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Introduction

Adipose tissue serves a major role in regulating energy metabolism in the body. When lipolysis of adipose tissue is stimulated by catecholamines during exercise or fasting, stored triglycerides (TG) are hydrolyzed and fatty acids are used as an energy source. Approximately two-thirds of the fatty acids produced during lipolysis bind to glycerol-3-phosphate in adipocytes to generate micro-lipid droplets (1–3). The re-esterification process is critical for facilitating lipolysis in adipocytes. Moreover, fatty acid re-esterification has been reported to buffer the toxic effects of fatty acids, and prevent endoplasmic reticulum stress and mitochondrial damage (4,5). Therefore, promotion of fatty acid re-esterification is important for efficient lipolysis and homeostasis.

Catecholamines have been shown to elevate the intracellular AMP/ATP ratio and oxygen consumption rate (OCR) in adipocytes (6,7), suggesting the activation of mitochondrial respiration. In addition to mitochondrial activity, catecholamine stimulation has been reported to increase glucose uptake and glycolysis in adipocytes (8,9). ATP is essential for the generation of cAMP, and phosphorylation and activation of lipases in the lipolysis process. Therefore, increased ATP utilization resulting from the activation of lipases seems to be met by the enhancement of energy synthesis via glycolysis and mitochondrial respiration to maintain intracellular ATP levels. Veliova et al (10) reported that the inhibition of fatty acid re-esterification in brown adipocytes can suppress cellular ATP demand and decrease OCR. These data indicated that ATP is used not only to activate lipases but also to promote fatty acid re-esterification during lipolysis.

Based on these data, it is likely that the energy state of lipolysis influences lipolytic activity via lipase activation and fatty acid re-esterification. Therefore, the present study aimed to clarify the effect of intracellular energy state on lipolytic activity and fatty acid re-esterification during lipolysis.

Materials and methods

Materials

All reagents were obtained from Nacalai Tesque, Inc., unless otherwise indicated.

Cell culture

3T3-L1 cells (Japanese Collection of Research Bioresources Cell Bank) were maintained as previously described (11). Briefly, 3T3-L1 cells were grown in growth medium (GM) consisting of DMEM (FUJIFILM Wako Pure Chemical Corporation), 10% FBS (MP Bio Japan K.K.) and 1% penicillin-streptomycin in a humidified atmosphere containing 5% CO2 at 37°C. For differentiation, confluent cells were cultured in GM supplemented with 0.5 µM IBMX (MilliporeSigma), 0.25 µM dexamethasone and 1 µg/ml human insulin (Eli Lilly and Company) for 48 h. Subsequently, the medium was replaced with differentiation medium, which comprised GM and 1 µg/ml human insulin, and was replenished with differentiation medium every 48 h until day 7.

Lipolytic stimulation

Lipolytic stimulation was applied to the differentiated 3T3-L1 adipocytes. After washing with PBS, the cells were incubated with glucose-free, phenol red-free DMEM (Thermo Fisher Scientific, Inc.) containing 1% BSA and 13.8 mM glucose in the presence of 1 µM isoproterenol at 37°C for up to 4 h. To inhibit the mitochondrial respiratory chain, oligomycin A (0.5–10 µM), rotenone (0.5–5 µM) (MilliporeSigma), chlorpromazine (10–100 µM) or 2,4-dinitrophenol (DNP; 10–100 µM) were added to the medium 10 min before isoproterenol stimulation. Aliquots of the medium were collected 4 h after isoproterenol stimulation, and the concentrations of glycerol, glucose and lactate were measured using a glycerol assay kit (cat. no. F6428; Sigma-Aldrich; Merck KGaA), glucose CII test Wako kit (cat. no. 439–90901; FUJIFILM Wako Pure Chemical Corporation) and N-assay L Lac kit (cat. no. 30171000; Nittobo Medical Co., Ltd.), respectively. Glycerol was measured in the culture medium as an index of lipolysis because no discernible decline in TG levels was observed with 4-h isoproterenol stimulation in our preliminary study. Similarly, since free fatty acids (FFAs) are intracellularly re-esterified, an elevation in FFA concentration in the medium is an inappropriate index of lipolysis. Furthermore, adipocytes lack expression of glycerol kinase, the enzyme responsible for metabolizing and re-esterifying glycerol, leading to the excretion of generated glycerol outside the cell. Consequently, glycerol alone is presented as a suitable indicator of lipolysis in the present study.

