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Pharmacological inhibition of galectin-1 by lactulose alleviates weight gain in diet-induced obese rats
Pharmacological inhibition of galectin-1 by lactulose alleviates weight gain in diet-induced obese rats Rajib Mukherjee, Jong Won Yun PII: DOI: Reference:
S0024-3205(16)30068-6 doi: 10.1016/j.lfs.2016.02.018 LFS 14703
To appear in:
Life Sciences
Received date: Revised date: Accepted date:
13 November 2015 31 January 2016 5 February 2016
Please cite this article as: Mukherjee Rajib, Yun Jong Won, Pharmacological inhibition of galectin-1 by lactulose alleviates weight gain in diet-induced obese rats, Life Sciences (2016), doi: 10.1016/j.lfs.2016.02.018
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ACCEPTED MANUSCRIPT Revised research article for submission to Life Sciences (Ms. No. LFS-D-15-01110)
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Pharmacological inhibition of galectin-1 by lactulose alleviates weight gain
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in diet-induced obese rats
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Rajib Mukherjee, Jong Won Yun*
Department of Biotechnology, Daegu University, Kyungsan, Kyungbuk, 712-714,
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Republic of Korea,
Running title: Lactulose reduces body weight gain
*Corresponding author at: Department of Biotechnology, Daegu University, Kyungsan, Kyungbuk 712-714, Republic of Korea. E-mail address: [email protected] (J.W. Yun) Phone: +82-53-850-6556, Fax: +82-53-850-6559
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ACCEPTED MANUSCRIPT ABSTRACT Aims: Galectin-1 (GAL1) is an important member of the lectin family with a carbohydrate
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recognition domain and has recently been demonstrated to be involved in adipose metabolism.
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inhibitor lactulose under high fat diet (HFD)-induced obesity.
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In the present study, we investigated the effects of targeted inhibition of GAL1 by its binding
Main methods: Effects of targeted inhibition of GAL1 by lactulose on lipid metabolism were
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investigated in vitro and in vivo. Changes in lipogenic capacity in lactulose-treated adipocytes were demonstrated by Oil Red O staining, triglyceride quantification and major adipogenic
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marker expression patterns. After lactulose treatment in Sprague-Dawley rats, various important body weight parameters, food efficiency, plasma metabolic parameters (glucose,
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ALT, free fatty acid, triglycerides, leptin, and insulin) and metabolic protein expression
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patterns were evaluated.
Key findings: Lactulose treatment reduced adipogeneis and fat accumulation in vitro by
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down-regulation of major adipogenic transcription factors such as C/EBPα and PPARγ. In vivo treatment of lactulose to 5-week-old Sprague-Dawley male rats significantly alleviated
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HFD-induced body weight gain and food efficiency as well as improved plasma and other metabolic parameters. In addition, lactulose treatment down-regulated major adipogenic marker proteins (C/EBPα and PPARγ) in adipose tissue as well as stimulated expression of proteins involved in energy expenditure and lipolysis (ATP5B, COXIV, HSL, and CPT1). Significance: In conclusion, reduced adipogenesis and increased energy expenditure mediated by lactulose treatment synergistically contribute to alleviation of HFD-induced body weight gain. Therefore, pharmaceutical targeting of GAL1 using lactulose would be a novel therapeutic approach for the treatment of obesity. Keywords: Adipogenesis, Adipose tissue, Galectin-1, Lactulose, Obesity.
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ACCEPTED MANUSCRIPT 1. Introduction Metabolic diseases such as type 2 diabetes, cardiovascular diseases, atherosclerosis, and
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cancer have been shown to be connected with obesity, which has emerged as a leading health
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hazard over recent decades [1]. Obesity and related metabolic complications are regulated
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through complex multi-protein networks, and these novel protein networks can be targeted for efficient obesity management [2,3]. Recently, multiple novel marker proteins from
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important obesity regulatory networks have been manipulated using specific inhibitors, and these inhibitors show great potential as anti-obesity drugs [4,5].
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Galectins (GALs) are animal lectins with carbohydrate recognition domains (CRDs) and multiple functions in mammals [6]. Depending on their functions and molecular structures,
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GAL family proteins can be divided into 15 different types and are involved in various intra
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or extra cellular functions such as cell-cell adhesion and intracellular vesicle transport [7].
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GAL1 is a prominent member of the GAL family, which contains a single CRD and forms symmetric homodimers using hydrophobic faces [8]. GAL1, an important endogenous member of the GAL family, is involved in regulation of the antigen-specific T cell response,
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apoptosis in thymocytes or activated T cells, as well as modulation of T cell receptor signaling through CD6 [9,10,11]. In connection with obesity, up-regulation of GAL1 has been observed in subcutaneous adipose tissues of obese patients and diet-induced obese mice models [12]. In addition, GAL1 is secreted at higher concentrations during adipocyte differentiation, demonstrating its involvement as a growth-modulating factor in adipocyte development [12,13]. Taken together, the adipocyte-specific roles of GAL1 led us to investigate its potential role as a positive regulator of adipogenesis and lipogenesis. Therefore, the current study was designed to identify a targeted inhibitor of GAL1 in order to determine whether or not pharmacological inhibition of GAL1 can alleviate body weight gain in a diet3
ACCEPTED MANUSCRIPT induced obese animal model. To date, several different types of glycoconjugates or synthetic chemicals have been
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identified based on their divergent binding capability towards GAL family proteins [14,15].
