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Effect of liraglutide on proliferation and differentiation of human adipose stem cells
Accepted Manuscript Title: Effect of liraglutide on proliferation and differentiation of human adipose stem cells Author: Giulia Cantini, Alessandra Di Franco, Jinous Samavat, Gianni Forti, Edoardo Mannucci, Michaela Luconi PII: DOI: Reference:
S0303-7207(14)00424-9 http://dx.doi.org/doi: 10.1016/j.mce.2014.12.021 MCE 8995
To appear in:
Molecular and Cellular Endocrinology
Received date: Revised date: Accepted date:
7-12-2014 27-12-2014 28-12-2014
Please cite this article as: Giulia Cantini, Alessandra Di Franco, Jinous Samavat, Gianni Forti, Edoardo Mannucci, Michaela Luconi, Effect of liraglutide on proliferation and differentiation of human adipose stem cells, Molecular and Cellular Endocrinology (2015), http://dx.doi.org/doi: 10.1016/j.mce.2014.12.021. 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.
Effect of liraglutide on proliferation and differentiation of human adipose stem cells Giulia Cantini1§, Alessandra Di Franco1§, Jinous Samavat1, Gianni Forti1, Edoardo Mannucci2*, Michaela Luconi1* 1
Endocrinology Unit, Dept. Experimental and Clinical Biomedical Sciences, University of Florence, Italy 2 Diabetes Agency, Azienda Ospedaliera-Universitaria Careggi, Florence, Italy §These authors equally contributed to the study
Running title: GLP-1 effects on human adipose stem cells
Keywords: Glucagone-Like-Peptide-1, adipogenesis, adipose stem cells, weight loss
*Correspondence: Dr. Edoardo Mannucci, M.D. Diabetes Agency Azienda Ospedaliero-Universitaria Careggi, Via delle Oblate 4, 50141 Florence-Italy Tel:+39 055 7945476 Fax:+39 055 7949742 [email protected] and Prof. Michaela Luconi, Ph.D. Endocrinology Unit Dept. Experimental and Clinical Biomedical Sciences University of Florence Viale Pieraccini 6, 50139 Florence-Italy Tel:+39 055 4271369 Fax:+39 055 4271371 [email protected]
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Highlights 1. Liraglutide interfers with human adipose stem cell proliferation and differentiation in vitro.
2. Liraglutide inhibits lipid accumulation in differentiating cells but stimulates adiponectin expression.
3. This in vitro effect on adipocyte precursors may contribute to the drug effect in obese patients.
ABSTRACT Glucagon-Like Peptide-1 (GLP-1) receptor agonists, used as glucose-lowering drugs, also induce weight loss by inhibiting food intake. The present study was aimed at the assessment of the in vitro effects of the GLP-1 receptor agonist liraglutide on proliferation and differentiation of human adipose stem cells (ASC) obtained from subcutaneous adipose tissue of morbidly obese subjects undergoing bariatric surgery. Liraglutide (10-100 nM) significantly inhibited ASC proliferation and viability, with a maximum effect at 6 days of culture (45% and 50%, for liraglutide 10 and 100 nM, respectively); the effect was reverted by exendin 9-39. Glucose uptake was significantly reduced by liraglutide in a dose dependent manner. Treatment with liraglutide reduced intracellular lipid accumulation in differentiating ASC, together with FABP-4 mRNA expression (-18%, -23%, -46%, for 1 nM, 10 nM and 100 nM, respectively), whereas it stimulated adiponectin (APN) expression (1.86, 2.64, 2.28 fold increase, for 1 nM, 10 nM and 100 nM, respectively). Liraglutide exerts effects on human adipose cell precursors, inhibiting proliferation and differentiation, while stimulating the expression of the insulin-sensitizing adipokine APN. These
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effects could contribute to the actions of GLP-1 receptor agonists on body weight and insulin sensitivity.
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1.INTRODUCTION Glucagon-Like Peptide-1 (GLP-1) is a gastrointestinal hormone, mainly produced after meals, which stimulates insulin secretion, and inhibits glucagon release in a dose-dependent fashion (Nauck et al, 2013). GLP-1 receptor agonists are currently used for the treatment of type 2 diabetes. It has been observed that these drugs also produce a relevant weight loss (Monami et al, 2009); for this reason, some of the molecules of this class are currently under development as a treatment for obesity in non-diabetic individuals, with interesting results from clinical trials (Astrup et al, 2009; 2012). Mechanisms of weight loss with GLP-1 and its analogues are probably complex and not completely elucidated. The stimulation of GLP-1 receptors in the hypothalamus, and particularly in paraventricular nuclei (PVN), is capable of inducing satiety and inhibiting food intake in rodents (Turton et al, 1996; Dalvi et al, 2012). Interestingly, GLP-1- producing neurons localized in the nucleus of the solitary tract and activated by food consumption, project their terminations to PVN, constituting a feedback loop inhibiting further food intake after meals (Larsen et al 1997). In addition, peripherally-secreted GLP-1 is capable of crossing the blood-brain barrier, at least in the hypothalamus, stimulating central receptors (Kastin et al, 2002; Holst 2004). It has also been observed that GLP-1 and its short-acting receptor agonists delay gastric emptying, possibly contributing to the inhibition of food intake; this could be due to either central or peripheral effects (van Bloemendaal et al 2014; Dailey et al, 2013). However, the relevance of this mechanism for the regulation of body weight is questionable. In fact, long acting formulations of GLP-1 receptor agonists (exenatide long-acting release and liraglutide), which have no relevant effect on gastric emptying, produce a weight loss similar to that of shorter acting agonists (Buse et al, 2009; Drucker et al, 2008; Blevins et al 2011). GLP-1 and its receptor agonists have shown additional extra-glycemic effects on many organs and tissues different from pancreatic islets and central nervous system, including endothelium and myocardium (Herzlinger et al, 2013). The possibility of a direct effect of GLP-1 receptor agonists 4 Page 4 of 23
on the adipose tissue and on pre-adipocytes has not been thoroughly explored so far. In 3T3-L1 mouse cells, a line of immortalized fibroblasts with some functional characteristics of preadipocytes, GLP-1 and liraglutide reduced apoptosis and stimulated adipocyte differentiation (Challa et al, 2012; Yang et al, 2013a), although an opposite effect was described in human mesenchymal stem cells from bone marrow (Sanz et al, 2010). In mature adipocytes obtained from rat adipose tissue and from 3T3-L1 adipocytes differentiated in vitro, GLP-1 receptor agonists induce an increase in glucose metabolism and lipogenesis (Sancho et al, 2005; Gao et al, 2007; Yang et al 2013b). The aim of the present study was to evaluate the potential in vitro effects of liraglutide on a model of human adipose stem cells (ASC) obtained from human subcutaneous adipose tissue of obese subjects, which can be differentiated in vitro to mature adipocytes.
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2.MATERIALS AND METHODS 2.1.Materials Media and sera for cell cultures were purchased from Sigma-Aldrich (Milan, Italy) and tissue plasticware was obtained from Corning (Milan, Italy). AdipoRed and MTS (CellTiter 96® Aqueous One Solution Cell proliferation assay) were purchased from Lonza (Milan, Italy) and Promega (Madison, WI, USA), respectively. Liraglutide was kindly provided by Novo Nordisk (Bagsvaert, Danmark), while exendin fragment 9-39 was purchased from Sigma-Aldrich (Milan, Italy). Other reagents for cell cultures, fluorescence microscopy were obtained from Sigma-Aldrich (Milan, Italy), except where differently indicated. [3H]-Thymidine was provided by Perkin Elmer (Waltham, Massachusetts, USA).
2.2.Subjects and Ethics Statement Subcutaneous adipose tissue samples were obtained from 11 subjects (4 males and 7 females: mean age±SD: 52±15 yrs; BMI±SD: 39.9±7.7 kg/m2), undergoing bariatric surgery at AOU Careggi Hospital, following written informed consent. All the protocols were approved by the Local Ethical Committee of AOU Careggi Hospital (Ref. Protocol 58-11). A total of 20 independent S-ASC populations have been obtained from the stromal vascular fraction (SVF) derived from multiple fragments of subcutaneous adipose tissue of the same subjects. We excluded all subjects affected by insulin-resistance, cancer, infections, inflammation or taking thiazolidinediones or steroids.
