Submit or Track your Manuscript LOG-IN

Effects of Energy Levels on Autophagy, Adipogenic Differentiation and Lipid Metabolism in Subcutaneous and Visceral Pre-Adipocytes


Effects of Energy Levels on Autophagy, Adipogenic Differentiation and Lipid Metabolism in Subcutaneous and Visceral Pre-Adipocytes

Jie Chen, Qinghua Qiu, Yanjiao Li, Xianghui Zhao, Lanjiao Xu, Xiaowen Xiong, Qingqi Wen, Mingren Qu and Kehui Ouyang*

Jiangxi Province Key Laboratory of Animal Nutrition/Engineering Research Center of Feed Development, Animal Nutrition and Feed Safety Innovation Team, Institute of South Grassland Research, Jiangxi Agricultural University, Nanchang, Jiangxi, 330045, China

Jie Chen and Qinghua Qiu contributed equally to this work as first authors.


This study aimed to investigate the differences in energy levels on autophagy, adipogenic differentiation and fat metabolism between subcutaneous and visceral pre-adipocytes. C57BL/6J male mouse primary subcutaneous and visceral pre-adipocytes were isolated and in-vitro adipogenic differentiation was performed in high-glucose medium (4500 mg/L glucose, HGM) or low-glucose medium (1000 mg/L glucose, LGM). Lipid contents in adipocytes were determined by oil red-O staining. Autophagy, adipogenic differentiation and fat metabolism were evaluated by western blot. Subcutaneous pre-adipocytes proliferation rate, differentiation degree and lipid accumulation were higher than that of visceral pre-adipocytes both in HGM and LGM. In HGM group, autophagy marker genes i.e., microtubule-associated protein 1A/1B-light chain 3 (LC-3) and lipolysis protein i.e., hormone-sensitive lipase (HSL) expression in visceral pre-adipocytes were higher than that in subcutaneous pre-adipocytes, but adipogenesis transcriptional factors such as sterol regulatory element-binding protein 1c (SREBP-1c) and peroxisome proliferator-activated receptor-gamma (PPAR-γ) were lower than that in subcutaneous pre-adipocytes. Moreover, the LGM group increased the expressions of LC-3, reduced the p62 in both subcutaneous and visceral pre-adipocytes, and decreased the expression of the PPAR-γ, SREBP-1c, and HSL. It is shown that triglyceride accumulation of pre-adipocytes in LGM was more than that of HGM. There were differences in autophagy, adipogenesis differentiation and lipolysis between subcutaneous and visceral pre-adipocytes in vitro. In addition, the low energy levels on the adipogenesis differentiation and lipid metabolism of subcutaneous pre-adipocytes were greater than that of visceral pre-adipocytes, but not the responses to autophagy.

Article Information

Received 13 May 2021

Revised 18 June 2021

Accepted 30 June 2021

Available online 10 May 2022

(early access)

Published 22 February 2023

Authors’ Contribution

JC and KO conceptualization. JC methodology. JC, QQ and KO validation. JC and QQ formal analysis. JC, QQ, YL, XZ, LX, XW, QW, MQ and KO investigation. JC, QQ and KO data curation. JC writing original draft preparation. JC, QQ and KO writing review and editing. JC, QQ and KO visualization. QQ and KO supervision. KO project administration. KO funding acquisition.

Key words

Subcutaneous pre-adipocytes, Visceral pre-adipocytes, Autophagy, Lipid accumulation, Energy levels


* Corresponding author:

0030-9923/2023/0003-1009 $ 9.00/0

Copyright 2023 by the authors. Licensee Zoological Societ of Pakistan.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (


The growth rate of livestock is fast during the fattening period and high production could be obtained by supplying high energy (Scheffler et al., 2014). However, feeding high-energy diets generally increases the fat deposition of the subcutaneous and visceral tissues in varying degrees, which is thought to be a waste and resultant inefficiency in production and economic benefits (Krieger et al., 2018; Bachmann et al., 2018). Numerous animal studies have demonstrated that energy restriction reduces the overall fat deposition of fattening animals and reduces the subcutaneous and visceral tissue in varying degrees (Abdullah and Musallam, 2007; Khanal et al., 2016). Fat deposition consists of two steps: first, pre-adipocytes differentiate into mature adipocytes, and then triglycerides are accumulated in mature adipocytes through fat synthesis and fat degradation. It has been reported in previous studies that different concentrations of glucose affect pre-adipocytes differentiation (Xie et al., 2008; Krishna et al., 2020). However, the relationship between higher fat deposition and high differentiation of pre-adipocytes is still not clear. Moreover, the difference in energy levels on pre-adipocytes differentiation of subcutaneous and visceral is still unknown.

Animal adipose deposition is generally determined by processes that regulate adipocyte number and size (Macotela et al., 2012). The pre-adipocytes undergo differentiation in response to adipogenic signal factors. Furthermore, pre-adipocytes undergo differentiation to active major transcriptional signals, such as sterol regulatory element-binding protein 1c (SREBP-1c) and peroxisome proliferator-activated receptor-gamma (PPAR-γ), that sequentially turns on adipogenic process (Rosen and Macdougald, 2006). Moreover, increased adipogenesis increase adipocyte number, that is, proliferation and differentiation of pre-adipocytes. The size of adipocytes was due to lipogenesis and lipolysis with the help of fatty acid synthase (FAS) and hormone-sensitive lipase (HSL), respectively (Boström et al., 2005; Fujimoto and Parton, 2011). Previous study has reported that subcutaneous pre-adipocytes and visceral pre-adipocytes have the potential of differentiation, but there are biological differences between them because of different origin and growth microenvironment (Fraser et al., 2006). Therefore, clarifying the differences in fat deposition between subcutaneous and visceral pre-adipocytes differentiation could be helpful to reveal the regulation of lipogenesis and lipolysis of subcutaneous and visceral adipocytes by dietary energy in animal production.

