Animal Models

Inhibition of adipogenesis in 3T3-L1 cells and suppression of abdominal fat accumulation in high-fat diet-feeding C57BL/6J mice after downregulation of hyaluronic acid

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Abstract

Objective:

Adipogenesis can be spatially and temporally regulated by extracellular matrix (ECM). We hypothesized that the regulation of hyaluronic acid (HA), a component of the ECM, can affect adipogenesis in fat cells. The effects of HA on adipogenesis were investigated in vitro in 3T3-L1 cells and in vivo in high-fat diet-feeding C57BL/6J mice.

Methods:

We investigated the effects of HA by degradation of pre-existing or synthesized HA and artificial inhibition of HA synthesis in adipogenesis.

Results:

In vitro adipogenesis in 3T3-L1 cells was inhibited by treating them with exogenous hyaluronidase (HYAL) and with 4-methylumbelliferone, which inhibited the synthesis of HA in a concentration-dependent manner. In vivo, abdominal fat accumulation in high-fat diet-feeding C57BL/6J mice was suppressed by exogenous HYAL 104 IU injections, which was associated with reduction of lipid accumulation in liver and increase of insulin sensitivity.

Conclusion:

Changes in the ECM such as accumulation of high molecular weight of HA by HAS and degradation of HA by endogenous HYAL were essential for adipogenesis both in vitro and in vivo.

Introduction

Obesity is defined as a condition in which excessive fat accumulates in the body, which can result in many health-related problems. Obesity is one of the main factors responsible for the increased incidence of cardiovascular disease, Type 2 diabetes and several types of cancer. The resultant increases in morbidity and reductions in life expectancy are both personally and economically damaging.1 As a result, obesity is a serious public health problem, and mechanisms to control obesity have been actively studied worldwide. Though several drugs have been developed to treat obesity, an ideal pharmacological therapy has not yet been implemented. Therefore, a novel approach for the treatment of obesity is needed.

Adipocyte differentiation has previously been studied in vitro, using specific cell lines to establish ideal conditions for differentiation.2, 3, 4, 5 Likewise, adipocytes studied using in vitro systems shared common characteristics and mechanisms of differentiation with adipocytes in vivo.6, 7 For example, it has been demonstrated that peroxisome proliferator-activated receptor gamma (PPARγ), which is a transcription factor of the ligand-dependent nuclear receptor type,8 and CCAAT-enhancer-binding proteins (C/EBPs), which are a family of transcription factors of the leucine zipper type,9 play important roles in adipogenesis. In addition, the extracellular matrix (ECM) was found to be involved in adipogenesis through changes in protein composition and dynamics.10

During adipogenesis, regulation of the expression of various genes that are specifically involved in the formation of ECM and cytoskeletal elements is necessary for the transformation of pre-adipocytes into mature adipocytes.11, 12, 13 Several reports have examined the relationship between the ECM and the differentiation of 3T3-L1 cells, including changes in the protein composition and reorganization of the ECM during 3T3-L1 differentiation.14, 15 Demeulemeester et al.16 reported that weight gain in C57BL/6J mice was reduced by treatment with a matrix metalloproteinase (MMP) inhibitor. Therefore, appropriate regulation of the ECM has a role in adipose tissue development in vivo as well as in vitro. Hyaluronic acid (also known as hyaluronan or hyaluronate (HA)) is a critical component of the ECM, and is found both at the cell surface and within the cells. HA is a negatively charged, non-sulfated, glycosaminoglycan (GAG) composed of repeating disaccharides of D-glucuronic acid and N-acetyl-glucosamine. HA is synthesized as a large unbranched polymer (molecular weight ranging from 20 kDa to >2 MDa) on the cytoplasmic side of the plasma membrane by HA synthase (HAS), which is an integral membrane glycosyltransferase, and is subsequently extruded to the extracellular surface.17, 18 Despite its simple structure, HA has numerous functions, as it is progressively degraded by enzymes of the hyaluronidase (HYAL) family at the cellular level.19 Recently, it has been suggested that the specific functions of HA fragments are polymer-size-dependent,19 though the location and the concentration of the polymers as well as of various HA-size-specific binding proteins are also variables.20 Native high molecular weight (Mw) HA acts as a scaffold on which other macromolecules are assembled and has a remarkable ability to retain water, thus contributing to the organization and maintenance of the integrity and hydrodynamic properties of the ECM.21 In 1993, it was reported first by Calvo et al.22 that HA existed in the extracellular media of differentiated 3T3-L1 pre-adipocyte cultures and after that a few papers regarding the relationship between HA and adipogenesis have been published.23, 24 Interestingly, it was shown by Allingham et al. that the expression of genes for the biosynthesis and degradation of HA was positively involved in the differentiation of adipocytes.23 Also, HA-modified matrix has been studied in vitro and in vivo for adipose tissue engineering.25, 26, 27 Therefore, it has been expected that HA is important in adipogenesis.

