Activation of PPARγ at an Early Stage of Differentiation Enhances Adipocyte Differentiation of MEFs Derived from Type II Diabetic TSOD Mice and Alters Lipid Droplet Morphology

Abstract

Type 2 diabetic Tsumura, Suzuki, obese, diabetes (TSOD) mice gradually gain weight as compared to corresponding Tsumura, Suzuki, non-obesity (TSNO) control mice, and develop insulin resistance. Although development of type 2 diabetes mellitus is associated with dysfunction of adipocytes, little is known about the properties of adipocytes from TSOD mice. Therefore, we attempted to remove intracorporeal factors and elucidate inherent properties of adipocytes of TSOD mice using adipocytes differentiated from mouse embryonic fibroblasts (MEFs) in vitro. Here, we show that MEFs of TSOD have low potency for differentiation into adipocytes. The percentage of Oil red O-stained cells and levels of adipogenic markers in cells differentiated from MEFs of TSOD are lower than those in cells differentiated from MEFs of TSNO. We further show that treatment with an agonist of peroxisome proliferator-activated receptor-γ (PPARγ) (rosiglitazone) at an early stage of differentiation increases the percentage of Oil red O-stained cells in TSOD-MEFs differentiated into adipocytes. Moreover, the lipid droplet size in those adipocytes is larger than that in the adipocytes differentiated from MEFs of TSNO. Although persistent treatment of MEFs of TSOD with rosiglitazone during differentiation increases the percentage of Oil red O-stained cells, the lipid droplet size in adipocytes treated as such does not reach the size of those treated in early stage only. Thus, activation of PPARγ by its agonist at an early stage of differentiation compensates for the low potency toward adipogenic differentiation of, and accelerates formation of enlarged lipid droplets in adipocytes derived from, MEFs of TSOD mice.

The worldwide population with type 2 diabetes mellitus (T2DM) is increasing, and the number of patients reached 415 million in 2015.¹⁾ Since T2DM is associated with vascular and other end-organ disease,²⁾ prevention of T2DM is a major public health issue. It is well known that obesity is a major risk factor for T2DM.³⁾ Notably, incident of T2DM is correlated with abdominal visceral adipose tissue mass in humans.⁴⁾ Therefore, understanding of roles of adipose tissue in the development of T2DM may lead to better methods for prevention of T2DM.

Adipose tissue plays important roles in energy storage and release, and secretes various cytokines. Previous studies show that adipose-specific knockout of mitochondrial transcription factor A enhances energy expenditure and protects from diet-induced obesity and insulin resistance in mice.⁵⁾ Moreover, adipose-specific glucose transporter type 4 (GLUT4) knockout mice exhibit insulin resistance and diabetes via secretion of retinol binding protein-4.⁶⁾ Thus, adipose functions are closely associated with the development of insulin resistance and T2DM. However, since T2DM is a complex illness resulting from a variety of genetic and environmental factors, single gene knockout mice provide limited information on the roles of adipose tissue in the development of T2DM. Therefore, more suitable model of human T2DM have been needed for such a kind of research.

In 1992, the Tsumura, Suzuki, obese, diabetes (TSOD) mouse has been established as an animal model of human T2DM by the selective breeding of male mice of ddY strain with heavy body and urinary glucose.⁷⁾ The mice exhibit overeating and gradually gain weight as compared to corresponding control mice, Tsumura, Suzuki, non-obesity (TSNO), and high blood glucose and urinary glucose are frequently observed in male mice of TSOD. Quantitative trait locus (QTL) analysis reveal that body weight and blood glucose levels in male mice of TSOD are affected by combination of multiple genetic loci (e.g., NIDD4, 5, 6).⁸⁾ Several studies examine the roles of adipose tissue in development of T2DM using the mice and show that insulin-dependent glucose uptake⁹⁾ and GLUT4 translocation to the plasma membrane¹⁰⁾ is impaired in the adipose tissue. However, since these properties may be developed by various intracorporeal factors such as hormones and cytokines, the inherent properties of adipocytes and its specific roles in development of T2DM in TSOD mice are not fully understood. Therefore, to remove intracorporeal factors and further evaluate the properties of adipocytes of TSOD mice, we attempted to differentiate mouse embryonic fibroblasts (MEFs) derived from TSOD mice into adipocytes in vitro.

