Effects of Adra2α expression of adipose stem cells on the treatment of type 2 diabetic mice

Abstract

Background

Adipose stem cell (ASC) therapy has been tested as a new option for the treatment of type 2 diabetes (T2D). Our previous transcriptome sequencing analysis showed that the adrenergic α2 receptor (Adra2α) was highly expressed in ASCs from T2D mice compared to healthy controls. This study aims to explore the role of Adra2α on the characterization and therapeutic function of ASCs.

Methods

Clonidine (an Adra2α agonist) or si-RNA was used to observe Adra2α on ASCs proliferation, migration, growth factors (HGF, TGF-β1 and VEGF) expression and secretion. T2D mice were treated with non-treated control or Adra2α knockdown T2D ASCs (namely NC ASCs or KD ASCs). Mice glucose levels, insulin sensitivity and other metabolic indicators were measured and compared.

Results

Treatment of ASCs with Clonidine reduced the proliferation, migration, and growth factors expression and secretion of ASCs, while Adra2α knocking down ASCs showed opposite effects. This translated in vivo when T2D + KD ASCs could improve hyperglycemia and insulin resistance, reduce fat content in adipose tissues and livers, suppress body inflammation, and increase pancreatic β cell mass in T2D mice compared to NC ASCs.

Conclusions

Adra2α plays a critical role in regulating the proliferation, migration, and expression of growth factors of ASCs. Suppression of Adra2α expression in T2D ASCs restored/improved their therapeutic effects.

Introduction

Diabetes mellitus is one of the top ten causes of death worldwide [1]. Type 2 diabetes (T2D) accounts for more than 90% of all diabetic patients [2]. T2D is a metabolic disease caused by insulin resistance and β-cell dysfunction [3]. T2D also causes many complications including nephropathy, cardiovascular disease, delayed wound healing and others [4]. At present, people with diabetes can only rely on drugs that have side effects, more effective treatments are urgently needed.

Mesenchymal stem cells (MSCs) opened a new door for treating diabetes. Among them, adipose-derived stem cells (ASCs) are a promising therapeutic tool due to their convenient acquisition and rapid proliferation [5, 6]. Many groups, including our own, have demonstrated the significant therapeutic effects of ASCs on diabetes, which could repair pancreatic β-cell function, improve immune dysfunction, and promote diabetic wound healing [7,8,9,10].

Some disease conditions, including T2D, reduce the therapeutic effects of ASCs [9]. In a previous study, we performed RNAseq analysis comparing gene expression in ASCs harvested from healthy or T2D mice and found that Adra2α expression was significantly elevated in T2D ASCs [11]. Adrenoceptors are stress hormone receptors and mediate various biological effects of catecholamine hormones such as epinephrine and norepinephrine. Adrenoceptors consist of α1-adrenoceptors (α1A, α1B, α1D), α2-adrenoceptors (α2A, α2B, α2C) and β-adrenoceptors (β1, β2, β3) [12, 13]. Adra2α is present throughout the central and peripheral nervous system, and is responsible for the transmission of sympathetic nerve activity to the peripheral nervous system [14]. Adra2α is known to mediate the suppression of insulin secretion via adrenaline [15]. Adra2α knockout mice present with enhanced insulin secretion [16]; While animals with β cell-specific overexpression of Adra2α are glucose-intolerant [17]. In T2D animals, treatment with Adra2α agonist Clonidine increased blood glucose, while treatment with Adra2α antagonist showed the opposite effects [18,19,20]. Rosengren and colleagues also demonstrated that Adra2α is a coupled receptor of pancreatic β cells, increased Adra2α expression in those cells inhibits insulin secretion and contributes to the formation of T2D, and these effects could be counteracted by Adra2α antagonists [15]. However, to our knowledge, the role of Adra2α expression on ASCs has not been studied. In this study, we explored the effects of Adra2α on ASCs from T2D mice in the treatment of T2D, which will help us to better understand the feasibility of autologous ASCs transplantation in T2D patients.