In this study, we examined if the regulation of HA inhibited adipogenesis and thus lipid accumulation based on this association between HA and adipogenesis. We observed the inhibitory effects of HA on adipogenesis in 3T3-L1 cells in vitro, and the prevention of insulin resistance and non-alcoholic fatty liver disease (NAFLD) caused by excess accumulation of abdominal fat in HFD-feeding C57BL/6J mice in vivo by artificially reducing HA levels.

Materials and methods

Culture and differentiation of 3T3-L1 pre-adipocytes

3T3-L1 pre-adipocytes were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA). The pre-adipocytes were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Hyclone, South Logan, UT, USA), and supplemented with 10% newborn calf serum (Hyclone) and antibiotics (Hyclone) at 37 °C in 5% CO2. To induce adipocyte differentiation, the pre-adipocytes were plated at a density of 2 × 105 cells per well on six-well plates. After confluence, cells were incubated in DMEM supplemented with 10% fetal bovine serum (Hyclone) and antibiotics, and treated with DMI (1 μM dexamethasone (Sigma-Aldrich, St Louis, MO, USA), 0.5 mM 3-iso-butyl-1-methylxantine (Sigma-Aldrich) and 1 μg ml−1 insulin (Roche Diagnostics GmbH, Mannheim, Germany)) for 3 days followed by treatment with insulin alone. To maintain artificially low levels of HA during adipogenesis, we used testicular type hyaluronidase (PH-20; HYAL; Sigma-Aldrich) and 4-methylumbelliferone (4-MU; Sigma-Aldrich), which inhibited the HA synthesis.28 3T3-L1 cells were treated with various concentrations of HYAL and 4-MU after DMI induction, respectively. Quantification of HA in culture media was measured using Hyaluronan Enzyme-Linked Immunosorbent Assay kit (HA-ELISA; Echelon, Salt Lake City, UT, USA), according to the manufacturer’s instructions. For subsequent experiments, the cells and cultured media were harvested at the following times: during the contact-inhibited phase (growth arrest) prior to induction of differentiation (days 2 and 3), during the clonal expansion phase (day 6), post-differentiation (day 8), and during terminal differentiation (day 10).23 On day 10, cells were stained with Oil Red O (Sigma-Aldrich).

Transfection of small interfering RNA

Small interfering RNAs (siRNAs) were used to silence mouse hyaluronic acid synthase 2 (HAS2) expression. Scramble control and HAS2-specific siRNAs were synthesized by Genolution Pharmaceuticals, Inc. (Seoul, Korea). The sense sequence of HAS2-specific siRNA was 5′-CUGAAACUCCCAUAGAAUAUU-3′. The sense sequence of control non-specific scramble RNA was 5′-CCUCGUGCCGUUCCAUCAGGUAGUU-3′. Cells plated at a density of 2 × 105 cells per well in a six-well plate were transfected with 40 pmol of scramble RNA or HAS2-specific siRNAs using RNAiMAX (Invitrogen, Carlsbad, CA, USA) as previously described.29 Cells were treated with siRNA for 6 h, and then the medium was exchanged. After 48 h, cells were processed using differentiation protocols. Transfection was carried out in duplicated wells and repeated at least three times.