Many reports revealed that autophagy plays a key role in differentiation of pre-adipocytes (Christian et al., 2013). In the process of pre-adipocyte differentiation, cytoplasm’s other organelles are gradually degraded and triglycerides gradually occupy those organelles, for further mature adipocytes formation. The mechanism by which cytoplasm is removed is not elucidated; therefore, autophagy is one of the best well-characterized processes to examine degradation of intracellular proteins and organelles in cytoplasm (Mizushima et al., 2008). It has been reported that autophagy is a lysosome-mediated self-degradation pathway that widely exists in eukaryotic cells (Klionsky et al., 2016). It has also been reported that autophagy plays a key role in the process of differentiation and examining the responses of differentiation; thus, autophagy could be a possible marker (Dong and Czaja, 2011; Goldman et al., 2011). In a recent study, its similar impairment of adipocyte differentiation was observed in 3T3-L1 pre-adipocytes in which microtubule -associated protein 1A/1B-light chain-3 (LC-3) was removed (Hwang and Lee, 2020), or the autophagy process was blocked by chemical inhibitors (Park et al., 2017). Another study has also reported that removal of gene 5 or 7 related to autophagy in the mice reduces lipid biosynthesis and decreases overall body mass of fat tissues (Zhang et al., 2009). A recent experiment has proven that energy levels have a great regulatory effect on autophagy (Sugita, 2020), but energy levels in the subcutaneous and visceral pre-adipocytes differentiation and autophagy difference are still unknown. Therefore, in this study, mouse primary subcutaneous and visceral pre-adipocytes were cultured in vitro for adipogenesis differentiation with high-glucose or low-glucose, to explore the effect of energy levels on autophagy, adipogenesis differentiation, adipogenesis, and lipolysis between subcutaneous and visceral pre-adipocytes.


Subcutaneous and visceral primary pre-adipocytes isolation and culture

The 5 week old C57BL/6J male mice were dislocated to death and soaked in ice-cold 1 × phosphorus buffer saline (PBS). The subcutaneous pre-adipocytes were collected from groin, and the visceral pre-adipocytes was collected from epididymis. The isolation and culture of mouse pre-adipocytes were performed as previously described as Wei et al. (2015) with minor modifications. Briefly, subcutaneous and visceral adipose tissue was rinsed with 1 × PBS and cut with scissors into approximately 1 mm3 section under sterile conditions, followed by digestion with 1% type 1 collagenase (Solarbio, China) and 0.25% trypsin (Solarbio, China) at 37 for 50 min in a shaking water bath. Then, complete medium (Dulbecco’s modified Eagle’s high-glucose medium, HGM, 4500 mg/L glucose) supplemented with 10% fetal bovine serum (FBS) and antibiotics (500 μg/mL penicillin and 500 μg/mL streptomycin) was added to stop further digestion. The mixture was filtered through a 150 μm and a 400 μm cell strainer, and centrifuged at 1600 rpm for 10 min. Then, the precipitation was re-suspended with erythrocyte lysate (Solarbio, China) and kept for 10 min. Obtained precipitation was centrifuged at 1200 rpm for 10 min. The pellet containing stromal vascular cells was washed by 1 × PBS, re-suspended in complete medium and incubated at 37 in a humidified atmosphere containing 5% CO2. All cell culture experiments were carried out in triplicate per mouse.

Determination of growth curve of subcutaneous and visceral pre-adipocytes

The subcutaneous and visceral primary pre-adipocytes were seeded in 96-well culture plate (n = 6) for 24 h. Then, 10 μL of cell counting kit-8 (CCK-8) (Solarbio, China) solution was added to each well and incubated for 4 h, followed by absorbance measurement at 450 nm by a microplate reader (Thermo Fisher, USA) (day 1). After then, every two days the number of pre-adipocytes was determined by CCK-8 until day 15. The growth curve of pre-adipocytes represented the total succinate dehydrogenase activity per 5 × 103 cells.

Differentiation of subcutaneous and visceral pre-adipocytes in HGM or LGM

The subcutaneous and visceral primary pre-adipocytes were seeded in 6-well culture plates and separated into several groups: subcutaneous pre-adipocytes in high-glucose medium (SH), subcutaneous pre-adipocytes in low-glucose medium (Dulbecco’s modified Eagle’s low-glucose medium (LGM, 1000 mg/L glucose) (SL), visceral pre-adipocytes in high-glucose medium (VH), visceral pre-adipocytes in low-glucose medium (VL). The differentiation method was carried out as described in previous study (Kaneko et al., 2018). For adipocyte differentiation, 100% confluent cells were further incubated for the complete cell cycle arrest and for the start of clonal expansion in HGM or LGM that was supplemented with 10% FBS and 1% antibiotics, which was further supplemented with 1.72 μM insulin (Solarbio, China), 0.5 mM 1-methyl-3-isobutylxanthine (Solarbio, China), and 1 μM dexamethasone (Solarbio, China) (day 0). Two days later, the cells were further incubated in HGM or LGM supplemented with 1.72 μM insulin, for two days. After that, the cells were replaced by the fresh HGM or LGM every two days until the cells were completely differentiated.

