Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Metabolic and Pancreatic Effects of Bone Marrow Mesenchymal Stem Cells Transplantation in Mice Fed High-Fat Diet

  • Patricia de Godoy Bueno ,

    Contributed equally to this work with: Patricia de Godoy Bueno, Ângela Merice de Oliveira Leal

    Affiliation Department of Physiological Science, Center of Biological Sciences and Health, Federal University of São Carlos, São Carlos, São Paulo, Brazil

  • Juliana Navarro Ueda Yochite ,

    ‡ These authors also contributed equally to this work.

    Affiliation Department of Biochemistry and Immunology, Ribeirao Preto Medical School, University of São Paulo, Ribeirao Preto, São Paulo, Brazil

  • Graziela Fernanda Derigge-Pisani ,

    ‡ These authors also contributed equally to this work.

    Affiliation Department of Physiological Science, Center of Biological Sciences and Health, Federal University of São Carlos, São Carlos, São Paulo, Brazil

  • Kelen Cristina Ribeiro Malmegrim de Farias ,

    ‡ These authors also contributed equally to this work.

    Affiliation Department of Clinical, Toxicological and Bromatological Analyses, Faculty of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirao Preto, São Paulo, Brazil

  • Lucimar Retto da Silva de Avó ,

    ‡ These authors also contributed equally to this work.

    Affiliation Department of Medicine, Federal University of São Carlos, São Carlos, São Paulo, Brazil

  • Júlio César Voltarelli †,

    † Deceased.

    ‡ These authors also contributed equally to this work.

  • Ângela Merice de Oliveira Leal

    Contributed equally to this work with: Patricia de Godoy Bueno, Ângela Merice de Oliveira Leal

    angelaleal@ufscar.br

    Affiliation Department of Medicine, Federal University of São Carlos, São Carlos, São Paulo, Brazil

Metabolic and Pancreatic Effects of Bone Marrow Mesenchymal Stem Cells Transplantation in Mice Fed High-Fat Diet

  • Patricia de Godoy Bueno, 
  • Juliana Navarro Ueda Yochite, 
  • Graziela Fernanda Derigge-Pisani, 
  • Kelen Cristina Ribeiro Malmegrim de Farias, 
  • Lucimar Retto da Silva de Avó, 
  • Júlio César Voltarelli, 
  • Ângela Merice de Oliveira Leal
PLOS
x

Abstract

The purpose of this study was to investigate the effects of multiple infusions of allogeneic MSCs on glucose homeostasis and morphometry of pancreatic islets in high- fat diet (HFD) fed mice. Swiss mice were fed standard diet (C group) or HFD (HFD group). After 8 weeks, animals of HFD group received sterile phosphate-buffered saline infusions (HFD-PBS) or four infusions of MSCs one week apart (HFD-MSCs). Fasting glycemia (FG) was determined weekly and glucose (GTT) and insulin (ITT) tolerance tests were performed 4, 8, 12, and 16 weeks after the infusions of MSCs. The MSCs transplanted mice were classified as responder (FG < 180 mg/dL, 72.2% of transplanted mice) or non-responder (FG > 180mg/dL, 28.8%) Seven weeks after MSCs infusions, FG decreased in HFD-MSCs responder mice compared with the HFD-PBS group. Sixteen weeks post MSCs infusions, GTT and ITT areas under the curve (AUC) decreased in HFD-MSCs responder mice compared to HFD-PBS group. Serum insulin concentration was higher in HFD-PBS group than in control animals and was not different compared with the other groups. The relative volume of α-cells was significantly smaller in HFD-PBS group than in C group and significantly higher in HFD-MSCs-NR than in HFD-PBS and HFD-MSCs-R groups. Cell apoptosis in the islets was higher in HFD-PBS group than in C group, and lower in HFD-MSCs responder mice than in HFD-PBS group and non-responder animals. The results demonstrate the ability of multiple infusions of MSCs to promote prolonged decrease in hyperglycemia and apoptosis in pancreatic islets and increase in insulin sensitivity in HFD fed mice.

Introduction

Type 2 Diabetes Mellitus (T2D), the most common form of diabetes (approximately 90% of cases) is caused basically by two pathogenic mechanisms-insulin resistance and secretory dysfunction/decrease of pancreatic β-cells and currently there are experimental, clinical and epidemiological evidences of the involvement of immune and inflammatory mediators in these two mechanisms [1]. Insulin resistance is closely related with obesity. The progression of obesity to insulin resistance and to T2D involves the adaptive expansion of β-cells and increase of insulin secretion, and if this compensation is inadequate, glucose intolerance and T2D develop, with subsequent decline of pancreatic β-cell mass [2,3].

The treatment of T2D is complex and requires nutritional counseling, exercise, several oral drugs and, often, multiple daily insulin injections. Still, treatment of T2D can only ameliorate hyperglycemia or temporarily improve the response to insulin in target tissues. In addition, adherence to therapy is usually low and most patients maintain hyperglycemia, which is the major factor responsible for the onset of the chronic and severe complications of diabetes [4]. Therefore, the development of new preventive and therapeutic strategies for T2D is essential. The interest in regenerative therapeutics for T2D was initially motivated by the importance of preserving β-cell mass and function.

The regenerative cellular therapy, in particular with multi/pluripotent cells, has been investigated as a potential therapeutic strategy for T2D [5]. Among them, mesenchymal stem cells (MSCs) due to their immunoregulatory properties are relevant therapeutic candidates [6,7].

Bone marrow (BM) is an important source of easily obtained adult stem cells that include hematopoietic stem cells, mesenchymal stromal stem cells, and endothelial progenitor cells [8]. MSCs are one of the most important multipotent adult stem cells, which can be extensively culture-expanded, are undifferentiated, self-renewable, have low immunogenicity and their clinical utilization involves few ethical concerns [6].

MSCs are able to modify the microenvironment of injured tissues contributing to tissue repair and regeneration through the secretion of cytokines, anti-inflammatory and anti-apoptotic molecules with trophic and immunomodulatory functions [9,10,11,12].