Fat accumulation induced by a high-fat diet feeding and HYAL injections

Male C57BL/6J Jms Slc (5.5 weeks old) mice were purchased from Central Lab Animal Inc. (Seoul, Korea). Upon arrival, mice were fed a Rodfeed (normal-fat diet, NFD; DBL, Eumsung, Korea) for 1 week. The animals were housed in individual cages in a temperature-controlled room with a 12-h light/dark cycle. When mice were 6.5 weeks of age, they were divided into five groups (n=5 in all groups except group B, in which n=4). Mice in one of two groups fed NFD were injected with phosphate-buffered saline (PBS) as a negative control for fat accumulation, while the other group was injected with 104 IU of HYAL (BMI Korea, Jeju, Korea) as a negative control for high-dose HYAL injection. Three groups were given a high-fat diet containing 60% fat (HFD; Research Diets Inc., New Brunswick, NJ, USA). Two groups of mice fed HFD were injected with 102 IU and 104 IU of HYAL, respectively. The third group was injected with PBS every 3 days as a positive control for HFD-induced fat accumulation. Food intake was measured every 3 days, and body weight was monitored every 6 days for 2 months.

Intraperitoneal glucose tolerance test (IPGTT)

IPGTTs were performed in all mice at 13.5 weeks of age. After overnight fasting, animals received an intraperitoneal injection of D-glucose (2 g kg−1) in 0.9% NaCl.30 A drop of blood was taken from the tail vein before the glucose injection (0), as well as 15, 30, 60, 90, and 120 min after the injection in order to determine blood glucose levels using a glucometer (Accu-Chek Active; Roche Diagnostics GmbH).

Micro-CT

At 14 weeks of age, the mice were imaged using Micro-CT (Skyscan model 1076; Skyscan, Kontich, Belgium) under anesthesia. The resolution of the micro-CT was 35 μm.

Sample and serum analysis

At 14.5 weeks of age, the mice were put under deep anesthesia after overnight fasting. Blood samples were taken from the inferior vena cava, and serum samples were collected by centrifuging at 14 000 r.p.m. for 30 min after being kept at room temperature for 1 h. Aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), total cholesterol (T.cho), triglycerides (TG), high-density lipoprotein (HDL-C), low-density lipoprotein (LDL-C) and insulin levels were analysed by the Korea Animal Medical Science Institute (KAMESI, Seoul, Korea). The weights of fat tissues and organs were also measured.

Quantitative real-time PCR

Total RNA was prepared directly from cells or tissues using Tri reagent (Molecular Research Center, Inc., Cincinnati, OH, USA) according to the manufacturer’s instructions. Moloney murine leukemia virus (M-MLV) reverse transcriptase (Superbio Co., Daejeon, Korea) was used for cDNA synthesis, and qReal-time PCR was performed using SYBR green premix (Takara bio Inc., Shiga, Japan). Mouse β-2 microglobulin (or β2M) mRNA or 36B4 was used as the internal standard for error correction between samples. Oligonucleotide sequences for each primer are listed in Supplementary Table 1. qReal-time PCR amplification of each cDNA was performed independently in triplicate.31

Western blot

After terminal differentiation, 3T3-L1 cells were lysed using Pro-prep (Intron Biotechnology, Seongnam, Korea). Western blot was followed as described by Waki et al.32 Antibodies against PPARγ, Fabp4 (or aP2), and HRP-conjugated anti-goat IgG were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and antibodies against β-actin and HRP-conjugated anti-mouse IgG were purchased from Abcam (Cambridge, UK) and Cell Signaling Technology, Inc. (Beverly, MA, USA), respectively.

H&E staining

Epididymal fat and liver tissue were fixed with 4% paraformaldehyde, dehydrated, and embedded in paraffin. Sections 10 μm thick were subjected to hematoxylin and eosin (H&E) staining.33

Statistical analysis

Data were analysed using GraphPAD Prism statistical software (version 5.01; GraphPAD Software Inc., La Jolla, CA, USA), and were expressed as means±s.e.m. Significant differences between groups were determined using the Student’s t-test and one-way ANOVAs followed by post hoc Dunnett’s multiple comparisons tests, or two-way ANOVAs with Bonferroni correction.

Results

Downregulation of HA concentration by exogenous HYAL and 4-MU during induction of adipogenesis in 3T3-L1 cells

Prior to investigating the downregulation of HA during adipogenesis, we confirmed that cell viability was not affected by exogenous HYAL or 4-MU up to concentrations of 200 μM (Supplementary Figures 1a and 1b). When exogenous HYAL and 4-MU were provided continuously in cultured media after DMI induction, HA concentrations were reduced compared with the untreated condition during adipogenesis (Figures 1a and b). In the positive control group (PBS), HA concentration increased 11-fold (between days 2 and 5). However, HA concentration decreased to 54% and to 8% after treatment with exogenous HYAL 100 and 200 IU, respectively, compared to PBS treatment (100%). In addition, HA concentrations after treatment with 100 and 200 μM 4-MU were reduced to 38% and to 6%, respectively. Thus, exogenous HYAL and 4-MU were suitable for downregulation of HA levels during adipogenesis in 3T3-L1 cells.