The ratio of lactate to glucose was calculated as follows: Lactate/glucose (mg/dl)=LCconditioned/(GCFresh-GCconditioned), where GCfresh refers to the glucose concentration in the fresh medium, and LCconditioned and GCconditioned correspond to the lactate and glucose concentrations in the conditioned medium, respectively.

Western blotting

3T3-L1 adipocytes were washed twice with PBS and then proteins were extracted using ice-cold radioimmunoprecipitation buffer containing 0.25 M Tris-HCl (pH 7.4), 0.75 M NaCl, 0.25% deoxycholic acid, 5% NP-40, 5 mM EDTA, 1% protease inhibitor cocktail and 1% phosphatase inhibitor cocktail. The homogenates were centrifuged at 15,000 × g for 5 min at 4°C. The supernatants were collected, and protein concentrations were measured using a bicinchoninic acid assay kit. Samples were prepared in 4X Laemmli sample buffer and 30 µg proteins were separated by SDS-PAGE on 10% gels, before being transferred to PVDF membranes (Thermo Fisher Scientific, Inc.). After transfer, the membranes were blocked for 1 h at room temperature in Tris-buffered saline with 0.05% Tween 20 supplemented with 5% nonfat milk. The membranes were incubated overnight at 4°C with anti-phosphorylated-cAMP-dependent protein kinase A (PKA) substrate antibody (cat. no. 9624; Cell Signaling Technology, Inc.). The membranes were then washed and incubated with horseradish peroxidase-conjugated anti-rabbit IgG (1:5,000; cat. no. 7074; Cell Signaling Technology, Inc.) at room temperature for 1 h. Enhanced chemiluminescence (MillporeSigma) was used to detect the protein bands. Images were captured using a chemiluminescence detector (LAS500; GE Healthcare Bio-Sciences) and band intensities were semi-quantified using ImageJ 1.52 (National Institutes of Health). Equal protein loading was confirmed by staining the blots with Coomassie brilliant blue (FUJIFILM Wako Pure Chemical Corporation).

Glucose uptake

Glucose uptake was measured as previously described (9). Briefly, 3T3-L1 cells were washed with PBS and incubated for 2 h in glucose-free, phenol red-free DMEM containing 1% BSA and 1 µM isoproterenol in the presence of 2-deoxy-glucose (2DG) (MilliporeSigma) and glucose (1:1 molar ratio) at 37°C. After stimulation of lipolysis, the cells were washed three times with cold PBS and harvested with 0.1 M HCl. Samples were boiled at 95°C for 15 min and centrifuged at 3,000 × g for 5 min at 4°C. The supernatants were neutralized with 0.1 M NaOH and used for measurement of cellular 2DG6P. The concentration of 2DG6P was measured as previously described (12).

Biochemical analyses

To measure the cellular ATP concentration, 3T3-L1 adipocytes were treated as described for lipolytic stimulation. After incubation, the cells were washed twice with cold PBS and harvested with 0.1 M HCl. After neutralization with 0.1 M NaOH, the samples were centrifuged at 3,000 × g for 5 min at 4°C and the supernatants were used to measure cellular ATP. The ATP concentration was measured using the Cellno ATP Assay reagent (cat. no. CA2-10; TOYO B-Net Co., Ltd.) according to the manufacturer's instructions.

Statistical analysis

Data are expressed as the mean ± SEM. Statistical analyses were performed using Bell Curve for Excel version 3.10 (Social Survey Research Information). Statistical analysis for multiple comparisons was performed using one-way or two-way ANOVA followed by Dunnett's post hoc test or Tukey's post hoc test, respectively. An unpaired Student's t-test was used for comparisons between two groups. P<0.05 was considered to indicate a statistically significant difference.