Evaluation of accumulated lipid contents in adipogenesis

Cells were washed twice with 1 × PBS solution, fixed in 4% paraformaldehyde 150 μL/well for 30 min at 4 , and then fixative was discarded, washed with twice 1 × PBS. Cells were stained in the oil red-O (Solarbio, China) working solution (3:2, Saturated oil red-O solution: water) 120 μL/well protected from light for 30 min at 25 and washed twice with 1 × PBS. To quantify lipid accumulation, isopropanol/water (6:4) solution was added to the stained culture-well, and the dye was extracted with gentle shaking for 40 min at room temperature. After that, the absorbance at 510 nm was measured with a microplate reader. Accumulated lipid content in adipocytes was expressed as the amount of intracellular lipid accumulated per 5×103 cells.

Western blot analysis

The cells were collected and washed with ice-cold 1 × PBS and lysed in the Radio-Immunoprecipitation Assay buffer (50 mM Tris–HCl, pH 7.2, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) (Solarbio, China) containing protease inhibitor cocktail (100 ×) (CWBIO, China) on ice for 10 min. Cell lysates were centrifuged at 12,000 rpm for 15 min at 4 . The supernatant was then collected for SDS-PAGE analysis. Protein concentration was determined using the protein assay kit (CWBIO, China). Total proteins (20 μg) were separated on a 10% or 12% SDS-PAGE unless indicated otherwise and transferred to nitrocellulose membrane using a wet transfer system (Bio-Rad, USA) for 90 min at 200 mA. The membrane was blocked for 1.5 h at room temperature in the 5% skim milk/TBS/T (50 Mm Tris-HCL, pH 7.6, 0.1 M NaCl, and 0.1% tween-20) solution. The incubation with antibodies against LC-3 (rabbit polyclonal, 1:600, 14600-1-AP, ProteintechTM), p62 (rabbit polyclonal, 1:4000, 18420-1-AP, ProteintechTM), SREBP-1c (rabbit polyclonal, 1:2000, 14088-1-AP, ProteintechTM), PPAR-γ (rabbit polyclonal, 1:1000, 16643-1-AP, ProteintechTM), FAS (rabbit polyclonal, 1:700, 10624-1-AP, ProteintechTM), HSL (rabbit polyclonal, 1:1000, 17333-1-AP, ProteintechTM) and GAPDH (mouse monoclonal, 1:70000, 60004-1-Ig, ProteintechTM) were dealed overnight at 4 ℃ in TBS/T. The membrane was then incubated with secondary antibodies (anti-rabbit IgG, 1:7000, zb-2305, ZSGB-BIOTM) for 1.5 h at room temperature. Finally, the membrane was washed thrice in TBS/T for 10 min and analyzed with Versa DocTM 4000 MP (Bio-Rad, USA). Semi-quantitative analysis of the data was performed using Image J (NIH). Protein band density was quantified and normalized to the GAPDH.

Statistical analysis

All data were obtained from triplicate measurements. The data are expressed as mean ± SD. The double-tailed t-test was used to analyze differences between groups after checking normal distribution. For comparisons among groups, one-way ANOVA was used, followed by the Duncan test. Statistical were calculated with SPSS 22.0 for Windows (SPSS Inc., Chicago IL, USA). Differences were considered significant at when 0.01 < p < 0.05 (* or #) and p < 0.01 (** or ##).


Morphology and growth curve of subcutaneous and visceral primary pre-adipocytes

As shown in Figure 1A and 1B, mouse subcutaneous and visceral primary pre-adipocyte adherent cells were observed to be fusiform, polygonal, or irregular, and the primary cells of subcutaneous and visceral had no difference in morphology. As shown in Figure 1C, the growth curves of subcutaneous and visceral pre-adipocytes were S-shaped and experienced latent period (days 1-5), logarithmic growth period (days 5-11), plateau growth period (days 11-13), and aging period (days 13-15). The proliferation rate of subcutaneous pre-adipocytes was faster than that of viscera after day 7.


Differentiation and lipid evaluation of subcutaneous and visceral pre-adipocytes

To investigate the effect of energy levels on subcutaneous and visceral pre-adipocytes differentiation, intracellular lipids were observed (Fig. 2A and 2B) and quantified (Fig. 2E) after oil red-O staining (Fig. 2C and 2D). Results showed that a large amount of reflective grease was observed on day 10. In addition, the concentration of triglyceride in subcutaneous pre-adipocytes was more than visceral in both HGM and LGM (SH > VH, p < 0.01; SL > VL, p < 0.01). Although the size of adipocytes was greater in HGM, the total amount of triglycerides was lower than LGM (SH < SL, p < 0.01; VH < VL, p < 0.05). The results showed that low glucose promotes the accumulation of triglycerides in subcutaneous and visceral pre-adipose during differentiation and has a promoting effect on visceral pre-adipocytes (VL - VH (0.039) > SL - SH (0.027), p > 0.05).


Autophagy-related protein content in pre-adipocytes during differentiation

As shown in Figure 3, our results showed that autophagy-related protein LC-3 expression increased by differentiation time. The LC-3 protein in SH was increased at day 0, 2 and 6, but reduced at day 4, 8, and 10 compared with VH. The SL was lower than VL on day 2, 8 and 10. In energy levels, the LC-3 protein in LGM was higher than the HGM both in subcutaneous and visceral pre-adipocytes during differentiation. The p62 gene is a negative regulatory gene of autophagy, and its expression is negatively correlated with autophagy to some extent. The protein expression of p62 has reached the maximum on day 6 and decreased during differentiation (Fig. 3D). The p62 protein in SH was higher than VH on day 2, 4 and 10. The SL of p62 protein was higher than VL on day 4 and 6. In energy levels, the p62 protein in HGM was higher than LGM both in subcutaneous and visceral pre-adipocytes during differentiation. Our results indicated that low energy stimulated autophagy of subcutaneous and visceral pre-adipocyte during differentiation. It was shown that the enhancement of subcutaneous pre-adipocytes was lower than that of viscera on autophagy during differentiation (Table I).