2.3.Isolation and culture of human adipose-derived stem cells from SAT Subcutaneous adipose stem cell (S-ASC) isolation and characterization was performed as described elsewhere (Baglioni et al, 2009; 2012), from SVF derived from mechanic and enzymatic processing of adipose tissue biopsies obtained during bariatric surgery. Briefly, 24 hours after SVF plating onto 100-mm cell culture dishes in complete culture medium [DMEM containing 20% fetal bovine 6 Page 6 of 23
serum (FBS), 100 µg/ml streptomycin, 100 U/ml penicillin, 2 mM L-glutamine, and 1 µg/ml amphotericin-B], non-adherent contaminating cells were removed. Confluent cells were trypsinized and expanded in T75 flasks (passage 1, P1) by culturing at 37°C in humidified atmosphere with 5% CO2. Confluent and homogeneous fibroblast-like cell populations, named ASC, were obtained after 2–3 weeks of culture.
2.4.Experimental protocols In all the experiments, only cells at early culture passages were used (P1–P3). Cells plated at different density according to the experiment, were grown to subconfluence or to confluence for the adipogenesis induction, were subjected to the different treatment protocols, after an overnight starvation by FBS lowering from 20% to 5%. Each experiment was performed at least 3 times with the indicated replicates using ASC populations derived from at least 3 independent subjects.
2.5.ASC Viability and Proliferation Assays Cell count. Seeded cells (7x103 ASC) were treated for the indicated times, then trypsinized and counted in the haemocytometer. Mean cell number was obtained by counting 4 replicates for each point in each experiment. Dead cells were excluded by trypan blue exclusion test. MTS assay. Five hundred cells were seeded in 96-well plates. After 24-hour starvation in low serum medium they were treated for the indicated times and cell number evaluated by MTS assay (Promega), according to the manufacturer’s instructions. Sample absorbance was measured on an ELISA plate reader (Wallac 1420, Perkin Elmer) at 490 nm wavelength. DNA synthesis assay-[3H]Thymidine uptake. DNA synthesis was evaluated according to the amount of [3H]TdR incorporated into trichloroacetic acid (TCA)-precipitated materials. Cells grown till 70% confluence, were 24 h-starved, then treated with liraglutide (10 and 100 nM) for 24 and 48 hours, pulsing them with 0.5 µCi/ml [3H]TdR (6.7 Ci/mmol) for 4 hours before stopping 7 Page 7 of 23
proliferation in ice-cold 10% TCA. After washing in 5% TCA, cells were solubilized in 0.25 N NaOH,at 37°C and radioactivity was measured by scintillation counter. Experiments were performed in quadruplicate in 3 independent cell populations.
2.6. Glucose uptake measurement Ten thousand S-ASC were seeded in 12-well plates and grown at confluence. S-ASC were washed twice with PBS and overnight incubated in serum free and low glucose medium (DMEM with 0.55 mM glucose). Cells were left untreated (Ctrls) or treated for 1 hour with increasing concentrations of insulin (10-100-1000 nM) in the presence or absence of liraglutide 10-100nM. After washing with PBS, cells were incubated with Hepes buffer (140 mM NaCl, 20 mM Hepes-Na pH 7.4, 2.5 mM MgSO4, 1mM CaCl2, 5 mM KCl) containing 2-deoxy-[3H]D-glucose [1 μCi/μl] for 10 minutes at 37°C. Cells were washed twice in PBS cold, then lysed in 100 mM NaOH for 1 hour at 37°C. Radioactivity was measured by a scintillation counter. Data were normalized on protein content measured by Bradford Reagent (Sigma-Aldrich, Milan, Italy), according to the manifacturer’s instructions.
2.7. In vitro adipose differentiation S-ASC (105 cells) were cultured in a 6-well plate in 10% FBS-DMEM, 0.5 mM 3-isobutyl-1methylxanthine, 1 mM dexamethasone, 200 mM indomethacin and 10 mM insulin (DIM cocktail) for 2 weeks, then shifted to 10% FBS-DMEM containing 1.7 mM insulin for another week (Baglioni et al, 2009; 2012).
2.8. Intracellular triglyceride staining techniques Oil Red O: adipose differentiation was assessed by Oil Red O according to the manufacturer’s instructions and evaluated by light microscopy. 8 Page 8 of 23
AdipoRed spectrofluorimetric quantification: adipose differentiation was assessed by AdipoRed staining according to the manufacturer’s instructions. AdipoRed fluorescence emission was measured with an ELISA plate reader (Wallac 1420, Perkin Elmer) at 485/572 nm excitation/emission. Specific absorbance of the differentiated adipocytes was calculated as fold increase on unspecific absorbance of the undifferentiated S-ASC cultured for the same time interval in 10% FBS-DMEM. Each point obtained in 10 replicates from 3 independent experiments. AdipoRed fluorescence microscopy: S-ASC grown on glass coverslips were subjected to adipose differentiation in the absence or presence of liraglutide, then treated with AdipoRed staining as described above. After fixation in 4% paraformaldehyde, cells were counterstained with DAPI [(1:2000), Sigma-Aldrich, Milan, Italy]. Fluorescence images were acquired with a Leica DM4000 epifluorescence microscope (Leica Microsystems GmbH, Wetzlar, Germany).
2.9. RNA isolation and quantitative real-time RT-PCR RNA isolation from cells was performed as previously detailed (Baglioni et al, 2009; 2012) and quantitative real-time RT-PCR was carried out using primers and probes for FABP4, APN and GAPDH genes (Taqman Gene Expression Assay, Life Technologies, respective codes: HS00609791_m1, HS00605917_m1, FAM MGB 4325934-1301038). The amount of target, normalized to the endogenous reference gene (GAPDH) and relative to a calibrator (Stratagene, La Jolla, CA, USA) was calculated by 2-∆∆Ct.
2.10. Statistical Analysis Statistical analysis was performed using SPSS 20.0 software (SPSS Inc. Chicago, IL, USA). The Kolmogorov–Smirnov test was used to verify the normal distribution of data which were expressed as mean ±SE. One way ANOVA followed by post-hoc Dunnett’s test was applied for multiple comparisons, while Student’s t test was applied for statistical analysis of two classes of data. A p<0.05 value was considered statistically significant. 9 Page 9 of 23
3. RESULTS 3.1. Liraglutide inhibited cell proliferation and metabolism of ASC The effects of liraglutide in vitro on cell viability in S-ASC were evaluated by MTS assay. ASC exponential growth was significantly inhibited by liraglutide and can be observed in a time dependent manner (Fig.1A). In particular, the inhibitory effects of the GLP-1 analogue was significantly different for all doses starting from day 5 of treatment. A dose response effect was not evident except for the highest dose (5000 mM) at 7 days. The effect of liraglutide on S-ASC was confirmed and more evident as assessed by direct cell count experiments in cells treated with 10 and 100 nM liraglutide up to 15 days (Fig.1B). Reduction in cell number exerted by liraglutide at both concentrations was maximal after 6 days (Fig.1C). Finally, the inhibitory effect on cell proliferation was assessed by [3H]-thymidine incorporation after 24 and 48 hour incubation with 10 and 100 nM liraglutide (Fig.1D). No significant difference in the inhibitory effect of liraglutide was evident between 10 and 100 nM (Fig.1). In order to study the putative effect of the GLP-1 analogue on cell metabolism, the ability of liraglutide to interfere with glucose uptake was evaluated in cultured S-ASC. Glucose uptake experiments were performed in S-ASC after stimulation with increasing doses of insulin (1-100 nM) in the presence or absence of different concentrations of liraglutide (10-1000 nM), (Fig.2). Insulin treatment induced an increase in glucose uptake (Fig.2, dashed line), which was significantly inhibited by increasing concentrations of the GLP-1 analogue with a maximal effect with liraglutide 1000 nM.