Figure 1
figure1

HA concentration after exogenous HYAL and 4-MU treatment. (a and b) HA concentration in culture media as measured by HA-ELISA kits was decreased by exogenous HYAL and 4-MU in a concentration-dependent manner, respectively. Data are expressed as mean±s.e.m., and were analysed using one-way ANOVAs with Bonferroni correction (###P<0.001 between PBS and concentration HYAL 100 IU or 4-MU 100 μM, ***P<0.001 between PBS and concentration HYAL 200 IU or 4-MU 200 μM).

Reduction in HA levels and inhibition of adipogenesis in 3T3-L1

We treated 3T3-L1 cells with exogenous HYAL or 4-MU, which inhibited the formation and accumulation of lipid droplets in a concentration-dependent manner (Figures 2a and b). Adipogenesis was inhibited by up to 96% (P<0.0001) and up to 80% (P<0.0001) with exogenous HYAL 200 IU and 200 μM 4-MU treatment, respectively (Supplementary Figures 2a and b). However, as the timing of treatment with exogenous HYAL and 4-MU were delayed, their abilities to suppress adipogenesis were poor (Supplementary Figures 2c and d). Adipogenesis was inhibited most effectively in the case in which exogenous HYAL and 4-MU were administered after DMI induction. We wondered whether the reduction of HA during DMI induction was involved in clonal expansion but it was revealed that clonal expansion was independent of HA reduction (Supplementary Figures 3a and b). We also investigated whether these inhibitory effects corresponded to the expression profiles of adipogenic marker genes in 3T3-L1. At the mRNA level, the expression of adipogenic master marker genes such as PPARγ and C/EBPα8, 34, 35 decreased with exogenous HYAL and 4-MU in a concentration-dependent manner (Figures 2c and d). Fabp4 and AdipoQ induced by PPARγ and C/EBPα36 showed a similar pattern of expression to that of PPARγ and C/EBPα (Figures 2c and d). It was also determined that downregulation of HA by exogenous HYAL and 4-MU resulted in inhibition of differentiation of pre-adipocytes into mature adipocytes at the protein level as well (Figure 2e).

Figure 2
figure2

Effects of exogenous HYAL and 4-MU on adipogenesis. (a and b) Adipogenesis in 3T3-L1 cells depended on the treatment concentration of exogenous HYAL or 4-MU (upper box: scanning of six-well plates, lower box: view under inverted microscope). (c and d) The mRNA expression levels of PPARγ, C/EBPα, Fabp4, and AdipoQ measured by qReal-time PCR decreased with HYAL or 4-MU treatment in a concentration-dependent manner. The experiments were repeated in triplicate. (e) The protein expression level of PPARγ and Fabp4 as confirmed by western blot were also reduced by HYAL or 4-MU treatment. The data are expressed as mean±s.e.m. and were analysed using one-way ANOVAs with Dunnett’s multiple comparisons tests (*P<0.05, **P<0.001, ***P<0.0001).

The effects of HYAL on adipogenesis prompted us to investigate the roles of HAS2, which is a core enzyme on HA accumulation in adipocyte differentiation.23 To show the effects of HAS2 in adipogenesis, siRNA-mediated HAS2 silencing was performed. siRNAs were transiently transfected into 3T3-L1 cells and differentiated into adipocytes for 5 days. Consistent with the effects of HYAL, lipid accumulation was significantly impaired by silencing HAS2 expression compared with the control non-specific scramble siRNA transfected cells (Figures 3a and c). The degree of inhibitory effects on HAS2 expression by introducing two different doses of siRNAs mirrors the degree of anti-lipogenic effects during adipocyte differentiation (Figure 3b). Furthermore, the expression of adipocyte markers such as PPARγ and Fabp4 was also dose dependently inhibited by silencing HAS2 expression (Figures 3d and e). Taken together, these data demonstrate that inhibition of HA accumulation by pharmacological, enzymatic and genetical suppression affects lipid accumulation and adipocyte differentiation.