Materials and methods

Animals

Male C57BL/6(J) mice (4 weeks old) were purchased from Jinan Pengyue Experimental Animal Technology Co., Ltd (Jinan, China). All animal experiments involved in this study were approved by the Institutional Animal Care and Use Committee at Qingdao Agricultural University.

Induction of T2D mice

Animal cages were numbered. Mice were fed with a standard chow diet for one week, then were randomly divided into two groups, with one fed with the standard chow diet (10% of calories from fat), and the other with a high-fat diet (HFD) (60% of calories from fat), respectively. Mice’s body weight and blood glucose levels were measured weekly. When the non-fasting blood glucose of HFD group reached > 180 mg/mL, streptozotocin (STZ, Sigma-Aldrich, St. Louis, MO, USA) was intraperitoneally injected at a dose of 40 mg/kg daily for three consecutive days. Glucose tolerance test (GTT) and insulin tolerance test (ITT) were performed as previously described [8]. Blood glucose level area under the curve (for GTT) and above the curve (for ITT) were calculated.

Culture of mouse ASCs

To relieve the mice, the rapid cervical dislocation method was performed correctly by a well-trained person to sacrifice the mice, and no anesthetic was used during the animal experiments. White fat pads around the epididymis area were collected to isolate ASCs according to the previously published protocol [8]. In brief, adipose tissues were minced and digested with collagenase I (Sigma-Aldrich, St. Louis, MO, USA). The digestion was stopped with a complete cell culture medium made of DMEM, 10% fetal bovine serum (FBS, Gibco, Langley, OK, USA) and 1% Penicillin–streptomycin (PS, Sangon Biotech Co., Ltd., Shanghai, China). The mixture was centrifuged, and the cell pellet was resuspended and cultured in complete cell culture medium [21].

Scratch test to measure cell migration

ASCs were seeded in six-well plates at a density of about 1 × 10⁵/cm². About 24 h later, when the cells reached about 80–90% confluence, a 10 µL pipette was used to make scratches of equal size (marked as time 0 h). Cells were photographed at 12 h, 24 h, 36 h and 48 h later, the width of cell scratch was calculated using the Image J software. The percentage of the scratch areas re-occupied by migrating cells were calculated as described [22].

Determining growth curve using a cell counting kit 8 (CCK-8)

ASCs were inoculated in 96-well plates (3 × 10³ cells/well), and 100 µL cell culture medium was added to each well. After 24 h of ASC culture, 10 µL CCK-8 solution was added under dark conditions and then the ASCs were incubated in the incubator for 1 h. The OD value was detected at the wavelength of 450 nm for 6 consecutive days, and then a cell growth curve was drawn [22].

Measurements of growth factor secreted by ASCs by the sandwich enzyme-linked immunosorbent assay (ELISA)

The concentrations of transforming growth factor (TGF-β), hepatocyte growth factor (HGF), and vascular endothelial growth factor (VEGF) secreted by ASCs were measured by ELISA according to the manufacturer’s recommendations (Langton Biotechnology Co., Ltd., Shanghai, China) [22].

SiRNA transfection

Mouse Adra2α mRNA region-specific synthetic small interference (Si)RNA oligonucleotides (Quanyang Biotechnology Co. Ltd, Shanghai, China) were used to deplete Adra2α expression in ASCs. Briefly, ASCs were seeded into 24-well plates at a density of about 8 × 10⁴ cells/well. When ASCs reached about 70% confluence, cells were transfected with the siRNA for Adra2α according to the manufacturer's protocols. The transfection efficiency was observed by fluorescence microscope about 10 h after siRNA transfection. 24 h later, RNA was extracted to determine transfection efficiency or used for other experiments.

Mice treatments

T2D mice were randomly divided into three groups. Group 1 received 2 × 10⁶ normal C57BL/6 ASCs (named: “T2D + C57 ASCs”), group 2 received non-treated control T2D ASCs (called “T2D + NC ASCs”) and group 3 received Adra2α knockdown T2D ASCs (called “T2D + KD ASCs”). Cells were injected via the tail vein. C57BL/6 (fed normal chow) and T2D mice receiving PBS were used as healthy and T2D controls, respectively.