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Lactulose and lactulose amines are small-sized inhibitors of GAL1 that are known to inhibit
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migration and invasion as well as induce apoptosis in endothelial cells and small cell lung cancer cells [14]. But, lactulose binding to different members of galectin is dependent on
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thermodynamic interaction between galactose binding site and lactulose. This difference in interaction can affect specificity of lactulose to other members of galectin like GAL3, GAL12
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[16]. Lactulose (4-O-β-D-galactopyranosyl-D-fructose) is a semi-synthetic disaccharide comprised of fructose and galactose bonded together by a β-1,4-glycosidic bond, which is
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insensitive to mammalian digestive enzymes [17]. Due to its non-metabolizable
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characteristics, lactulose can readily pass through the stomach and small intestine, thereby
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providing a perfect medium for beneficial bacteria in the large intestine, specially members of bifidobacteria [18]. Oral administration of lactulose is already known to be beneficial for insulin resistance by improving insulin signaling and reducing free fatty acids as well as
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blood glucose levels in obese patients [19,20]. Intake of lactulose is a well-known medication for functional constipation [21] and has shown a beneficial impact on non-alcoholic steatohepatitis in rats when administered orally [22]. Colonic fermented products of lactulose are also known to reduce lipolysis in obese subjects by adjusting blood free fatty acid levels, improving free fatty acid-dependent insulin resistance [20]. In addition, lactulose-containing food preparations improve insulin signaling and have positive effects on blood glucose levels in obese patients [19]. However, no direct evidence has linked lactulose to anti-obesity effects until now. The goal of this study was therefore to examine whether or not lactulose efficiently 4
ACCEPTED MANUSCRIPT inhibits GAL1, thereby reducing adipogenesis and lipogenesis in both cultured white and brown adipocytes as well as diet-induced obese rats, in order to evaluate pharmaceutical
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targeting of GAL1 using lactulose as a novel therapeutic approach for treatment of obesity.
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2. Materials and methods 2.1. Adipocyte culture and differentiation
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3T3-L1 and HIB1B cells that derived from mice white and brown preadipocytes, respectively were cultured and differentiated using previously described protocols [23].
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Briefly, adipocytes were cultured on appropriate plates until confluent, after which growth medium was changed to differentiation cocktail and maturation medium. Maturation medium
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was changed every 2 days. 3T3-L1 cell line was purchased from the Korean Cell Line Bank
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(KCLB10092.1), and HIB1B cell line was a kind gift from Dr. Kwang-Hee Bae [24]. Unless
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otherwise stated, both adipocytes were separately treated with 250 or 500 µM of lactulose in maturation medium for 4 days before further analysis. Cytotoxicity of lactulose on both adipocytes was assessed by MTT [3'-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
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bromide] (Generay Biotech, Shanghai, China) using a previously described protocol [25].
2.2. Oil Red O staining and quantification of triglycerides (TG) 3T3-L1 and HIB1B adipocytes were matured for 4 days and used for Oil Red O staining as well as quantification of TG according to previously described methods [23]. Briefly, adipocytes were fixed with 10% formalin for 1 h at room temperature and stained by a mixture of Oil Red O and water at a 6:4 ratio for 10 min, followed by washing three times with deionized water. For TG quantification, mature cells were washed twice with PBS and harvested to prepare cell lysates using RIPA buffer (Sigma, St. Louis, MO, USA). TG content 5
ACCEPTED MANUSCRIPT was measured according to the manufacturer’s instructions using a TG test kit (Asan Pharm.
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Co., Yeongcheon, Korea), and absorbance was measured at 550 nm.
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2.3. Quantitative real-time RT-PCR
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Mature adipocytes were harvested and total RNA isolated using a RNA isolation kit (RNA-spin, iNtRON Biotechnology, Seongnam, Korea), after which 1 µg of RNA was
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converted into cDNA using Maxime RT premix (iNtRON Biotechnology). Transcription level of each gene was quantitatively determined by employing Fast start universal SYBR Green
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mastermix (Roche, Basel, Switzerland) with real-time RT-PCR (Stratagene 246 mx 3000p QPCR System, Agilent Technologies, Santa Clara, CA, USA) and normalized to the level of
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β-actin. Sequences of primer sets used in this study are listed in Table S1 in Supplementary
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Materials.
2.4. Immunoblot analysis
Lysates from both white adipose tissue (WAT) and brown adipose tissue (BAT) were
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prepared in RIPA Buffer (Sigma), followed by immunoblotting using monoclonal or polyclonal antibodies against GAL1, Peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding protein α (C/EBPα), Cytochrome c oxidase IV (COX IV), Hormone sensitive lipase (HSL), ATP synthase subunit beta, mitochondrial (ATP5B), Carnitine O-palmitoyltransferase 1 (CPT1), Uncouplin protein-1 (UCP1), and β-actin (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) according to common laboratory protocols [23]. Band intensities of target proteins were normalized using β-actin to calculate relative intensities of protein bands using ImageMaster software (GE Healthcare, Little Chalfont, Buckinghamshire, UK). 6
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2.5. Animal experiments
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Five-week-old Sprague-Dawley (SD) male rats were acclimatized with normal chow for
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1 week and then divided into four groups viz. control rats fed a normal diet (NC), HFD-fed
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control rats (HC), HFD-fed rats treated with lactulose (LT) by oral administration (LT-OR), and HFD-fed rats treated with lactulose by intraperitoneal injection (LT-IP). PBS or lactulose
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solubilized in PBS was administered either by oral gavage or intraperitoneal injection every 4th day to rats at a dose of 5 mg/kg body weight for 8 weeks. ND and HFD groups contained
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12% and 60% fat as an energy source, respectively (Korea Lab., Hanam, Korea). All animal experiments were approved by the Committee for Laboratory Animal Care and Use of Daegu
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University.
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2.6. Quantification of plasma TG, FFA, leptin, AST, and ALT Blood samples were collected from abdominal aortas and collected into EDTA tubes (BD, Franklin Lakes, NJ, USA) to isolate plasma by centrifugation (3,000×g, 10 min), followed by
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storage at -80ºC until further analysis. Common plasma biochemical characterizations were performed using commercial ELISA kits for AST-ALT (Asan Pharm. Co., Seoul, Korea), cholesterol (Biovision Inc. Milpitas, CA, USA), FFA, leptin (Enzo Life Sciences Inc., Farmingdale, NY, USA), plasma TG (Cayman Chemical Company, Ann Arbor, MI, USA), free glycerol (Sigma), and insulin (ALPCO, Salem, NH, USA). All ELISA experiments were carried out in triplicate using individual plasma samples to quantify plasma parameters.