Several studies have shown that MSCs transplantation decreased blood glucose levels and promoted regeneration of pancreatic islet of diabetic animals [13,14,15,16,17,18,19,20,21]. However, these findings were still not adequate to explain the therapeutic contribution of MSC to T2D. Most pre-clinical studies of type 2 diabetes use transgenic manipulations or streptozotocin-induced diabetes as experimental models to evaluate the metabolic effects MSCs infusions [14,22,23]. Nevertheless, these animal models do not reflect the pathogenesis of the human disease which is complex and closely associated with obesity. However, so far, the effects of MSCs infusion in the high fat diet (HFD)-induced diabetes model have been unknown.

Morphometric studies of pancreatic islets have been made since the 50's and since then have helped unravel the complex relationship between the different cell types that compose them as well as their relationship with physiological and pathological conditions, especially diabetes mellitus type 1 and type 2 [24,25].

The purpose of this study was to investigate the effects of multiple infusions of allogeneic BM MSCs on glucose homeostasis and morphometry of pancreatic islets in HFD-induced hyperglycemia in Swiss mice.

Materials and Methods

Animals and experimental groups

Four week-old male Swiss mice (State University of Campinas Central Breeding Center, Campinas, São Paulo, Brazil), were acclimated in individual cages under controlled temperature, humidity and lighting (12-h dark/light cycle) and with free access to water and standard rodent chow. After 7 days, the animals were randomly assigned into 2 groups, Control group (C) fed standard rodent chow and High-fat diet group (HFD) fed 60% of Kcal as fat (PragSoluções, São Paulo, Brazil). After 8 weeks, animals of HFD group were randomly divided into 2 groups: HF mice, which received sterile phosphate-buffered saline (PBS) infusions (HFD-PBS) and HF mice, which received multiple MSCs infusions (HFD-MSCs), (Fig 1).

thumbnail
Fig 1. Experimental groups and study design.

Eight weeks after high fat-diet or standard diet regimen (control group), mice received 4 weekly intraperitoneal (i.p) infusions of PBS (HFD-PBS group) or 5–8 x 106 BM-MSC (HFD-MSC group). Fasting glycemia (FG) was determined every week and glucose (GTT) and insulin (ITT) tolerance tests were performed in the 9th, 17th, 21st, 25th, and 29th week of the experimental period.

https://doi.org/10.1371/journal.pone.0124369.g001

Animal protocols were approved by the Ethic Committee of the Federal University of São Carlos (Approval ID number 053/2008).

Fasting glycemia, serum insulin determinations and glucose (GTT) and insulin (ITT) tolerance tests

Fasting glycemia (FG) was determined after 8 weeks of the diet regimen. The animals from which FG was ≥ 180 mg/dL were considered hyperglycemic [26]. Fasting glycemia was also determined weekly after MSCs infusions.

GTT and ITT were performed after 8 weeks of the diet regimen and 4, 8, 12, and 16 weeks after the MSCs infusions. After overnight fasting, unanesthetized mice were injected with 1.5g of 50% glucose solution per kg of body weight (BW) by intraperitoneal (i.p) route. Blood samples were collected before glucose injection and 30, 60, 90, and 120 min after the injection. ITT was performed after overnight fasting, in unanesthetized mice injected i.p. with human insulin 0.75 U/kg BW. Blood samples were collected before injection and 15, 30, 60, and 90 min after insulin challenge.

Blood glucose samples were collected from the tail vein. Blood glucose levels were measured by Accu-Check glucose meter (Roche Diagnostic, Indianapolis, USA) and serum insulin concentration was determined by ELISA (Millipore Corporation, Billerica, MA, U.S.A.) according to the manufacturer’s protocol.

Isolation, culture and characterization of bone marrow MSCs

The bone marrow cells were isolated from the tibias and femurs of male Wistar rats, aged 6–8 weeks. After washing and centrifugation, cells were resuspended in a α-minimum medium (Gibco, Auckland, New Zealand) supplemented with 15% of fetal bovine serum (Hyclone, Logan, UT, USA) and 100 μg/ml penicillin (Gibco), 100 μg/ml streptomycin (Gibco) and 2 mM L‐glutamine (Gibco), and plated in a 150x20mm dish (density of 5 x 106 nucleated cells/dish). The cells were incubated in a humidified atmosphere containing 95% air and 5% CO2 at 37°C. The non-adherent cells were removed by changing the medium after 3-day culture. Confluent primary cultures were washed with PBS and lifted by incubation with trypsin (Sigma‐Aldrich, Saint Louis, MO, USA) at 37°C for 5 minutes. RPMI1640 (Gibco), supplemented with 10% fetal bovine serum (HyClone, Logan, UT, Canada) was added to neutralize the excess trypsin. The cells were centrifuged and seeded into a 150x20mm dish (density of 5x106 nucleated cells/dish). Subsequent passages were performed similarly until the fifth passage.

To characterize MSCs, one aliquot of trypsinised MSCs from the fifth passage was stained with phycoerythrin (PE)-conjugated monoclonal antibodies against CD31, CD45 or fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies against CD 11b, CD44, and CD29 (Becton-Dickinson/BD, San Jose, CA, USA) for 15 minutes at room temperature. Stained cells were washed and analyzed immediately on a FACSort flow cytometer using CellQuest software (BD).

Adherent cells were further characterized according to their in vitro osteogenic and adipogenic differential potential. Osteogenic differentiation was induced by culturing confluent MSCs for 3 weeks in α-minimum medium (Gibco) supplemented with 7.5% fetal bovine serum (Hyclone, Logan, UT, USA), 1μM dexamethasone, 200 μM ascorbic acid (Sigma‐Aldrich), 10mM β-glycerophosphate (Sigma‐Aldrich). To observe calcium deposition, cultures were analyzed by von Kossa staining [27]. Adipogenic differentiation was induced by culturing confluent MSCs for 2 weeks in α-minimum medium (Gibco) supplemented with 15% fetal bovine serum (Hyclone, Logan, UT, USA), 100mM dexamethasone (Prodome, Campinas, SP, Brazil) 10μg/mL insulin (Sigma‐Aldrich), and 100μM indomethacin (Sigma‐Aldrich). The cells were then analyzed by Sudan II-Scarlat staining [28].

Multiple administration of MSCs

Bone marrow MSCs between fourth to fifth passages were used for multiple infusions in HFD mice. HFD-MSCs group received four i.p. infusions of 5 – 8x106MSCs resuspended in 200μL of PBS, with one week of interval. HFD-PBS group received 200μL of PBS i.p. (Fig 1).