3.2.Liraglutide interfered with in vitro adipose differentiation. Our group had previously demonstrated that cultured S-ASC can be differentiated in vitro towards the adipose lineage when maintained in the appropriate inductive medium, resulting in formation of adipocytes with small lipid droplets (Baglioni et al, 2009; 2012). After adipogenic differentiation in the absence or presence of 10 nM and 100 nM liraglutide, Oil Red O (Fig.3A-F) and AdipoRed 10 Page 10 of 23
(Fig.3G-I) staining of intracellular triglycerides showed a reduction of differentiated adipocytes in liraglutide-treated wells. Quantitative evaluation of AdipoRed staining confirmed a statically significant lower level of fluorescent intensity when S-ASC were differentiated in the presence of liraglutide, with a similar inhibitory effect obtained with the 2 doses of liraglutide (Fig.3M). Interestingly, when adipogenic markers were evaluated by Taqman analysis of gene expression, FABP4 (a marker of overall adipocyte differentiation) was reduced, whereas adiponectin (APN) was concomitantly increased by liraglutide (Tab.1).
3.3.Exendin 9-39 treatment reverted the liraglutide inhibitory effects on cell viability and adipocyte differentiation. To confirm the specificity of the anti-proliferative effects exerted by liraglutide in S-ASC, we repeated the experiments in the presence of 10 nM exendin 9-39, the antagonist of GLP-1 receptor, which reverted the effects of liraglutide on cell viability, assessed either through MTS assay (Fig.4A) or cell counts (Fig.4B). In addition, exendin 9-39 abolished the effects of both doses of liraglutide on adipogenesis, measured with AdipoRed staining (Fig.5).
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4.DISCUSSION The present data show that liraglutide is capable of inhibiting in vitro proliferation and differentiation of human adipose stem cells derived from obese subjects.. This cell effect, which is evident within a few days of exposure, may contribute to the regulation of body weight observed in obese patients. The effect appears to be specific and probably mediated by the GLP-1 receptor, as it is inhibited by the GLP-1 receptor antagonist exendin 9-39. The effect of liraglutide seems already maximal at 10 nM with no significant increase for higher doses. This dose range (10-100 nM) is compatible with the 8-12 h peak obtained after a single subcutaneous injection of 1.2-1.8 mg in both healthy and diabetic subjects (Watson et al, 2010; Jiang et al, 2011). These results are partly discordant from those previously reported in 3T3-L1 mouse cells (Challa et al, 2012; Yang et al, 2013a), a line of immortalized fibroblasts with some of the functional characteristics of pre-adipocytes, showing that liraglutide and GLP-1 stimulates proliferation and adipocyte differentiation. This discrepancy could be due to differences between species (humans vs. rodents) or between cell types (pre-adipocytes ex vivo vs. immortalized fibroblasts). Interestingly, GLP-1 has been reported to inhibit adipocyte differentiation from human mesenchymal stem cells from bone marrow (Sanz et al, 2010), suggesting that the adipogenesis-inhibitory effect may be specific in humans. Moreover, our model of adipose precursors is derived from obese subjects and should represent the real target of liraglutide in obese treated patients, compared to the healthy adipocytes studied in the other papers where liraglutide may act in a different manner. In the present study, liraglutide was also found to inhibit insulin-induced glucose uptake in human ASC. This result could seem surprising, considering that GLP-1 and its receptor agonists are usually reported to exert insulin-sensitizing effects, both in vivo and in several tissues in vitro. Previous papers reported a stimulatory effect of GLP-1R agonists on glucose uptake and metabolism (Sancho et al, 2005; Gao et al, 2007; Yang et al 2013b) as well as lipogenesis (Sancho et al, 2005). Conversely, lipolysis inhibition is described in mature adipocytes isolated from rats (Sancho et al, 2005) and obtained from mouse 3T3-L1 precursors (Montrose-Rafizadeh et al, 1997), 12 Page 12 of 23
as well as in human myocytes (Luque et al, 2002). The opposite effect observed in our ASC model may be due to a different action of GLP-1R agonists on stem cells and mature cells. In fact, in our precursor model liraglutide significantly reduces cell glucose-uptake maybe due to inhibition of both proliferation and differentiation, whereas in fully differentiated mature cells such as mature adipocytes, skeletal muscle as well as cardiomyocytes, this molecule acts as an insulin sensitizer. In this respect, the reduction of insulin-induced glucose uptake may be viewed as an overall inhibition of metabolic activity related to the reduction of cell proliferation and differentiation in adipocyte precursors. Moreover, the observed difference may be due to liraglutide action in precursors derived from obese instead of healthy subjects. The adipose tissue does not play a relevant role in overall glucose uptake in vivo, but it contributes to the regulation of glucose metabolism and insulin sensitivity through secretion of adipocytokines. The expression of adiponectin, a well-known adipocyte hormone which improves insulin sensitivity, appears to be increased by liraglutide in ASC, thus suggesting that liraglutide not only limits the differentiation rate of adipose cells obtained from dysfunctional tissue of obese subjects, but also ameliorates the functional properties of the obtained adipocytes. This result is in line with observations from clinical trials, showing that both exenatide (Bunck et al, 2010) and liraglutide (Yang et al, 2013c) increase circulating adiponectin levels in patients with type 2 diabetes. However, it should be considered that the increase of adiponectin in clinical trials could have been determined by weight loss (Calvani et al, 2004; Rossmeislovà et al, 2013), whereas the present data, obtained in vitro, suggest a direct effect of GLP-1 receptor stimulation on adiponectin synthesis. Interestingly, treatment with the synthetic GLP-1 receptor agonist exendin-4 stimulates adiponectin secretion in mature 3T3-L1 adipocytes (Kim Chung et al, 2009). Our findings suggest that in obese subjects, liraglutide reduces proliferation and differentiation of adipose stem cells, while ameliorating functionality of the few adipocytes obtained following in vitro adipogenesis. Some limitations of the present study should be considered when interpreting its results. Adipocyte precursors were obtained from morbidly obese subjects; the possibility that the corresponding cells 13 Page 13 of 23
from healthy individuals have a different behavior should be taken into account, but goes beyond the object of our study. The experiments reported were performed on primary cultures of adipose stem cells, which could have been contaminated by other cell types (in particular, endothelial cells) thus possibly interfering with the results (Baglioni et al, 2009; 2012). Furthermore, the number of patients from whom the cells were obtained is relatively limited (n=11), and the possibility of interindividual variations cannot be completely ruled out. Despite these limitations, the present data highlight a further and previously unknown mechanism which could contribute to the effect of GLP-1 receptor agonists in obese subjects. The molecular pathways and the clinical impact of this effect deserve further investigations.
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DECLARATION OF INTEREST Dr. E. Mannucci has received consultancy fees from Novo Nordisk, Eli Lilly, AstraZeneca, and GSK; speaking fees from Novo Nordisk, Eli Lilly, AstraZeneca, and Sanofi; research grants from Novo Nordisk, Eli Lilly and AstraZeneca. The other authors have no conflicts to declare.
ACKNOWLEDGMENTS We thanks Dr. Tommaso Mello (University of Florence) for fluorescence image acquisition. This study was supported by an unrestricted research grant by Novo Nordisk and by the Italian Ministry of University and Research (PRIN 2011 prot. 2010C8ERKX).