2.7. Statistical analysis Results are expressed as the mean ± S.D. Statistical significances were calculated using 7
ACCEPTED MANUSCRIPT one-way (0 μM vs. 250, 500 μM of lactulose) or two-way ANOVA (HC vs. NC, LT-OP, LT-
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OR), followed by Tukey’s post-hoc test (for more than two groups), where p<0.05 or p<0.01.
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3. Results
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3.1. Lactulose treatment reduces lipid content and lipogenic markers in cultured adipocytes Lactulose treatment attenuated TG content in both 3T3-L1 and HIB1B adipocytes, and
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Oil Red O staining also demonstrated reduced oil droplet accumulation in lactulose-treated adipocytes (Fig. 1A, B). MTT assay demonstrated no cytotoxicity for lactulose in adipocytes
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at doses ranging from 125 to 750 μM (Fig. 1C). Key adipogenic markers such as CEBPα/Cebpa and PPARγ/Pparg were also dose-dependently down-regulated upon lactulose
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treatment (Fig. 1D, E).
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3.2. Lactulose-treated rats exhibit attenuated body weight, adipose tissue weight, and improved glucose tolerance
Treatment of HFD-fed rats with lactulose for 8 weeks (5 mg/kg of lactulose, every 4th
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day) by either oral administration (LT-OR) or intraperitoneal injection (LT-IP) resulted in 15.6% and 11.6% weight reductions, respectively (Fig. 2A). Improved glucose tolerances in lactulose-treated rats as demonstrated by IPGTT were indicative of improved metabolic conditions (Fig. 2B). Lactulose treatment also significantly attenuated WAT and liver weights as well as food efficiency (total body weight gain/ total food consumed), whereas BAT and kidney weights exhibited no significant changes (Fig. 2C, D). In addition, TG content of both WAT and BAT were significantly reduced after lactulose treatment, demonstrating the antilipogenic effects of lactulose (Fig. 2E, F). Unchanged weight of BAT may also indicate less prominent effect of lactulose on BAT physiology. 8
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3.3. Lactulose treatment alleviates obesity-related plasma parameters
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To confirm the toxicity of lactulose, plasma levels of two major liver toxicity parameters
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such as AST and ALT were determined. As shown in Fig. 3A, AST and ALT levels were not
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significantly altered after lactulose treatment. Plasma levels of free fatty acids (FFA), TG, leptin, and insulin were also significantly reduced in lactulose-treated obese rats (Fig. 3B, C,
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D), which is in agreement with the results of reduced fat mass. Moreover, plasma insulin level was reduced upon lactulose treatment, reflecting improved metabolic conditions (Fig.
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3E).
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3.4. Lactulose treatment alters regulation of major metabolic proteins in adipose tissues
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To elucidate the molecular mechanism behind the positive effects of lactulose in lipid
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metabolism, expression levels of metabolic marker proteins in WAT and BAT isolated from lactulose-treated rats were determined. Lactulose treatment markedly suppressed GAL1 expression and consequently attenuated expression levels of major adipogenic proteins
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(C/EBPα and PPARγ) in both WAT and BAT (Fig. 4). But, GAL3 and GAL12 expression were not reduced after lactulose treatment which may indicate specificity of lactulose towards GAL1 (Supplementary Fig. 1). Interestingly, lactulose treatment also augmented expression of key proteins involved in oxidative phosphorylation (ATP5B, COXIV), lipolysis (HSL), βoxidation (CPT1), and thermogenesis (UCP1), demonstrating stimulated energy expenditure upon lactulose treatment (Fig. 4).
4. Discussion In the present study, we hypothesized that GAL1 may play important regulatory roles in 9
ACCEPTED MANUSCRIPT lactulose-dependent improvement of lipid and glucose metabolism, as lactulose is a known GAL1 inhibitor. To test this hypothesis, both in vitro and in vivo experiments were performed
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using cultured adipocytes and HFD-induced obese rats, respectively. Our in vitro data
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demonstrate that reduced oil droplet accumulation and TG content in lactulose-treated
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adipocytes resulted from compromised lipogenic capacity, as evidenced by down-regulation of major adipogenic transcription factors PPARγ and C/EBPα at both the mRNA and protein
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levels. Interestingly, reduced adipogenic capacity of lactulose-treated adipocytes cannot be attributed to toxicity since lactulose treatment had no effect on cell viability. Hence, we
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believe that down-regulation of adipogenic markers is the main mechanism behind reduced adipogenic capacity of lactulose-treated adipocytes. Moreover, we observed that lactulose
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treatment either by oral administration or intraperitoneal injection significantly reduced HFD-
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induced obesity, further demonstrating the anti-obesity characteristics of lactulose, including
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reduction of food intake and lipogenesis as well as stimulation of catabolic protein expression. Furthermore, immunoblotting revealed that lactulose treatment significantly reduced important adipogenic markers such as C/EBPα and PPARγ along with its primary target
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GAL1. Adipogenic transcription factors such as C/EBPα and PPARγ form essential regulatory networks for lipogenic pathways as well as modulate downstream protein networks related to adipose tissue remodeling and metabolic syndrome [26,27]. In our study, lactulose treatment down-regulated its primary target, GAL1, which is also reported to be highly expressed in adipose tissues of obese patients, indicating its stimulatory role in obesity [12]. In contrast, lactulose treatment stimulated expression of major energy expenditure protein such as ATP5B, COX IV, which is involved in oxidative phosphorylation [28], thermogenic protein UCP1 in BAT [29], and HSL, a lipase involved in hydrolysis of intracellular tri-(di)acylglycerol and cholesteryl ester hydrolysis [30]. Taken together, we 10
ACCEPTED MANUSCRIPT postulate that lactulose treatment not only attenuates lipogenic capacity but also stimulates catabolic pathway proteins in adipose tissues to improve total energy balance upon HFD
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feeding. The stimulated expression of HSL and UCP1 can also be resulted from β-adrenergic
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receptor activation in adipose tissue. On the other hand, lactulose-dependent attenuation in
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triglyceride content can be resulted from reduced lipogenic proteins or stimulated expression of lipid catabolic proteins. Further studies will be needed to confirm the effect of lactulose on
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total lipogenesis or lipolysis and any other secondary mechanisms.