Pancreas immunohistochemistry

Sixteen weeks after the last MSCs infusion, animals were euthanized and pancreas was rapidly removed, fixed in 10% neutral-buffered formalin, dehydrated through graded ethanol passages, cleared in xylene and embedded in paraffin wax. Five μm sections were deparaffinized and rehydrated in a graded series of ethanol washes. Subsequently, 3 discontinuous sections of 5μm pancreas sample were obtained and stained by hematoxylin and eosin (HE) and immunodetection of insulin, glucagon, Ki67 and caspase. Only for Ki67 and caspase detection, antigen retrieval was performed using citrate buffer. Endogenous peroxidase was quenched by hydrogen peroxide 3% (Peroxidase-Blocking reagent, DAKO Cytomation, Fort Collins, CO, USA). Then, sections were incubated with PBS/ BSA 1% to prevent unspecific staining. Next, primary antibodies were applied to the sections. In this study, four primary antibodies diluted in PBS/1%BSA were used: 1) rabbit polyclonal anti-insulin (dilution 1:100; Santa Cruz Biotechnology, CA, USA), 2) mouse monoclonal anti-glucagon (dilution 1:2000; Abcam, Cambridge, UK), 3) rabbit polyclonal anti-caspase (dilution 1:500; Abcam, Cambridge, UK) or 4) rabbit monoclonal anti-Ki67 (dilution 1:100; Abcam, Cambridge, UK). Sections were then incubated with LSABTM+ Kit/HRP (DAKO Cytomation, Fort Collins, CO, USA). The slides were stained with 3.3’diaminobenzidine (DAB—DAKO) according to the manufacturer’s instructions. Finally the sections were and counterstained with Harrys hematoxylin stain. Images were captured and analyzed by a system composed of a video cam (Carl Zeiss AxionCam-MRc) coupled to an Axion Vision Vert A1 microscope (Car Zeiss GmbH, Jena, German), linked to a microcomputer with a Axiovision 4.8 software. Captures were performed using X20 objective magnification.

Determination of islet size and quantification of β and α cells volume

For morphometric pancreas analysis, fifteen islets from each mouse were randomly chosen. Each islet was evaluated to obtain the total islet area and the area positive for insulin or glucagon within each islet using Image-Pro Plus software (Media Cybernetics, Silver Spring, MD). Then, the relative volume of β-cell or α-cell was calculated as the percentage area positive for insulin or glucagon within the islets [25].

Proliferation and apoptosis

Pancreatic islet apoptosis and proliferation was expressed as caspase-3 or Ki67 positive cells, respectively, per islet area. Software Image-Pro Plus (Media Cybernetics, Silver Spring, MD) was used to measure areas and Image J (National Institutes of Health, Bethesda, MD, USA) was used to count the positive cells.

Statistical analysis

Data are presented as mean ± SEM. Statistical analysis was done by unpaired and paired Student’s t tests and analysis of variance (ANOVA). Tukey’s multiple comparisons test was used for post hoc analysis of between-group comparisons. Data are presented as means ± SEM. P values < 0.05 were considered statistically significant.

All statistical analyses were performed by GraphPad Prism 5.0 software (GraphPad software, Inc., California, USA)

Results

Characterization of allogeneic bone marrow derived MSCs

Allogeneic BM-MSCs expressed typical mesenchymal cell markers (Fig 2A) as previously described [29]. MSCs stained positive for CD44 and CD29 while negative for CD31, CD45 and CD11b. MSCs were further characterized by their osteogenic and adipogenic differential potential (Fig 2B2D).

thumbnail
Fig 2. Bone marrow-derived MSCs isolated from Wistar rats.

Immunologic phenotypes of MSCs (A). Passage five plastic adherent cells cultured in alpha-MEM supplemented with 15% fetal bovine serum (B) and differentiated into adipogenic (C) or osteogenic (D) lineages.

https://doi.org/10.1371/journal.pone.0124369.g002

Metabolic characteristic of the animals

Eight weeks after the HFD regimen, mice fed high-fat diet had significantly higher FG than that of control animals (210.5±6.8 vs 133.22±4.8 mg/dL, p <0.05) until the end of the experimental period. These animals had significantly higher glycemia response to both glucose and insulin injections than that of control mice (Fig 3A and 3B).

thumbnail
Fig 3. Glucose Tolerance Test (GTT) (A) and Insulin Tolerance Test (ITT) glycemia responses (B) increase in high fat diet-fed mice.

Glycemia AUC, area under the curve (AUC) analysis of glycemia profile of GTT and ITT after eight weeks of diet regimen. Values are expressed as mean ± SEM (10–28 mice/group). * p < 0.05, control group (C) vs. high fat diet group (HFD).

https://doi.org/10.1371/journal.pone.0124369.g003

MSCs multiple injections decrease blood glucose levels and improve glucose and insulin response

Sixteen weeks after BM-MSCs fourth infusion, 72.2% (n = 13) of the animals exhibited fasting glycemia lower than 180 mg/dL (henceforth called responders or HFD-MSCs-R), while 27.8% (n = 5) did not respond to MSCs therapy—non-responders (or HFD-MSCs-NR). Fig 4 shows the evolution of fasting glycemia in each group throughout the entire experimental period. Fasting glycemia of HFD-MSCs-R mice was significantly lower compared HFD-PBS since the fourth week post- MSCs infusion. Post-treatment fasting glycemia of HFD-MSCs-R was significantly lower than their pre-treatment values (140.5 ± 22.4 vs 214.0 ± 20.8, p< 0.05, Fig 5).

thumbnail
Fig 4. Fasting glycemia decrease since the 7th week post-MSCs infusion.

Fasting glycemia of HFD-MSCs-R mice was significantly lower compared to HFD-PBS since the 7th week post- MSC infusion. Values are expressed as mean ± SEM (5–13 mice/group). * p < 0.05, control group (C) vs. high fat diet group (HFD-PBS); # p < 0.05, HFD-MSCs-R vs. HFD-PBS.

https://doi.org/10.1371/journal.pone.0124369.g004

thumbnail
Fig 5. Fasting glycemia decrease post-MSCs infusion.

