Abstract
The fat-derived factor, adiponectin, is considered a salutary circulating factor. We recently demonstrated that native adiponectin binds T-cadherin and promotes intracellular biogenesis and secretion of the exosome. Exosomes play important roles in various aspects of homeostasis, including glucose and energy metabolism. However, it remains unclear whether and how the promotion of exosome production by adiponectin in vivo is beneficial for glucose and lipid metabolism. In the present study, overexpression of human adiponectin in mice resulted in an increased number of circulating exosomes, but it did not significantly improve glucose metabolism, change body weights, or change triglyceride clearance under a high-fat diet. Multiple small doses of streptozotocin increased blood glucose and decreased triglyceride clearance similarly in both wild-type and transgenic mice. Thus, these results indicated that human adiponectin overexpression in mice increases plasma exosomes but does not significantly influence glucose and lipid metabolism.
SMALL VESICLES delimited by a lipid bilayer released from the cells are called extracellular vesicles [1]. Extracellular vesicles are characterized into two groups as follows: ectosomes, which are generated and shed directly from the plasma membrane; or exosomes, which are released by the exocytosis of multivesicular bodies (MVBs), in which the limiting membrane of MVBs is inwardly budded and internal luminal vesicles (ILVs) are formed into the internal space of this endosome [2]. Currently, exosomes are considered to function in cellular waste disposal, alternative lysosomal autolysis and cell-to-cell intercellular communications for paracrine and endocrine purposes. Although the specific roles of exosome cargos or cell-to-cell communications between specific cell types have been increasingly studied, the overall effects of circulating exosomes have not been studied.
Adipose tissue secretes over 600 proteins, including adiponectin (APN) [3-5]. APN is a beneficial secreted factor that improves whole-body insulin sensitivity as demonstrated using genetically modified animals and recombinantly produced APN [6-8]. The genetic loss of APN in mice has been reported to have various effects on insulin sensitivity and whole-body glucose metabolism, ranging from no effect to significant difference, depending on the diet conditions and source of KO animals [9-13]. The metabolic effects of constitutive overexpression of APN have been studied with a collagenous domain-truncated mutant of APN [14]. Genome-wide Mendelian randomization studies in human subjects have suggested a limited causal effect of plasma APN on glucose metabolism, although the individual difference in plasma adiponectin levels due to genetic variations were less than 4% [15-18].
Native APN in the plasma specifically binds T-cadherin with high affinity and undergoes endocytosis into MVBs, which enhance ILV biogenesis [19]. The loss of APN or T-cadherin in mice affects the number of circulating exosomes as evaluated by exosome marker contents in semipurified small extracellular vesicle fractions [19]. The overexpression, but not loss, of APN significantly decreases vascular ceramides under ceramide-accumulating hypertensive conditions [19]. The adenovirus-mediated overexpression of APN or peroxisome proliferator-activated receptor γ (PPARγ) agonist-mediated increase in circulating APN increases exosome secretion in vivo from systemically injected mesenchymal stem cells, which improves the protection from load-induced heart failure in mice [20].
To elucidate the overall effects of circulating exosomes, specifically, exosomes generated through the APN/T-cadherin system, we investigated the effects of transgenic (Tg) APN overexpression on glucose metabolism in mice.
Materials and Methods
Animals
A fertilized egg from a human adiponectin (APN) transgenic (Tg) mouse #11 was kindly gifted by Dr Otabe (Kurume University, Japan) [21]. For each experiment, male hemizygote Tg mice and their wild-type littermates were generated and used. This study was approved by the Ethics Review Committee for Animal Experimentation of Osaka University School of Medicine and conducted under the Guide for the Care and Use of Laboratory Animals published by the United States National Institute of Health.