Unexpectedly, we found reduced expression of several adipogenic markers in cells differentiated from MEFs of TSOD mice vs. MEFs differentiated from TSNO mice. Therefore, MEFs derived from TSOD have a lower propensity toward adipocyte differentiation than MEFs from TSNO mice. Further analysis revealed that lower potential toward adipocyte differentiation is reversed by peroxisome proliferator-activated receptor-γ (PPARγ), a key regulator of adipogenic differentiation. When PPARγ agonist treatment occurs at an early stage of differentiation, MEFs of TSOD more readily differentiate into adipocytes, similar to MEFs of TSNO mice. Intriguingly, the adipocytes exhibit enlarged lipid droplets. Thus, our results reveal new insight into the properties of adipocytes of TSOD mice.

MATERIALS AND METHODS

Preparation of MEFs

To prepare MEFs, E14 embryos were obtained from TSOD or TSNO pregnant mice (11 weeks of age; institute for animal reproduction, Ibaraki, Japan). In brief, embryos were washed twice with phosphate buffered saline (PBS), and then the head and internal organs of each embryo were removed with scissors. To determine sex of embryos, genomic DNA was extracted from the head of embryos using Geno Plus™ Genomic DNA Extraction Miniprep System (Viogene-BioTek, Taipei, Taiwan). Amplification of Sox17, which is coded on the X chromosome, and Zfy, which is coded on the Y chromosome, were performed by PCR using T100™ thermal cycler (Bio-Rad, Hercules, CA, U.S.A.) with Tks Gflex DNA polymerase (TaKaRa, Otsu, Japan) and the specific primers: Sox17, 5′-ccc tta agg ccg tag tac aggtgcagagc-3′ (sense) and 5′-GCC GCG TGG CCA TGG ATG GC-3′ (antisense); Zfy, 5′-CCT ATT GCA TGG ACT GCA GCT TAT G-3′ (sense) and 5′-GAC TAG ACA TGT CTT AAC ATC TGT CC-3′ (antisense).¹¹⁾ Reaction mixtures were incubated for an initial denaturation at 95°C for 5 min followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 56°C for 30 s and extension at 72°C for 1 min. The products of PCR were subjected to electrophoresis on a 2% agarose gel containing 0.5 µg/mL ethidium bromide (Nippon Gene, Toyama, Japan). When both Sox17 and Zfy was amplified from genomic DNA, we determined the embryo as male. Dissected male embryos were digested for 24 h at 4°C in Dulbecco’s-PBS(−) containing 0.25% Trypsin (Life Technologies, Carlsbad, CA, U.S.A.) and 0.02% ethylenediaminetetraacetic acid. The cells were dispersed and cultured in Dulbecco’s modified Eagle’s medium containing 4.5 mg/L D-glucose (DMEM-high glucose; Life Technologies) supplemented with 10% fetal bovine serum (FBS, Biowest, Nuaillé, France), 50 units/mL penicillin and 50 µg/mL streptomycin (Sigma-Aldrich, St. Louis, MO, U.S.A.). In each experiment, we obtained embryos from at least two pregnant TSOD or TSNO mice, and MEFs are derived from single embryo. All experiments proceeded in accordance with the Guide for the Care and Use of Laboratory Animals at Teikyo University and were approved by the Animal Care and Use Committee at Teikyo University (approval numbers, 12-011).

Differentiation into Adipocytes and Treatment with Rosiglitazone

For differentiation of MEFs into mature adipocytes, post-confluent cells (designated day 0) were induced to differentiate by addition of 0.5 mM 3-isobuthyl-1-methylxanthine (Sigma-Aldrich), 1 µM dexamethasone (Sigma-Aldrich) and 5 µg/mL bovine insulin (Sigma-Aldrich) in the media until day 4. Then, cells were cultured with DMEM-high glucose containing 10% FBS and 5 µg/mL insulin for 2 d. After day 6, the differentiated state was maintained with 0.5 µg/mL insulin until indicated periods. When PPARγ was activated by the rosiglitazone, 1 nM rosiglitazone (Wako Pure Chemical Industries, Ltd., Osaka, Japan) was added into the media during differentiation.