H&E staining

Adipose tissue, liver and pancreas of each mouse were harvested and embedded in paraffin for tissue sections. Hematoxylin–eosin (H&E) staining was performed using standard methods as previously described [8]. Sections were observed under a light microscope.

Pancreas immunofluorescence analysis

Pancreas tissue sections were stained with the anti-insulin antibody (Shanghai Bio-Platform Technology Company, Shanghai, China) for immunohistochemistry. Insulin⁺ area in sections was quantified as described [21].

RT-PCR analysis

Total RNA was extracted from cells or tissues using an RNA extraction kit (Aidlab Biotechnology Co., Ltd., Beijing, China). Total RNA was then transcribed into cDNA with reverse transcriptase. The following primer pairs were used to amp:

β-actin: F: 5'-GGCTGTATTCCCCTCCATCG-3’, R: 5'-CCAGTTGGTAACAATGCCATGT-3’;

VEGF: F: 5'-GAGGTCAAGGCTTTTGAAGGC-3', R: 5'-CTGTCCTGGTATTGAGGGTGG-3’;

HGF: F: 5'-CTAGCCTCTGTCCCTTACCCA-3’, R: 5'-TGGAGCACTTTCTGTTGAGGT-3’;

TGF-β: F: 5'-TCTGCATTGCACTTATGCTGA-3’, R: 5'-AAAGGGCGATCTAGTGATGGA-3’;

Adra2α: F: 5'-GTGACACTGACGCTGGTTTG-3’, R: 5'-CCAGTAACCCATAACCTCGTTG-3’;

TNF-α: F: 5'-GACGTGGAACTGGCAGAAGAG-3’, R: 5'-TTGGTGGTTTGTGAGTGTGAG-3’;

IL-6: F: 5'-TAGTCCTTCCTACCCCAATTTCC-3’, R: 5'-TTGGTCCTTAGCCACTCCTTC-3’;

INSR: F: 5'-TCAAGACCAGACCCGAAGATT-3’, R: 5'-TCTCGAAGATAACCAGGGCATAG-3’;

PPAR-γ: F: 5'-CTCCAAGAATACCAAAGTGCGA-3’, R: 5'-GCCTGATGCTTTATCCCCACA-3’;

F4/80: F: 5'-TGACTCACCTTGTGGTCCTAA-3’, R: 5'-CTTCCCAGAATCCAGTCTTTCC-3’;

Nod2: F: 5'-TGGACACAGTCTGGAACAAGG-3’, R: 5'-CAGGACCCATACAGTTCAAAGG-3’;

GAPDH: F: 5'-CACGGCAAATTCAACGGCACAG-3’, R: 5'-AGACACCAGTAGACTCCACGACATA-3’.

Western blot analysis

Cells were lysed with RIPA Tissue/Cell lysate buffer (Beijing Solaibao Technology Co., Ltd., Beijing, China). Proteins within a sample were quantified with a trace protein concentration assay kit (Shenggong Co., Ltd., Shanghai, China) and resuspended in Loading Buffer. The samples were prepared with a 15% separation gel and a 5% concentration gel before transferring onto a PVDF membrane. Then, the samples were blocked with 10% BSA, washed with TBST buffer and incubated with primary antibody at 4℃ overnight. After washing with TBST three times, the samples were incubated with the secondary goat anti-rabbit (IgG) antibody for 1 h at room temperature. Finally, an ECL luminescent solution (Nanjing Vazyme Biotech Co., Ltd., Nanjing, China) was added for exposure and analysis.

Statistical analysis

Data presented were from at least three independent experiments. The data were expressed as mean ± standard deviation (SD). Student’s t-test or ANOVA tests were used to compare the difference among groups, *P < 0.05 indicated a significant difference.