Results

Inhibition of the mitochondrial respiratory chain reduces ATP concentration and catecholamine-stimulated lipolysis in adipocytes

To investigate the effect of intracellular ATP levels on catecholamine-stimulated lipolysis, 3T3-L1 cells were cultured in medium containing electron transport chain inhibitors, and catecholamine-stimulated lipolytic activity was evaluated. Oligomycin A and rotenone, inhibitors of ATP synthase and complex I, respectively, decreased intracellular ATP levels (Fig. 1A and B). However, no significant decrease in ATP was observed in response to 10–50 µM of DNP and chlorpromazine, a mitochondrial uncoupler and an inhibitor of mitochondrial complex I, respectively (Fig. 1C and D). In addition, microscopic observations revealed abnormalities in the shape of the adipocytes cultured with chlorpromazine or DNP at a concentration of 100 µM, suggesting that high concentrations of these compounds were cytotoxic (data not shown). Therefore, oligomycin A and rotenone were used in subsequent experiments. The addition of oligomycin A and rotenone reduced the amount of glycerol released in response to isoproterenol stimulation (Fig. 2A and B). These results suggested that the intracellular energy state affects the capacity of adipocytes for catecholamine-stimulated lipolysis.

Figure 1. Effects of mitochondrial respiratory chain inhibition on ATP levels in 3T3-L1 adipocytes. 3T3-L1 adipocytes were cultured in DMEM with 1 µM isoproterenol with/without (A) oligomycin A, (B) rotenone, (C) chlorpromazine and (D) DNP for 4 h. Cells were lysed with 0.1 M HCl and neutralized, and the supernatants were subsequently used to measure cellular ATP content. All data are presented as the mean ± SEM, n=3-11. A one-way ANOVA with Dunnett's post hoc test was performed for statistical analysis. **P<0.01 vs. Con group. Con, control; DNP, 2,4-dinitrophenol.

Figure 2. Inhibition of the mitochondrial respiratory chain reduces catecholamine-stimulated lipolysis in 3T3-L1 adipocytes. 3T3-L1 adipocytes were cultured in DMEM with 1 µM isoproretenol with/without (A) oligomycin A or (B) rotenone for 4 h. Aliquots of the medium were collected, and the glycerol concentration was measured. All data are presented as the mean ± SEM, n=3-6. A one-way ANOVA with Dunnett's post hoc test was performed for statistical analysis. **P<0.01 vs. Con group. Con, control.

Decreased intracellular ATP levels do not affect lipolytic signaling

When lipolysis is stimulated by catecholamines, ATP is used in signaling pathways that activate downstream lipases. To investigate the effect of the inhibition of mitochondrial ATP synthesis on lipolytic signaling, the phosphorylation levels of PKA substrates were evaluated following the addition of oligomycin A and rotenone. Isoproterenol treatment markedly increased phosphorylation of PKA substrates. By contrast, oligomycin A and rotenone did not affect the phosphorylation levels of PKA substrates (Fig. 3A-D). These findings indicated that intracellular ATP levels do not affect lipolytic signaling activation.

Figure 3. PKA substrate phosphorylation levels in response to treatment with Iso and mitochondrial respiration chain inhibitors in 3T3-L1 adipocytes. 3T3-L1 adipocytes were cultured in DMEM with 1 µM Iso with/without Oligo and Rot for 4 h. Representative western blot using PKA substrate antibody following treatment with (A) Oligo and (B) Rot. Semi-quantification of PKA substrates following treatment with (C) Oligo and (D) Rot. CBB staining was used as a loading control. All data are presented as the mean ± SEM, n=3-5. A two-way ANOVA with Tukey's post hoc test was performed for statistical analysis. *P<0.05, **P<0.01 vs. same treatment in non-Iso group. CBB, Coomassie brilliant blue; Con, Control; Iso, isoproterenol; Oligo, oligomycin A; PKA, cAMP-dependent protein kinase A; Rot, rotenone.

Inhibition of mitochondrial ATP synthesis causes preferential utilization of glucose for ATP synthesis over fatty acid re-esterification

The present study indicated that suppression of ATP production via inhibition of the mitochondrial respiratory chain decreased lipolysis but did not affect PKA signaling. This suggests that factors other than lipase activation trigger the decrease in lipolytic activity. Fatty acid re-esterification also requires ATP during lipolysis (13). Therefore, it is possible that inhibition of mitochondrial ATP synthesis reduces lipolysis by suppressing fatty acid re-esterification.

The inhibition of the electron transport chain enhances glycolysis, another major energy-producing pathway, in various cell types (13–15). Therefore, the present study evaluated the glucose uptake and lactate concentration as the end products of the glycolytic system. The results showed that inhibition of the mitochondrial respiratory chain increased the reduction rate of glucose concentration in the medium and glucose uptake (Fig. 4A-D), and increased lactate production (Fig. 4E and F). To assess whether glucose taken up into the cell was utilized for ATP production, the ratio of lactate production to glucose reduction was calculated, based on the fact that up to two lactate molecules are produced from one glucose molecule in the glycolytic system. The lactate/glucose ratio increased with the addition of the inhibitors, indicating that most of the glucose taken up into the cell may be preferentially used by the glycolytic system for ATP production rather than supplying substrates for re-esterification (Fig. 4G and H).