Oil Red O Staining

MEFs derived from TSOD and TSNO mice were differentiated into adipocytes until indicated time points, and then washed twice with warm PBS followed by fixation with 10% formaldehyde (Wako Pure Chemical Industries, Ltd.) for 30 min. After equilibration with 60% isopropanol, cells were stained for 1 h with Oil red O solution (Oil red O dye dissolved in 60% isopropanol). Then, stained cells were washed three times with 60% isopropanol and once with MilliQ water. After microscopic observation and taking photos, cells were collected with a cell scraper. Then, intracellular Oil red O dye was extracted from the cells with 100% isopropanol. The amount of intracellular Oil red O dye was determined by measuring at 540 nm with spectrophotometer (UV-1800; Shimadzu, Kyoto, Japan). Quantification of the lipid droplet size was performed using five sections from each culture dish with Image J software (National Institutes of Health, Bethesda, MD, U.S.A.) as described in a previous publication.¹²⁾ The size of each lipid droplet was determined by the number of pixels encompassed by the droplet; the area of one pixel is equivalent to 3×10⁻² µm².

Quantitative RT-PCR (qRT-PCR)

MEFs derived from TSOD and TSNO mice were induced to differentiation, and then total RNAs were isolated from the cells at the indicated time points using the RNAiso Plus (TaKaRa) according to the manufacturer’s instructions. cDNAs were synthesized from 1 µg of each RNA using the PrimeScript II reverse transcriptase (TaKaRa). PCR was performed using the SYBR Premix Ex Taq II (TaKaRa) with Applied Biosystems 7500 Real-Time PCR system (Applied Biosystems, Foster City, CA, U.S.A.) as described in Ishibashi et al. The primers are as follows: PPARγ, 5′-CCC CTG CTC CAG GAG ATC TAC-3′ (sense) and 5′-GCA ATC AAA GAA GGA ACA CGT TGT-3′ (antisense)¹³⁾; Fatty acid binding protein 4 (FABP4), 5′-AAG TGG GAG TGG GCT TTG C-3′ (sense) and 5′-TGG TGA CCA AAT CCC CAT TT-3′ (antisense)¹²⁾; CCA AT enhancer-binding protein α (C/EBPα), 5′-GAC CAT TAG CCT TGT GTG TAC TGT ATG-3′ (sense) and 5′-TGG ATC GAT TGT GCT TCA AGT T-3′ (antisense)¹⁴⁾; C/EBPβ, 5′-TGG ACA AGC TGA GCG ACG AG-3′ (sense) and 5′-GAA CAA GTT CCG CAG GGT GC-3′ (antisense)¹⁵⁾; C/EBPδ, 5′-TCC ACG ACT CCT GCC ATG TA-3′ (sense) and 5′-GCG GCC ATG GAG TCA ATG-3′ (antisense)¹⁶⁾; 18S rRNA, 5′-GGC GTC CCC CAA CTT CTT A-3′ (sense) and 5′-GGG CAT CAC AGA CCT GTT ATT G-3′ (antisense).¹³⁾

RESULTS

Low Potential for Adipocyte Differentiation of MEFs Derived from TSOD Mice

MEFs derived from TSOD or TSNO mice were induced differentiation into adipocytes as above in the absence of PPARγ agonist, rosiglitazone. Since TSOD mice exhibit obesity,⁷⁾ we expected that MEFs derived from TSOD would readily differentiate into adipocytes. To examine the differentiation degree, we stained cells with Oil red O and measured both the number of stained cells (differentiation rate) and the amount of incorporated Oil red O (triglyceride content). However, unexpectedly, the number of Oil red O-stained cells in differentiated MEFs derived from TSOD mice was lower than that in MEFs derived from TSNO mice (Figs. 1A, B). Moreover, the amount of incorporated Oil red O was lowered in cells differentiated from MEFs of TSOD mice (Fig. 1C). Adipogenic marker expression (PPARγ and FABP4)¹⁷⁾ increased until day 8 in differentiating MEFs of TSNO and TSOD (data not shown). However, at 8 d and beyond induction to differentiation, the levels of both markers in cells differentiated from MEFs of TSOD mice was lower than that in cells differentiated from MEFs of TSNO mice (Fig. 1D). Taken together, these results suggest that MEFs of TSOD mice exhibit lower potential toward adipocyte differentiation than MEFs of TSNO mice under similar culture conditions in the absence of specific agonist compounds.