Results

Generation of T2D mice

T2D mice were generated by HFD feeding in combination with multiple low dose STZ injection. After 19 weeks of continuous standard chow or HFD diet Fig. 1 feeding, mice in the HFD group showed much heavier body weights (38.4 ± 1.44 g v.s. 29.87 ± 1.28 g) than that in the chow group (Fig. 1 A and B). The average non-fasting blood glucose levels of the HFD group was above 180 ± mg/dL, which was significantly higher than the chow group (Fig. 1C). After three consecutive injections of STZ, HFD mice had blood glucose levels > 200 mg/mL for at least three days consecutively. Glucose tolerance test (GTT) and insulin resistance test (ITT) showed impaired glucose tolerance (Fig. 1D and E) and reduced insulin sensitivity compared to chow mice (Fig. 1F and G).

Fig.1

Comparison of normal control mice and T2D mice. A Image of T2D (HFD diet) and C57BL/6 normal control mice (Chow diet) at 19 weeks. B, C Changes of body weight and blood glucose of normal control mice (Chow mice) and T2D mice. D Blood glucose levels of Chow and T2D mice during GTT. E Area statistics under the curve of GTT. F Blood glucose levels of T2D and Chow mice during ITT. G Area statistics up the curve of ITT. C57BL/6 (Chow) mice, n ≥ 3, T2D mice, n ≥ 3. *P < 0.05; **P < 0.01, One-way ANOVA test. T2D mice: HFD diet combined with STZ injection; C57BL/6 (Chow) mice: mice fed standard normal diet

Knocking down expression of Adra2α in T2D ASCs using siRNA transfection

To assess the role of Adra2α in ASCs, we Fig. 2 used siRNA transfection to knock down the expression of Adra2α in T2D ASCs and used GFP as a reporter for gene knockdown (Fig. 2A and B). Our data showed that expression of Adra2α was decreased by at least 80% in Adra2α knockdown (KD) cells compared with non-treated control (NC) ASCs at 24 h post-transfection as indicated by RT-PCR analysis (Fig. 2C). Western blot confirmed a much-reduced Adra2α protein measured at 48 h after transfection in KD ASCs compared with control T2D (NC) ASCs (Fig. 2D and E). These data show the successful knocking down of Adra2α in T2D ASCs.

Fig.2

ASCs siRNA. A, B Micrographs of T2D ASCs after siRNA transfection under light (A) and fluorescence (B) microscope. C Detecting the expression of Adra2α in Adra2α knockdown T2D ASCs (KD ASCs) and non-treated control T2D ASCs (NC ASCs) by quantitative PCR. D Western blot analysis of Adra2α expression of KD ASCs and NC ASCs. Blots were cropped, and the full-length blots are presented in Supplementary Fig. 1. E Western Blot results statistics. Data are mean ± SEM of at least three individual experiments. At least 3 mice were included in each group. *P < 0.05, **P < 0.01, One-way ANOVA test

The impact of Adra2α on ASC migration and proliferation

To study the impact of Adra2α in ASC characterization, we performed the cell migration scratch assay. We first compared KD ASCs with NC ASCs. The Fig. 3 scratch (wound) healing rate of KD ASCs reached about 80% at 36 h and 100% at 48 h, while the healing rate in the NC ASCs group was only about 70% at 48 h (Fig. 3A and B). This result indicate that Adra2α suppresses T2D ASCs migration, and knockdown of Adra2α enhance cell migration.

Fig.3

Effects of Adra2α knockdown on ASCs migration and proliferation. A Migrating images of KD ASCs and NC ASCs. B ASCs scratch (wound) closure rate. C Growth curves of KD ASCs and NC ASCs. Data are representative of at least 3 individual experiments. At least 3 mice were used in each group. *P < 0.05, **P < 0.01, One-way ANOVA test

The impact of Adra2α knockdown on ASC proliferation was analyzed by comparing the growth curve of NC ASCs and KD ASCs. As shown in (Fig. 3C), KD ASCs showed faster proliferation compared to NC ASCs, suggesting Adra2α inhibits cell growth.