Figure 3
figure3

Inhibition of adipogenesis by knockdown of HAS2. (a) Knockdown of HAS2 expression in 3T3-L1 cells with transfection of siRNA inhibited lipid accumulation as assessed by Oil Red O staining. 3T3-L1 cells were induced to differentiate into adipocytes for 5 days. (b) Transient transfection of 3T3-L1 cells with siRNA targeted to HAS2 dose dependently reduces the expression of HAS2. (c) Quantification of lipid accumulation of differentiated cells by measuring the extracted dye at 520 nm. (d, e) Knockdown of HAS2 inhibits the expression of adipocyte markers. Expression of PPARγ (d) and its target gene Fabp4 (e) was measured by real-time PCR. NC: growth media, scr: scrambled siRNA-transfected cells in differentiating condition, siHAS2: HAS2-specific siRNA-transfected cells in differentiating condition. Data shown represent the mean±s.e.m. Statistical significance was determined relative to scrambled RNA-transfected cells by the Student’s t-test (*P<0.05; **P<0.005; ***P<0.0005).

Suppression of increases in body weight and body fat by HYAL treatment in HFD-feeding C57BL/6J mice

To confirm the effects of downregulation of HA in vivo, we used an animal model of HFD-feeding C57BL/6J mice. We intended to decrease HA levels in the mice using IP injections of HYAL every 3 days while providing a HFD. We prepared 5 groups of mice (n=5 in all groups except group B, where, n=4 ). Group A, fed NFD and injected with PBS; group B, fed NFD and injected with HYAL 104 IU; group C, fed HFD and injected with PBS; group D, fed HFD and injected with HYAL 102 IU; and group E, fed HFD and injected with HYAL 104 IU. Body weight and food intake were monitored every 6 days and every 3 days, respectively. These data showed that weight gain decreased with injections of HYAL (Figure 4a) without significant differences in food intake (Supplementary Figure 4a). At last measurement, the body weight and the net weight gained in group C and group E were as follows: group C (32.60±0.81 g, 12.20±0.49 g); and group E (29.80±0.58 g, 10.40±0.51 g). These data indicate that body weight and net weight gained in group E were down by 9% and 15%, respectively, compared with group C. The distribution and volume of abdominal fat in group E were determined using micro-CT (resolution=35 μm) for comparison with that of control groups (Figure 4b). Abdominal fat in group E, especially epididymal fat (Epi fat, black arrows), was lesser than that of the control groups. In comparing the abdominal views and the abdominal fat, Epi fat (white arrows) and retroperitoneal fat (RP fat) appeared to be inhibited by injections of HYAL 104 IU (Figure 4c). The weights of Epi fat in groups C and E were 1.05±0.19 and 0.60±0.04 g, respectively (P<0.01). Thus, expansion of Epi fat was suppressed by up to 40% by injections of HYAL 104 IU. There were no differences, however, between the weights of organs in each group (Supplementary Figure 4b). The tendency for increased body fat to be suppressed by exogenous HYAL treatment was somewhat reflected in the total cholesterol (T.cho) and low-density lipoprotein (LDL-C) levels (Figure 4d) in serum. The T.cho levels in groups E and C were 151.50±3.12 and 168.75±8.07 mg dl−1, respectively (P<0.05). In addition, it appeared that the size of Epi fat cells in group E as seen on H&E staining was smaller than that of group C, indicating that the degree of lipid accumulation in fat cells was reduced by HYAL 104 IU injection (Figure 4e).

Figure 4
figure4

The effects of HYAL treatment on C57BL/6 J mice. (a) Weight gain was decreased by injections of HYAL 104 IU compared with the positive controls. Body weight was monitored every 6 days for 8 weeks. Groups A (empty circle) and B (gray circle) were negative controls for fat accumulation and high dose HYAL, respectively. Group C (black square) was a positive control for fat accumulation. Groups D (gray triangle) and E (black inverted triangle) were induced obesity groups that were simultaneously injected with HYAL 102 IU and104 IU, respectively (n=5 in all groups except group B, n=4 in group B). (b) Injections of HYAL 104 IU inhibited adipogenesis in body fat, especially epididymal fat (Epi fat, black arrows). Abdominal fat tissue was measured by micro-CT (resolution=35 μm). (c) Increases in body fat were suppressed by injections of HYAL 104 IU. The volume and weight of Epi fat (white arrows) and retroperitoneal fat (RP fat) were diminished in HYAL-injected mice. (d) Total cholesterol (T.cho) in serum showed a mild decrease after HYAL injection. Total cholesterol (T.cho), triglyceride (TG), high density lipoprotein (HDL-C), and low density lipoprotein (LDL-C) levels were evaluated by serum analysis. (e) Fat cells were smaller in size than those of the positive controls on H&E staining after paraformaldehyde fixation (scale bar=50 μm). The data are expressed as mean±s.e.m. and were analysed using two-way ANOVAs with Bonferroni correction for body weight, and one-way ANOVAs with Dunnett’s multiple comparisons tests for the weight of fat, serum analysis, and qReal-time PCR (*P<0.05, **P<0.001). The full colour version of this figure is available at International Journal of Obesity online.