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16. Jiang, J., Zhang, J., Jacobsen, L.V., Hu, P. 2011. The pharmacokinetics, pharmacodynamics, and tolerability of liraglutide, a once-daily human GLP-1 analogue, after multiple subcutaneous administration in healthy Chinese male subjects. Clin. Pharmacol. 51,1620-7. 17. Kastin, A.J., Akerstrom, V. and Pan, W., 2002. Interactions of glucagon-like peptide-1 (GLP-1) with the blood–brain barrier. J. Mol. Neurosci. 18, 7–14. 18. Kim Chung, T., Hosaka, T., Yoshida, M., Harada, N., Sakaue, H., Sakai, T., Nakaya, Y., 2009. Exendin-4, a GLP-1 receptor agonist, directly induces adiponectin expression through protein kinase A pathway and prevents inflammatory adipokine expression. Biochem. Biophys. Res. Commun. 390, 613-8. 19. Larsen, P.J., Tang-Christensen, M., Holst, J.J., Orskov, C., 1997. Distribution of glucagonlike peptide-1 and other preproglucagon-derived peptides in the rat hypothalamus and brainstem. Neuroscience 77, 257–270 20. Luque, M.A., González, N., Márquez, L., Acitores, A., Redondo, A., Morales, M., Valverde, I., Villanueva-Peñacarrillo, M.L, 2002. Glucagon-like peptide-1 (GLP-1) and glucose metabolism in human myocytes. J. Endocrinol. 173, 465-73. 21. Monami, M., Marchionni, N., Mannucci, E., 2009 Glucagon-like peptide-1 receptor agonists in type 2 diabetes: a meta-analysis of randomized clinical trials. Eur. J. Endocrinol. 160. 909-17. 22. Montrose-Rafizadeh, C., Yang, H., Wang, Y., Roth, J., Montrose, M.H., Adams, L.G., 1997. Novel signal transduction and peptide specificity of glucagon-like peptide receptor in 3T3L1 adipocytes. J. Cell. Physiol. 172, 275-83 23. Nauck, M.A., 2013. A critical analysis of the clinical use of incretin-based therapies: The benefits by far outweigh the potential risks. Diabetes Care 36, 2126-32. 24. Rossmeislová, L., Malisová, L., Kracmerová, J., Tencerová, M., Kovácová, Z., Koc, M., Siklová-Vítková, M., Viquerie, N., Langin, D., Stich, V., 2013. Weight loss improves the
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adipogenic capacity of human preadipocytes and modulates their secretory profile. Diabetes 62, 1990-5. 25. Sancho. V,, Trigo, M.V., González, N., Valverde, I., Malaisse, W.J., VillanuevaPeñacarrillo, M.L., 2005. Effects of glucagon-like peptide-1 and exendins on kinase activity, glucose transport and lipid metabolism in adipocytes from normal and type-2 diabetic rats. J. Mol. Endocrinol. 35, 27-38. 26. Sanz, C., Vázquez, P., Blázquez, C., Barrio, P.A., Alvarez Mdel, M., Blázquez, E., 2010. Signaling and biological effects of glucagon-like peptide 1 on the differentiation of mesenchymal stem cells from human bone marrow. Am. J. Physiol. Endocrinol. Metab. 298, E634-43. 27. Turton, M.D., O'Shea, D., Gunn, I., Beak, S.A., Edwards, C.M., Meeran, K., Choi, S.J., Taylor, G.M., Heath, M.M., Lambert, P.D., Wilding, J.P., Smith, D.M., Ghatei, M.A., Herbert, J., Bloom, S.R., 1996. A role for glucagon-like peptide-1 in the central regulation of feeding. Nature 379, 69-72. 28. van Bloemendaal, L., Ten Kulve, J.S., la Fleur, S.E., Ijzerman, R.G., Diamant, M., 2014. Effects of glucagon-like peptide 1 on appetite and body weight: focus on the CNS. J. Endocrinol. 221, T1-16. 29. Watson, E., Jonker, D.M., Jacobsen, L.V., Ingwersen, S.H. 2010. Population pharmacokinetics of liraglutide, a once-daily human glucagon-like peptide-1 analog, in healthy volunteers and subjects with type 2 diabetes, and comparison to twice-daily exenatide. J. Clin. Pharmacol. 50,886-94. 30. Yang, J., Ren, J., Song, J., Liu, F., Wu, C., Wang, X., Gong, L., Li, W., Xiao, F., Yan F., Hou, X., Chen, L. 2013. Glucagon-like peptide 1 regulates adipogenesis in 3T3-L1 preadipocytes. Int. J. Mol. Med. 31, 1429-35a.
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31. Yang, J., Ren, J., Song, J., Liu, F., Wu, C., Wang, X., Gong, L., Li, W., Xiao, F., Yan, F., Hou, X., Chen, L., 2013. Glucagon-like peptide 1 regulates adipogenesis in 3T3-L1 preadipocytes. Int. J. Mol. Med. 31, 1429-35b. 32. Yang, M., Liu, R., Li, S., Luo, Y., Zhang, Y., Zhang, L., Liu, D., Wang, Y., Xiong, Z., Boden, G., Chen, S., Li, L., Yang, G., 2013. Zinc-α2-glycoprotein is associated with insulin resistance in humans and is regulated by hyperglycemia, hyperinsulinemia, or liraglutide administration: cross-sectional and interventional studies in normal subjects, insulinresistant subjects, and subjects with newly diagnosed diabetes. Diabetes Care 36, 1074-82c.
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FIGURE LEGENDS
Fig.1: Liraglutide inhibitory effects on cell viability and proliferation. A: Results are expressed as mean±SE MTS absorbance fold increase (FI) over day 0 (A) or haemocytometer cell counting (B) at the different time points and in the absence (Ctrl) or presence of liraglutide. Mean values of all Ctrl time points were significantly different vs. day 1, p<0.001, as evaluated with ANOVA analysis. Difference significance between liraglutide-stimulated vs. respective controls was evaluated by ANOVA analysis (F and p values are indicated) followed by Dunnett’s post-hoc test: * p<0.05,** p<0.01, § p<0.001 vs. respective controls. Experiments were performed in 6 different S-ASC populations. C: Bar-graph shows the viability percentage inhibition exerted by liraglutide 10 nM and 100 nM vs. respective Ctrls at the indicated time points, calculated from panel B. D: Data represent mean±SE cpm after a 4h pulse of [3H]-thymidine in ASC stimulated for 24 and 48 hours with liraglutide 10 and 100 nM. Percentage of inhibition vs. controls is indicated. Difference significance between liraglutide-stimulated vs. controls was evaluated by ANOVA analysis (F and p values are indicated) followed by Dunnett’s post-hoc test: ** p<0.01, § p<0.001 vs. respective controls. Experiments were performed in 3 different S-ASC populations.
Fig.2: Inhibitory effects of liraglutide on glucose uptake after insulin stimulation. Glucose uptake was performed in S-ASC after stimulation with increasing doses of insulin (1-10100 nM) in the absence (dot line) or presence (bar-graphs) of different concentrations of liraglutide (10-100-1000 nM). Results represent mean±SE glucose uptake fold increase (FI) vs. Ctrl without insulin. All mean values of insulin treated cells were significantly different vs. non-stimulated Ctrl, p<0.001 as evaluated with ANOVA analysis. Difference significance between liraglutide-stimulated vs. nonstimulated cells was evaluated by ANOVA analysis (F and p values are indicated) followed
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by Dunnett’s post-hoc test: * p<0.05 and ** p<0.001 vs. respective controls without liraglutide. Experiments were performed in 3 different S-ASC populations.
Fig.3: Liraglutide interferes with adipogenic differentiation induced in vitro. Oil Red O and AdipoRed fluorescence staining of intracellular triglyceride depots in adipocytes obtained from in vitro-differentiation of S-ASC in the absence (ADIPO) or presence (ADIPO+LIRA) of liraglutide 10 and 100 nM. Representative microscopy images of in vitrodifferentiated adipocytes stained with Oil Red O (red A-F) and AdipoRed (green G-I), with DAPI counterstaining (blue J-L). No Oil Red O or AdipoRed staining was present in liraglutide-untreated undifferentiated ASC controls (not shown). Magnification (10x A-C, 20x D-L), inset (40x). Bargraphs represent mean±SE absorbance fold increase (FI) vs. ASC after AdipoRed staining evaluated spectrofluorimetrically (M). Difference significance between liraglutide-stimulated vs. untreated cells (ADIPO) was valuated by ANOVA analysis (F and p values are indicated) followed by Dunnett’s post-hoc test: * p<0.05 vs. ADIPO.
