The therapeutic effects of adipose-derived mesenchymal stem cells on obesity and its associated diseases in diet-induced obese mice

Obesity is a global public health concern associated with increased risk of several comorbidities. Due to the limited effectiveness of current therapies, new treatment strategies are needed. Our aim was to examine the effect of adipose-derived mesenchymal stem cells (AD-MSCs) on obesity and its associated diseases in a diet-induced obese (DIO) animal model. C57BL6 mice were fed with either high fat diet (HFD) or CHOW diet for 15 weeks. Obese and lean mice were then subjected to two doses of AD-MSCs intraperitoneally. Mice body weight and composition; food intake; blood glucose levels; glycated hemoglobin (HbA1c), intraperitoneal glucose tolerance test and atherogenic index of plasma (AIP) were measured. Pro-inflammatory cytokines, tumor necrosis factor-α and interleukin-6, were also determined. AD-MSCs treatment reduced blood glucose levels, HbA1c and AIP as well as improved glucose tolerance in DIO mice. In addition, MSCs caused significant attenuation in the levels of inflammatory mediators in HFD-fed mice. Taken together, AD-MSCs were effective in treating obesity-associated diabetes in an animal model as well as protective against cardiovascular diseases as shown by AIP, which might be partly due to the attenuation of inflammatory mediators. Thus, AD-MSCs may offer a promising therapeutic potential in counteracting obesity-related diseases in patients.


Results
Generation of HFD-induced obese and diabetic mice. 8-week-old male C57BL/6 mice were allowed to adapt for 1 week to the new environment. Then, mice were fed for 15 weeks with either a high-fat diet (HFD) to generate diet-induced obese (DIO) mice or a standard CHOW diet to produce lean mice (Fig. 1a). Food intake was measured after 12 weeks of feeding. Nevertheless, there was no significant difference in food intake between DIO and CHOW mice (Fig. 1b). Regarding body weight, mice fed the HFD displayed significantly higher body weights than mice fed with the CHOW diet after only 3 weeks of feeding (p < 0.01). This increase in body weight in the HFD-fed mice continued throughout the 15-week feeding period to reach 48.63 ± 1.46 g in DIO mice as compared to 31.75 ± 0.73 g (p < 0.001) in CHOW mice at 24 weeks of age as shown in Fig. 1c. As presented in Fig. 1d-f, there were alterations in body composition parameters upon high fat dieting, as determined using Minispec LF110 body composition analyzer. At baseline, no significant differences in parameters were observed. However, after 9 weeks of HFD feeding, there was significant elevation (p < 0.001) in fat mass percentage in the HFD group (Fig. 1d). At week 21, DIO mice had 46.4% body fat mass as compared to 19.5% in CHOW mice (p < 0.001). On the other hand, lean mass percentage decreased significantly in DIO mice at weeks 18 and 21 (p < 0.001) (Fig. 1e). However, free fluid content was not significantly different between both mice groups at all time points (Fig. 1f). It can be concluded that DIO mice continued to gain weight over the time period of the study and that was mainly due to the increase in total body fat mass rather than lean mass.
Blood glucose levels were also measured regularly every 2 weeks in both mice groups. After only 2 weeks on HFD, blood glucose significantly increased from 135.5 ± 1.33 mg/dl to 152.5 ± 2.25 mg/dl (p < 0.01) and remained elevated to reach 187.7 ± 5.6 mg/dl on week 21 as compared to 140.5 ± 1.99 mg/dl in CHOW-fed mice (p < 0.001) (Fig. 2a). However, blood glucose measurements showed no significant increase in the control group throughout www.nature.com/scientificreports/ the 15-week feeding period. This indicates that blood glucose levels were altered by the HFD thus inducing hyperglycemia. A two-hour intraperitoneal glucose tolerance test (IPGTT) was also performed after 15 weeks of diet feeding to assess glucose homeostasis in mice. After 6 h of fasting (t = 0 min), there was a significant increase (P < 0.05) in blood glucose levels in DIO mice (199 ± 8.4 mg/dl) versus control mice (136.6 ± 3.63 mg/ dl) (Fig. 2b). Now in order to determine the body's ability to metabolize glucose, a 2 g/kg of glucose bolus was injected intraperitoneally (IP). DIO mice showed a rise in glucose levels (374 ± 15.5 mg/dl) after 15 min of infusion with a peak occurring after 30 min (423 ± 12.6 mg/dl) (p < 0.001) (Fig. 2b). Two hours later, both groups showed a reduction in glucose levels with the DIO group displaying significantly (p < 0.001) higher blood glucose level as compared with the CHOW-fed mice with a mean of 294 ± 24 mg/dl and 184.3 ± 18.14 mg/dl, respectively. These data suggest that the HFD caused glucose intolerance in mice.