Treatments
Male hemizygote Tg mice and their wild-type littermates were fed either normal chow (NC) (Oriental Yeast) or a high-fat diet (HFD) (60% energy from fat; Research Diets, #D12492) from 8 weeks of age for 6 weeks for evaluation of glucose metabolism. Streptozotocin (STZ) (Sigma–Aldrich #0130) dissolved in 0.1 mol/L citrate buffer was intraperitoneally injected into 8-week-old male mice at 50 mg/kg for 5 consecutive days after 4 h of fasting [22]. After the treatment, blood glucose was measured twice per week at 9:00 AM, and the mice were fed ad libitum.
Systemic tests
Glucose metabolism: We performed an oral glucose tolerance test (OGTT) and insulin tolerance test (ITT). Glucose (2 g/kg body weight) was orally administered after overnight fasting for OGTT. Human insulin (Novo Nordisk; 0.75 U/kg body weight) was intraperitoneally administered after 4 h of fasting for ITT. Blood glucose concentrations were measured using monitoring kits (Sanwa Kagaku Kenkyusho).
Lipid tolerance test: Mice were fasted overnight and then orally gavaged with 20% intralipidTM (15 μL/g; Sigma I141). Tail venous blood samples were collected, and triglyceride contents in plasma were measured using a LabAssayTM Triglyceride kit (Fujifilm #LABTRIG-M1). Glycerol and nonesterified fatty acid (NEFA) were measured with a Glycerol Assay Kit (Sigma #MAK117-1KT) and a LabAssayTM NEFA kit (Fujifilm #LABNEFA-M1), respectively.
Quantitative RT‒PCR
Total RNA isolation from the liver and cDNA synthesis were performed with TRI Reagent® (Sigma, #T9424) and ReverTra Ace® qPCR RT Master Mix (TOYOBO, #FSQ-201), respectively, according to the manufacturers’ instructions. Quantitative real-time PCR was performed using Power SYBR® Green Master Mix (Applied Biosystems, #4368577) with a LightCycler® 96 System (Roche). The results for each sample were normalized to the respective mouse RPLP0 mRNA levels. The primers used in this study were as follows: human APN, 5'-AAC ATG CCC ATT CGC TTT AC-3' (forward) and 5'- ATT ACG CTC TCC TTC CCC AT-3' (reverse); and mouse RPLP0, 5'-GGC CAA TAA GGT GCC AGC T-3' (forward) and 5'- TGA TCA GCC CGA AGG AGA AG-3' (reverse).
Adiponectin ELISA
Serum mouse and human APN levels were measured using mouse/rat APN ELISA kits and human APN ELISA kits (Otsuka Pharmaceutical Co.), respectively. According to the manufacturer’s instructions, human APN has some cross-reactivity for the mouse/rat APN ELISA kit, and mouse APN has no cross-reactivity for the human APN ELISA kit. Thus, human APN Tg mouse serum principally shows pseudo-high mouse APN levels.
Gel filtration analysis
The gel filtration analysis was performed as described previously [23]. Wild-type and human Tg APN mouse sera (1 mL) were separated on a HiLoad 16/60 Superdex 200 prep grade gel-filtration column (Cytiva, #17106901) using an AKTA pure 25 M1 (Cytiva) with a 1.0 mL/minute flow rate at room temperature (25°C). The running buffer consisted of 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 1 mM CaCl2. Each 1-mL fraction was collected and analysed.
Plasma exosome isolation and analysis
Exosome isolation from mouse plasma was performed using two strategies. (1) For nanoparticle tracking analysis, a MagCapture Exosome Isolation Kit PS (FUJIFILM-Wako, #293-77601) was used to isolate exosomes from plasma according to the manufacturer’s instructions. The eluted fractions were analysed with a NanoSight LM10-HS apparatus and NTA2.3 software (Malvern). (2) For analysis of exosome-specific proteins, we used a combination of polymer-based precipitation and ultracentrifugation. The plasma sample was mixed with thrombin (500 U/mL) for 10 min to remove fibrin, and the sample was centrifuged at 12,000 × g for 20 minutes. Exosomes were precipitated from cleared plasma using the ExoQuick (SBI, #EXOQ5A-1) polymer-based exosome precipitation kit according to the manufacturer’s instructions. The precipitated vesicles were washed twice with 500 μL of phosphate buffered saline containing 0.005% Tween 20 by ultracentrifugation at an average of 110,000 × g for 2 hours (Optima TLX with TLA120.1 rotor, Beckman Coulter). The supernatant was discarded, and the pellet was directly solubilized in Laemmli SDS sample buffer.