Fig. 1. Low Potential for Adipocyte Differentiation of MEFs Derived from TSOD Mice

MEFs derived from TSNO or TSOD mice were induced to differentiate into adipocytes as described under Materials and Methods. (A) Cells were stained with Oil red O and representative images are shown. (B) The number of Oil red O-stained cells were expressed as a percentage of total number of cells counted from at least three sections of photo obtained from each MEF. (C) The amount of incorporated Oil red O dye was measured at 540 nm with spectrophotometer. Results represent the mean±S.D., n=3. (D) The expression levels of PPARγ and FABP4 on day 8 were assessed with qRT-PCR. Results represent the mean±S.D., n=3. Asterisks indicate the significant difference (* p<0.05; ** p<0.01) calculated by Student’s t-test.

Differential Expression of Adipocyte Markers of Cells Derived from MEFs of TSOD vs. TSNO Mice at an Early Stage of Adipocyte Differentiation

It is known that expression levels of C/EBPβ and δ are increased at the initial stages of MEFs differentiation to adipocytes followed by an increase of PPARγ and C/EBPα expression.^18,19) As shown in Fig. 2 (upper panels), there was no difference between cells differentiated from MEFs of TSNO and TSOD mice in expression of C/EBPβ or δ. In contrast, expression levels of PPARγ and C/EBPα were lower in cells differentiated from MEFs of TSOD vs. TSNO mice at day 4 (Fig. 2, lower panels). These results suggest that MEFs of TSOD may initiate differentiation appropriately, but have a block in early stage of differentiation.

Fig. 2. Differential Expression of Adipocyte Markers of Cells Derived from MEFs of TSOD vs. TSNO Mice at Early Stages of Adipocyte Differentiation

MEFs derived from TSNO or TSOD mice were induced toward adipocyte differentiation until day 4. The expression levels of C/EBPα, β, δ and PPARγ were assessed with qRT-PCR at days 0, 1, 2 and 4. Results represent the mean±S.D., n=3 (C/EBPα, β, δ) or 6 (PPARγ). Asterisks indicate the significant difference (* p<0.05; ** p<0.01) calculated by Student’s t-test.

Treatment with Rosiglitazone at an Early Stage of Adipogenic Differentiation Accelerates Differentiation of MEFs Derived from TSOD Mice

It is well known that PPARγ acts as a master regulator of adipocyte differentiation²⁰⁾ and activation of PPARγ induces expression of itself and various molecules including FABP4 and C/EBPα.²¹⁾ Thus, we hypothesized that insufficient activation of PPARγ might be involved in the lower potential of TSOD-derived MEFs toward adipocyte differentiation. To test this hypothesis, MEFs of TSOD were induced toward adipocyte differentiation in the presence of a PPARγ agonist (rosiglitazone). Treatment with rosiglitazone from days 0 to 8 increased the number of Oil red O-stained cells (Figs. 3A, B), the content of Oil red O (Fig. 3C) and expression levels of PPARγ and FABP4 (Fig. 3D) in cells differentiated from MEFs of TSOD mice.

Fig. 3. Role of PPARγ Activation at Early Stages of Adipocyte Differentiation of MEFs Derived from TSOD Mice

MEFs derived from TSNO or TSOD mice were induced toward adipocyte differentiation until day 8. During differentiation into adipocytes, cells were treated with DMSO or PPARγ agonist (rosiglitazone) from days 0 to 8 (days 0–8), days 0 to 2 (days 0–2), days 2 to 4 (days 2–4) or days 4 to 6 (days 4–6). (A) Cells were stained with Oil red O and representative images are shown. (B) The number of Oil red O-stained cells were expressed as a percentage of total number of cells counted from at least three sections of photo obtained from each MEF. (C) The amount of incorporated Oil red O dye was measured as described in Fig. 1B. (D) The expression levels of PPARγ and FABP4 were assessed with qRT-PCR. Results represent the mean±S.D., n=6–14. Asterisks indicate the significant difference (* p<0.05; ** p<0.01; *** p<0.001; NS, not significant) calculated by Student’s t-test.