Sixteen weeks after the 4th infusion of BM-MSCs, 72.2% (n = 13) of the animals attained fasting glycemia lower than 180 mg/dL (responders). Post- infusion fasting glycemia of these mice was significantly lower than their pre-infusion values. * p < 0.05, pre-vs. post-infusion of BM-MSCs fasting glycemia.

https://doi.org/10.1371/journal.pone.0124369.g005

Body weight of HFD fed animals was significantly higher than control animals beginning at the 8th week until the end of the experimental period. At the end, the body weight of HFD-PBS, HFD-MSCs-R and HFD-MSCs-NR was not different (Table 1).

Serum insulin levels were significantly higher in HFD-PBS than in C (Fig 6). However, there was no difference among HFD-PBS, HFD-MSCs-R and HFD-MSCs-NR groups.

thumbnail
Fig 6. Serum insulin levels increase in high-fat-diet fed mice.

Values are expressed as mean ± SEM (5–13 mice/group). * p < 0.05, control group (C) vs. high fat diet infused with PBS (HFD-PBS).

https://doi.org/10.1371/journal.pone.0124369.g006

Glycemia response to glucose injection (GTT) was significantly higher in HFD-PBS than in C in the fourth week post-MSCs or PBS infusions. However, there was no difference among HFD-PBS, HFD-MSCs-R and HFD-MSCs-NR groups. The same pattern was observed in the 8th and 12th weeks post-MSCs or PBS infusions (data not shown). In the 16th week post-MSCs or PBS infusions, the glycemia response to glucose injection was significantly lower in HFD-MSCs-R than in HFD-PBS group (Fig 7).

thumbnail
Fig 7. Glucose Tolerance Test (GTT) glycemia response decrease post-MSCs infusion.

Only in the 16th week post-MSC infusions, the glycemia response to glucose injection was significantly lower in HFD-MSCs-R when compared to HFD-PBS group. Values are expressed as mean ± SEM (5–13 mice/group). * p < 0.05, control group (C) vs. HFD-PBS; # p < 0.05, HFD-MSCs-R vs. HFD-PBS.

https://doi.org/10.1371/journal.pone.0124369.g007

Glycemia response to insulin injection (ITT) was significantly higher in HFD-PBS than in C at the fourth week post-MSCs or PBS infusions. Nevertheless, there was no difference among HFD-PBS, HFD-MSCs-R and HFD-MSCs-NR groups. The same pattern was observed in the 8th and 12th weeks post-MSCs or PBS infusions (data not shown). In the 16th week post-MSCs or PBS infusions, the glycemia response to insulin was significantly lower in both HFD-MSCs-R and HFD-MSCs-NR than in HFD-PBS group (Fig 8).

thumbnail
Fig 8. Insulin Tolerance Test (ITT) glycemia response decrease post-MSCs infusion.

Only in the 16th week post-MSC infusions, the glycemia response to insulin injection was significantly lower in both HFD-MSCs-R and HFD-MSCs-NR compared to HFD-PBS group. Values are expressed as mean ± SEM (5–13 mice/group). * p < 0.05, control group (C) vs. HFD-PBS; # p < 0.05, HFD-MSCs-R vs. HFD-PBS; & p < 0.05, HFD-MSCs-NR vs. HFD-PBS.

https://doi.org/10.1371/journal.pone.0124369.g008

Pancreatic islet analysis

Pancreatic islets showed no morphological changes, maintaining their typical round or oval shapes. No inflammatory cells (insulitis) were observed in the pancreatic islets (Fig 9)

thumbnail
Fig 9. Morphological features of pancreatic islet by hematoxylin and eosin staining.

Representative images of pancreas section stained for hematoxylin and eosin of control group (A) and HFD-PBS (B), HFD-MSCs-R (C), and HFD-MSCs-NR (D) groups.

https://doi.org/10.1371/journal.pone.0124369.g009

According to the morphometric analysis of the islets, the total islet areas and the relative volumes of β-cells were not different among the groups (Fig 10A10E). However, the relative volumes of α-cells were significantly smaller in HFD-PBS group than in C and significantly higher in HFD-MSCs-NR than in HFD-PBS and HFD-MSCs-R groups (Fig 11A11E).

thumbnail
Fig 10. Islet areas and relative volumes of β-cell did not differ among the groups.

Representative images of pancreas section stained for insulin of control group (A) and HFD-PBS (B), HFD-MSCs-R (C), and HFD-MSCs-NR (D) groups. Quantitative data correspond to mean ± SEM (5–13 mice/group) (E).

https://doi.org/10.1371/journal.pone.0124369.g010

thumbnail
Fig 11. Relative volumes of islet α-cell decrease in HFD-PBS and increase in HFD-MSCs-NR.

Representative images of pancreas section stained for glucagon of control group (A) and HFD-PBS (B), HFD-MSCs-R (C), and HFD-MSCs-NR (D) groups. Quantitative data correspond to mean ± SEM (5–13 mice/group) (E). * p < 0.05, control group (C) vs. HFD-PBS; + p < 0.05, HFD-MSCs-NR vs. HFD-PBS; & p < 0.05, HFD-MSCs-NR vs. HFD-MSCs-R.

https://doi.org/10.1371/journal.pone.0124369.g011

Apoptosis in islet cells was significantly higher in HFD-PBS group than in C and in HFD-MSCs-NR group compared with HFD-PBS and HFD-MSCs-R groups. However, the number of apoptotic cells was reduced in HFD-MSCs-R group when compared with HFD-PBS group (Fig 12A12E).

thumbnail
Fig 12. Islet apoptosis increases in HFD-PBS and HFD-MSCs-NR and decreases in HFD-MSCs-R.

Representative images of pancreas section stained for caspase-3 of control group (A) and HFD-PBS (B), HFD-MSCs-R (C), and HFD-MSCs-NR (D) groups. Quantitative data correspond to mean ± SEM (5–13 mice/group) (E). * p < 0.05, control group (C) vs. HFD-PBS; # p < 0.05, HFD-MSCs-R vs. HFD-PBS; + p < 0.05, HFD-MSCs-NR vs. HFD-PBS; & p < 0.05, HFD-MSCs-NR vs. HFD-MSCs-R.

https://doi.org/10.1371/journal.pone.0124369.g012

Regarding islet cellular proliferation, it was significantly smaller in HFD-PBS group than in C. No other difference among the groups was observed (Fig 13A13E).

thumbnail
Fig 13. Islet proliferation decreases in high fat diet-fed mice.