Western blotting
Whole serum, fractionated serum samples, or isolated plasma exosomes were incubated at 100°C for 5 minutes in Laemmli SDS sample buffer with 2-mercaptoethanol. The samples were separated with 4%–20% gradient SDS‒PAGE gels (Bio-Rad, #5671095) and transferred onto nitrocellulose membranes. The membranes were blocked with Blocking One (Nacalai, #03953-95) for 60 minutes at room temperature, incubated with primary antibodies using Can Get Signal Solution 1 (TOYOBO, #NKB-201) overnight at 4°C, and incubated with secondary HRP-conjugated antibodies using Can Get Signal Solution 2 (TOYOBO, #NKB-301) for 60 minutes at room temperature. The following antibodies were used: anti-mouse APN antibody (R&D, #AF1119); anti-alpha-tubulin antibody (Cell Signalling, #2125S); anti-ALIX antibody (Santa Cruz, #sc-53538); anti-Tsg101 antibody (Abcam, #ab125011); anti-syntenin antibody (Abcam, #ab19903); HRP-conjugated anti-Goat IgG antibody (Thermo, #811620); and HRP-conjugated anti-rabbit IgG antibody (GE Healthcare, #NA934V).
Recombinant adiponectin
Recombinant APN was produced in mice by adenoviral infection of human or mouse APN cDNA. APN was purified with a T-cadherin resin as described previously from the resultant serum containing recombinant APN [23].
Statistical analysis
Data are presented as the mean ± SE with scatter plots. p < 0.05 was considered statistically significant. All analyses were performed using JMP Pro 15.1.0 for Windows (SAS Institute).
Results
Effect of chronic overexpression of human adiponectin on innate (mouse) adiponectin
Human adiponectin (APN) transgenic (Tg) mice were generated to express human APN solely in the liver using a serum amyloid protein promoter (Fig. 1A) [21]. As maternal APN affects foetal growth and metabolism in the dam, we compared hemizygote Tg mice with their littermate wild-type (WT) mice throughout the studies. We confirmed human APN expression in the liver with qPCR (Fig. 1B) and in serum with human APN enzyme-linked immunosorbent assay (ELISA) (Fig. 1C). As Otabe et al. described previously [21], the innate mouse APN levels measured using mouse APN ELISA were elevated more than threefold in Tg mice compared to WT mice (Fig. 1D).
Due to the possibility of the cross-reactivity of human APN to the mouse APN ELISA kit used in the present study, we performed western blot analysis using the mouse-specific anti-APN antibody (Fig. 1E). Each sample was heated and reduced to assess APN in serum as a monomer [24]. Consistent with the ELISA measurement above (Fig. 1D), the amount of total mouse APN in the serum was significantly increased in Tg mice (Fig. 1E and 1F).
Circulating APN forms 18-mers, hexamers, and trimers before secretion [24-26]. To assess which fraction of APN was increased in the mice, we performed gel filtration analysis, and we evaluated the fractionated serum by western blot analysis. As shown in Fig. 1G, both 18-mer and hexamer mouse APN were increased in Tg mice compared to WT mice, whereas trimer mouse APN was not changed. Mouse APN can be increased by its increased expression in adipose tissue where APN is originally produced. The expression levels of mouse APN in subcutaneous adipose tissue did not differ between WT and Tg mice (Fig. 2A). Moreover, the levels of total mouse APN (Fig. 2B and 2C) and the levels of 18mer, hexamer, and trimer mouse APN (Fig. 2D and 2E) were not changed in subcutaneous white adipose tissue between WT and Tg mice.