To focus on the temporal relationship of PPARγ activation with adipocyte differentiation in the early stages, MEFs from TSOD mice were treated with rosiglitazone from days 0 to 2 (0–2), days 2 to 4 (2–4) or days 4 to 6 (4–6). Intriguingly, days 0–2 appeared to be most important; the number of Oil red O-stained cells (Figs. 3A, B), the content of Oil red O (Fig. 3C) and expression levels of PPARγ (Fig. 3D) were similar in cells differentiated from MEFs of TSOD mice with rosiglitazone from days 0 to 8 and days 0 to 2; treatment from days 2–4 or 4–6 increased the number of Oil red O-stained cells, but not to the same degree (Fig. 3B). However, expression levels of PPARγ were similar regardless of treatment from days 0–2, 2–4 or 4–6 (Fig. 3D). These results suggest that the number of cells differentiating into adipocytes was different based on the timing of activation of PPARγ. Intriguingly, the propensity of adipocytes differentiated from MEFs of TSOD mice with rosiglitazone from days 0 to 8 and days 0 to 2 will not same propensity, because the content of Oil red O seemed to be higher in cells treated with rosiglitazone from days 0 to 2 (Fig. 3C). Therefore, the droplet size (i.e., amount of Oil red O per cell; a marker of hypertrophic adipocytes) might be different as well prompting the studies below.

Compensation of PPARγ Activation Develops Enlarged Lipid Droplets in Adipocytes Differentiated from MEFs of TSOD Mice

At day 8, there was no difference in the lipid droplet size among adipocytes differentiated from MEFs of TSNO and TSOD with or without rosiglitazone (Fig. 4, left panel). However, when cells were treated with rosiglitazone from days 0 to 2 and differentiated into adipocytes until day 16, the average size of lipid droplets in cells differentiated from MEFs of TSOD mice were larger than those in cells differentiated from MEFs of TSNO mice (Fig. 4, right panel). Moreover, adipocytes differentiated from MEFs of TSOD mice with rosiglitazone days 0 to 2 had larger lipid droplets than adipocytes differentiated from MEFs of TSNO mice without rosiglitazone. On the other hand, adipocytes differentiated from MEFs of TSOD mice with rosiglitazone from days 0 to 16 had small lipid droplets compared with cells differentiated from MEFs of TSNO mice without rosiglitazone.

Fig. 4. Brief, Early PPARγ Activation Results in Enlarged Lipid Droplets in Adipocytes Differentiated from MEFs of TSOD Mice; Persistent Activation Does Not

Quantitation of lipid droplet size. MEFs of TSNO or TSOD were differentiated into adipocytes until days 8 or 16 in the presence of DMSO or rosiglitazone from days 0 to 2 (days 0–2), days 0 to 8 (days 0–8) or days 0 to 16 (days 0–16). Then, cells were stained with Oil red O and average size of lipid droplet was measured using Image J software as described in Materials and Methods. Results represent the mean±S.D., n=3. Asterisks indicate the significant difference (* p<0.05; NS, not significant) calculated by Student’s t-test.

Taken together, these results suggest that MEFs derived from TSOD mice have a lower potential for adipocytes differentiation than MEFs derived from TSNO mice. This lack of differentiation is most likely NOT due to initiation of adipocyte formation, but poor adipocyte development that can be overcome early (days 0–2 of differentiation) by PPARγ agonist treatment. Such treatment restores the percentage of MEFs differentiating into adipocytes, but changes the phenotype to one of enlarged lipid droplets. If PPARγ agonist treatment is continued beyond days 2 to 16, the lipid droplets of TSOD MEF-derived adipocytes do not become enlarged.

DISCUSSION

In the present study, we have shown that MEFs derived from TSOD mice are less likely to differentiate into adipocytes than MEFs derived from TSNO mice. Activation of PPARγ by its agonist, rosiglitazone, at an early stage of differentiation compensates for this deficiency but leads to enlarged lipid droplets in the derived adipocytes.