Representative images of pancreas section stained for Ki67 of control group (A) and HFD-PBS (B), HFD-MSCs-R (C), and HFD-MSCs-NR (D) groups. Quantitative data correspond to mean ± SEM (5–13 mice/group) (E). * p < 0.05, control group (C) vs. HFD-PBS.

https://doi.org/10.1371/journal.pone.0124369.g013

Apoptosis in islets was positively correlated with fasting glycemia (r = 0.56; p = 0.002), glycemia response to glucose (r = 0.59; p = 0.001) and insulin (r = 0.42; p<0.05) injections (Fig 14A, 14B and 14C). Cell proliferation was negatively correlated with fasting glycemia (r = 0.39; p<0.05) (Fig 14D).

thumbnail
Fig 14. Correlation analysis.

Islet apoptosis was positively correlated with fasting glycemia (A) and glucose tolerance test (GTT) (B) and insulin tolerance test (ITT) glycemia responses (C). Islet proliferation was negatively correlated with fasting glycemia (D).

https://doi.org/10.1371/journal.pone.0124369.g014

Discussion

The results show that multiple infusions of allogeneic BM-MSCs are able to promote prolonged decrease in glucose intolerance and apoptosis in pancreatic islets and increase in insulin sensitivity in hyperglycemic HFD fed mice. To the best of our knowledge this is the first report of the effects of BM-MSCs infusion in pancreatic and metabolic parameters in exclusively HFD fed hyperglycemic mice.

The HFD fed rodent is one of the most widely used models for studying the metabolic derangements caused by obesity in humans, including glucose intolerance, insulin resistance and type 2 diabetes mellitus. It had been previously described that Swiss mice fed HFD for 8 weeks develop obesity, insulin resistance and hyperglycemia as observed in the present study [30]. This animal model resembles more closely the human type 2 diabetes background since it has no transgenic manipulations or drug-induced β cell toxicity, such as caused by streptozotocin.

Previous studies have reported beneficial effects of stem cells transplantation on the metabolic status in both types 1 and 2 diabetic patients and in rodent models of diabetes [5,13,14,15,16,17,20,22,31,32,33,34,35,36,37,38,39]. The beneficial effects have been attributed to immunological and regenerative properties of different origin stem cells, including umbilical cord cells [40,41,42,43]. However, most pre-clinical studies use streptozotocin-induced diabetes models to evaluate the metabolic effects of single or multiple intravenous MSCs infusions. To the best of our knowledge this is the first report of the effects of BM-MSCs infusion in pancreatic and metabolic parameters in exclusively HFD fed hyperglycemic mice.

Several studies suggest that MSCs may be autologously, allogeinic and even xenogeinic transplanted since the infusion of MSCs in immunocompetent animal models did not lead to rejection of the same and provided good therapeutic results [17,38,42,44,45].

In the present study, the progressive decrease of fasting glycemia in HFD-MSCs-R animals was observed since the 7th week post-MSCs infusions and maintained until the 16th week post-MSCs infusions. At this time, there was no difference in glucose levels between C animals and HFD-MSC-R animals. It was previously demonstrated that MSCs infusion in only STZ or STZ+HFD diabetes animal models is accompanied by decrease of hyperglycemia as observed in the present study, however in most of the studies, the experimental period was shorter than the one in the present study, even though in most of the studies, the experimental period was shorter than in the present study [13,14,15,16,17,20,22,38] and there was increase of plasma insulin levels[14,15,20,22,38].

In spite of the consistent decrease of hyperglycemia promoted by the multiple MSCs infusions, morphometric analysis of the islets showed that the total islet area and the relative volume of β-cell did not change post-MSCs infusions, as opposed to previous reports using other animal models [13,14,15,16,22]. This result associated with the unchanged insulin levels and the decreased glycemia response to both, glucose and insulin injections suggest the increase of insulin sensitivity of peripheral target tissues in the animals treated with MSCs, as recently reported by others [14,38]. Indeed, it was demonstrated that MSC infusion increased expression of GLUT4 and elevated phosphorilation of insulin receptor 1 and protein kinase B in insulin target tissues [14,39]. The mechanisms, however, are poorly understood. It is speculated that the migration of MSCs to different organs [46], in special to damaged tissues [47], could lead to modulation of insulin resistance and local inflammation, which are typical mechanisms involved in pathogenesis of obesity and type 2 diabetes. Most evidences suggest that the beneficial effects of BM-MSCs transplantation are not associated with trans-differentiation of MSCs into pancreatic β cells but rather related to MSC-derived paracrine factors such as cytokines and growth factorsresponsible for immunossupression, differentiation, angiogenesis and stimulation of endogenous cells [48,49,50]. The migration and homing ability of MSCs to injured tissues enable the pacrine effects of MSCs [51]. Even though we did not evaluate MSCs migration and homing analysis in the present study, previous studies have shown that MSCs injected intraperitoneally migrate to various organs such as lung, spleen, pancreas, kidneys, brain, heart, thymus, liver, among others [52,53].

The data regarding the pattern of α-cells and glucagon in pathologic states such as obesity, insulin resistance and type 2 diabetes in both humans and rodents are highly controversial [54,55,56,57,58,59,60]. In the present study, the volume of α-cells decreased in HFD fed mice. This finding could be associated with increased insulin levels since insulin is a potent α-cell inhibitor [59,60,61,62]. In addition, recent evidences point to the conversion of pancreatic α-cells to β-cells in conditions of stress [63,64]. A substantial proportion of α-cells becoming β-cells could explain the decrease in α-cells volume in the presence of unaltered β-cell observed in HFD. Nevertheless, this hypothesis does not explain the increase in α-cells volume observed in HFD-MSCs-NR group in comparison with HFD-MSCs-R group.

Beta-cell apoptosis has been considered an important mechanism in the pathogenesis of type 2 diabetes and increased islet apoptosis in HFD fed mice was demonstrated [65]. In the present study, not only did apoptosis increase in HFD fed mice but also proliferation decreased in these animals. Although islet proliferation did not change after MSCs infusions, islet apoptosis decreased in HFD-MSCs-R group. This result is confirmed by an in vitro study that demonstrates decreased caspase 3 expression and new expression of Ki67 in islet cell nuclei by electrofusion between dispersed islet cells and MSCs [66]. In addition, it was recently demonstrated in vitro that MSCs co-cultured with pancreatic islets release trophic factors that increase the survival of the islets and lead to expression of Pdx1 [67]. Altogether these data emphasize the cytoprotective property of MSCs [11]. Interestingly, islet apoptosis was positively correlated with fasting glycemia and glucose response to both glucose and insulin injections and islet proliferation was negatively correlated with fasting glycemia. These findings demonstrated the importance of the entire islet cell population and metabolic modulation.