Effect of chronic overexpression of human adiponectin on the quantity of plasma exosomes
We previously reported that APN enhances exosome biogenesis and that the transient overexpression of mouse APN using recombinant adenovirus increases plasma exosome levels in vivo [19]. To assess the plasma exosome level in human APN Tg mice (chronic hyperadiponectinemia mice), we analysed exosomes isolated from WT and Tg mouse plasma using polymer-based precipitation and ultracentrifugation as described previously [19]. As shown in Fig. 3A and 3B, an approximately 1.5–2.0-fold increase in the plasma exosome levels determined by the exosome-specific markers (ALIX, Tsg101, and syntenin) was observed in Tg mice compared to their WT littermates. We also performed nanoparticle tracking analysis to assess the number of exosomes purified from WT and Tg mouse plasma (Fig. 3C). Consistent with the western blot results, the plasma exosomes were significantly increased by 50% in Tg mice compared to WT mice (Fig. 3C and 3D).
High-fat diet-induced obesity and glucose tolerance in mice
We next evaluated the metabolic impact of higher circulating exosomes in human APN Tg mice under high-fat diet conditions (Fig. 4A). The body weights of Tg mice increased similarly to those of WT littermates with no statistically significant difference between Tg and WT littermates (Fig. 4B). After 6 and 12 weeks of high-fat diet feeding, we evaluated glucose tolerance (Fig. 4A) and found no statistically significant difference between Tg and WT littermates (Fig. 4C and 4D). We also evaluated insulin-induced glucose lowering and found no statistically significant difference (Fig. 4E). Recently, it has been reported that triglyceride clearance is altered by acute loss of APN under high-fat diet conditions [27]. To investigate differences in lipid metabolism, we conducted oral fat loading (Fig. 4A), which increased plasma triglycerides in both mouse groups (Fig. 4F). There were significantly higher triglycerides in Tg mice than in WT mice at the peak of 3 hr after loading. Because the appearance of triglycerides depends on absorption and clearance, we next measured plasma glycerol levels to evaluate triglyceride clearance, which indicated that there was no significant difference between Tg and WT mice (Fig. 4G). However, the appearance of nonesterified fatty acids (NEFAs) in the plasma was relatively delayed in Tg mice compared to WT mice (Fig. 4H), which suggested a delayed clearance of triglycerides in Tg mice. Finally, to evaluate the nonspecific effects of transgene expression in the liver where human APN is specifically expressed under the serum amyloid protein promoter, we conducted a pyruvate tolerance test and found no difference between Tg and WT mice (Fig. 4I).
Triglyceride clearance is mainly catalysed by lipoprotein lipase, which is controlled by insulin. Due to higher insulin levels, high-fat diet conditions may not similarly affect lipoprotein lipase expression in various tissues [27]. We examined streptozotocin-induced diabetes to determine whether there are any differences in lipoprotein lipase (LPL) availability between Tg and WT mice under insulin-deficient conditions (Fig. 5A). Body weight and blood glucose increased in both groups with no significant difference between Tg and WT mice (Fig. 5B and 5C). We also tested oral fat loading (Fig. 5D and 5F). The plasma triglycerides were similar, but there was a weak tendency for higher triglycerides in Tg mice than in WT mice at the peak of 2 hr after loading (Fig. 5D). Both plasma glycerol and NEFA increased similarly (Fig. 5E and 5F), which suggested that the higher APN in the Tg mice does not improve but rather tends to delay triglyceride clearance.