The mechanism of insufficient activation of PPARγ in MEFs derived from TSOD mice remains unclear. During adipocyte differentiation, activity of PPARγ is regulated by its expression and binding with its ligands.²²⁾ It is known that PPARγ enhances C/EBPα expression, and C/EBPα positively regulates expression of PPARγ.²³⁾ As shown in Fig. 2, induction of C/EBPα is repressed in cells differentiated from MEFs of TSOD mice vs. those of TSNO mice, suggesting that repression of C/EBPα expression may be partially involved in low levels of expression of PPARγ. However, our results are not likely due only to repression of C/EBPα expression. There is no difference in expression levels of C/EBPα between MEFs of TSOD vs. TSNO mice during day 0 to 2 (Fig. 2), although expression levels of PPARγ seem to be lowered in MEFs of TSOD mice in the period. Therefore, we considered other possibilities and hypothesized that PPARγ in MEFs of TSOD mice exhibit lower affinity for ligands or activation after ligand binding than MEFs of TSNO mice. Previously, it has been reported that mutations in ligand-binding of PPARγ in patients with T2DM reduce ligand-affinity and impair transcriptional activity of PPARγ.²⁴⁾ Further analysis of regulation of PPARγ activity will help us determine which of these possibilities are more likely to explain the insufficient activation of PPARγ in MEFs of TSOD mice.

TSOD mice exhibit obesity and their adipose tissue has hypertrophic adipocytes.²⁵⁾ Since adipose-specific PPARγ knockout mice exhibit reduced fat mass and are resistant to high fat diet-induced obesity,²⁶⁾ activation of PPARγ will occur during development of obesity in TSOD mice. As shown in Fig. 4, persistent (days 0–16) activation of PPARγ with rosiglitazone during differentiation enhances adipocyte differentiation, although the lipid droplet size in the cells is smaller than that in adipocytes differentiated from MEFs of TSNO mice. This effect of PPARγ agonist on adipocytes is consistent with a previous report.²⁷⁾ On the other hand, limited (days 0–2 only) exposure to rosiglitazone at an early stage of differentiation develops enlarged lipid droplets in adipocytes differentiated from MEFs of TSOD mice (Fig. 4). Moreover, since expression levels of FABP4, a transcriptional target of PPARγ,²⁸⁾ is lowered in adipocytes differentiated from MEFs of TSOD with rosiglitazone from days 0 to 2 compared with those from days 0 to 8 (Fig. 3D), activation of PPARγ will be decreasing toward the late stages of differentiation. These results suggest that temporary activation of PPARγ enables MEFs of TSOD to differentiate into adipocytes and develops enlarged lipid droplets, which will occur during development of obesity in TSOD mice. This hypothesis is supported by the observation that TSOD mice dramatically gain weight around 5 weeks of age and the rate of weight gain is similar to that of TSNO mice after 5 weeks of age.^10,29) In the period, obesity is mainly developed by overeating which is caused by hypothalamic neuropeptides and the properties of adipocytes may synergistically promote the development of obesity. It will lead to development of T2DM. Therefore, exploration of intrinsic PPARγ agonists secreted from other tissues or the adipocyte itself may be of value in further determining the mechanisms of T2DM in TSOD mice.

How might adipocytes differentiated from MEFs of TSOD mice develop lipid droplets of different size than TSNO mice in vitro? Lipid droplet size is generally correlated with content of triglyceride in the droplets. Since expression levels of both adipose tissue triglyceride lipase and hormone-sensitive lipase is lowered in adipose tissue of TSOD mice,³⁰⁾ expression of these lipases would be expected to be lowered in adipocytes differentiated from MEFs of TSOD mice with rosiglitazone at early stage of differentiation.

In conclusion, we show for the first time that MEFs of TSOD mice exhibit a lower potential toward adipocyte differentiation, which is restored by activation of PPARγ at an early stage of differentiation. Moreover, PPARγ agonist treatment of MEFs from TSOD mice results in adipocytes that form enlarged lipid droplets. Further study of the regulation of PPARγ activity at the early stages of differentiation is likely to elucidate important mechanisms that may contribute to obesity and T2DM.

Acknowledgment

This work was supported in part by the TSOD Mouse Research Fund for E. N and N.O.

Conflict of Interest

The authors declare no conflict of interest.

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