Reductions in lipid accumulation in the liver after HYAL treatment

Liver damage is a serious problem brought about by NAFLD. We analysed the liver tissue from mice in each group to confirm whether the symptoms of NAFLD were reduced by HYAL treatment. On comparing the color of the liver tissue, the livers of mice in group E appeared healthier than those of group C (Figure 5a). This was clearly shown on H&E staining, as the lipid spots in livers from group E were fewer and smaller than that of group C (Figure 5b). The degree of liver injury was estimated numerically through serum levels of liver enzymes, including aspartate aminotransferase (AST), alanine transaminase (ALT) and alkaline phosphatase (ALP)37 (Figure 5c). Enzyme levels in group E were somewhat lower than those of group C, and there was no evidence of damage to normal livers from injection of HYAL 104 IU. Thus, our results showed that the accumulation of lipid in the liver was decreased by treatment with HYAL 104 IU, and that NAFLD did not develop in mice that received these injections due to the absence of progression of obesity.

Figure 5
figure5

The effects of HYAL injections on NAFLD on C57BL/6 J mice. (a and b) The accumulation of lipid in the liver was reduced by treatment with HYAL 104 IU. The livers were histologically analysed using H&E staining after paraformaldehyde fixation (scale bar=50 μm). (c) Hepatotoxicity caused by progression of obesity was alleviated by HYAL 104 IU injections. Liver enzymes including aspartate aminotransferase (AST), alanine transaminase (ALT) and alkaline phosphatase (ALP) were measured by serum analysis (n=4 in each group). The data are expressed as mean±s.e.m. and were analysed using one-way ANOVAs with Dunnett’s multiple comparisons tests (*P<0.05).

Effects of HYAL treatment on insulin resistance caused by HFD-induced abdominal obesity in C57BL/6J mice

Obesity is significantly associated with metabolic syndromes such as Type 2 diabetes and insulin resistance is a typical symptom of Type 2 diabetes. To investigate whether insulin resistance was improved with exogenous HYAL treatment, we performed IPGTTs and measured insulin levels in the serum. In group C, blood glucose peaked at 30 min (524.00±26.95 mg dl−1) and was maintained at a higher level at 90 min (338.60±25.33 mg dl−1) than in group A (Figure 6a). In addition, the insulin level of group C was 10.80±1.73 μIU ml−1, which was much higher than that of group A (9.00±0.71 μIU ml−1) (Figure 6b). Thus, insulin resistance appeared to be greater in the HFD-feeding mice group C. On the other hand, blood glucose in group E peaked at 15 min (477.20±17.96 mg dl−1) and subsequently decreased over time. There was a significant difference in blood glucose levels at 60 min (P<0.05) between groups C and E. In addition, the area under the curve for group E was 23% less than that of group C, based on the IPGTT graph (Figure 6a). The insulin level for group E was 4.18±0.46 μIU ml−1, and there were no significant differences between groups A and E (Figure 6b). Collectively, these results indicate that insulin resistance was alleviated after treatment with of HYAL 104 IU.

Figure 6
figure6

The effects of HYAL injections on insulin resistance in C57BL/6J mice. (a) Blood glucose levels in the HFD-fed, HYAL 104 IU-injected mice were more smoothly regulated than the positive controls during intraperitoneal glucose tolerance tests (IPGTT) (n=5 in each group except group B, n=4 in group B). Groups A (empty circle) and B (gray circle) were negative controls for obesity and high dose HYAL, respectively. Group C (black square) was a positive control for obesity, and Group E (black inverted triangle) had induced obesity and was injected with HYAL 104 IU. (b) HYAL 104 IU injections decreased insulin levels in the group fed HFD, but the insulin level of the negative control was not affected by HYAL. The values were expressed as mean±s.e.m. and analysed using two-way ANOVAs with Bonferroni correction (*P<0.05).