Table I. Average protein expression content of subcutaneous and visceral pre-adipocytes in high-glucose medium and low-glucose medium during differentiation.

Protein name


















































Note: The values were shown as means ± SD, different letters in the same row means significant between the treatments (p < 0.05), same letter in the same row means no significant between the treatments (p > 0.05). The data come from western blot gray value analysis, which is the relative expression analysis of differentiation day 0 to 10. Subcutaneous pre-adipocyte in high-glucose medium (SH), subcutaneous pre-adipocyte in low-glucose medium (SL), visceral pre-adipocyte in high-glucose medium (VH), visceral pre-adipocyte in low-glucose medium (VL). High-glucose medium supplemented 4500 mg/L glucose, low-glucose medium supplemented 1000 mg/L glucose. (SL-SH)/SH and (VL-VH)/VH was represents the low-glucose medium relative high-glucose medium of target expression, value > 0 represents promotion, value < 0 represents inhibition.


Protein content of adipogenic transcription factors in pre-adipocytes during differentiation

As shown in Figure 4, during the differentiation period, the SREBP-1c and PPAR-γ protein expression was increased initially and then decreased during differentiation. The SREBP-1c protein expression in SH was higher than VH during differentiation, and the SL also was higher than VL except on day 4 and 10. In energy levels, the SREBP-1c protein expression in HGM was higher than the LGM in both subcutaneous and visceral pre-adipocytes. Low glucose decreased the expression of SREBP-1c in subcutaneous and visceral pre-adipocytes during differentiation, especially in subcutaneous pre-adipose. The PPAR-γ protein expression results showed that (Fig. 4D), the protein contents of PPAR-γ were not different in subcutaneous and visceral pre-adipocytes during differentiation (only at day 6, the SL was lower than VL). But in energy levels, the PPAR-γ protein in HGM was higher than the LGM on day 4 and 6; however, no difference was observed on other days. Our results indicated that low energy inhibited adipogenic transcription factors of SREBP-1c and PPAR-γ in subcutaneous and visceral pre-adipocyte during differentiation, and the inhibition of subcutaneous pre-adipocytes was higher than that of viscera pre-adipocytes (Table I). The results showed that low glucose decreased the differentiation degree of subcutaneous and visceral pre-adipocytes, especially on subcutaneous pre-adipocytes.

Protein content of adipogenesis and lipolysis in pre-adipocytes during differentiation

The results of the pre-adipocytes differentiation period and fat metabolism-related protein expression are shown in Figure 5. The FAS protein expression was increased by differentiation time. The FAS protein expression in SH was increased at day 0, 6 and 10, but reduced at day 4 and 8 compared with VH. The SL was increased on day 8 and 10, reduced on day 2 and 4 compared with VL. In energy levels, the FAS protein in HGM was increased at day 4 and 10 of pre-adipocytes and reduced at day 2 and 8 in



subcutaneous compared with the LGM. There was no difference between HGM and the LGM in visceral pre-adipocytes except on day 2. These results indicated that low glucose inhibited adipogenesis of FAS in subcutaneous expression but stimulated visceral pre-adipocyte during differentiation (Table I). The lipolysis protein expression of HSL was increased during differentiation (Fig. 5D). The HSL protein expression in SH was lower than VH except on day 6, and the SL was also lower than VL except day 6 and 8. In energy levels, the HSL protein expression in LGM was lower than HGM, indicating that low energy was inhibiting lipolysis both in subcutaneous and visceral pre-adipocyte during differentiation, and inhibition of subcutaneous pre-adipocytes was higher than that of viscera pre-adipocytes (Table I). These results showed that low glucose significantly decreased the expression of lipolysis in subcutaneous and visceral pre-adipocytes during differentiation, and weakened the process of fat degradation, especially on subcutaneous pre-adipocytes.


As a mature cell line, 3T3-L1 has been well studied in adipocytes (Fan et al., 2019), but its properties and characteristics are far from those of primary adipocytes. They do not represent the function of subcutaneous and visceral pre-adipocytes. In the present study, we extracted mouse subcutaneous and visceral pre-adipocytes culture in vitro. Subcutaneous and visceral pre-adipocytes were not different in cell morphology as Toyoda et al. (2009) have described, no significant difference in morphology was observed between subcutaneous and visceral. In terms of cell proliferation, subcutaneous and visceral pre-adipocytes were different in the logarithmic growth and decline phases. This may explain why the number of subcutaneous adipocytes was greater than viscera. In addition, subcutaneous pre-adipocytes lipid accumulation was more than viscera, which was the same as that of HGM and LGM differentiation as previous study in 3T3-L1 was described (Lustig et al., 2017). From the stained with oil red-O dyes photographed, the lipid droplets formed by HGM differentiation were more extensive than that by LGM, but the LGM differentiation of triglycerides accumulation was more than HGM differentiation, in both subcutaneous and visceral. These results were not consistent with cattle and cows, feeding high concentrate diet could significantly increase fat deposition in Hanwoo Cattle muscle (Reddy et al., 2017), and high energy group increased total visceral adipose and abdominal adipose of cow (Drackley et al., 2014). The lipid droplet volume of pre-adipocyte fusion induced by high glucose is larger than that induced by low glucose. We speculate that the rate of β-oxidation may be more intense in high glucose environment, although we did not detect the index of β-oxidation. This may be a possible explanation for the contradiction between the volume of lipid droplets and total triglycerides induced by different energy levels.