Discussion
Human APN Tg mice have been reported to have both higher human APN and mouse APN in their plasma as well as prolonged healthy longevity under high-fat diet conditions [21]. Higher mouse APN in addition to transgene-derived human APN was observed in the present study. Furthermore, we demonstrated an increase in high-molecular-weight multimer and hexamer APN similar to that in T-cadherin-deficient mice [28]. There was no change in the de novo production of mouse APN in adipose tissues. Therefore, these findings suggested that the increase in mouse APN levels in high-molecular-weight multimer fractions may arise from the competition of T-cadherin-mediated clearance by transgene-derived human APN.
Here, we used hemizygote Tg mice by crossing male hemizygote Tg mice with wild-type female C57BL/6J mice to prevent the inhibition of foetal growth by maternal APN [29]. In these conditions, we observed no substantial difference in growth between Tg and wild-type mice up to 8 weeks old; however, when the mice were mated within Tg mice, the offspring grew more slowly as demonstrated previously [21]. Such differences could affect the results of this study.
We previously reported that the APN/T-cadherin system regulates plasma exosome levels. APN deficiency decreases plasma exosomes, and APN overexpression by adenovirus infection increases plasma exosomes [19]. In the present study, we evaluated plasma exosome levels in human APN Tg mice by two different methods. The quantification of typical exosome markers in the fractions obtained following polyethylene glycol-mediated precipitation and two rounds of ultracentrifugation gave higher intensity bands of ALIX, Tsg101, and syntenin in the plasma exosome fractions of Tg mice compared to WT mice. Phosphatidylserine binding resin-mediated purification and subsequent quantification of exosomes resulted in significantly higher vesicle numbers in the plasma of Tg mice compared to WT mice. Therefore, these findings clearly demonstrated that an increase in APN increases the number of exosomes in plasma.
Exosome biogenesis mechanisms have long been studied, and various molecules have been revealed to play roles in different aspects of exosome production as follows: intraluminal biogenesis by the syntenin-ALIX-endosomal sorting complexes required for transport (ESCRT)-III pathway [30]; ESCRT-independent ceramide pathway [31]; tetraspanin-mediated pathway [32]; and APN/T-cadherin pathway [19]. Among them, to our knowledge, the APN/T-cadherin system is the only in vivo demonstrated mechanism to regulate the systemic concentration of exosomes by regulating IVL biogenesis in T-cadherin-expressing cells, including endothelial cells [19], skeletal muscle cells, heart muscle cells [33], and tissue residential stem/stromal cells [6, 20, 34, 35]. The present study further supported the importance of the APN/T-cadherin system in whole-body exosome regulation in vivo.
Exosomes produced from tissues and cells affect various metabolic processes [6]. However, whether the change in the total number of exosomes in vivo through the APN/T-cadherin system affects glucose and lipid metabolism remains unclear. The present study demonstrated the approximate 1.5-fold increase in the total number of plasma exosomes in human APN Tg mice did not cause a significant improvement in glucose and lipid metabolism under high-fat diet and streptozotocin treatment conditions, respectively.
We utilized human APN Tg mice and found that both human and mouse APN levels were increased in their plasma. Because plasma exosomes were increased in these mice, it is likely that both species of APN, or the sum of them, enhanced exosome biogenesis and secretion.
Numerous studies have shown that APN is a beneficial protein that improves glucose tolerance and insulin resistance [6, 7, 36, 37]. Additionally, a decrease in blood APN levels has been observed in patients with obesity, mainly with visceral obesity, and type 2 diabetes [38-41]. Based on these observations, it has been suggested that APN has beneficial effects on both glucose and lipid metabolism [36, 37]. Salutary metabolic effects have been investigated [42], especially for the globular form of APN (the proteolytically activated form of APN), which has led to the discovery of the APN receptor, AdipoR [43]. Peptide and small-molecule agonists of APN have been investigated for clinical uses [44-46]. In this sense, the present study did not exclude the possibility that human APN is not proteolytically transformed into its active globular form in mice and thereby cannot show additive effects on glucose and lipid metabolism. The mechanism of this transactivation of APN in vivo in a physiological setting has not been elucidated, nor the presence of its resultant active globular form in biological fluids has been demonstrated, although proteolytic generation of active globular form from full-length APN was suggested in vitro [47, 48].