Fig.4: Exendin 9-39 reverts the liraglutide inhibitory effect on cell viability. S-ASC were treated with liraglutide at the indicated doses (10-100nM) in the presence or absence of exendin 9-39 (10 nM) for the indicated time. A: Bar-graphs represent mean±SE MTS absorbance fold increase (FI) over day 0 of untreated (Ctrl) or treated cells with liraglutide (10-100nM) or a combination of liraglutide (10-100 nM) and exendin 9-39 (10 nM). B: Histograms represent mean±SE cell counting at each day of indicated treatment. Difference significance between liraglutide-stimulated vs. untreated cells was evaluated by ANOVA analysis (F and p values are indicated) followed by Dunnett’s post-hoc test: * p<0.05 and ** p<0.001 vs. respective liraglutide-untreated controls. Student’s t test was used to evaluate the statistical significance of exendin 9-39 effect: § p<0.001 exendin 9-39 vs. respective liraglutide doses. Experiments were performed in 4 different S-ASC populations. 22 Page 22 of 23
Fig.5: Exendin 9-39 interferes with liraglutide inhibition of in vitro-induced adipose differentiation. S-ASC were differentiated in vitro towards adipocytes in the absence or presence of liraglutide (10100nM) alone or combined with 10 nM exendin 9-39. At the end of differentiation, cells were stained with AdipoRed and absorbance evaluated spectrofluorimetrically. Bar-graphs represent mean±SE AdipoRed absorbance percentage over liraglutide-untreated adipocytes
(% ADIPO
differentiation). Statistical significance of exendin 9-39 reversion effect was evaluated by Student’s t test: *p<0.05, **p<0.005 exendin 9-39 vs. respective liraglutide doses. Data were obtained in 4 SASC independent populations.
Tab.1: Liraglutide interferes with adipogenesis but positively regulates adiponectin expression. Taqman analysis of FABP4 and APN expression normalized on GAPDH in S-ASC differentiated in vitro towards adipocytes in the absence or presence of liraglutide (LIRA 1-100 nM). Data are expressed in mean±SE fold increase on liraglutide-untreated adipocytes (ADIPO) from n=3 independent experiments performed in duplicates. F and p values from ANOVA analysis are indicated followed by post hoc Dunnett’s test: *p<0.05, **p<0.005, ***p<0.001 vs. liraglutide untreated adipocytes (ADIPO). ADIPO+1 nM LIRA
ADIPO+10 nM LIRA
ADIPO+100 nM LIRA
FABP4
F=5.176 p=0.008
0.82±0.07
0.67±0.09*
0.54±0.16**
APN
F=8.991 p=0.001
1.86±0.11
2.64±0.43***
2.28±0.26**
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S0303-7207(14)00424-9 http://dx.doi.org/doi: 10.1016/j.mce.2014.12.021 MCE 8995
To appear in:
Molecular and Cellular Endocrinology
Received date: Revised date: Accepted date:
7-12-2014 27-12-2014 28-12-2014
Please cite this article as: Giulia Cantini, Alessandra Di Franco, Jinous Samavat, Gianni Forti, Edoardo Mannucci, Michaela Luconi, Effect of liraglutide on proliferation and differentiation of human adipose stem cells, Molecular and Cellular Endocrinology (2015), http://dx.doi.org/doi: 10.1016/j.mce.2014.12.021. 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.
Effect of liraglutide on proliferation and differentiation of human adipose stem cells Giulia Cantini1§, Alessandra Di Franco1§, Jinous Samavat1, Gianni Forti1, Edoardo Mannucci2*, Michaela Luconi1* 1
Endocrinology Unit, Dept. Experimental and Clinical Biomedical Sciences, University of Florence, Italy 2 Diabetes Agency, Azienda Ospedaliera-Universitaria Careggi, Florence, Italy §These authors equally contributed to the study
Running title: GLP-1 effects on human adipose stem cells
Keywords: Glucagone-Like-Peptide-1, adipogenesis, adipose stem cells, weight loss
*Correspondence: Dr. Edoardo Mannucci, M.D. Diabetes Agency Azienda Ospedaliero-Universitaria Careggi, Via delle Oblate 4, 50141 Florence-Italy Tel:+39 055 7945476 Fax:+39 055 7949742 [email protected] and Prof. Michaela Luconi, Ph.D. Endocrinology Unit Dept. Experimental and Clinical Biomedical Sciences University of Florence Viale Pieraccini 6, 50139 Florence-Italy Tel:+39 055 4271369 Fax:+39 055 4271371 [email protected]
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Highlights 1. Liraglutide interfers with human adipose stem cell proliferation and differentiation in vitro.
2. Liraglutide inhibits lipid accumulation in differentiating cells but stimulates adiponectin expression.
3. This in vitro effect on adipocyte precursors may contribute to the drug effect in obese patients.
ABSTRACT Glucagon-Like Peptide-1 (GLP-1) receptor agonists, used as glucose-lowering drugs, also induce weight loss by inhibiting food intake. The present study was aimed at the assessment of the in vitro effects of the GLP-1 receptor agonist liraglutide on proliferation and differentiation of human adipose stem cells (ASC) obtained from subcutaneous adipose tissue of morbidly obese subjects undergoing bariatric surgery. Liraglutide (10-100 nM) significantly inhibited ASC proliferation and viability, with a maximum effect at 6 days of culture (45% and 50%, for liraglutide 10 and 100 nM, respectively); the effect was reverted by exendin 9-39. Glucose uptake was significantly reduced by liraglutide in a dose dependent manner. Treatment with liraglutide reduced intracellular lipid accumulation in differentiating ASC, together with FABP-4 mRNA expression (-18%, -23%, -46%, for 1 nM, 10 nM and 100 nM, respectively), whereas it stimulated adiponectin (APN) expression (1.86, 2.64, 2.28 fold increase, for 1 nM, 10 nM and 100 nM, respectively). Liraglutide exerts effects on human adipose cell precursors, inhibiting proliferation and differentiation, while stimulating the expression of the insulin-sensitizing adipokine APN. These
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effects could contribute to the actions of GLP-1 receptor agonists on body weight and insulin sensitivity.
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1.INTRODUCTION Glucagon-Like Peptide-1 (GLP-1) is a gastrointestinal hormone, mainly produced after meals, which stimulates insulin secretion, and inhibits glucagon release in a dose-dependent fashion (Nauck et al, 2013). GLP-1 receptor agonists are currently used for the treatment of type 2 diabetes. It has been observed that these drugs also produce a relevant weight loss (Monami et al, 2009); for this reason, some of the molecules of this class are currently under development as a treatment for obesity in non-diabetic individuals, with interesting results from clinical trials (Astrup et al, 2009; 2012). Mechanisms of weight loss with GLP-1 and its analogues are probably complex and not completely elucidated. The stimulation of GLP-1 receptors in the hypothalamus, and particularly in paraventricular nuclei (PVN), is capable of inducing satiety and inhibiting food intake in rodents (Turton et al, 1996; Dalvi et al, 2012). Interestingly, GLP-1- producing neurons localized in the nucleus of the solitary tract and activated by food consumption, project their terminations to PVN, constituting a feedback loop inhibiting further food intake after meals (Larsen et al 1997). In addition, peripherally-secreted GLP-1 is capable of crossing the blood-brain barrier, at least in the hypothalamus, stimulating central receptors (Kastin et al, 2002; Holst 2004). It has also been observed that GLP-1 and its short-acting receptor agonists delay gastric emptying, possibly contributing to the inhibition of food intake; this could be due to either central or peripheral effects (van Bloemendaal et al 2014; Dailey et al, 2013). However, the relevance of this mechanism for the regulation of body weight is questionable. In fact, long acting formulations of GLP-1 receptor agonists (exenatide long-acting release and liraglutide), which have no relevant effect on gastric emptying, produce a weight loss similar to that of shorter acting agonists (Buse et al, 2009; Drucker et al, 2008; Blevins et al 2011). GLP-1 and its receptor agonists have shown additional extra-glycemic effects on many organs and tissues different from pancreatic islets and central nervous system, including endothelium and myocardium (Herzlinger et al, 2013). The possibility of a direct effect of GLP-1 receptor agonists 4 Page 4 of 23
on the adipose tissue and on pre-adipocytes has not been thoroughly explored so far. In 3T3-L1 mouse cells, a line of immortalized fibroblasts with some functional characteristics of preadipocytes, GLP-1 and liraglutide reduced apoptosis and stimulated adipocyte differentiation (Challa et al, 2012; Yang et al, 2013a), although an opposite effect was described in human mesenchymal stem cells from bone marrow (Sanz et al, 2010). In mature adipocytes obtained from rat adipose tissue and from 3T3-L1 adipocytes differentiated in vitro, GLP-1 receptor agonists induce an increase in glucose metabolism and lipogenesis (Sancho et al, 2005; Gao et al, 2007; Yang et al 2013b). The aim of the present study was to evaluate the potential in vitro effects of liraglutide on a model of human adipose stem cells (ASC) obtained from human subcutaneous adipose tissue of obese subjects, which can be differentiated in vitro to mature adipocytes.