Unexpectedly, oral administration of lactulose induced higher body weight reduction than
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intraperitoneal injection. Earlier evidence has indicated that the beneficial effects of lactulose on glucose or lipid metabolism are achieved by oral dose only, and the mechanism of action
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was believed to be improved absorption of calcium and magnesium to block fat absorption in
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the gut [31,32]. Lactulose is a non-metabolizable synthetic carbohydrate that is absorbed in
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very minute quantities across the mucosa of the gastrointestinal tract through passive diffusion [33]. However, beneficial gut bacteria species such as Bifidobacteria and other lactic acid bacteria efficiently utilize lactulose to produce lactic acid and acetic acid to inhibit
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growth of pathogenic bacteria like Salmonella [18]. Additionally, lactulose is a prebiotic substance with known growth stimulatory activity for Bifidobacteria as well as other lactic acid bacteria, and these bacteria show significant anti-obesity effects [34,35]. Current data demonstrate that lactulose has major anti-obesity activity independent of treatment method. However, we believe that lactulose-dependent down-regulation of lipogenesis through GAL1 action and stimulation of beneficial bacterial growth in the gut work synergistically to cause body weight reduction upon oral administration. Taken together, current data suggest that lactulose not only improves the gut microenvironment as reported previously but also directly inhibits GAL1-dependent lipogenesis to improve total energy balance during HFD exposure. 11
ACCEPTED MANUSCRIPT Another surprising finding of this study is down-regulation of GAL1 upon treatment of lactulose, a binding inhibitor of GAL1. This result drove us to hypothesize that lactulose may
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regulate mRNA or protein expression using some secondary mechanism, as GAL1 has pre-
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mRNA splicing activity that can be inhibited using carbohydrate-based inhibitors [36]. On the
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other hand, GAL1 has been reported to be involved in regulation of ubiquitination of histone H2B lysine-123, an important transcription factor that controls transcription in yeast [37].
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Hence, we postulate that lactulose binding to GAL1 may negatively modulate transcriptional regulation of important targets in adipose tissue. However, these dynamic hypotheses need to
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be further tested to verify the effect of lactulose on GAL1-dependent pre-mRNA splicing and
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protein ubiquitination-mediated regulation of major adipose tissue markers.
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5. Conclusions
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In conclusion, current data suggest that lactulose targets GAL1 to alleviate lipogenesis in adipose tissue and may work synergistically with the prebiotic effects of lactulose to improve
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HFD-induced obesity and related metabolic complications.
Conflicts of interest
The authors declared no conflicts of interest.
Authors’ contribution RM carried out the experimental works, data analyses and interpretation and JWY was responsible for the planning of this study and wrote manuscript.
Acknowledgements 12
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This study was supported by Daegu University Research Grant 2014.
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[34] Y. Bouhnik, A. Attar, F.A. Joly, M. Riottot, F. Dyard, B. Flourie, Lactulose ing estion increases faecal bifidobacterial counts: a randomised double-blind study in h ealthy humans, Eur J Clin Nutr 58 (2004) 462-466. [35] H.M. An, S.Y. Park, K. Lee do, J.R. Kim, M.K. Cha, S.W. Lee, H.T. Lim, K.J. Kim, N.J. Ha, Antiobesity and lipid-lowering effects of Bifidobacterium spp. in high fat diet-induced obese rats, Lipids Health Dis 10 (2011) 116. [36] R.J. Patterson, K.C. Haudek, P.G. Voss, J.L. Wang, Examination of the role of galectins in pre-mRNA splicing, Methods Mol Biol 1207 (2015) 431-449. [37] A. Shukla, S.R. Bhaumik, H2B-K123 ubiquitination stimulates RNAPII elongation independent of H3-K4 methylation, Biochem Biophys Res Commun 359 (2007) 214-220.
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ACCEPTED MANUSCRIPT Figure captions Fig. 1. Effect of lactulose (LT) treatment on relative triglyceride (TG) content (A), Oil Red O
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staining indicating oil droplet accumulation, where bar represents 50 µm in 3T3-L1 and 100
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µm in HIB1B adipocytes, respectively (B), and cell viability of 3T3-L1 and HIB1B adipocytes (C). Lactulose significantly reduces major lipogenic markers at mRNA (D) and
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protein levels (E). Statistical significances between control (0 μM of lactulose) and treatment groups (250 and 500 μM of lactulose) were calculated using one-way ANOVA followed by
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Tukey’s post-hoc test, where *p<0.05 or †p<0.01.
Fig. 2. Effects of lactulose (LT) treatment (5 mg/kg, every 4th day) either by oral
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administration (LT-OR) or intraperitoneal injection (LT-IP) on HFD-induced body weight gain with whole body images of each group (inside panels: NC, HC, LT-OR, LT-OP from left) (A), intraperitoneal glucose tolerance test (B), food efficiency (C), metabolic tissue weights
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(D), TG content of adipose tissues (E, F). NC and HC refer to normal controls and HFD
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controls, respectively. Statistical significances between high fat control (HC) and other three groups (NC, LT-OP, LT-OR) were calculated using two-way ANOVA followed by Tukey’s
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post-hoc test, where *p<0.05 or †p<0.01 (n= 6).
Fig. 3. Effect of lactulose (LT) treatment on AST and ALT as toxicity markers (A), free fatty
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acid (B), plasma TG (C), leptin (D), and insulin (E) levels in LT-treated rats by oral administration (LT-OR) or intraperitoneal injection (LT-IP), where NC and HC refer to normal controls and HFD controls, respectively. Statistical significances between high fat control (HC) and other three groups (NC, LT-OP, LT-OR) were calculated using two-way ANOVA followed by Tukey’s post-hoc test, where *p<0.05 or †p<0.01 (n= 6).