Effect of AD-MSCs on body weight and composition.
We examined the impact of IP injection of AD-MSCs on obesity and related comorbidities. After chronic HFD feeding (15 weeks), six mice of DIO and CHOW groups were injected with 4.2 × 10 7 AD-MSCs/kg, while the control groups were injected with DMEM/ F12 medium. A second dose followed after 10 weeks. After 15 weeks of feeding at baseline (day 0), the body weights of both DIO groups, HFD + MEDIA and HFD + ADMSCs, were significantly high (p < 0.001) and reached around 49.35 ± 1.42 g and 49.28 ± 2.3 g, respectively, as compared with the control groups, ND + media and ND + ADMSCs. However, at day 112, the body weights of both DIO groups (HFD + MEDIA and HFD + ADM-SCs) decreased and reached 37.75 ± 2.6 g and 37.36 ± 2.47 g, but there were neither significant differences between HFD + MEDIA and ND + MEDIA groups, nor between HFD + ADMSCs and ND + ADMSCs groups (Fig. 3a). Body composition analysis, a method used to measure the percentages of fat mass, lean mass and free fluids, was performed. DIO groups had significantly (p < 0.001) higher percentages of fat mass (52% and 46.7%) after chronic HFD feeding (day 7) as compared to controls (23%); however, after AD-MSCs treatments, the percentages of fat mass decreased significantly in HFD group from 46.73 ± 4.02% to 32.27 ± 2.59% in a trend similar to that of HFD + MEDIA group (Fig. 3b). On the other hand, the lean mass % (Fig. 3c) and fluid content % (Fig. 3d) were significantly lower in HFD-fed mice compared to CHOW-fed controls at baseline. Fifteen weeks after the first AD-MSCs administration, no significant changes were observed in both parameters in HFD-fed mice compared to controls.
In addition to blood glucose and HbA1c tests, IPGTT was performed 8 weeks after first injection. HFD + MEDIA group compared to control groups showed a significant increase (p < 0.001) in glucose level > 340 mg/dl after 30 min of glucose injection which dropped to around 201.75 ± 5.8 mg/dl after 2 h. However, HFD + ADMSCs group showed a significant difference (p < 0.001) in glucose levels compared to control groups with the peak occurring at 30 min with a mean of 332.6 ± 6.84 mg/dl and reaching 163 ± 4.02 mg/dl at 2 h (Fig. 5a). 6 weeks after second injection, no significant differences in glucose levels among HFD + ADMSCs and control groups were observed (Fig. 5b). As a conclusion, AD-MSCs provided a positive effect on glycemic status and enhanced glucose disposal. www.nature.com/scientificreports/  www.nature.com/scientificreports/ Atherogenic index of plasma (AIP) levels. AIP is a very strong marker of cardiovascular diseases 15 . The effect of AD-MSCs on AIP was assessed by measuring HDL and TG values at the end of the study. Cardiovascular risk or AIP was the highest in HFD + MEDIA group as shown in Fig. 6. However, treatment of HFD group with AD-MSCs was sufficient to significantly reduce AIP to levels similar to those of normal diet groups.

Evaluation of TNF-α and IL-6 levels.
Pro-inflammatory cytokines, TNF-α and IL-6, were measured at the end of the experiment. High levels of TNF-α and IL-6 were detected in both HFD and HFD + MEDIA groups; however, AD-MSCs transplantation caused a significant decrease in the protein levels (Fig. 7). Control groups (ND + MEDIA and ND + ADMSCs) showed normal levels of both cytokines.