In conclusion, the results demonstrate the ability of MSCs to promote prolonged decrease in hyperglycemia and apoptosis in pancreatic islets and increase in insulin sensitivity in HFD fed mice.

Acknowledgments

We thank Dr. Heloisa Sobreiro Selistre de Araujo for the assistance with microscopy and Dr. Azair Liane Matos do Canto de Souza for the assistance with animals.

Author Contributions

Conceived and designed the experiments: PGB AMOL JNUY. Performed the experiments: PGB JNUY. Analyzed the data: PGB GFDP. Contributed reagents/materials/analysis tools: KCRM AMOL LRSA JCV. Wrote the paper: PGB AMOL JNUY.

References

  1. 1. Stumvoll M, Goldstein BJ, van Haeften TW. Type 2 diabetes: principles of pathogenesis and therapy. Lancet. 2005; 365: 1333–1346. pmid:15823385
  2. 2. Asghar Z, Yau D, Chan F, Leroith D, Chan CB, Wheeler MB. Insulin resistance causes increased beta-cell mass but defective glucose-stimulated insulin secretion in a murine model of type 2 diabetes. Diabetologia. 2006; 49: 90–99. pmid:16362284
  3. 3. Butler AE, Janson J, Soeller WC, Butler PC. Increased beta-cell apoptosis prevents adaptive increase in beta-cell mass in mouse model of type 2 diabetes: evidence for role of islet amyloid formation rather than direct action of amyloid. Diabetes. 2003; 52: 2304–2314. pmid:12941770
  4. 4. Zenari L, Marangoni A. What are the preferred strategies for control of glycaemic variability in patients with type 2 diabetes mellitus? Diabetes Obes Metab. 2013; 15 Suppl 2: 17–25. pmid:24034516
  5. 5. Hess D, Li L, Martin M, Sakano S, Hill D, Strutt B, et al. Bone marrow-derived stem cells initiate pancreatic regeneration. Nat Biotechnol. 2003; 21: 763–770. pmid:12819790
  6. 6. Si YL, Zhao YL, Hao HJ, Fu XB, Han WD. MSCs: Biological characteristics, clinical applications and their outstanding concerns. Ageing Res Rev. 2011; 10: 93–103. pmid:20727988
  7. 7. Aggarwal S, Pittenger MF. Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood. 2005; 105: 1815–1822. pmid:15494428
  8. 8. Levesque JP, Winkler IG, Larsen SR, Rasko JE. Mobilization of bone marrow-derived progenitors. Handb Exp Pharmacol. 2007: 3–36. pmid:17554502
  9. 9. Nauta AJ, Fibbe WE. Immunomodulatory properties of mesenchymal stromal cells. Blood. 2007; 110: 3499–3506. pmid:17664353
  10. 10. Park KS, Kim YS, Kim JH, Choi B, Kim SH, Tan AH, et al. Trophic molecules derived from human mesenchymal stem cells enhance survival, function, and angiogenesis of isolated islets after transplantation. Transplantation. 2010; 89: 509–517. pmid:20125064
  11. 11. Yeung TY, Seeberger KL, Kin T, Adesida A, Jomha N, Shapiro AM, et al. Human mesenchymal stem cells protect human islets from pro-inflammatory cytokines. PLoS One. 2012; 7: e38189. pmid:22666480
  12. 12. Stagg J. Immune regulation by mesenchymal stem cells: two sides to the coin. Tissue Antigens. 2006; 69: 1–9.
  13. 13. Ezquer FE, Ezquer ME, Parrau DB, Carpio D, Yanez AJ, Conget PA. Systemic administration of multipotent mesenchymal stromal cells reverts hyperglycemia and prevents nephropathy in type 1 diabetic mice. Biol Blood Marrow Transplant. 2008; 14: 631–640. pmid:18489988
  14. 14. Si Y, Zhao Y, Hao H, Liu J, Guo Y, Mu Y, et al. Infusion of mesenchymal stem cells ameliorates hyperglycemia in type 2 diabetic rats: identification of a novel role in improving insulin sensitivity. Diabetes. 2012; 61: 1616–1625. pmid:22618776
  15. 15. Ezquer F, Ezquer M, Simon V, Conget P. The antidiabetic effect of MSCs is not impaired by insulin prophylaxis and is not improved by a second dose of cells. PLoS One. 2011; 6: e16566. pmid:21304603
  16. 16. Dinarvand P, Hashemi SM, Soleimani M. Effect of transplantation of mesenchymal stem cells induced into early hepatic cells in streptozotocin-induced diabetic mice. Biol Pharm Bull. 2010; 33: 1212–1217. pmid:20606315
  17. 17. Lee RH, Seo MJ, Reger RL, Spees JL, Pulin AA, Olson SD, et al. Multipotent stromal cells from human marrow home to and promote repair of pancreatic islets and renal glomeruli in diabetic NOD/scid mice. Proc Natl Acad Sci U S A. 2006; 103: 17438–17443. pmid:17088535
  18. 18. Lin P, Chen L, Yang N, Sun Y, Xu YX. Evaluation of stem cell differentiation in diabetic rats transplanted with bone marrow mesenchymal stem cells. Transplant Proc. 2009; 41: 1891–1893. pmid:19545751
  19. 19. Zhou H, Tian HM, Long Y, Zhang XX, Zhong L, Deng L, et al. Mesenchymal stem cells transplantation mildly ameliorates experimental diabetic nephropathy in rats. Chin Med J (Engl). 2009; 122: 2573–2579. pmid:19951572
  20. 20. Boumaza I, Srinivasan S, Witt WT, Feghali-Bostwick C, Dai Y, Garcia-Ocana A, et al. Autologous bone marrow-derived rat mesenchymal stem cells promote PDX-1 and insulin expression in the islets, alter T cell cytokine pattern and preserve regulatory T cells in the periphery and induce sustained normoglycemia. J Autoimmun. 2009; 32: 33–42.