Fig. 4. Lactulose (LT) treatment significantly affects expression of major adipogenic and energy expenditure proteins in epididymal WAT and BAT depots of LT-treated rats by oral administration (LT-OR) or intraperitoneal injection (LT-IP), where NC and HC refer to normal controls and HFD controls, respectively. Band densities were normalized using βactin in each tissue to calculate relative intensities (%). Statistical significances between high fat control (HC) and other three groups (NC, LT-OP, LT-OR) were calculated using two-way ANOVA followed by Tukey’s post-hoc test, where *p<0.05 or †p<0.01 (n= 6). 17
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S0024-3205(16)30068-6 doi: 10.1016/j.lfs.2016.02.018 LFS 14703
To appear in:
Life Sciences
Received date: Revised date: Accepted date:
13 November 2015 31 January 2016 5 February 2016
Please cite this article as: Mukherjee Rajib, Yun Jong Won, Pharmacological inhibition of galectin-1 by lactulose alleviates weight gain in diet-induced obese rats, Life Sciences (2016), doi: 10.1016/j.lfs.2016.02.018
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Revised research article for submission to Life Sciences (Ms. No. LFS-D-15-01110)
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Pharmacological inhibition of galectin-1 by lactulose alleviates weight gain
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in diet-induced obese rats
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Rajib Mukherjee, Jong Won Yun*
Department of Biotechnology, Daegu University, Kyungsan, Kyungbuk, 712-714,
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Republic of Korea,
Running title: Lactulose reduces body weight gain
*Corresponding author at: Department of Biotechnology, Daegu University, Kyungsan, Kyungbuk 712-714, Republic of Korea. E-mail address: [email protected] (J.W. Yun) Phone: +82-53-850-6556, Fax: +82-53-850-6559
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ACCEPTED MANUSCRIPT ABSTRACT Aims: Galectin-1 (GAL1) is an important member of the lectin family with a carbohydrate
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recognition domain and has recently been demonstrated to be involved in adipose metabolism.
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inhibitor lactulose under high fat diet (HFD)-induced obesity.
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In the present study, we investigated the effects of targeted inhibition of GAL1 by its binding
Main methods: Effects of targeted inhibition of GAL1 by lactulose on lipid metabolism were
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investigated in vitro and in vivo. Changes in lipogenic capacity in lactulose-treated adipocytes were demonstrated by Oil Red O staining, triglyceride quantification and major adipogenic
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marker expression patterns. After lactulose treatment in Sprague-Dawley rats, various important body weight parameters, food efficiency, plasma metabolic parameters (glucose,
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ALT, free fatty acid, triglycerides, leptin, and insulin) and metabolic protein expression
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patterns were evaluated.
Key findings: Lactulose treatment reduced adipogeneis and fat accumulation in vitro by
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down-regulation of major adipogenic transcription factors such as C/EBPα and PPARγ. In vivo treatment of lactulose to 5-week-old Sprague-Dawley male rats significantly alleviated
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HFD-induced body weight gain and food efficiency as well as improved plasma and other metabolic parameters. In addition, lactulose treatment down-regulated major adipogenic marker proteins (C/EBPα and PPARγ) in adipose tissue as well as stimulated expression of proteins involved in energy expenditure and lipolysis (ATP5B, COXIV, HSL, and CPT1). Significance: In conclusion, reduced adipogenesis and increased energy expenditure mediated by lactulose treatment synergistically contribute to alleviation of HFD-induced body weight gain. Therefore, pharmaceutical targeting of GAL1 using lactulose would be a novel therapeutic approach for the treatment of obesity. Keywords: Adipogenesis, Adipose tissue, Galectin-1, Lactulose, Obesity.
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ACCEPTED MANUSCRIPT 1. Introduction Metabolic diseases such as type 2 diabetes, cardiovascular diseases, atherosclerosis, and
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cancer have been shown to be connected with obesity, which has emerged as a leading health
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hazard over recent decades [1]. Obesity and related metabolic complications are regulated
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through complex multi-protein networks, and these novel protein networks can be targeted for efficient obesity management [2,3]. Recently, multiple novel marker proteins from
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important obesity regulatory networks have been manipulated using specific inhibitors, and these inhibitors show great potential as anti-obesity drugs [4,5].
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Galectins (GALs) are animal lectins with carbohydrate recognition domains (CRDs) and multiple functions in mammals [6]. Depending on their functions and molecular structures,
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GAL family proteins can be divided into 15 different types and are involved in various intra
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or extra cellular functions such as cell-cell adhesion and intracellular vesicle transport [7].
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GAL1 is a prominent member of the GAL family, which contains a single CRD and forms symmetric homodimers using hydrophobic faces [8]. GAL1, an important endogenous member of the GAL family, is involved in regulation of the antigen-specific T cell response,
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apoptosis in thymocytes or activated T cells, as well as modulation of T cell receptor signaling through CD6 [9,10,11]. In connection with obesity, up-regulation of GAL1 has been observed in subcutaneous adipose tissues of obese patients and diet-induced obese mice models [12]. In addition, GAL1 is secreted at higher concentrations during adipocyte differentiation, demonstrating its involvement as a growth-modulating factor in adipocyte development [12,13]. Taken together, the adipocyte-specific roles of GAL1 led us to investigate its potential role as a positive regulator of adipogenesis and lipogenesis. Therefore, the current study was designed to identify a targeted inhibitor of GAL1 in order to determine whether or not pharmacological inhibition of GAL1 can alleviate body weight gain in a diet3
ACCEPTED MANUSCRIPT induced obese animal model. To date, several different types of glycoconjugates or synthetic chemicals have been
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identified based on their divergent binding capability towards GAL family proteins [14,15].
