin-stimulated glucose uptake (data not shown). Guanosine prevents the inhibitory effects of MPA in 3T3-L1 cells In differentiating 3T3-L1 cells, 1 lM MPA blocked MCE and markedly reduced induction of C/EBPa, although C/EBPb and PPARc were relatively unaffected, whilst co-treatment with 60 lM guanosine reversed these effects (Fig. 2D and E). Consistent with several reports in other cell types, IMPDH levels were increased by MPA and reduced by guanosine (Fig. 2E). As before, HPRT expression was induced upon differentiation and this was unaffected by treatment. Moreover, the inhibitory effects of MPA on lipid accumulation and G3PDH activity were ameliorated by co-treatment with guanosine (Fig. 2F and G). In summary, these data demonstrate that IMPDH expression and activity are increased transiently during differentiation of 3T3-L1 cells and inhibition of IMPDH activity blocks differentiation, while co-treatment with guanosine abrogates this inhibition. Mycophenolate mofetil (MMF) induces loss of weight and fat in dietinduced obese mice We next examined the effects of inhibiting IMPDH activity on adipose tissue in vivo, in a mouse model of diet-induced obesity.
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Fig. 1. IMPDH expression and activity increase transiently during 3T3-L1 preadipocyte differentiation. 3T3-L1 preadipocytes were differentiated and harvested at indicated times. (A) MCE and total protein accumulation. Data are expressed as fold increase compared to day 0, and represent the mean + SE of three independent experiments. (B) Photomicrographs showing accumulation of lipid during differentiation (day 0–9). (C) Western blot analysis of cell lysates probed with antibodies as indicated. Data are representative from three independent experiments. (D) Quantitation of IMPDH expression and activity during differentiation. Data are expressed as fold increase of the value at day 0 and are representative of three independent experiments.
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Tubulin Fig. 2. MPA blocks differentiation of 3T3-L1 preadipocytes. 3T3-L1 preadipocytes were differentiated in the presence of vehicle (0) or increasing concentrations of MPA. (A) Photomicrographs showing lipid accumulation on day 8. (B) G3PDH activity in cells harvested on day 8. Results are expressed as percentage of control (vehicle) and represent the mean + SE of four independent experiments. (C) Effects of 1 lM MPA, 60 lM guanosine (GUA) or both (M + G) on intracellular GTP and ATP levels in non-differentiated (ND) or differentiated (CON, MPA, GUA and M + G) cells harvested on days 0, 1, 3 and 6. Results are expressed as the mean + SE of three independent experiments. (*p < 0.05 MPA vs. CON). (D and E) 3T3-L1 preadipocytes were untreated (control; CON) or treated with 1 lM MPA, 60 lM guanosine (GUA), or both (M + G) and harvested on day 3 of differentiation. (D) MCE as determined by SYTO 60. Results are presented as increase in cell number compared with non-differentiated post-confluent cells and represent the mean + SE of three independent experiments. (*p < 0.05 MPA vs. CON). (E) Western blot analysis of cell lysates probed with antibodies as indicated. Data are representative from three individual experiments. ND, non-differentiated post-confluent cells. (F and G) Cells were treated as described above with MPA and/or GUA from days 0–6 and harvested on day 8. (F) Lipid accumulation was assessed morphologically (left panel) and by Oil Red-O assay (right panel). Data are presented as percentage of control and represent the mean + SE of six independent experiments. (G) G3PDH activity showing the mean + SE of six (CON; MPA) or three (GUA; M + G) independent experiments. (**p < 0.01 vs. control; ##p < 0.01 vs. MPA).