To confirm the role of Adra2α in cell migration, C57(BL/6) ASCs were treated with an Adra2α agonist, Clonidine, at 10 or 20 μM during a scratch test. Non-treated Fig. 4 cells were used as controls. Our data show that cells treated with the higher dose of clonidine showed the slowest recovery rate (Fig. 4 A and B), suggesting that activation of Adra2α inhibits the migration of ASCs in a dose-dependent manner. We next compared the growth curves of ASCs treated with 10 or 20 μM Clonidine. The results showed the proliferation rates of ASCs were reduced when Clonidine (especially 20 μM) was added into the cell culture medium compared to non-treated controls (0 μM) (Fig. 4C), suggesting that activation of Adra2α reduces cell proliferation.

Fig.4

Effects of Clonidine on ASCs migration and proliferation. A Images of ASCs migration when ASCs were cultured with different concentrations of Adra2α agonist Clonidine (0, 10, 20 uM). B ASCs scratch (wound) closure rate. C Growth curves of ASCs with different concentrations of Clonidine (0, 10, 20 uM). Data are representative of at least 3 individual experiments. At least 3 mice were used in each group. *P < 0.05, **P < 0.01, One-way ANOVA test

Effects of Adra2α on growth factors secretion and expression

ASCs exert protective effects via their paracrine secretion of growth factors, including HGF, TGF-β1 and VEGF, etc. We next evaluated the role of Adra2α in regulating growth factors expression and secretion in ASCs treated with agonist or siRNA. First, the impact of Adra2α expression on growth factors secretion and expression in ASCs was assessed in Adra2α knockdown T2D ASCs. When T2D ASCs were transfected 72 h, cells supernatant was collected for ELISA and RNA was extracted for RT-PCR. The results showed that the secretion and Fig. 5 expression of growth factors were significantly increased in Adra2α KD ASCs compared to NC ASCs (Fig. 5A-F).

Fig.5

Effects of Adra2α on ASCs growth factors expression and secretion. Secretion (A-C) and expression (D-F) of VEGF, HGF and TGF-β of KD ASCs and NC ASCs. Secretion (G-I) and expression (J-L) of VEGF, HGF and TGF-β of ASCs with different concentrations of Clonidine. Data are mean ± SEM of at least three individual experiments. At least 3 mice were included in each group. *P < 0.05, **P < 0.01, One-way ANOVA test

Then, C57(BL/6) ASCs were treated with 0, 10 and 20 μM Clonidine. The concentrations of HGF, TGF-β1 and VEGF in the cell culture supernatant was detected at 72 h after initial cell seeding by ELISA. In the meantime, RNA was extracted from cells and the mRNA expression of these three growth factors was detected by RT-PCR. The results showed that both the secretion and expression of growth factors of ASCs were significantly decreased in the Clonidine-treated groups in a dose-dependent manner compared to control cells (Fig. 5G-I). These data suggest that Adra2α suppresses the mRNA expression and secretion of HGF, TGF-β1 and VEGF in ASCs.

ASC infusion reduces hyperglycemia with no impact on mouse body weight in the T2D mice

We next injected C57(BL/6) ASCs, non-treated control T2D ASCs (NC ASCs) or Adra2α knockdown T2D ASCs (KD ASCs) into the T2D mice. Chow and T2D mice receiving PBS were used as controls. Mouse weights and blood glucose levels were measured and compared. Our data showed that there was no significant difference in mouse Fig. 6 body weights in those receiving ASCs or PBS 4 weeks after treatment (Fig. 6A). In contrast, T2D mice receiving C57(BL/6), NC or KD ASCs all showed decreased blood glucose levels compared to T2D + PBS groups (Fig. 6B).

Fig.6

Effects of ASCs injection on body weight and blood glucose of T2D mice. A Changes in body weight of 5 groups of mice (Chow-fed mice receiving PBS; T2D mice receiving PBS; T2D mice receiving C57BL/6, NC or KD ASCs, namely CHOW, T2D, T2D + C57 ASCs, T2D + NC ASCs and T2D + KD ASCs groups). B Non-fasting blood glucose levels of 5 groups of mice. Blood glucose levels (C) and areas under the curve (D) during GTT of 5 groups of mice at 2 weeks after cells treatment. Blood glucose levels (E) and areas up the curve (F) during ITT of 5 groups of mice at 2 weeks after cells infusion. Blood glucose levels (G) and areas under the curve (H) during the GTT of 5 groups of mice at 4 weeks after cells infusion. Blood glucose levels (I) and areas above the curve (J) during the ITT at 4 weeks after cells infusion. n ≥ 3 per group; *p < 0.05, **p < 0.01, Student’s t test. Error bars represent SD