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Lactulose and lactulose amines are small-sized inhibitors of GAL1 that are known to inhibit
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migration and invasion as well as induce apoptosis in endothelial cells and small cell lung cancer cells [14]. But, lactulose binding to different members of galectin is dependent on
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thermodynamic interaction between galactose binding site and lactulose. This difference in interaction can affect specificity of lactulose to other members of galectin like GAL3, GAL12
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[16]. Lactulose (4-O-β-D-galactopyranosyl-D-fructose) is a semi-synthetic disaccharide comprised of fructose and galactose bonded together by a β-1,4-glycosidic bond, which is
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insensitive to mammalian digestive enzymes [17]. Due to its non-metabolizable
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characteristics, lactulose can readily pass through the stomach and small intestine, thereby
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providing a perfect medium for beneficial bacteria in the large intestine, specially members of bifidobacteria [18]. Oral administration of lactulose is already known to be beneficial for insulin resistance by improving insulin signaling and reducing free fatty acids as well as
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blood glucose levels in obese patients [19,20]. Intake of lactulose is a well-known medication for functional constipation [21] and has shown a beneficial impact on non-alcoholic steatohepatitis in rats when administered orally [22]. Colonic fermented products of lactulose are also known to reduce lipolysis in obese subjects by adjusting blood free fatty acid levels, improving free fatty acid-dependent insulin resistance [20]. In addition, lactulose-containing food preparations improve insulin signaling and have positive effects on blood glucose levels in obese patients [19]. However, no direct evidence has linked lactulose to anti-obesity effects until now. The goal of this study was therefore to examine whether or not lactulose efficiently 4
ACCEPTED MANUSCRIPT inhibits GAL1, thereby reducing adipogenesis and lipogenesis in both cultured white and brown adipocytes as well as diet-induced obese rats, in order to evaluate pharmaceutical
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targeting of GAL1 using lactulose as a novel therapeutic approach for treatment of obesity.
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2. Materials and methods 2.1. Adipocyte culture and differentiation
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3T3-L1 and HIB1B cells that derived from mice white and brown preadipocytes, respectively were cultured and differentiated using previously described protocols [23].
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Briefly, adipocytes were cultured on appropriate plates until confluent, after which growth medium was changed to differentiation cocktail and maturation medium. Maturation medium
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was changed every 2 days. 3T3-L1 cell line was purchased from the Korean Cell Line Bank
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(KCLB10092.1), and HIB1B cell line was a kind gift from Dr. Kwang-Hee Bae [24]. Unless
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otherwise stated, both adipocytes were separately treated with 250 or 500 µM of lactulose in maturation medium for 4 days before further analysis. Cytotoxicity of lactulose on both adipocytes was assessed by MTT [3'-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
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bromide] (Generay Biotech, Shanghai, China) using a previously described protocol [25].
2.2. Oil Red O staining and quantification of triglycerides (TG) 3T3-L1 and HIB1B adipocytes were matured for 4 days and used for Oil Red O staining as well as quantification of TG according to previously described methods [23]. Briefly, adipocytes were fixed with 10% formalin for 1 h at room temperature and stained by a mixture of Oil Red O and water at a 6:4 ratio for 10 min, followed by washing three times with deionized water. For TG quantification, mature cells were washed twice with PBS and harvested to prepare cell lysates using RIPA buffer (Sigma, St. Louis, MO, USA). TG content 5
ACCEPTED MANUSCRIPT was measured according to the manufacturer’s instructions using a TG test kit (Asan Pharm.
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Co., Yeongcheon, Korea), and absorbance was measured at 550 nm.
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2.3. Quantitative real-time RT-PCR
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Mature adipocytes were harvested and total RNA isolated using a RNA isolation kit (RNA-spin, iNtRON Biotechnology, Seongnam, Korea), after which 1 µg of RNA was
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converted into cDNA using Maxime RT premix (iNtRON Biotechnology). Transcription level of each gene was quantitatively determined by employing Fast start universal SYBR Green
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mastermix (Roche, Basel, Switzerland) with real-time RT-PCR (Stratagene 246 mx 3000p QPCR System, Agilent Technologies, Santa Clara, CA, USA) and normalized to the level of
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β-actin. Sequences of primer sets used in this study are listed in Table S1 in Supplementary
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Materials.
2.4. Immunoblot analysis
Lysates from both white adipose tissue (WAT) and brown adipose tissue (BAT) were
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prepared in RIPA Buffer (Sigma), followed by immunoblotting using monoclonal or polyclonal antibodies against GAL1, Peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding protein α (C/EBPα), Cytochrome c oxidase IV (COX IV), Hormone sensitive lipase (HSL), ATP synthase subunit beta, mitochondrial (ATP5B), Carnitine O-palmitoyltransferase 1 (CPT1), Uncouplin protein-1 (UCP1), and β-actin (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) according to common laboratory protocols [23]. Band intensities of target proteins were normalized using β-actin to calculate relative intensities of protein bands using ImageMaster software (GE Healthcare, Little Chalfont, Buckinghamshire, UK). 6
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2.5. Animal experiments
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Five-week-old Sprague-Dawley (SD) male rats were acclimatized with normal chow for
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1 week and then divided into four groups viz. control rats fed a normal diet (NC), HFD-fed
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control rats (HC), HFD-fed rats treated with lactulose (LT) by oral administration (LT-OR), and HFD-fed rats treated with lactulose by intraperitoneal injection (LT-IP). PBS or lactulose
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solubilized in PBS was administered either by oral gavage or intraperitoneal injection every 4th day to rats at a dose of 5 mg/kg body weight for 8 weeks. ND and HFD groups contained
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12% and 60% fat as an energy source, respectively (Korea Lab., Hanam, Korea). All animal experiments were approved by the Committee for Laboratory Animal Care and Use of Daegu
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University.