By suppressing APN by a tetracycline-controlled genetic modification in mice, it has been reported that triglyceride clearance mediated by LPL is significantly decreased after loss of APN under insulin-deficient conditions [27]. To investigate whether the large increase in APN may change LPL activity and thereby improve lipid metabolism, we administered streptozotocin to human APN Tg mice. However, we could not find significant improvement in either glucose or lipid metabolism in these mice. Unfortunately, the mechanism through which APN regulates LPL activity has not been addressed [27].
Basic research using APN-deficient mice and supplementation with recombinant APN has also shown little effect on glucose metabolism [11, 49]. Recently, a Mendelian randomization study has been conducted as a method for showing causal relationships in humans [15, 17]. These genome-wide clinical studies have suggested that changes in APN levels due to genetic variances have a limited effect on the risk of obesity and type 2 diabetes in humans [15, 17, 18]. The present study has demonstrated that chronic hyperadiponectinemia induced by transgenic overexpression increases plasma exosomes without significantly improving glucose and lipid metabolism. It has been reported that human APN Tg mice have a longer lifespan than WT mice under high-fat diet conditions [21]. Although significant metabolic improvement was not observed under high-fat diet conditions, increased exosome production may be associated with the mechanism of lifespan extension in human APN Tg mice, which remains to be investigated.
Abbreviations
APN, Adiponectin; MVBs, multivesicular bodies; ILVs, internal luminal vesicles; Tg, transgenic; ESCRT, endosomal sorting complexes required for transport; LPL, lipoprotein lipase; OGTT, oral glucose tolerance test; ITT, insulin tolerance test; STZ, streptozotocin; NEFA, nonesterified fatty acid; ELISA, enzyme-linked immunosorbent assay
Acknowledgements
We thank all members of the Third Laboratory (Adiposcience Laboratory, Department of Metabolic Medicine, Osaka University) for their helpful discussion of the project. We also thank the staff of the Centre for Medical Research and Education (Graduate School of Medicine, Osaka University) for their excellent technical support and assistance.
Authors’ Contributions
K.K., S.K., and S.F. designed the research protocol, performed the biochemical experiments, performed the cellular experiments, performed the in vivo experiments, analysed the data, and co-wrote the manuscript. K.F., T.O., and E.K. performed the in vivo experiments. M.I., T.S., and Y.K. performed and assisted with the biochemical measurements. K.F. and Y.K. assisted with the data analysis. S.O. supplied hAPN Tg mice. Y.F., H.N., and N.M. contributed to editing the manuscript. S.K. and S.F. directed the project, and I.S. supervised the project and finalized the manuscript.
Funding
This work was supported in part by Grant-in-Aid for Scientific Research (Nos. JP19K08980 to N.M., JP19K09023 to H.N., and JP19K08978 to S.K.); Grant-in-Aid for Challenging Research (Pioneering) (No. JP20K20606 to I.S.); Grant-in-Aid for Early-Career Scientists (Nos. JP21K16353 to Y.F. and JP21K16340 to S.F.); a joint research grant with Kowa Pharmaceutical (to I.S.); a joint research grant with Rohto Pharmaceutical (to I.S.); and the Uehara Memorial Foundation (to I.S.). The authors declare that these funding agencies had no role in the study design, data collection, data analysis, decision to publish, or preparation of the manuscript.
Disclosure
S.K. belongs to the department endowed by Takeda Pharmaceutical Company, Rohto Pharmaceutical Co., Ltd., Sanwa Kagaku Kenkyusho Co., Ltd., FUJI OIL HOLDINGS INC., and Kobayashi Pharmaceutical Co., Ltd. N.M. belongs to the department endowed by Kowa Pharmaceutical Co., Ltd.
The funders had no role in the study design, data collection, analysis, decision to publish, or preparation of the manuscript.
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