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2.MATERIALS AND METHODS 2.1.Materials Media and sera for cell cultures were purchased from Sigma-Aldrich (Milan, Italy) and tissue plasticware was obtained from Corning (Milan, Italy). AdipoRed and MTS (CellTiter 96® Aqueous One Solution Cell proliferation assay) were purchased from Lonza (Milan, Italy) and Promega (Madison, WI, USA), respectively. Liraglutide was kindly provided by Novo Nordisk (Bagsvaert, Danmark), while exendin fragment 9-39 was purchased from Sigma-Aldrich (Milan, Italy). Other reagents for cell cultures, fluorescence microscopy were obtained from Sigma-Aldrich (Milan, Italy), except where differently indicated. [3H]-Thymidine was provided by Perkin Elmer (Waltham, Massachusetts, USA).
2.2.Subjects and Ethics Statement Subcutaneous adipose tissue samples were obtained from 11 subjects (4 males and 7 females: mean age±SD: 52±15 yrs; BMI±SD: 39.9±7.7 kg/m2), undergoing bariatric surgery at AOU Careggi Hospital, following written informed consent. All the protocols were approved by the Local Ethical Committee of AOU Careggi Hospital (Ref. Protocol 58-11). A total of 20 independent S-ASC populations have been obtained from the stromal vascular fraction (SVF) derived from multiple fragments of subcutaneous adipose tissue of the same subjects. We excluded all subjects affected by insulin-resistance, cancer, infections, inflammation or taking thiazolidinediones or steroids.
2.3.Isolation and culture of human adipose-derived stem cells from SAT Subcutaneous adipose stem cell (S-ASC) isolation and characterization was performed as described elsewhere (Baglioni et al, 2009; 2012), from SVF derived from mechanic and enzymatic processing of adipose tissue biopsies obtained during bariatric surgery. Briefly, 24 hours after SVF plating onto 100-mm cell culture dishes in complete culture medium [DMEM containing 20% fetal bovine 6 Page 6 of 23
serum (FBS), 100 µg/ml streptomycin, 100 U/ml penicillin, 2 mM L-glutamine, and 1 µg/ml amphotericin-B], non-adherent contaminating cells were removed. Confluent cells were trypsinized and expanded in T75 flasks (passage 1, P1) by culturing at 37°C in humidified atmosphere with 5% CO2. Confluent and homogeneous fibroblast-like cell populations, named ASC, were obtained after 2–3 weeks of culture.
2.4.Experimental protocols In all the experiments, only cells at early culture passages were used (P1–P3). Cells plated at different density according to the experiment, were grown to subconfluence or to confluence for the adipogenesis induction, were subjected to the different treatment protocols, after an overnight starvation by FBS lowering from 20% to 5%. Each experiment was performed at least 3 times with the indicated replicates using ASC populations derived from at least 3 independent subjects.
2.5.ASC Viability and Proliferation Assays Cell count. Seeded cells (7x103 ASC) were treated for the indicated times, then trypsinized and counted in the haemocytometer. Mean cell number was obtained by counting 4 replicates for each point in each experiment. Dead cells were excluded by trypan blue exclusion test. MTS assay. Five hundred cells were seeded in 96-well plates. After 24-hour starvation in low serum medium they were treated for the indicated times and cell number evaluated by MTS assay (Promega), according to the manufacturer’s instructions. Sample absorbance was measured on an ELISA plate reader (Wallac 1420, Perkin Elmer) at 490 nm wavelength. DNA synthesis assay-[3H]Thymidine uptake. DNA synthesis was evaluated according to the amount of [3H]TdR incorporated into trichloroacetic acid (TCA)-precipitated materials. Cells grown till 70% confluence, were 24 h-starved, then treated with liraglutide (10 and 100 nM) for 24 and 48 hours, pulsing them with 0.5 µCi/ml [3H]TdR (6.7 Ci/mmol) for 4 hours before stopping 7 Page 7 of 23
proliferation in ice-cold 10% TCA. After washing in 5% TCA, cells were solubilized in 0.25 N NaOH,at 37°C and radioactivity was measured by scintillation counter. Experiments were performed in quadruplicate in 3 independent cell populations.
2.6. Glucose uptake measurement Ten thousand S-ASC were seeded in 12-well plates and grown at confluence. S-ASC were washed twice with PBS and overnight incubated in serum free and low glucose medium (DMEM with 0.55 mM glucose). Cells were left untreated (Ctrls) or treated for 1 hour with increasing concentrations of insulin (10-100-1000 nM) in the presence or absence of liraglutide 10-100nM. After washing with PBS, cells were incubated with Hepes buffer (140 mM NaCl, 20 mM Hepes-Na pH 7.4, 2.5 mM MgSO4, 1mM CaCl2, 5 mM KCl) containing 2-deoxy-[3H]D-glucose [1 μCi/μl] for 10 minutes at 37°C. Cells were washed twice in PBS cold, then lysed in 100 mM NaOH for 1 hour at 37°C. Radioactivity was measured by a scintillation counter. Data were normalized on protein content measured by Bradford Reagent (Sigma-Aldrich, Milan, Italy), according to the manifacturer’s instructions.
2.7. In vitro adipose differentiation S-ASC (105 cells) were cultured in a 6-well plate in 10% FBS-DMEM, 0.5 mM 3-isobutyl-1methylxanthine, 1 mM dexamethasone, 200 mM indomethacin and 10 mM insulin (DIM cocktail) for 2 weeks, then shifted to 10% FBS-DMEM containing 1.7 mM insulin for another week (Baglioni et al, 2009; 2012).
2.8. Intracellular triglyceride staining techniques Oil Red O: adipose differentiation was assessed by Oil Red O according to the manufacturer’s instructions and evaluated by light microscopy. 8 Page 8 of 23
AdipoRed spectrofluorimetric quantification: adipose differentiation was assessed by AdipoRed staining according to the manufacturer’s instructions. AdipoRed fluorescence emission was measured with an ELISA plate reader (Wallac 1420, Perkin Elmer) at 485/572 nm excitation/emission. Specific absorbance of the differentiated adipocytes was calculated as fold increase on unspecific absorbance of the undifferentiated S-ASC cultured for the same time interval in 10% FBS-DMEM. Each point obtained in 10 replicates from 3 independent experiments. AdipoRed fluorescence microscopy: S-ASC grown on glass coverslips were subjected to adipose differentiation in the absence or presence of liraglutide, then treated with AdipoRed staining as described above. After fixation in 4% paraformaldehyde, cells were counterstained with DAPI [(1:2000), Sigma-Aldrich, Milan, Italy]. Fluorescence images were acquired with a Leica DM4000 epifluorescence microscope (Leica Microsystems GmbH, Wetzlar, Germany).
2.9. RNA isolation and quantitative real-time RT-PCR RNA isolation from cells was performed as previously detailed (Baglioni et al, 2009; 2012) and quantitative real-time RT-PCR was carried out using primers and probes for FABP4, APN and GAPDH genes (Taqman Gene Expression Assay, Life Technologies, respective codes: HS00609791_m1, HS00605917_m1, FAM MGB 4325934-1301038). The amount of target, normalized to the endogenous reference gene (GAPDH) and relative to a calibrator (Stratagene, La Jolla, CA, USA) was calculated by 2-∆∆Ct.