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2.6. Quantification of plasma TG, FFA, leptin, AST, and ALT Blood samples were collected from abdominal aortas and collected into EDTA tubes (BD, Franklin Lakes, NJ, USA) to isolate plasma by centrifugation (3,000×g, 10 min), followed by
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storage at -80ºC until further analysis. Common plasma biochemical characterizations were performed using commercial ELISA kits for AST-ALT (Asan Pharm. Co., Seoul, Korea), cholesterol (Biovision Inc. Milpitas, CA, USA), FFA, leptin (Enzo Life Sciences Inc., Farmingdale, NY, USA), plasma TG (Cayman Chemical Company, Ann Arbor, MI, USA), free glycerol (Sigma), and insulin (ALPCO, Salem, NH, USA). All ELISA experiments were carried out in triplicate using individual plasma samples to quantify plasma parameters.
2.7. Statistical analysis Results are expressed as the mean ± S.D. Statistical significances were calculated using 7
ACCEPTED MANUSCRIPT one-way (0 μM vs. 250, 500 μM of lactulose) or two-way ANOVA (HC vs. NC, LT-OP, LT-
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OR), followed by Tukey’s post-hoc test (for more than two groups), where p<0.05 or p<0.01.
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3. Results
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3.1. Lactulose treatment reduces lipid content and lipogenic markers in cultured adipocytes Lactulose treatment attenuated TG content in both 3T3-L1 and HIB1B adipocytes, and
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Oil Red O staining also demonstrated reduced oil droplet accumulation in lactulose-treated adipocytes (Fig. 1A, B). MTT assay demonstrated no cytotoxicity for lactulose in adipocytes
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at doses ranging from 125 to 750 μM (Fig. 1C). Key adipogenic markers such as CEBPα/Cebpa and PPARγ/Pparg were also dose-dependently down-regulated upon lactulose
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treatment (Fig. 1D, E).
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3.2. Lactulose-treated rats exhibit attenuated body weight, adipose tissue weight, and improved glucose tolerance
Treatment of HFD-fed rats with lactulose for 8 weeks (5 mg/kg of lactulose, every 4th
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day) by either oral administration (LT-OR) or intraperitoneal injection (LT-IP) resulted in 15.6% and 11.6% weight reductions, respectively (Fig. 2A). Improved glucose tolerances in lactulose-treated rats as demonstrated by IPGTT were indicative of improved metabolic conditions (Fig. 2B). Lactulose treatment also significantly attenuated WAT and liver weights as well as food efficiency (total body weight gain/ total food consumed), whereas BAT and kidney weights exhibited no significant changes (Fig. 2C, D). In addition, TG content of both WAT and BAT were significantly reduced after lactulose treatment, demonstrating the antilipogenic effects of lactulose (Fig. 2E, F). Unchanged weight of BAT may also indicate less prominent effect of lactulose on BAT physiology. 8
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3.3. Lactulose treatment alleviates obesity-related plasma parameters
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To confirm the toxicity of lactulose, plasma levels of two major liver toxicity parameters
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such as AST and ALT were determined. As shown in Fig. 3A, AST and ALT levels were not
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significantly altered after lactulose treatment. Plasma levels of free fatty acids (FFA), TG, leptin, and insulin were also significantly reduced in lactulose-treated obese rats (Fig. 3B, C,
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D), which is in agreement with the results of reduced fat mass. Moreover, plasma insulin level was reduced upon lactulose treatment, reflecting improved metabolic conditions (Fig.
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3E).
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3.4. Lactulose treatment alters regulation of major metabolic proteins in adipose tissues
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To elucidate the molecular mechanism behind the positive effects of lactulose in lipid
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metabolism, expression levels of metabolic marker proteins in WAT and BAT isolated from lactulose-treated rats were determined. Lactulose treatment markedly suppressed GAL1 expression and consequently attenuated expression levels of major adipogenic proteins
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(C/EBPα and PPARγ) in both WAT and BAT (Fig. 4). But, GAL3 and GAL12 expression were not reduced after lactulose treatment which may indicate specificity of lactulose towards GAL1 (Supplementary Fig. 1). Interestingly, lactulose treatment also augmented expression of key proteins involved in oxidative phosphorylation (ATP5B, COXIV), lipolysis (HSL), βoxidation (CPT1), and thermogenesis (UCP1), demonstrating stimulated energy expenditure upon lactulose treatment (Fig. 4).
4. Discussion In the present study, we hypothesized that GAL1 may play important regulatory roles in 9
ACCEPTED MANUSCRIPT lactulose-dependent improvement of lipid and glucose metabolism, as lactulose is a known GAL1 inhibitor. To test this hypothesis, both in vitro and in vivo experiments were performed
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using cultured adipocytes and HFD-induced obese rats, respectively. Our in vitro data
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demonstrate that reduced oil droplet accumulation and TG content in lactulose-treated
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adipocytes resulted from compromised lipogenic capacity, as evidenced by down-regulation of major adipogenic transcription factors PPARγ and C/EBPα at both the mRNA and protein
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levels. Interestingly, reduced adipogenic capacity of lactulose-treated adipocytes cannot be attributed to toxicity since lactulose treatment had no effect on cell viability. Hence, we
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believe that down-regulation of adipogenic markers is the main mechanism behind reduced adipogenic capacity of lactulose-treated adipocytes. Moreover, we observed that lactulose
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treatment either by oral administration or intraperitoneal injection significantly reduced HFD-
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induced obesity, further demonstrating the anti-obesity characteristics of lactulose, including
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reduction of food intake and lipogenesis as well as stimulation of catabolic protein expression. Furthermore, immunoblotting revealed that lactulose treatment significantly reduced important adipogenic markers such as C/EBPα and PPARγ along with its primary target
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GAL1. Adipogenic transcription factors such as C/EBPα and PPARγ form essential regulatory networks for lipogenic pathways as well as modulate downstream protein networks related to adipose tissue remodeling and metabolic syndrome [26,27]. In our study, lactulose treatment down-regulated its primary target, GAL1, which is also reported to be highly expressed in adipose tissues of obese patients, indicating its stimulatory role in obesity [12]. In contrast, lactulose treatment stimulated expression of major energy expenditure protein such as ATP5B, COX IV, which is involved in oxidative phosphorylation [28], thermogenic protein UCP1 in BAT [29], and HSL, a lipase involved in hydrolysis of intracellular tri-(di)acylglycerol and cholesteryl ester hydrolysis [30]. Taken together, we 10
ACCEPTED MANUSCRIPT postulate that lactulose treatment not only attenuates lipogenic capacity but also stimulates catabolic pathway proteins in adipose tissues to improve total energy balance upon HFD
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feeding. The stimulated expression of HSL and UCP1 can also be resulted from β-adrenergic
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receptor activation in adipose tissue. On the other hand, lactulose-dependent attenuation in
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triglyceride content can be resulted from reduced lipogenic proteins or stimulated expression of lipid catabolic proteins. Further studies will be needed to confirm the effect of lactulose on
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total lipogenesis or lipolysis and any other secondary mechanisms.