Effects of ASC injection on glucose tolerance and insulin sensitivity of T2D mice

GTT and ITT were performed at 2 weeks after cell infusion. T2D mice showed a dramatic reduction in glucose clearance compared to chow controls. In contrast, T2D mice receiving C57(BL/6), NC or KD ASCs showed significantly increased glucose clearance compared to T2D controls (Fig. 6C and D), suggesting ASC therapy promotes glucose clearance. Similarly, T2D mice showed impaired insulin sensitivity compared to chow controls, while T2D receiving C57(BL/6), NC or KD ASCs showed significantly increased insulin sensitivity compared to T2D controls (Fig. 6E and F), although no significant differences were observed among different types of ASCs. GTT and ITT were repeated at 4 weeks after cell therapy. Our results showed that T2D mice receiving C57(BL/6), NC or KD ASCs still showed significantly faster glucose clearance (Fig. 6G and H) and insulin sensitivity than T2D controls (Fig. 6I and J). All three types of ASCs could improve glucose clearance and insulin sensitivity in T2D mice, and the effects lasted for at least 4 weeks after treatment. It is worth noting that KD T2D ASCs showed a better trend in their glucose tolerance and insulin-sensitizing effects compared to NC ASCs, although the differences between these two groups were not significant.

Effects of ASCs infusion on pancreatic β cell mass

We next assessed the effects of ASC treatments on pancreatic β cell mass in pancreases harvested from treated mice using H&E staining and immunofluorescent staining for insulin at 4 weeks after cell injection. H&E staining showed that T2D + C57(BL/6) ASCs and T2D + KD ASCs had better therapeutic effects on islet β cell injury in T2D Fig. 7 mice than T2D + NC ASCs (Fig. 7A and B). As the immunofluorescence experiments showed, β cell mass in the ASCs treatment groups was also increased compared to T2D + PBS group, especially the KD ASCs and C57(BL/6) ASCs treatment groups. Although β cell mass in the T2D + NC ASCs group was larger than the T2D + PBS group, but the difference was not significant, suggesting Arda2 knockdown improved the protective effects of T2D ASC on β cell mass (Fig. 7C and D).

Fig.7

Effects of ASCs infusion on pancreatic β cell mass in T2D mice. A HE staining of pancreatic tissues of 5 groups of mice; B Islet area statistics; C immunofluorescence of pancreatic tissues of 5 groups of mice. Red represents insulin + cells. Blue stains for nuclei. Scale bars, 100 mm. D Statistics of insulin + area in pancreases of 5 groups of mice. E–G Expression of inflammatory factors F4/80 (E), TNF-α (F) and NOD2 (G) in pancreatic tissues of 5 groups of mice at 4 weeks after cells treatment. At least 3 mice were included in each experiment. *p < 0.05 and **p < 0.01, One-way ANOVA test. Error bars represent SD

To investigate the effects of ASCs on the inflammation of the pancreas, we measured the expression of inflammatory factors in pancreatic tissues using RT-PCR analysis. Our results showed that the expression of inflammatory factors F4/80, TNF-α and Nod2 in the cell-treated groups decreased significantly compared to T2D + PBS group, especially the expression of Nod2 and TNF-α (Fig. 7E-G).

Effects of ASCs infusion on adipose tissues

To investigate the effects of ASCs on adipose tissues of T2D mice, adipose tissues from five groups of mice were used for H&E staining. The Fig. 8 results showed T2D mice had significantly enlarged fat bubble areas compared to chow controls. Treatment with ASCs decreased the fat bubble size of adipose tissue compared to T2D mice treated with PBS group, T2D + C57(BL/6) ASCs and T2D + KD ASCs treatment groups also showed the most dramatic effects, while the T2D + NC ASCs group showed a trend of better effect (Fig. 8A and B).