Many reports have revealed that the autophagic process is required for pre-adipocytes adipogenesis (Dong and Czaja, 2011; Armi et al., 2013). Autophagy plays important role in recycling intracellular energy resources in response to nutrient-depleted conditions and removing cytotoxic proteins and organelles under stressful conditions (Fujimoto and Parton, 2011). Our results showed that the expression of autophagy-related genes fluctuated with the induction of differentiation time, and the expression of autophagy protein LC-3 increased gradually. Our result was consistent with that of Hahm et al. (2014), the protein of LC-3 was gradually increased by differentiation day. Our results showed that during differentiation of LGM, the protein expression of LC-3 in subcutaneous and visceral pre-adipocytes was higher than that in HGM, which was consistent with previous results that alleged that low energy could induce autophagy (Yoshizaki et al., 2012). The low glucose (1000 mg/L) used in this study was aimed to fulfill minimum requirement for cell growth; thus, it can be assumed that autophagy occurs due to lack of energy for cell differentiation or pre-adipocytes faced survival stress (Mizushima et al., 2004). Furthermore, the LGM differentiation of lipolysis and adipogenesis transcriptional factors was lower than HGM, which may be caused by autophagy. The LGM was used in this study not only enhanced the degree of autophagy of pre-adipocytes, but also increased the accumulation of triglycerides, indicating that increasing autophagy in the process of differentiation can increase the differentiation of pre-adipocytes. Moreover, we found that visceral autophagy was higher than subcutaneous pre-adipocyte during differentiation, with Choi et al. (2020) in mouse result was similar, reduced adipose tissue mass was more prominent in visceral compared to subcutaneous white adipose tissue by activating autophagy. Although we did not detect the expression of Beclin1, another important regulatory pathway of autophagy, we speculate that the expression of Beclin1 gene in visceral pre-adipocytes may also be higher than that in subcutaneous pre-adipocytes (Yang et al., 2014). Results represent that visceral pre-adipocytes may be has more sensitivity to autophagy. Moreover, our study found that the protein expressions of LC3 in subcutaneous was higher than visceral at day 0. These findings have already been explained in the 3T3 pre-adipocytes expression with differentiation time (Hahm et al., 2018; Zutphen et al., 2014), who partially explained that the subcutaneous pre-adipocytes proliferation is more than that of viscera, and after receiving the differentiation signal, the cell state changes lead to subcutaneous pre-adipocytes cycle arrest (Wei et al.,2018). We hypothesized that the higher lipolysis of visceral pre-adipocytes during differentiation might be due to their higher degree of autophagy, that is, autophagy enhances the role of visceral lipolysis. It may also inhibit the expression of SREBP-1c (Zhang et al., 2013), leading to the difference in triglycerides accumulation between subcutaneous and visceral pre-adipocytes differentiation.

Pre-adipocytes differentiation was a crucial and strictly regulated process that occurs principally in adipose tissue, in which key transcription factors, such as PPAR-γ and SREBP-1c, sequentially turn on adipogenic process (Ameer et al., 2014). Our study results show that the protein expression of SREBP-1c in subcutaneous pre-adipocyte was higher than that in viscera, and there was no difference in the expression of PPAR-γ protein between subcutaneous and visceral. We speculate that it is precisely because PPAR-γ is an important transcription factor of adipogenesis and a decisive switch that determines the differentiation of pre-adipocytes into mature adipocytes, so its role in the process of adipogenic differentiation of pre-adipocytes in different parts should be consistent, so there is no significant difference in PPAR - γ protein expression between subcutaneous and visceral pre-adipocytes (Wajchenberg et al., 2002). D-allulose (a rare sugar with almost zero energy) could decrease intracellular lipid accumulation during 3T3-L1 adipocyte differentiation, and along with reducing PPAR-γ mRNA expression, but the effect on SREBP gene was not significant (Moon et al., 2020). The subcutaneous and visceral pre-adipocyte SREBP-1c gene in LGM was lower than that of HGM as previously described in offspring mouse (Vithayathil et al., 2018), and it may be regulated by autophagy. This indicated that low glucose reduced the adipogenic differentiation of subcutaneous pre-adipocytes, but this effect was minor in viscera. However, LGM differentiation accumulated lipid more than HGM while reduced adipogenesis transcriptional factors and inhibited the lipolysis. These results explored that the adipocytes differentiation was different with triglyceride accumulation (Thounaojam et al., 2011). Pre-adipocyte differentiation was regulated by many genes, and triglyceride accumulation is the result of the dynamic action of lipogenesis and lipolysis. Although the degree of adipogenic differentiation can affect the triglyceride accumulation of cells, but the accumulation of more triglycerides does not mean that the degree of adipogenic differentiation is very high.

According to previous reports, lipid accumulation was caused by subtle differences between lipogenesis and lipolysis (Shuster et al., 2012; Poussin et al., 2008). In the process of pre-adipocyte adipogenic differentiation, day 0-2 was considered a determinant of cellular behaviors at the late stage, and day 2-10 can be regarded as the process of pre-adipocyte accumulation of triglyceride. Our results showed that FAS expression in subcutaneous and visceral was not different in HGM and LGM. An interesting phenomenon is that the expression of FAS in subcutaneous pre-adipocyte was higher than that of visceral on day 0. This may be an explanation that the adipogenic differentiation potential of subcutaneous pre-adipocytes is higher than that of visceral pre-adipocytes. In addition, a previous study found that primary pre-adipocytes have the potential to differentiate automatically even without the addition of inducers consistent (Abuna et al., 2016). Although FAS protein expression in viscera was higher than subcutaneous in the early stage (day 2 and 4) of differentiation time, but the early stage is not the peak of triglyceride accumulation, and pre-adipocytes must be degraded other organelles such as mitochondria before they began to accumulate triglycerides (Drackley et al., 2014). Adipogenic differentiation is a continuous process, and the accumulation of triglycerides is affected by adipogenesis and lipolysis, which is not determined by the difference in gene expression at every moment. Lipolysis is a catabolic pathway that promotes triglycerides mobilization in a step-wise fashion by increasing HSL (Wang et al., 2013). Our results showed that the expression of HSL in visceral was higher (day 2 and 10) than subcutaneous at HGM differentiation, indicating that visceral pre-adipocyte lipolysis maybe related to autophagy (Harada et al., 2003). Although lipolysis is not the main metabolic pathway in pre-adipocyte adipogenic differentiation stage, fat deposition is the result of the combined action of adipogenesis and lipolysis.