Discussion
Obesity is a serious public health concern that increases the chances of developing numerous diseases such as diabetes, cardiovascular diseases and other comorbidities 4 . Throughout the past half century, scientific progression has allowed management of obesity and its associated diseases via several measures 16 . However, all these management options are not without limitations; thus, there is an urgent need for new interventions. Stem cell therapy seems to be promising as an alternative strategy to manage obesity and its related problems 17,18 . As a matter of fact, mesenchymal stem cells have become attractive candidates due to the vital role they play in adipogenesis and hence have been proposed as a novel therapeutic option 19 . The differentiation potential of MSCs, relative ease of isolation and expansion coupled with their immunomodulatory, anti-inflammatory and homing properties have made MSCs extensively studied both in vitro and in vivo for the treatment of many diseases 17 .
The study presented here was undertaken to determine the effect of AD-MSCs intervention on body weight, body  www.nature.com/scientificreports/ composition, hyperglycemia, glucose tolerance and cardiovascular risk in high fat diet-induced obese mouse model as well as its mechanism of action. In our work, after 15-week chronic feeding period, DIO mice demonstrated substantial increase in body weight with a weight gain of 110%. This was associated with significantly elevated fat mass (27.9% increase) and allevaited lean mass and free fluids. In contrast, mice fed with the CHOW diet remained healthy throughout the study. Herein, it was essential to inquire whether weight gained on a HFD was related to an increase in energy intake as a result of hyperphagia. Therefore, we measured the food intake in experimental mice over 24 h. No significant change in food intake was observed in DIO and control groups. Nevertheless, other studies like the one presented by Licholai et al. concluded that HFD induces an overconsumption 20 . In parallel, DIO mice gradually developed hyperglycemia with blood glucose levels exceeding 187 mg/dl and impaired glucose tolerance at the end of experimental feeding; whereas, control mice remained lean and normoglycemic with no metabolic abnormalities. This is consistent with previous studies [21][22][23] .
To test our hypothesis, we examined the impact of IP injection of human AD-MSCs on body weight and composition in both DIO mice and their controls. One of the strengths of our study is the utilization of human cells rather than murine MSCs in animal disease model, as this will more closely mimic the human milieu and help accelerate the translation of stem cell therapy to clinical practice. A previous review demonstrated the effectiveness of human MSCs administered across several different cross-species models in 88 (93.6%) out of 94 experimental studies 24 .
The body weight of both DIO groups (HFD + ADMSC and HFD + MEDIA) decreased significantly with time to reach weight similar to the normal diet groups after 16 weeks (112 days) of treatment. Despite no change in overall body weight at the end of the treatment period, stem cell therapy was sufficient to reduce body fat mass in DIO mice with slight increase in lean mass and free fluids. This was similar to other studies showing that administration of MSCs did not affect body weight in DIO animals 25 . However, it contradicts with others demonstrating that AD-MSCs can induce a decrease in body weight in HFD-fed mice 26,27 . Herein, we have also shown that AD-MSCs can ameliorate hyperglycemia induced by the HFD and improve glucose tolerance and glucosylated Hb. In 2019, a study conducted by Shree et al. concluded that in obese/diabetic mice, AD-MSCs effectively decreased blood glucose levels and improved its tolerance in the experimental subjects 28 . It is well known that obesity is associated with increased risk of developing CVD. Therefore, to determine the effect of AD-MSC implantation on CVD risk in DIO mice, we measured AIP. AIP is a valuable novel marker to predict the risk of developing dyslipidemia and associated diseases such as cardiovascular diseases 15 . Reduced levels of AIP in HFD group treated with AD-MSCs showed the beneficial aspect and the possible anti-atherosclerotic effects of AD-MSCs. Our study agreed with a number of previous studies showing an improvement in serum lipid profile after AD-MSCs transplantation 2,14,28 . Moreover, when measuring inflammatory cytokines, it was striking that AD-MSCs treatment decreased TNF-α and IL-6 serum levels to concentrations similar to that of ND groups. This could explain the mechanism by which MSCs exert their hypoglycemic and cardioprotective effects by suppressing inflammatory markers, TNF-α and IL-6, that both play a role in the progression of diabetes and atherosclerosis 29,30 . Similar to our work, other laboratories have shown attenuation in inflammatory markers upon administration of MSCs in HFD-fed animal models 31,32 .
It is critically important to understand that obesity is associated with macrophage accumulation in adipose tissue 33,34 . It has been reported that diet-induced obesity activates adipose tissue macrophages (ATM) into pro-inflammatory M1 macrophages that produce a variety of pro-inflammatory cytokines such as TNF-α and IL-6, which contribute to the development of diabetes and atherosclerosis 35,36 . In our study, in line with previous studies, MSC administration lowered the expression of pro-inflammatory cytokines 37,38 . Owing to their immunomodulatory and anti-inflammatory properties, we postulate that MSC infusion produced significant anti-diabetic effects via soluble factors in part through directing ATM into anti-inflammatory M2 state and subsequently suppressing the secretion of pro-inflammatory mediators, thus ameliorating the inflammatory microenvironment 32,39 .
In conclusion, our work showed effectiveness in treating obesity-associated diabetes as well as protective effect against CVD as shown by the AIP, which might be partly due to the attenuation of inflammatory mediators, www.nature.com/scientificreports/ TNF-α and IL-6. Despite the importance of our work in proving the functionality of using AD-MSCs for treating obesity-related diseases in an animal model, more future experiments should be conducted before moving forward into assessing AD-MSCs transplantation in treating obesity-concomitant diseases in humans.