  21. 21. Sordi V, Malosio ML, Marchesi F, Mercalli A, Melzi R, Giordano T, et al. Bone marrow mesenchymal stem cells express a restricted set of functionally active chemokine receptors capable of promoting migration to pancreatic islets. Blood. 2005; 106: 419–427. pmid:15784733
  22. 22. Hao H, Liu J, Shen J, Zhao Y, Liu H, Hou Q, et al. Multiple intravenous infusions of bone marrow mesenchymal stem cells reverse hyperglycemia in experimental type 2 diabetes rats. Biochem Biophys Res Commun. 2013; 436: 418–423. pmid:23770360
  23. 23. Hu J, Wang F, Sun R, Wang Z, Yu X, Wang L, et al. Effect of combined therapy of human Wharton's jelly-derived mesenchymal stem cells from umbilical cord with sitagliptin in type 2 diabetic rats. Endocrine. 2014; 45: 279–287. pmid:23686639
  24. 24. Hellman B. The total volume of the pancreatic islet tissue at different ages of the rat. Acta Pathol Microbiol Scand. 1959; 47: 35–50. pmid:14400892
  25. 25. Butler AE, Janson J, Bonner-Weir S, Ritzel R, Rizza RA, Butler PC. Beta-cell deficit and increased beta-cell apoptosis in humans with type 2 diabetes. Diabetes. 2003; 52: 102–110. pmid:12502499
  26. 26. Urban VS, Kiss J, Kovacs J, Gocza E, Vas V, Monostori E, et al. Mesenchymal stem cells cooperate with bone marrow cells in therapy of diabetes. Stem Cells. 2008; 26: 244–253. pmid:17932424
  27. 27. Laschke MW, Schank TE, Scheuer C, Kleer S, Shadmanov T, Eglin D, et al. In vitro osteogenic differentiation of adipose-derived mesenchymal stem cell spheroids impairs their in vivo vascularization capacity inside implanted porous polyurethane scaffolds. Acta Biomater. 2014.
  28. 28. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, et al. Multilineage potential of adult human mesenchymal stem cells. Science. 1999; 284: 143–147. pmid:10102814
  29. 29. Cheng CC, Lian WS, Hsiao FS, Liu IH, Lin SP, Lee YH, et al. Isolation and characterization of novel murine epiphysis derived mesenchymal stem cells. PLoS One. 2012; 7: e36085. pmid:22558340
  30. 30. De Souza CT, Araujo EP, Stoppiglia LF, Pauli JR, Ropelle E, Rocco SA, et al. Inhibition of UCP2 expression reverses diet-induced diabetes mellitus by effects on both insulin secretion and action. FASEB J. 2007; 21: 1153–1163. pmid:17209127
  31. 31. Voltarelli JC, Couri CE, Stracieri AB, Oliveira MC, Moraes DA, Pieroni F, et al. Autologous nonmyeloablative hematopoietic stem cell transplantation in newly diagnosed type 1 diabetes mellitus. JAMA. 2007; 297: 1568–1576. pmid:17426276
  32. 32. Voltarelli JC, Couri CE. Stem cell transplantation for type 1 diabetes mellitus. Diabetol Metab Syndr. 2009; 1: 4. pmid:19825196
  33. 33. Hasegawa Y, Ogihara T, Yamada T, Ishigaki Y, Imai J, Uno K, et al. Bone marrow (BM) transplantation promotes beta-cell regeneration after acute injury through BM cell mobilization. Endocrinology. 2007; 148: 2006–2015. pmid:17255204
  34. 34. Jiang R, Han Z, Zhuo G, Qu X, Li X, Wang X, et al. Transplantation of placenta-derived mesenchymal stem cells in type 2 diabetes: a pilot study. Front Med. 2011; 5: 94–100. pmid:21681681
  35. 35. Bhansali A, Upreti V, Khandelwal N, Marwaha N, Gupta V, Sachdeva N, et al. Efficacy of autologous bone marrow-derived stem cell transplantation in patients with type 2 diabetes mellitus. Stem Cells Dev. 2009; 18: 1407–1416. pmid:19686048
  36. 36. Ezquer F, Ezquer M, Contador D, Ricca M, Simon V, Conget P. The antidiabetic effect of mesenchymal stem cells is unrelated to their transdifferentiation potential but to their capability to restore TH1/TH2 balance and to modify the pancreatic microenvironment. Stem Cells. 2012; 30: 1664–1674. pmid:22644660
  37. 37. Gao X, Song L, Shen K, Wang H, Niu W, Qin X. Transplantation of bone marrow derived cells promotes pancreatic islet repair in diabetic mice. Biochem Biophys Res Commun. 2008; 371: 132–137. pmid:18420028
  38. 38. Ho JH, Tseng TC, Ma WH, Ong WK, Chen YF, Chen MH, et al. Multiple intravenous transplantations of mesenchymal stem cells effectively restore long-term blood glucose homeostasis by hepatic engraftment and beta-cell differentiation in streptozocin-induced diabetic mice. Cell Transplant. 2012; 21: 997–1009. pmid:22004871
  39. 39. Hughey CC, Ma L, James FD, Bracy DP, Wang Z, Wasserman DH, et al. Mesenchymal stem cell transplantation for the infarcted heart: therapeutic potential for insulin resistance beyond the heart. Cardiovasc Diabetol. 2013; 12: 128. pmid:24007410
  40. 40. Francese R, Fiorina P. Immunological and regenerative properties of cord blood stem cells. Clin Immunol. 2010; 136: 309–322. pmid:20447870
  41. 41. Jurewicz M, Yang S, Augello A, Godwin JG, Moore RF, Azzi J, et al. Congenic mesenchymal stem cell therapy reverses hyperglycemia in experimental type 1 diabetes. Diabetes. 2010; 59: 3139–3147. pmid:20841611
  42. 42. Fiorina P, Jurewicz M, Augello A, Vergani A, Dada S, La Rosa S, et al. Immunomodulatory function of bone marrow-derived mesenchymal stem cells in experimental autoimmune type 1 diabetes. J Immunol. 2009; 183: 993–1004. pmid:19561093
  43. 43. Fiorina P, Voltarelli J, Zavazava N. Immunological applications of stem cells in type 1 diabetes. Endocr Rev. 2011; 32: 725–754. pmid:21862682