Besides, our results found that LGM differentiation of pre-adipocytes reduces lipolysis along with induced autophagy, which is inconsistent with previous results that autophagy enhancement was along with an increase in lipolysis (Shin et al., 2014; Leu et al., 2018). We hypothesized that in the LGM differentiation process, both subcutaneous and visceral pre-adipocytes produce a high level of autophagy due to the lack of energy needed for cell differentiation (Sathyanarayan et al., 2017). This autophagy can degrade other organelles, but it may also indirectly inhibit the occurrence of lipolysis, indicating that the lipolysis gene of HSL in LGM was lower than HGM in subcutaneous and visceral pre-adipocytes differentiated. Moreover, our findings represent that the subcutaneous is more sensitive to changes in energy levels. Our study suggests that energy supply should not be restricted during the early cell development stage to ensure the normal biological development of cells. This study may also provide information to reduce targeted fat accumulation via improving certain lipolysis. Adipose differentiation is a complex process, but the detailed functional connection between autophagy and adipogenesis or lipolysis remains further investigated.


Current results showed differences in autophagy, adipogenic differentiation, and lipolysis between subcutaneous and visceral pre-adipocytes in vitro. At the stage of adipose differentiation, visceral pre-adipocytes attenuate lipid accumulation via stimulating lipolysis and inhibiting adipogenic transcriptional factors during adipogenesis as compared to subcutaneous pre-adipocytes. Low-energy enhanced the autophagy of pre-adipocytes and decreased the differentiation of subcutaneous pre-adipocytes and inhibited the lipolysis of subcutaneous and visceral pre-adipocytes. The effect of decreasing the energy levels on the adipogenic differentiation and lipid metabolism of subcutaneous pre-adipocytes was greater than that of visceral pre-adipocytes, except for autophagy. However, the relationships among energy levels induced autophagy, lipolysis, and adipose transcription factors need to be further studied.


This research was funded by National Natural Science Foundation of China, 31560648; Science and Technology Project of Education Department of Jiangxi Province, GJJ200451; Jiangxi Agriculture Research System, JXARS-13 and China Agriculture Research System of MOF and MARA, CARS-37. The APC was funded by the above funds.

Institutional review board statement

The experimental protocol was approved by the animal ethics committee of Jiangxi Agricultural University (JXAULL-2020-036).

Statement of conflicts of interest

The authors have declared no conflicts of interest.


Abdullah, A.Y. and Musallam, H.S., 2007. Effect of different levels of energy on carcass composition and meat quality of male black goats kids. Livest. Sci., 107: 70-80.

Abuna, R.P.F., De Oliveira, F.S., Santos, T.D.S., Guerra, T.R., Rosa, A.L. and Beloti, M.M., 2016. Beloti. Participation of TNF-α in inhibitory effects of adipocytes on osteoblast differentiation. J. cell. Physiol., 231: 204-214.

Ameer, F., Scandiuzzi, L., Hasnain, S., Kalbacher, H. and Zaidi, N., 2014. De novo lipogenesis in health and disease. Metabolism, 63: 895-902.

Armi, C.D., Devereaux, K.A. and Paolo, G.D., 2013. The role of lipids in the control of autophagy. Curr. Biol., 23: R33-45.

Bachmann, M., Bochnia, M., Mielenz N., Spilke, J., Souffrant, W.B., Azem, E., Schliffka, W. and Zeyner, A., 2018. Impact of α-amylase supplementation on energy balance and performance of high-yielding dairy cows on moderate starch feeding. Anim. Sci. J., 89: 367-376.

Boström, P., Rutberg, M., Ericsson, J., Holmdahl, P., Andersson, L., Frohman, M.A., Borén, J. and Olofsson, S.O., 2005. Cytosolic lipid droplets increase in size by microtubule dependent complex formation. Arterioscl. Thromb. Vasc. Biol., 25: 1945-9151.

Choi, C., Song, H.D., Son, Y., Choi, Y., K., Ahn, S.Y., Jung, Y.S., Yoon, Y.C., Kwon, S.W. and Lee, Y.H., 2020. Epigallocatechin-3-gallate reduces visceral adiposity partly through the regulation of beclin1-dependent autophagy in white adipose tissues. Nutrients, 12: E3072.

Christian, P., Sacco, J. and Adeli, K., 2013. Autophagy: Emerging roles in lipid homeostasis and metabolic control. Biochim. biophys. Acta Biomembranes, 1831: 819-824.

Dong, H. and Czaja, M.J., 2011. Regulation of lipid droplets by autophagy. Trends Endocrinol. Metab., 22: 234-240.

Drackley, J.K., Wallace, R.L., Graugnard, D., Vasquez, J., Richards, B.F. and Loor, J.J., 2014. Visceral adipose tissue mass in nonlactating dairy cows fed diets differing in energy density. J. Dairy Sci., 97: 3420-3430.

Fan, Y., Gan, M.L., Tan, Y., Chen, L., Shen, L.Y., Niu, L.L., Liu, Y.H., Tang, G.Q., Jiang, Y.Z., Li, X.W., Zhang, S.H., Bai, L. and Zhu, L., 2019. Mir-152 regulates 3t3-l1 pre-adipocyte proliferation and differentiation. Molecules, 24: 33-79.