Unexpectedly, oral administration of lactulose induced higher body weight reduction than
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intraperitoneal injection. Earlier evidence has indicated that the beneficial effects of lactulose on glucose or lipid metabolism are achieved by oral dose only, and the mechanism of action
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was believed to be improved absorption of calcium and magnesium to block fat absorption in
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the gut [31,32]. Lactulose is a non-metabolizable synthetic carbohydrate that is absorbed in
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very minute quantities across the mucosa of the gastrointestinal tract through passive diffusion [33]. However, beneficial gut bacteria species such as Bifidobacteria and other lactic acid bacteria efficiently utilize lactulose to produce lactic acid and acetic acid to inhibit
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growth of pathogenic bacteria like Salmonella [18]. Additionally, lactulose is a prebiotic substance with known growth stimulatory activity for Bifidobacteria as well as other lactic acid bacteria, and these bacteria show significant anti-obesity effects [34,35]. Current data demonstrate that lactulose has major anti-obesity activity independent of treatment method. However, we believe that lactulose-dependent down-regulation of lipogenesis through GAL1 action and stimulation of beneficial bacterial growth in the gut work synergistically to cause body weight reduction upon oral administration. Taken together, current data suggest that lactulose not only improves the gut microenvironment as reported previously but also directly inhibits GAL1-dependent lipogenesis to improve total energy balance during HFD exposure. 11
ACCEPTED MANUSCRIPT Another surprising finding of this study is down-regulation of GAL1 upon treatment of lactulose, a binding inhibitor of GAL1. This result drove us to hypothesize that lactulose may
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regulate mRNA or protein expression using some secondary mechanism, as GAL1 has pre-
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mRNA splicing activity that can be inhibited using carbohydrate-based inhibitors [36]. On the
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other hand, GAL1 has been reported to be involved in regulation of ubiquitination of histone H2B lysine-123, an important transcription factor that controls transcription in yeast [37].
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Hence, we postulate that lactulose binding to GAL1 may negatively modulate transcriptional regulation of important targets in adipose tissue. However, these dynamic hypotheses need to
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be further tested to verify the effect of lactulose on GAL1-dependent pre-mRNA splicing and
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protein ubiquitination-mediated regulation of major adipose tissue markers.
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5. Conclusions
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In conclusion, current data suggest that lactulose targets GAL1 to alleviate lipogenesis in adipose tissue and may work synergistically with the prebiotic effects of lactulose to improve
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HFD-induced obesity and related metabolic complications.
Conflicts of interest
The authors declared no conflicts of interest.
Authors’ contribution RM carried out the experimental works, data analyses and interpretation and JWY was responsible for the planning of this study and wrote manuscript.
Acknowledgements 12
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This study was supported by Daegu University Research Grant 2014.
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ACCEPTED MANUSCRIPT Figure captions Fig. 1. Effect of lactulose (LT) treatment on relative triglyceride (TG) content (A), Oil Red O
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staining indicating oil droplet accumulation, where bar represents 50 µm in 3T3-L1 and 100
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µm in HIB1B adipocytes, respectively (B), and cell viability of 3T3-L1 and HIB1B adipocytes (C). Lactulose significantly reduces major lipogenic markers at mRNA (D) and
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protein levels (E). Statistical significances between control (0 μM of lactulose) and treatment groups (250 and 500 μM of lactulose) were calculated using one-way ANOVA followed by
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Tukey’s post-hoc test, where *p<0.05 or †p<0.01.
Fig. 2. Effects of lactulose (LT) treatment (5 mg/kg, every 4th day) either by oral
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administration (LT-OR) or intraperitoneal injection (LT-IP) on HFD-induced body weight gain with whole body images of each group (inside panels: NC, HC, LT-OR, LT-OP from left) (A), intraperitoneal glucose tolerance test (B), food efficiency (C), metabolic tissue weights
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(D), TG content of adipose tissues (E, F). NC and HC refer to normal controls and HFD
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controls, respectively. Statistical significances between high fat control (HC) and other three groups (NC, LT-OP, LT-OR) were calculated using two-way ANOVA followed by Tukey’s
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post-hoc test, where *p<0.05 or †p<0.01 (n= 6).
Fig. 3. Effect of lactulose (LT) treatment on AST and ALT as toxicity markers (A), free fatty
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acid (B), plasma TG (C), leptin (D), and insulin (E) levels in LT-treated rats by oral administration (LT-OR) or intraperitoneal injection (LT-IP), where NC and HC refer to normal controls and HFD controls, respectively. Statistical significances between high fat control (HC) and other three groups (NC, LT-OP, LT-OR) were calculated using two-way ANOVA followed by Tukey’s post-hoc test, where *p<0.05 or †p<0.01 (n= 6).
Fig. 4. Lactulose (LT) treatment significantly affects expression of major adipogenic and energy expenditure proteins in epididymal WAT and BAT depots of LT-treated rats by oral administration (LT-OR) or intraperitoneal injection (LT-IP), where NC and HC refer to normal controls and HFD controls, respectively. Band densities were normalized using βactin in each tissue to calculate relative intensities (%). Statistical significances between high fat control (HC) and other three groups (NC, LT-OP, LT-OR) were calculated using two-way ANOVA followed by Tukey’s post-hoc test, where *p<0.05 or †p<0.01 (n= 6). 17
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