2.10. Statistical Analysis Statistical analysis was performed using SPSS 20.0 software (SPSS Inc. Chicago, IL, USA). The Kolmogorov–Smirnov test was used to verify the normal distribution of data which were expressed as mean ±SE. One way ANOVA followed by post-hoc Dunnett’s test was applied for multiple comparisons, while Student’s t test was applied for statistical analysis of two classes of data. A p<0.05 value was considered statistically significant. 9 Page 9 of 23
3. RESULTS 3.1. Liraglutide inhibited cell proliferation and metabolism of ASC The effects of liraglutide in vitro on cell viability in S-ASC were evaluated by MTS assay. ASC exponential growth was significantly inhibited by liraglutide and can be observed in a time dependent manner (Fig.1A). In particular, the inhibitory effects of the GLP-1 analogue was significantly different for all doses starting from day 5 of treatment. A dose response effect was not evident except for the highest dose (5000 mM) at 7 days. The effect of liraglutide on S-ASC was confirmed and more evident as assessed by direct cell count experiments in cells treated with 10 and 100 nM liraglutide up to 15 days (Fig.1B). Reduction in cell number exerted by liraglutide at both concentrations was maximal after 6 days (Fig.1C). Finally, the inhibitory effect on cell proliferation was assessed by [3H]-thymidine incorporation after 24 and 48 hour incubation with 10 and 100 nM liraglutide (Fig.1D). No significant difference in the inhibitory effect of liraglutide was evident between 10 and 100 nM (Fig.1). In order to study the putative effect of the GLP-1 analogue on cell metabolism, the ability of liraglutide to interfere with glucose uptake was evaluated in cultured S-ASC. Glucose uptake experiments were performed in S-ASC after stimulation with increasing doses of insulin (1-100 nM) in the presence or absence of different concentrations of liraglutide (10-1000 nM), (Fig.2). Insulin treatment induced an increase in glucose uptake (Fig.2, dashed line), which was significantly inhibited by increasing concentrations of the GLP-1 analogue with a maximal effect with liraglutide 1000 nM.
3.2.Liraglutide interfered with in vitro adipose differentiation. Our group had previously demonstrated that cultured S-ASC can be differentiated in vitro towards the adipose lineage when maintained in the appropriate inductive medium, resulting in formation of adipocytes with small lipid droplets (Baglioni et al, 2009; 2012). After adipogenic differentiation in the absence or presence of 10 nM and 100 nM liraglutide, Oil Red O (Fig.3A-F) and AdipoRed 10 Page 10 of 23
(Fig.3G-I) staining of intracellular triglycerides showed a reduction of differentiated adipocytes in liraglutide-treated wells. Quantitative evaluation of AdipoRed staining confirmed a statically significant lower level of fluorescent intensity when S-ASC were differentiated in the presence of liraglutide, with a similar inhibitory effect obtained with the 2 doses of liraglutide (Fig.3M). Interestingly, when adipogenic markers were evaluated by Taqman analysis of gene expression, FABP4 (a marker of overall adipocyte differentiation) was reduced, whereas adiponectin (APN) was concomitantly increased by liraglutide (Tab.1).
3.3.Exendin 9-39 treatment reverted the liraglutide inhibitory effects on cell viability and adipocyte differentiation. To confirm the specificity of the anti-proliferative effects exerted by liraglutide in S-ASC, we repeated the experiments in the presence of 10 nM exendin 9-39, the antagonist of GLP-1 receptor, which reverted the effects of liraglutide on cell viability, assessed either through MTS assay (Fig.4A) or cell counts (Fig.4B). In addition, exendin 9-39 abolished the effects of both doses of liraglutide on adipogenesis, measured with AdipoRed staining (Fig.5).
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4.DISCUSSION The present data show that liraglutide is capable of inhibiting in vitro proliferation and differentiation of human adipose stem cells derived from obese subjects.. This cell effect, which is evident within a few days of exposure, may contribute to the regulation of body weight observed in obese patients. The effect appears to be specific and probably mediated by the GLP-1 receptor, as it is inhibited by the GLP-1 receptor antagonist exendin 9-39. The effect of liraglutide seems already maximal at 10 nM with no significant increase for higher doses. This dose range (10-100 nM) is compatible with the 8-12 h peak obtained after a single subcutaneous injection of 1.2-1.8 mg in both healthy and diabetic subjects (Watson et al, 2010; Jiang et al, 2011). These results are partly discordant from those previously reported in 3T3-L1 mouse cells (Challa et al, 2012; Yang et al, 2013a), a line of immortalized fibroblasts with some of the functional characteristics of pre-adipocytes, showing that liraglutide and GLP-1 stimulates proliferation and adipocyte differentiation. This discrepancy could be due to differences between species (humans vs. rodents) or between cell types (pre-adipocytes ex vivo vs. immortalized fibroblasts). Interestingly, GLP-1 has been reported to inhibit adipocyte differentiation from human mesenchymal stem cells from bone marrow (Sanz et al, 2010), suggesting that the adipogenesis-inhibitory effect may be specific in humans. Moreover, our model of adipose precursors is derived from obese subjects and should represent the real target of liraglutide in obese treated patients, compared to the healthy adipocytes studied in the other papers where liraglutide may act in a different manner. In the present study, liraglutide was also found to inhibit insulin-induced glucose uptake in human ASC. This result could seem surprising, considering that GLP-1 and its receptor agonists are usually reported to exert insulin-sensitizing effects, both in vivo and in several tissues in vitro. Previous papers reported a stimulatory effect of GLP-1R agonists on glucose uptake and metabolism (Sancho et al, 2005; Gao et al, 2007; Yang et al 2013b) as well as lipogenesis (Sancho et al, 2005). Conversely, lipolysis inhibition is described in mature adipocytes isolated from rats (Sancho et al, 2005) and obtained from mouse 3T3-L1 precursors (Montrose-Rafizadeh et al, 1997), 12 Page 12 of 23
as well as in human myocytes (Luque et al, 2002). The opposite effect observed in our ASC model may be due to a different action of GLP-1R agonists on stem cells and mature cells. In fact, in our precursor model liraglutide significantly reduces cell glucose-uptake maybe due to inhibition of both proliferation and differentiation, whereas in fully differentiated mature cells such as mature adipocytes, skeletal muscle as well as cardiomyocytes, this molecule acts as an insulin sensitizer. In this respect, the reduction of insulin-induced glucose uptake may be viewed as an overall inhibition of metabolic activity related to the reduction of cell proliferation and differentiation in adipocyte precursors. Moreover, the observed difference may be due to liraglutide action in precursors derived from obese instead of healthy subjects. The adipose tissue does not play a relevant role in overall glucose uptake in vivo, but it contributes to the regulation of glucose metabolism and insulin sensitivity through secretion of adipocytokines. The expression of adiponectin, a well-known adipocyte hormone which improves insulin sensitivity, appears to be increased by liraglutide in ASC, thus suggesting that liraglutide not only limits the differentiation rate of adipose cells obtained from dysfunctional tissue of obese subjects, but also ameliorates the functional properties of the obtained adipocytes. This result is in line with observations from clinical trials, showing that both exenatide (Bunck et al, 2010) and liraglutide (Yang et al, 2013c) increase circulating adiponectin levels in patients with type 2 diabetes. However, it should be considered that the increase of adiponectin in clinical trials could have been determined by weight loss (Calvani et al, 2004; Rossmeislovà et al, 2013), whereas the present data, obtained in vitro, suggest a direct effect of GLP-1 receptor stimulation on adiponectin synthesis. Interestingly, treatment with the synthetic GLP-1 receptor agonist exendin-4 stimulates adiponectin secretion in mature 3T3-L1 adipocytes (Kim Chung et al, 2009). Our findings suggest that in obese subjects, liraglutide reduces proliferation and differentiation of adipose stem cells, while ameliorating functionality of the few adipocytes obtained following in vitro adipogenesis. Some limitations of the present study should be considered when interpreting its results. Adipocyte precursors were obtained from morbidly obese subjects; the possibility that the corresponding cells 13 Page 13 of 23
from healthy individuals have a different behavior should be taken into account, but goes beyond the object of our study. The experiments reported were performed on primary cultures of adipose stem cells, which could have been contaminated by other cell types (in particular, endothelial cells) thus possibly interfering with the results (Baglioni et al, 2009; 2012). Furthermore, the number of patients from whom the cells were obtained is relatively limited (n=11), and the possibility of interindividual variations cannot be completely ruled out. Despite these limitations, the present data highlight a further and previously unknown mechanism which could contribute to the effect of GLP-1 receptor agonists in obese subjects. The molecular pathways and the clinical impact of this effect deserve further investigations.