Fraser, J.K., Wulur, I., Alfonso, Z. and Hedrick, M.H., 2006. Fat tissue: an underappreciated source of stem cells for biotechnology. Trends Biotechnol., 24: 150-154.

Fujimoto, T. and Parton, R.G., 2011. Not just fat: The structure and function of the lipid droplet. CSH Perspect. Biol., 3: 1-17.

Goldman, S.J., Zhang, Y. and Jin, S.F., 2011. Autophagic degradation of mitochondria in white adipose tissue differentiation. Antioxid. Redox. Signal., 14: 1971-1978.

Hahm, J.R., Noh, H.S., Ha, J.H., Roh, G.S. and Kim, D.R., 2014. Alpha-lipoic acid attenuates adipocyte differentiation and lipid accumulati|on in 3t3-l1 cells via ampk-dependent autophagy. Life Sci., 100: 125-132.

Harada, K., Shen, W.J., Patel, S., Natu, V., Wang, J., Osuga, J., Ishibashi, S. and Kraemer, F.B., 2003. Resistance to high-fat diet-induced obesity and altered expression of adipose-specific genes in HSL-deficient mice. Am. J. Physiol. Endoc. Metab., 285: 1182-1195.

Hwang, S.H. and Lee, M., 2020. Autophagy Inhibition in 3t3-l1 adipocytes breaks the crosstalk with tumor cells by suppression of adipokine production. Anim. Cells Syst., 24: 1650-1654.

Kaneko, H., Kobayashi, M., Mizunoe, Y., Yoshida, M., Yasukawa, H., Hoshino, S., Itagawa, R., Furuichi, T., Okita, N., Sudo, Y., Imae, M. and Higami, Y., 2018. Taurine is an amino acid with the ability to activate autophagy in adipocytes. Amino Acids, 50: 527-535.

Khanal, P., Johnsen, L., Axel, A.M.D., Hansen, P.W., Kongsted, A.H., Lyckegaard, N.B. and Nielsen, M.O., 2016. Long-term impacts of foetal malnutrition followed by early postnatal obesity on fat distribution pattern and metabolic adaptability in adult sheep. PLoS One, 11: e0156700.

Klionsky, D.J., Abdelmohsen, K., Abe, A., Abedin, M.J., Abeliovich, H., Arozena, A.A., Adachi, H., Adams, C.M., Adams, P.D., Adeli, K., Adhihetty, P.J., Adler, S.G., Agam, G., Agarwal, R., Aghi, M.K., Agenello, M., Agostinis, P., Aguilar, P.V.,……Zughaier, S.M., 2016. Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy, 12(2):443.

Krieger, J.P., Langhans, W. and Lee, S.J., 2018. Novel role of glp-1 receptor signaling in energy expenditure during chronic high fat diet feeding in rats. Physiol. Behav., 192: 194-199.

Krishna, M.S., Revathy, V.M. and Jaleel, A., 2020. Adipocytes utilize sucrose as an energy source-effect of different carbohydrates on adipocyte differentiation. J. cell. Physiol., 235: 891-899.

Leu, S.Y., Tsai, Y.C., Chen, W.C., Hsu, C.H., Lee, Y.M. and Cheng, P.Y., 2018. Raspberry ketone induces brown-like adipocyte formation through suppression of autophagy in adipocytes and adipose tissue. J. Nutr. Biochem., 56: 116-125.

Lustig, M., Moldovan, L.M.Y., Gefen, A. and Benayahu, D., 2017. Adipogenesis of 3T3L1 cells subjected to tensile deformations under various glucose concentrations. Comput. Method. Biomed., pp. 171-174.

Macotela, Y., Emanuelli, B., Mori, M.A., Gesta, S., Schulz, T.J., Tseng, Y.H. and Kahn, C.R., 2012. Intrinsic differences in adipocyte precursor cells from different white fat depots. Diabetes, 61: 1691-1699.

Mizushima, N., Levine, B., Cuervo, A.M. and Klionsky, D.J., 2008. Autophagy fights disease through cellular self-digestion. Nature, 451: 1069-1075.

Mizushima, N., Yamamoto, A., Matsui, M., Yoshimori, T. and Ohsumi, Y., 2004. In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker. Mol. Biol. Cell., 15: 1101-1111.

Moon, S., Kim, Y.H. and Choi, K., 2020. Inhibition of 3T3-L1 adipocyte differentiation by d-allulose. biotechnol. Bioproc. Engineer., 25: 22-28.

Park, P.J., Cho, J.Y. and Cho, E.G., 2017. Specific visible radiation facilitates lipolysis in mature 3t3-l1 adipocytes via rhodopsin-dependent β3-adrenergic signaling. Eur. J. Cell Biol., 96: 301-311.

Poussin, C., Hall, D., Minehira, K., Galzin, A.M., Tarussio, D. and Thorens, B., 2008. promotes autophagy/lipophagy of metabolism and extracellular matrix in visceral and subcutaneous fat of obese and rimonabant treated mice. PLoS One, 3: e3385.

Reddy, K.E., Jeong, J.Y., Baek, Y.C., Oh, Y.K., Kim, M., So, K.M., Kim, M.J., Kim, D.W., Park, S.K. and Lee, H.J., 2017. Early weaning of calves after different dietary regimens affects later rumen development, growth, and carcass traits in hanwoo cattle. Asian-Australas. J. Anim. Sci., 30: 1425-1434.

Rosen, E.D. and Macdougald, O.A., 2006. Adopocyte differentiation from the inside out. Nat. Rev. Mol. Cell Biol., 7: 885-896.