Fig.8

Effect of ASCs infusion on adipose tissues and livers of T2D mice. A HE staining of adipose tissue sections from 5 groups of mice. Scale bar = 100 μm. B Mean fat bubble diameters (μm) of epididymal adipocytes of mice from 5 groups of mice. Samples from at least 3 mice were analyzed. *p < 0.05 and **p < 0.01, Student’s t test. Error bars represent SD. C HE staining of liver tissue sections from 5 groups of mice. Scale bar = 100 μm. D Mean fat bubble diameters in livers. (E-I) Relative mRNA expressions of F4/80 (E) IL-6 (F), TNF-a (G), InsR (H) and PPARγ (I) in livers. Samples from at least 3 mice were analyzed. *p < 0.05 and **p < 0.01, Student’s t test. Error bars represent SD

Effects of ASC transplantation on livers

We next compared the impact of different ASCs on liver steatosis of T2D mice. Liver tissues from all five groups of mice were analyzed by H&E staining, and the presence of fat bubbles was measured. There were almost no obvious fat bubbles in the liver tissues of the chow mice, while fat bubble numbers of the T2D + PBS group were the highest, fat bubbles of the T2D + C57(BL/6) ASCs, T2D + NC ASCs and T2D + KD ASCs groups were significantly decreased compared to T2D + PBS group. Among all treatment groups, C57(BL/6) ASCs and Adra2α KD ASCs showed the best results (Fig. 8C and D).

We next measured the effects of ASCs on liver inflammation by measuring the expression of inflammatory factors. Our data showed that the T2D mice showed higher macrophage marker expression, including F4/80, IL-6 and TNF-α compared to chow controls. Meanwhile the markers decreased in the ASCs treatment groups compared with T2D + PBS groups. Especially for F4/80, the ASCs treatment groups were significantly lower than that of the T2D + PBS groups. For IL-6 and TNF-α, the expression of T2D + C57(BL/6) ASCs and T2D + KD ASCs groups was significantly lower than T2D + PBS group, although the expression of the T2D + NC ASCs group was also decreased, but the difference was not significant (Fig. 8E-G).

To evaluate the effects of ASC treatment on liver insulin resistance, we measured the expression of genes related to insulin resistance in the liver. As shown in Fig. 8, insulin receptor (InsR) expression was reduced in T2D mice compared to chow mice, but was significantly increased by cell therapy. Cell therapy groups also showed increased PPAR-γ expression, but the difference was insignificantly compared to T2D controls (Fig. 8H and I).

Discussion

We and others have found that ASCs may be a novel cell therapy tool for diabetes and its complications [8, 23] Human ASCs (hASCs) from diabetic patients showed decreased stem cell activity, differentiation function and angiogenesis due to diabetes-induced glycolipid toxicity [24]. We also found that the ASCs from diabetic donors secreted reduced amounts of growth factors and showed decreased ability to cure diabetes and diabetes complications compared to ASCs from healthy donors [8,9,10].

To understand the mechanisms of why T2D ASCs are inferior compared to ASCs from healthy control mice, RNAseq was performed in control and T2D ASCs. Our results identified that the expression of Adra2α was significantly increased in T2D ASCs [11], which led us to hypothesize that Adra2α upregulation was responsible for the reduced therapeutic effects of T2D ASCs. Findings in this study using Adra2α agonist and knockdown approaches confirmed the effects of Adra2α in maintaining ASC characteristics and in their therapeutic effects.

Adra2α has been shown to be involved in the etiology and pathogenesis of T2D, the enhanced expression of Adra2α in pancreatic β cells is associated with reduced insulin release and T2D [15]. Adra2α agonists can cause impaired insulin secretion and glucose tolerance in pancreatic β cells [17]. While Adra2α antagonists Yohimbine and Efaroxan can improve glucose tolerance and insulin secretion in T2D mice [25]. Adra2α knockdown can reduce visceral fat, improve hyperinsulinemia, glucose tolerance and insulin secretion [26]. These studies showed Adra2α was involved in regulating of blood glucose homeostasis [16]. Our data in this study add new evidence that Adra2α expression was upregulated in T2D ASCs, which showed increased Adra2α expression and had inferior therapeutic effects compared to ASCs from healthy control mice.