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DECLARATION OF INTEREST Dr. E. Mannucci has received consultancy fees from Novo Nordisk, Eli Lilly, AstraZeneca, and GSK; speaking fees from Novo Nordisk, Eli Lilly, AstraZeneca, and Sanofi; research grants from Novo Nordisk, Eli Lilly and AstraZeneca. The other authors have no conflicts to declare.
ACKNOWLEDGMENTS We thanks Dr. Tommaso Mello (University of Florence) for fluorescence image acquisition. This study was supported by an unrestricted research grant by Novo Nordisk and by the Italian Ministry of University and Research (PRIN 2011 prot. 2010C8ERKX).
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31. Yang, J., Ren, J., Song, J., Liu, F., Wu, C., Wang, X., Gong, L., Li, W., Xiao, F., Yan, F., Hou, X., Chen, L., 2013. Glucagon-like peptide 1 regulates adipogenesis in 3T3-L1 preadipocytes. Int. J. Mol. Med. 31, 1429-35b. 32. Yang, M., Liu, R., Li, S., Luo, Y., Zhang, Y., Zhang, L., Liu, D., Wang, Y., Xiong, Z., Boden, G., Chen, S., Li, L., Yang, G., 2013. Zinc-α2-glycoprotein is associated with insulin resistance in humans and is regulated by hyperglycemia, hyperinsulinemia, or liraglutide administration: cross-sectional and interventional studies in normal subjects, insulinresistant subjects, and subjects with newly diagnosed diabetes. Diabetes Care 36, 1074-82c.
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FIGURE LEGENDS
Fig.1: Liraglutide inhibitory effects on cell viability and proliferation. A: Results are expressed as mean±SE MTS absorbance fold increase (FI) over day 0 (A) or haemocytometer cell counting (B) at the different time points and in the absence (Ctrl) or presence of liraglutide. Mean values of all Ctrl time points were significantly different vs. day 1, p<0.001, as evaluated with ANOVA analysis. Difference significance between liraglutide-stimulated vs. respective controls was evaluated by ANOVA analysis (F and p values are indicated) followed by Dunnett’s post-hoc test: * p<0.05,** p<0.01, § p<0.001 vs. respective controls. Experiments were performed in 6 different S-ASC populations. C: Bar-graph shows the viability percentage inhibition exerted by liraglutide 10 nM and 100 nM vs. respective Ctrls at the indicated time points, calculated from panel B. D: Data represent mean±SE cpm after a 4h pulse of [3H]-thymidine in ASC stimulated for 24 and 48 hours with liraglutide 10 and 100 nM. Percentage of inhibition vs. controls is indicated. Difference significance between liraglutide-stimulated vs. controls was evaluated by ANOVA analysis (F and p values are indicated) followed by Dunnett’s post-hoc test: ** p<0.01, § p<0.001 vs. respective controls. Experiments were performed in 3 different S-ASC populations.
Fig.2: Inhibitory effects of liraglutide on glucose uptake after insulin stimulation. Glucose uptake was performed in S-ASC after stimulation with increasing doses of insulin (1-10100 nM) in the absence (dot line) or presence (bar-graphs) of different concentrations of liraglutide (10-100-1000 nM). Results represent mean±SE glucose uptake fold increase (FI) vs. Ctrl without insulin. All mean values of insulin treated cells were significantly different vs. non-stimulated Ctrl, p<0.001 as evaluated with ANOVA analysis. Difference significance between liraglutide-stimulated vs. nonstimulated cells was evaluated by ANOVA analysis (F and p values are indicated) followed
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by Dunnett’s post-hoc test: * p<0.05 and ** p<0.001 vs. respective controls without liraglutide. Experiments were performed in 3 different S-ASC populations.
Fig.3: Liraglutide interferes with adipogenic differentiation induced in vitro. Oil Red O and AdipoRed fluorescence staining of intracellular triglyceride depots in adipocytes obtained from in vitro-differentiation of S-ASC in the absence (ADIPO) or presence (ADIPO+LIRA) of liraglutide 10 and 100 nM. Representative microscopy images of in vitrodifferentiated adipocytes stained with Oil Red O (red A-F) and AdipoRed (green G-I), with DAPI counterstaining (blue J-L). No Oil Red O or AdipoRed staining was present in liraglutide-untreated undifferentiated ASC controls (not shown). Magnification (10x A-C, 20x D-L), inset (40x). Bargraphs represent mean±SE absorbance fold increase (FI) vs. ASC after AdipoRed staining evaluated spectrofluorimetrically (M). Difference significance between liraglutide-stimulated vs. untreated cells (ADIPO) was valuated by ANOVA analysis (F and p values are indicated) followed by Dunnett’s post-hoc test: * p<0.05 vs. ADIPO.
Fig.4: Exendin 9-39 reverts the liraglutide inhibitory effect on cell viability. S-ASC were treated with liraglutide at the indicated doses (10-100nM) in the presence or absence of exendin 9-39 (10 nM) for the indicated time. A: Bar-graphs represent mean±SE MTS absorbance fold increase (FI) over day 0 of untreated (Ctrl) or treated cells with liraglutide (10-100nM) or a combination of liraglutide (10-100 nM) and exendin 9-39 (10 nM). B: Histograms represent mean±SE cell counting at each day of indicated treatment. Difference significance between liraglutide-stimulated vs. untreated cells was evaluated by ANOVA analysis (F and p values are indicated) followed by Dunnett’s post-hoc test: * p<0.05 and ** p<0.001 vs. respective liraglutide-untreated controls. Student’s t test was used to evaluate the statistical significance of exendin 9-39 effect: § p<0.001 exendin 9-39 vs. respective liraglutide doses. Experiments were performed in 4 different S-ASC populations. 22 Page 22 of 23
Fig.5: Exendin 9-39 interferes with liraglutide inhibition of in vitro-induced adipose differentiation. S-ASC were differentiated in vitro towards adipocytes in the absence or presence of liraglutide (10100nM) alone or combined with 10 nM exendin 9-39. At the end of differentiation, cells were stained with AdipoRed and absorbance evaluated spectrofluorimetrically. Bar-graphs represent mean±SE AdipoRed absorbance percentage over liraglutide-untreated adipocytes
(% ADIPO
differentiation). Statistical significance of exendin 9-39 reversion effect was evaluated by Student’s t test: *p<0.05, **p<0.005 exendin 9-39 vs. respective liraglutide doses. Data were obtained in 4 SASC independent populations.
Tab.1: Liraglutide interferes with adipogenesis but positively regulates adiponectin expression. Taqman analysis of FABP4 and APN expression normalized on GAPDH in S-ASC differentiated in vitro towards adipocytes in the absence or presence of liraglutide (LIRA 1-100 nM). Data are expressed in mean±SE fold increase on liraglutide-untreated adipocytes (ADIPO) from n=3 independent experiments performed in duplicates. F and p values from ANOVA analysis are indicated followed by post hoc Dunnett’s test: *p<0.05, **p<0.005, ***p<0.001 vs. liraglutide untreated adipocytes (ADIPO). ADIPO+1 nM LIRA
ADIPO+10 nM LIRA
ADIPO+100 nM LIRA
FABP4
F=5.176 p=0.008
0.82±0.07
0.67±0.09*
0.54±0.16**
APN
F=8.991 p=0.001
1.86±0.11
2.64±0.43***
2.28±0.26**
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