Methods
Animals and diets. Twenty-four male C57BL/6 mice aged 8 weeks (25-28 g) were hosted in a controlled environment at the Animal Care Facility at the American University of Beirut with free access to water and food and a 12-h light/dark cycle with a temperature of 22 ± 2 °C. A period of seven days was used as an adaptation period before conduction of the experimentation. All experimental procedures were performed in agreement with the NIH Guidelines for the Use of Animals in Research and approved (18-12-510) by the Institutional Animal Care and Use Committee (IACUC) at the American University of Beirut. Our study was also carried out in accordance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines 40 . Mice were then randomly divided into two groups. The normal diet (ND) group was fed a normal CHOW diet with 10% of total calories from fat and the other group fed a HFD (Research Diets, Inc, D12492, New Jersey, USA) with 60% of calories from fat for a period of 15 weeks to generate the high fat diet-induced obese (DIO) mouse model.

Adipose-derived mesenchymal stem cells (AD-MSCs) culture. Human AD-MSCs (a kind gift from
Reviva Regenerative Medicine Center, Bsalim, Lebanon) were maintained in a DMEM/F12 medium with 10% FBS and 1% antibiotic-antimycotic at 37ºC and 5% CO 2 atmosphere. The medium was changed every 48 h and cells were split when they reached 80-90% confluence. At passage 4, cells were trypsinized and resuspended in DMEM/F12 media ready for animal treatment.
Experimental design. After 15 weeks of chronic feeding with the HFD or CHOW diet, the HFD was removed and half of the animals in each group (n = 6) were injected intraperitoneally (IP) with 4.2 × 10 7 cells/ kg AD-MSCs suspended in media (ND + ADMSCs and HFD + ADMSCs groups) and the other halves (n = 6) were injected with DMEM/F12 media as controls and labelled ND + MEDIA or HFD + MEDIA. A second treatment (4.2 × 10 7 cells/kg) was repeated 10 weeks after the first injection. Similarly, control groups were injected with an equal volume of DMEM/F12 medium. The experimental procedure design is summarized in Fig. 8. All animals were sacrificed 6 weeks after the second injection and serum collected. For serum collection, blood was withdrawn from the retro-orbital sinus, centrifuged at 12 g for 15 min at 4 °C, then serum stored at − 20 °C for further analysis. www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.