In vitro, Adra2α can affect many cell characteristics in many cell types (including pancreatic β cells described above). For example, Adra2α activation could promote the proliferation of breast cancer and pancreatic cancer cells [27, 28]. Adra2α affected proliferation and migration of bone marrow-derived mesenchymal stem cells through both α- and β-adrenergic receptors [29, 30]. Watanabe demonstrated that Clonidine could inhibit the expression and secretion of VEGF in human retinal pigment epithelial cell line (ARPE-19) [31]. Our previous findings showed that Adra2α can affect growth factors (HGF, VEGF and TGF-β1) expression and secretion of ASCs through G protein-cAMP signaling pathway [11]. In this study, our data showed that Adra2α activation could inhibit the proliferation, migration and growth factors secretion and expression of ASCs while its knockdown showed oppositive effects, this is consistent with other studies mentioned above.

In vivo, our previous study showed that Adra2α knockdown T2D ASCs could decrease inflammatory factor expression and promote wound healing compared to untreated T2D ASCs [11]. In this study, normal ASCs, non-treated control T2D ASCs (NC ASCs) and Adra2α knockdown T2DASCs (KD ASCs) were injected into T2D mice via the tail vein respectively. The results showed that the blood glucose of the cells treatment groups was significantly lower than the T2D + PBS group. In particular, the glucose tolerance and insulin tolerance of mice in T2D + KD ASCs group were better than T2D + NC ASCs group. This indicates that T2D ASCs can significantly improve glucose regulatory ability and insulin sensitivity of T2D mice after knockdown of Adra2α, and these effects can last for at least 4 weeks.

Maintaining blood glucose levels at a normal range requires sufficient pancreatic β cell mass [32]. In order to assess the effects of ASCs treatment on pancreatic β cells, pancreatic tissues were used to do HE staining and immunofluorescence tests. The results showed that T2D + C57(BL/6) ASCs and T2D + KD ASCs had better therapeutic effects on islet β cell injury in T2D mice than T2D + NC ASCs. In addition, obesity is the leading cause of insulin resistance and T2D [21], our results showed ASC infusion reduced fat deposition in adipose tissues and livers of T2D mice, especially the T2D + C57(BL/6) ASCs and T2D + KD ASCs. Since inflammation can adversely affect insulin sensitivity and worsen diabetic complications [33], the expression of inflammatory factors such as Nod2, TNF-α, F4/80 and IL-6 [21] was also measured. The findings revealed that both C57(BL/6) ASCs and T2D + KD ASCs yielded notable improvements in pancreas and liver inflammation among T2D mice, in contrast to T2D + NC ASCs.

Furthermore, the expression levels of insulin-sensitizing genes PPARγ and INSR, within the ASCs treatment cohorts exhibited a trend towards alignment with those observed in the chow group. Notably, the expression of INSR showed a particularly noteworthy restoration. These results suggest that ASCs may mitigate insulin resistance in T2D mice by attenuating inflammation and reinstating INSR expression in the liver of treated mice.

Our resutls consistently demonstrate that the therapeutic efficacy of Adra2α knockdown T2D ASCs markedly surpasses that of untreated T2D ASCs. Hence, the modulation of Adra2α expression emerges as a promising avenue for restoring or augmenting the therapeutic potential of T2D ASCs.

Conclusion

Our study provides compelling evidence for the novel role of Adra2α knockdown in ASCs in facilitating proliferation, migration and the secretion of growth factors in ASCs. Moreover, it elucidates its impact on regulating blood glucose levels and enhancing insulin sensitivity, as well as its potential in repairing pancreatic β cells and reducing adipose tissue and hepatic fat content in T2D mice. These findings hold promise for enhancing the therapeutic efficacy of ASCs derived from both T2D and healthy murine models.