Sathyanarayan, A., Mashek, M.T. and Mashek, D.G., 2017. ATGL promotes autophagy/lipophagy via SIRT1 to control hepatic lipid droplet catabolism. Cell Rep., 19: 1-9.

Scheffler, J.M., McCann, M.A., Greiner, S.P., Jiang, H., Hanigan, M.D., Bridges, G.A., Lake, S.L. and Gerrard, D.E., 2014. Early metabolic imprinting events increase marbling scores in fed cattle. J. Anim. Sci., 92: 320-324.

Shin, S., Choi, Y.M., Han, J.Y. and Lee, K., 2014. Inhibition of lipolysis in the novel transgenic quail model overexpressing G0/G1 Switch gene 2 in the adipose tissue during feed restriction. PLoS One, 9: e100905.

Shuster, A., Patlas, M., Pinthus, J.H. and Mourtzakis, M., 2012. The clinical importance of visceral adiposity: A critical review of methods for visceral adipose tissue analysis. Br. J. Radiol., 85: 1-10.

Sugita, K., 2020. Uncovering non-canonical autophagy in dermal dendritic cells in contact hypersensitivity: a key mechanism of immune tolerance. J. Allergy clin. Immun., 145: 1363-1364.

Thounaojam, M.C., Jadeja, R.N., Ramani, U.V., Devkar, R.V. and Ramachandran, A.V., 2011. Ramachandran Sida rhomboidea. roxb leaf extract down-regulates expression of PPARγ2 and leptin genes in high fat diet fed C57BL/6J mice and retards in vitro 3T3L1 pre-adipocyte differentiation. Int. J. mol. Sci., 12: 4661-4667.

Toyoda, M., Matsubara, Y., Lin, K., Sugimachi, K. and Furue, M., 2009. Characterization and comparison of adipose tissue-derived cells from human subcutaneous and omental adipose tissues. Cell Biochem. Funct., 27: 440-447.

Vithayathil, M.A., Gugusheff, J.R., Ong, Z.Y., Langley, S.C., Gibson, R.A. and Muhlhausler, B.S., 2018. Exposure to maternal cafeteria diets during the suckling period has greater effects on fat deposition and sterol regulatory element binding protein-1c (SREBP-1c) gene expression in rodent offspring compared to exposure before birth. . Nutr. Metab., 15: Article number 15.

Wajchenberg, B.L., Neto, D.G., Silva, M.E.R.D. and Santos, R.F., 2002. Depot-specific hormonal characteristics of subcutaneous and visceral adipose tissue and their relation to the metabolic syndrome. Horm. Metab. Res., 34: 616-621.

Wang, P.P., She, M.H., He, P.P., Chen, W.J., Laudon, M., Xu, X.X. and Yin, W.D., 2013. Piromelatine decreases triglyceride accumulation in insulin resistant 3T3-l1 adipocytes: role of ATGL and HSL. Biochimie, 95: 1650-1654.

Wei, S., Fu, X., Liang, X., Zhu, M.J., Jiang, Z., Parish, S.M., Dodson, M.V., Zan, L. and Du, M., 2015. Enhanced mitogenesis in stromal vascular cells derived from subcutaneous adipose tissue of wagyu compared with those of angus cattle. J. Anim. Sci., 93: 1015-1024.

Wei, S.G., Zheng, Y.Y., Zhang, M.M., Zheng, H. and Yan, P.S., 2018. Grape seed procyanidin extract inhibits idipogenesis and stimulates lipolysis of porcine adipocytes in vitro. J. Anim. Sci., 96: 2753-2762.

Xie, Y.H., Mo, Z.H., Chen, K., Yang, Y.B., Xing, X.W. and Liao, Y., 2008. Effects of different glucose concentrations on 3t3-l-1 cell differentiation and insig-1 and insig-2 mRNA expression. J. Cent. South Univ., 33: 238-244.

Yang, J., Lv, P.F., Zhang, X.Y., Deng, Y.J. and Yang, Y., 2014. Beclin 1 and its interaction protein expressi on in adipocyte differentiation and visceral and subcutaneous adipose tissue. J. clin. Endocrinol. Metab., 30: 1108-1114.

Yoshizaki, T., Kusunoki, C., Kondo, M., Yasuda, M., Kume, S., Morino, K., Sekine, O., Ugi, S., Uzu, T., Nishio, Y., Kashiwagi, A. and Maegawa, H., 2012. Autophagy regulates inflammation in adipocytes. Biochem. biophs. Res. Commun., 417: 352-357.

Zhang, C.B., He, Y.K., Okutsum M., Ongm L.C., Jinm Y., Zhengm L., Chowm P., Yu, S., Zhang, M. and Yan, Z., 2013. Autophagy is involved in adipogenic differentiation by repressesing proteasome-dependent ppar-γ2 degradation. Am. J. Physiol. Endocrinol. Metab., 305: E530-539.

Zhang, Y., Goldman, S., Baerga R., Zhao, Y., Komatsu, M. and Jin, S., 2009. Adipose-apecific deletion of autophagy-related gene 7 (atg7) in mice reveals a role in adipogenesis. Proc. natl. Acad. Sci. U.S.A., 106: 19860-19865.

Zutphen, T.V., Todde, V., Boer, R.D., Kreim, M., Hofbauer, H.F., Wolinski, H., Veenhuis, M., Klei, I.J.V.D. and Kohlwein, S.D., 2014. Lipid droplet autophagy in the yeast Saccharomyces cerevisiae. Mol. Biol. Cell, 25: 290-301.

To share on other social networks, click on any share button. What are these?

Pakistan Journal of Zoology


Vol. 55, Iss. 2, Pages 501-1002


Click here for more

Subscribe Today

Receive free updates on new articles, opportunities and benefits

Subscribe Unsubscribe