Discussion

Adipogenesis is regulated both spatially and temporally by sophisticated processes that maintain and alter the ECM via cell–cell and cell–environment interactions. The correlation between collagen and adipogenesis has been extensively studied in terms of the regulation of collagen synthesis and degradation. In these reports, adipogenesis was inhibited by the synthesis of certain types of collagen, and their degradation was artificially manipulated using ethyl-3,4-dihydroxybenzoate as a specific inhibitor of collagen synthesis and general inhibitor of MMP activity.38, 39 The ECM, however, includes a variety of components in addition to HA, and thus more sophisticated research approaches are necessary to understand the role of the ECM in adipogenesis. In this report we showed that the downregulation of HA in the ECM inhibited adipogenesis in vitro and in vivo and this finding would be a new angle to the adipogenesis research.

The relationship between adipogenesis and HA was shown via gene expression, as well as synthesis and degradation of HA during adipogenesis in 3T3-L1 cells.23 We focused on the effects of HA during adipogenesis both in vitro and in vivo, and the experiments were designed to downregulate HA during induction of adipogenesis. Downregulation of HA implies a reduction in high Mw HA (>2 MDa, the intact HA formation synthesized by HAS), the non-natural degradation of pre-existing or newly synthesized HA, and the artificial inhibition of HA synthesis. We first confirmed whether the HA concentration in culture media was reduced after treatment with exogenous HYAL and 4-MU in a concentration-dependent manner (Figure 1). Our results showed that treatment with HYAL and 4-MU suppressed the formation of lipid droplets and the accumulation of triglycerides, and reduced the expression of adipogenic marker genes in differentiated 3T3-L1 cells (Figure 2). We also confirmed that HA reduction by HAS2 knockdown inhibited adipogenesis (Figure 3). In addition, it was demonstrated that the abdominal fat accumulation and the serum lipid contents were reduced by exogenous HYAL 104 IU in HFD-feeding C57BL/6J mice (Figure 4).

Given that adipogenesis was inhibited by exogenous HYAL and 4-MU treatment in a concentration-dependent manner in vitro, we are able to predict the role of HA in adipogenesis. The function of HA is size (Mw)-dependent,19 and is thus determined by a regulated balance between synthesis by HAS and degradation by endogenous HYAL.40 It was confirmed that HAS2 and endogenous HYAL2 were primarily involved in adipogenesis in 3T3-L1 cells.25 HA concentration increased rapidly during the period from growth arrest to clonal expansion (Figure 1). However, HA accumulation did not affect clonal expansion (Supplementary Figures 3a and b). During this time frame, it was reported that HA synthesized by HAS2 had an average Mw of over 2 × 103 kDa, which is the longest chain synthesized by endogenous HAS and which corresponds to the HA Mw previously reported as being present during 3T3-L1 differentiation.23 It was previously thought that the accumulation of high Mw HA was required for the change in cellular morphology from a fibroblast-like shape to a round shape for the accumulation of lipid within the cytoplasm, similar to the early stages of differentiation of other cell types during morphogenesis.41, 42 This occurs in a hydrated environment, in which cells are prevented by structural barriers from undergoing morphogenetic changes and by receiving signals from HA and its associated factors.43 At the same time, expression of endogenous HYAL2 mRNA also increased (data not shown), which may be explained by the role of HYAL2 in the tethering of high Mw HA to CD44 on cell surfaces for intracellular signal transduction.44, 45 It has been reported that the HA-CD44-HYAL2 complex induces invagination of the plasma membrane, and that HYAL2 subsequently cleaves high Mw HA to a smaller 20 kDa size (50 disaccharide units), which is then internalized and delivered to the endosomes for signal transduction.40 The 20-kDa fragments of HA are highly angiogenic46 and induce transcription of MMPs.47 The details of HA-induced signal transduction for adipogenesis are unknown, though the results of our study suggest that HA fragments are involved in PPARγ-mediated adipogenesis, as well as in the angiogenesis and ECM alteration required for adipogenesis. A previous study suggested that multivalent binding of the HA ligand to CD44 at the cell surface may be an important first step in intracellular signaling by HA, and that it depended on the size of the HA ligand, as well as the quantity and density of cell surface CD44.48 The exogenous HYAL used in our experiments was testicular type HYAL; thus, it produced even-numbered oligosaccharides containing mostly tetrasaccharides as the smallest fragments, with N-acetylglucosamine at the reducing end, by random cleavage of β-N-acetyl-hexosamine (1→4) glycosidic bonds.41 It has been reported that HA oligomers having 6–18 sugars can bind monovalently to CD44, and that HA oligomers composed of 20–38 sugars can form divalent bonds with CD44.48 Both bonds have been shown to attenuate intracellular signaling of HA.49 As a result, it was supposed that HA tetrasaccharides produced by exogenous HYAL treatment cannot sufficiently stimulate intracellular signaling through monovalent binding with CD44. 4-MU is an indirect inhibitor of HA synthesis, which works by depleting UDP-glucuronic acid in cells. Therefore, the production of HA is downregulated by blocking elongation of HA at the extracellular surface.28 The common effects of exogenous HYAL and 4-MU treatment during adipogenesis included inhibition of timely accumulation of high Mw HA, resulting in a low-affinity interaction of HA ligand with CD44. Thus, downregulation of HA affected both morphogenesis and intracellular signal transduction of HA-induced adipogenesis.