Twice daily gavage with 60 mg/kg MMF, the pro-drug of MPA, for 28 days was associated with a 6% reduction in weight in obese animals maintained on a high-fat diet (vehicle treated animals 0.75 ± 0.6 g weight reduction, n = 6, p = 0.143; MMF-treated animals 1.6 ± 0.3 g weight reduction, n = 8, p = 0.03). Body composition analysis by DEXA revealed a 36% decrease in total body fat in MMF-treated animals (Fig. 3A), with no change in lean body mass (vehicle control animals: 0.5 ± 1.3 g reduction in lean mass; MMF: 1.5 ± 1.1 g increase; n.s.). Necropsy data confirmed significant MMF treatment-associated loss in epididymal fat and showed a trend toward retroperitoneal fat loss (Fig. 3B). In contrast there was no reduction in brown adipose tissue, liver or skeletal muscle mass (Fig. 3C). Weight loss was not associated with any alteration
in food intake (data not shown). Histology demonstrated a significant reduction in adipocyte size in response to MMF (Fig. 3D) and an estimated 15% reduction in total adipocyte number [10]. Treatment with MMF also decreased the appearance of hepatic steatosis (Fig. 3E). Control (vehicle gavage) animals showed a significant (p < 0.05) increase in fasting insulin over the 4 week study, consistent with a progressive decline in insulin sensitivity (Fig. 4A). This change was not seen in the MMF-treated animals. A similar trend was observed in fasting triglyceride levels (Fig. 4B). Fasting leptin was reduced in the MMF-treated animals, consistent with the observed fat loss (Fig. 4C). Fasting glucose, total cholesterol and adiponectin were not altered by MMF treatment (Fig. 4A–C). F4/80-positive macro-
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Fig. 3. Treatment with MMF reduces white adipose tissue in obese mice. Obese mice were treated twice daily with vehicle or 60 mg/kg MMF by oral gavage for 28 days. (A) Total body fat content as determined by DEXA at day 0 and 28 (*p = 0.02, MMF day 0 cf. 28). (B) Epididymal (Epid) and retroperitoneal (Retro) fat mass at day 28 (*p = 0.036, MMF cf. vehicle). (C) Brown adipose, liver and Quadricep tissue mass at day 28. Tissue weights are shown as % of total body weight. (D) Representative micrographs showing reduced adipocyte size (*p = 0.03, MMF cf. vehicle). (E) Representative micrographs showing reduced hepatic steatosis in MMF-treated animals. (n = 6 vehicle; n = 8 MMF).
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Fig. 4. Treatment with MMF reduces plasma insulin and leptin in obese mice. Obese mice were treated twice daily with vehicle or 60 mg/kg MMF by oral gavage for 28 days. Fasting biochemical parameters were measured by standard assay at day 0 and day 28. (A) Insulin and glucose. (B) Triglycerides and Cholesterol. (C) Leptin and adiponectin. (*p < 0.05, day 0 cf. day 28 within treatment; #p < 0.05, day 28 MMF cf. vehicle; n = 6 vehicle; n = 8 MMF).
phages were observed in adipose tissue of vehicle and MMF-treated mice, however, no difference was observed in the number of macrophages present following MMF treatment (data not shown). There were no clinical, biochemical or histological signs of toxicity in MMF-treated animals. Discussion The hypothesis that prompted these studies was based on observations implicating a role for IMPDH in lipid accumulation by virtue of its regulated association with lipid droplets [7]. In the current report we characterised key features of guanine metabolism during differentiation of 3T3-L1 cells and investigated the effects of IMPDH inhibition on adipose tissue in vivo. While we observed no association of IMPDH with lipid droplets during adi-
pogenesis our results demonstrated that inhibition of IMPDH blocked differentiation of 3T3-L1 cells and this effect could be prevented by guanosine supplementation. Consistent with this, treatment of diet-induced obese mice with the IMPDH inhibitor MMF reduced adipose tissue mass. Nucleotide biosynthesis is essential for a variety of cellular processes [3]. Dependence on the de novo and salvage guanine nucleotide biosynthesis pathways varies between cell types and contributes to the differential sensitivity to IMPDH inhibitors [3,11]. The relative importance of these pathways in adipogenesis had not been examined. We found that IMPDH expression and activity increased transiently during differentiation of the 3T3-L1 cells. In contrast, HPRT expression increased and was maintained upon adipocyte maturation suggesting the balance between the de novo and salvage pathways may change. We investigated the requirement for IMPDH activity during adipogenesis by employing the widely used inhibitor MPA. A major consequence of inhibition of IMPDH with MPA is a reduction in DNA synthesis, which leads to a block in proliferation, promoting