  44. 44. Zhang J, Li Y, Chen J, Cui Y, Lu M, Elias SB, et al. Human bone marrow stromal cell treatment improves neurological functional recovery in EAE mice. Exp Neurol. 2005; 195: 16–26. pmid:15904921
  45. 45. Kerkis I, Ambrosio CE, Kerkis A, Martins DS, Zucconi E, Fonseca SA, et al. Early transplantation of human immature dental pulp stem cells from baby teeth to golden retriever muscular dystrophy (GRMD) dogs: Local or systemic? J Transl Med. 2008; 6: 35. pmid:18598348
  46. 46. Gao J, Dennis JE, Muzic RF, Lundberg M, Caplan AI. The dynamic in vivo distribution of bone marrow-derived mesenchymal stem cells after infusion. Cells Tissues Organs. 2001; 169: 12–20. pmid:11340257
  47. 47. Le Blanc K, Pittenger M. Mesenchymal stem cells: progress toward promise. Cytotherapy. 2005; 7: 36–45. pmid:16040382
  48. 48. Xu YX, Chen L, Wang R, Hou WK, Lin P, Sun L, et al. Mesenchymal stem cell therapy for diabetes through paracrine mechanisms. Med Hypotheses. 2008; 71: 390–393. pmid:18538944
  49. 49. Choi JB, Uchino H, Azuma K, Iwashita N, Tanaka Y, Mochizuki H, et al. Little evidence of transdifferentiation of bone marrow-derived cells into pancreatic beta cells. Diabetologia. 2003; 46: 1366–1374. pmid:12898006
  50. 50. Burdon TJ, Paul A, Noiseux N, Prakash S, Shum-Tim D. Bone marrow stem cell derived paracrine factors for regenerative medicine: current perspectives and therapeutic potential. Bone Marrow Res. 2011; 2011: 207326. pmid:22046556
  51. 51. Sohni A, Verfaillie CM. Mesenchymal stem cells migration homing and tracking. Stem Cells Int. 2013; 2013: 130763. pmid:24194766
  52. 52. Meyerrose TE, De Ugarte DA, Hofling AA, Herrbrich PE, Cordonnier TD, Shultz LD, et al. In vivo distribution of human adipose-derived mesenchymal stem cells in novel xenotransplantation models. Stem Cells. 2007; 25: 220–227. pmid:16960135
  53. 53. Wilson T, Stark C, Holmbom J, Rosling A, Kuusilehto A, Tirri T, et al. Fate of bone marrow-derived stromal cells after intraperitoneal infusion or implantation into femoral bone defects in the host animal. J Tissue Eng. 2010; 2010: 345806. pmid:21350643
  54. 54. Li Z, Karlsson FA, Sandler S. Islet loss and alpha cell expansion in type 1 diabetes induced by multiple low-dose streptozotocin administration in mice. J Endocrinol. 2000; 165: 93–99. pmid:10750039
  55. 55. Fraulob JC, Ogg-Diamantino R, Fernandes-Santos C, Aguila MB, Mandarim-de-Lacerda CA. A Mouse Model of Metabolic Syndrome: Insulin Resistance, Fatty Liver and Non-Alcoholic Fatty Pancreas Disease (NAFPD) in C57BL/6 Mice Fed a High Fat Diet. J Clin Biochem Nutr. 2010; 46: 212–223. pmid:20490316
  56. 56. Henquin JC, Rahier J. Pancreatic alpha cell mass in European subjects with type 2 diabetes. Diabetologia. 2011; 54: 1720–1725. pmid:21465328
  57. 57. Meier JJ, Ueberberg S, Korbas S, Schneider S. Diminished glucagon suppression after beta-cell reduction is due to impaired alpha-cell function rather than an expansion of alpha-cell mass. Am J Physiol Endocrinol Metab. 2011; 300: E717–723. pmid:21285404
  58. 58. Schwasinger-Schmidt T, Robbins DC, Williams SJ, Novikova L, Stehno-Bittel L. Long-term liraglutide treatment is associated with increased insulin content and secretion in beta-cells, and a loss of alpha-cells in ZDF rats. Pharmacol Res. 2013; 76: 58–66. pmid:23891763
  59. 59. Kilimnik G, Zhao B, Jo J, Periwal V, Witkowski P, Misawa R, et al. Altered islet composition and disproportionate loss of large islets in patients with type 2 diabetes. PLoS One. 2011; 6: e27445. pmid:22102895
  60. 60. Gosmain Y, Masson MH, Philippe J. Glucagon: the renewal of an old hormone in the pathophysiology of diabetes. J Diabetes. 2013; 5: 102–109. pmid:23302052
  61. 61. Quesada I, Tuduri E, Ripoll C, Nadal A. Physiology of the pancreatic alpha-cell and glucagon secretion: role in glucose homeostasis and diabetes. J Endocrinol. 2008; 199: 5–19. pmid:18669612
  62. 62. Habener JF, Stanojevic V. alpha-cell role in beta-cell generation and regeneration. Islets. 2012; 4: 188–198. pmid:22847495
  63. 63. Thorel F, Herrera PL. [Conversion of adult pancreatic alpha-cells to beta-cells in diabetic mice]. Med Sci (Paris). 2010; 26: 906–909. pmid:21106168
  64. 64. Chung CH, Levine F. Adult pancreatic alpha-cells: a new source of cells for beta-cell regeneration. Rev Diabet Stud. 2010; 7: 124–131. pmid:21060971
  65. 65. Sone H, Kagawa Y. Pancreatic beta cell senescence contributes to the pathogenesis of type 2 diabetes in high-fat diet-induced diabetic mice. Diabetologia. 2005; 48: 58–67. pmid:15624098
  66. 66. Yanai G, Hayashi T, Zhi Q, Yang KC, Shirouzu Y, Shimabukuro T, et al. Electrofusion of mesenchymal stem cells and islet cells for diabetes therapy: a rat model. PLoS One. 2013; 8: e64499. pmid:23724055
  67. 67. Scuteri A, Donzelli E, Rodriguez-Menendez V, Ravasi M, Monfrini M, Bonandrini B, et al. A double mechanism for the mesenchymal stem cells' positive effect on pancreatic islets. PLoS One. 2014; 9: e84309. pmid:24416216