Our studies showed that the inhibition of adipogenesis by downregulation of HA in vitro was also reflected on abdominal fat accumulation in HFD-feeding C57BL/6J mice. Our results showed that the epididymal fat tissue included in abdominal fat failed to develop after treatment with exogenous HYAL due to inhibition of the accumulation of triglycerides in fat cells (Figure 4e). In the obese population, fat cell size is an important factor due to the ability of large fat cells to secrete more adipokines. In addition, the distribution of fat tissue is more significant than the total fat mass, as fat tissue has endocrine functions.50, 51 It has been shown that abdominal fat distribution is strongly correlated with the metabolic complications of obesity.50 Our results suggest that exogenous HYAL treatment decreased fat cell size and reduced abdominal fat tissue, eventually alleviating the insulin resistance and fatty liver caused by abdominal obesity.

To date, the effects of blocking HA have not been studied in adipogenesis. Our in vitro and in vivo studies highlight the potential therapeutic use of HA manipulation in obesity and its related metabolic diseases. However, the anti-obese effects in HFD-diet-induced obese mice and anti-adipogenic effects in 3T3-L1 cells are difficult to reconcile as inhibition of adipogenesis does not inhibit obesity. It has been believed that suppression of adipogenesis does not always correlate with inhibition of obesity as hypertrophy is the main contributing factor to adipose tissue expansion in obesity. However, recent remarkable studies by tracking adipocytes show that HFD-induced obesity can expand epididymal adipose tissue by both hypertrophy and hyperplasia. The studies show that de novo adipogenesis in epididymal adipose tissue is preferentially initiated in the prolonged HFD-induced obesity.52 In line with this, inhibition of hyperplasia can affect HFD-induced obesity similar to the effects by HA inhibition of the current study. It is also possible that the inhibition of obesity by suppression of HA can be attributed todecreased food intake. We failed to discriminate the food intake between control and HYAL-treated obese mice, suggesting that the energy consumption may not be a potential contributing factor. The increased energy expenditure by HA suppression is another possibility for the anti-obese effects of HA suppression. Collectively, it will be intriguing to elucidate the target tissues for the anti-obese effects of HA inhibition. The underlying mechanisms of a reduction in insulin resistance, fatty liver and inflammation would also be very interesting lines of study to be pursued.

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Acknowledgements

This study was supported by a grant from KAVITA Co. Mr. Hyenho Myeong from Korea BMI supplied the HYAL.

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Correspondence to S Lee.

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The authors declare no conflict of interest.

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Supplementary Information accompanies this paper on International Journal of Obesity website

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Ji, E., Jung, M., Park, J. et al. Inhibition of adipogenesis in 3T3-L1 cells and suppression of abdominal fat accumulation in high-fat diet-feeding C57BL/6J mice after downregulation of hyaluronic acid. Int J Obes 38, 1035–1043 (2014) doi:10.1038/ijo.2013.202

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Keywords

  • extracellular matrix
  • hyaluronic acid
  • adipogenesis
  • 3T3-L1 cells
  • high-fat diet-induced obesity

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