terminal differentiation in a range of neoplastic cell types [6]. We found that inhibition of IMPDH by MPA (1 lM) blocked DNA synthesis and terminal differentiation of the 3T3-L1 cells, highlighting the importance of MCE in this system [12,13]. Consistent with this, MPA treatment was most effective at reducing GTP levels during the period of MCE. Indeed, treatment of differentiating 3T3-L1 cells with MPA for 3 days during the period of MCE (days 0–2) reduced differentiation by 60–70% compared with only a 10–20% reduction when cells were treated with MPA post-MCE (days 3–5) (H.S. and J.P.W. – unpublished observations). Recent work from Arner and colleagues strengthens a model whereby acquisition of new adipocytes contributes to the dynamics of fat cell turnover, with increased rates of adipogenesis reported in obese subjects [1], providing further support for therapeutic strategies that target such processes [14]. Although inhibitors of IMPDH, including MMF and mizoribine, have been used extensively as immunosuppressive agents for a number of years we have been unable to find data describing their effects, or lack thereof, on adipose tissue mass in humans. Limited information comes from studies in rodents. Three reports suggest inhibiting IMPDH may reduce body mass although specific measurement of fat and lean body mass was not reported [15–17]. To investigate the potential effects of inhibiting IMPDH on adipose tissue in vivo we performed a study in a mouse model of diet-induced obesity treated with MMF. Treatment with MMF induced significant weight loss and this was largely due to a reduc-
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tion in white adipose tissue, with no change in brown adipose tissue, liver and muscle weight. In epididymal adipose tissue, where the effects of MMF were most marked, treatment with MMF reduced adipocyte size and number. Recent evidence indicates that adipose tissue in mice exhibits dynamic remodelling when animals are maintained on a high-fat diet, with marked changes in adipocyte cell number, cell death and cell size occurring after 8–20 weeks of high fat feeding [18]. Future studies will be required to determine which of these parameters are altered by MMF treatment. Circulating leptin levels were reduced in MMF-treated animals, consistent with the reduction in white adipose tissue. We also observed a relative reduction in fasting insulin in response to MMF, with no concomitant change in glucose levels, suggesting an improvement in insulin sensitivity. Other indicators of metabolic benefit induced by MMF included reduction in hepatic steatosis and a trend towards reduced triglyceride levels. MMF-treated animals showed no toxicity and food intake was similar to controls. Thus, the fat loss observed may relate to increased fuel oxidation/metabolic rate and/or a reduction in adipogenesis. Unlike leptin, total adiponectin levels were unchanged. Taking into account the reduction in adipose tissue this suggests that total adiponectin levels (adjusted for adipose tissue mass) were increased. Future studies will examine the mechanism underpinning the MMF-induced fat loss in more detail. The dose of MMF used in this study (total of 120 mg/kg/day) is similar to that used to achieve immunosuppression in murine studies [19,20]. Subsequent studies will also aim to determine if the dose relationship to fat loss and immunosuppression differ. Whilst obesity-related adipose tissue inflammation and insulin resistance is characterised by increased infiltration of macrophages [21,22], there was no difference in macrophage number in adipose tissue or liver of MMF-treated animals compared with control animals, suggesting MMF treatment does not affect inflammation in these tissues over the study period. Nevertheless, reduced adipocyte size and number correlate to metabolic improvements in obesity and recent evidence suggests that pro-inflammatory cytokines from the adipocyte can affect peripheral insulin sensitivity in the absence of changes to adipose tissue macrophages [23]. This provides a potential mechanism for the improved metabolic parameters. In summary, we have demonstrated that IMPDH inhibition blocks adipogenesis of the murine 3T3-L1 cell line and that MMF induces significant fat loss in diet-induced obese mice. These data suggest that inhibition of IMPDH may represent a novel therapeutic strategy to reduce the increase in fat mass which underpins obesity associated weight gain. Acknowledgments This work was funded by the Australian National Health and Medical Research Council, Diabetes Australia, the Lions Medical Research Foundation, the Princess Alexandra Foundation and the University of Queensland.
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