Figure 4. Mitochondrial respiratory chain inhibition increases glucose utilization and lactate production in 3T3-L1 adipocytes. 3T3-L1 adipocytes were cultured in DMEM with 1 µM Iso with/without Oligo or Rot. Glucose reduction was detected in the culture medium following lipolysis stimulated by 1 µM Iso with/without (A) Oligo or (B) Rot for 4 h. 2DG uptake during 2 h of Iso stimulation with (C) Oligo or (D) Rot. Adipocytes were stimulated with Iso, with or without Oligo or Rot, in culture medium containing 2DG and glucose (1:1 molar ratio). Lactate concentration was detected following lipolysis stimulated by Iso with/without (E) Oligo or (F) Rot. Aliquots of the medium were collected 4 h after isoproterenol stimulation, and the concentrations of glucose and lactate were measured. The ratio of lactate production to glucose reduction was calculated following lipolysis stimulated by Iso with/without (G) Oligo or (H) Rot for 4 h. Filled bars represent utilization of glucose in the glycolytic system. Dotted bars represent the contributions of fatty acid re-esterification. All data are presented as the mean ± SEM, n=3-8. Student's t-test was performed for statistical analysis. *P<0.05, **P<0.01 vs. Con group. 2DG, 2 deoxy-glucose; Con, Control; Iso, isoproterenol; Oligo, oligomycin A; Rot, rotenone.

Discussion

The present study aimed to investigate the relationship between the cellular energy state and lipolysis. The results indicated that the inhibition of mitochondrial ATP synthesis led to a decrease in catecholamine-stimulated lipolysis, whereas the activation level of lipolysis signaling remained unchanged. Conversely, glucose uptake and lactate production were increased by inhibition of the mitochondrial respiratory chain. These findings suggested that the preferential utilization of glucose for ATP production may have reduced lipolysis due to the suppressed re-esterification of fatty acids.

Obese individuals have been reported to exhibit resistance to lipolytic stimulation (16–18). Previous studies have attributed this, in part, to reduced mitochondrial function. Furthermore, a correlation between obesity and impaired adipocyte mitochondrial function, characterized by reductions in mitochondrial biogenesis, mitochondrial DNA content and activity of electron transport proteins, has been reported (19–21). These studies suggested an intracellular energy state and its relationship with lipolytic activity. The present results support the findings of previous studies and demonstrated the importance of energy state in lipolysis in adipocytes.

As reported previously, the rate of glucose uptake and utilization in adipocytes is enhanced during catecholamine stimulation (8,9). This increase in glucose utilization is believed to provide the substrates necessary for fatty acid re-esterification, which is important for promoting lipolysis. In the present study, the inhibition of the mitochondrial respiratory chain promoted glucose uptake, whereas lipolysis was inhibited. Furthermore, lactate production and the lactate/glucose ratio were increased by inhibiting the mitochondrial respiratory chain. These findings provide indirect evidence that the glucose taken up by the cells was predominantly allocated to ATP synthesis in the glycolytic system, rather than to fatty acid re-esterification. Nevertheless, these results do not directly establish the utilization of glucose for ATP synthesis, thereby warranting future investigations to directly evaluate this aspect using isotope-based analyses.

In the present study, DNP treatment did not induce significant changes in intracellular ATP levels. By contrast, DNP treatment has previously been reported to significantly decrease ATP levels in 3T3-L1 adipocytes (22,23). One possible reason for the disparity between the present findings and those of the previous study is the difference in culture conditions. Adipocytes were treated with DNP alone in previous studies, whereas DNP and catecholamines were administered simultaneously in the present study. Since catecholamine stimulation activates glucose uptake, it is plausible that ATP levels were maintained because of the augmented activation of the glycolytic system during inhibitor treatment.

Although the present study indicated that the decline in lipolysis resulting from the inhibition of the mitochondrial respiratory chain may be attributable to diminished re-esterification, other possible mechanisms remain to be considered. Catecholamine-stimulated lipolysis in adipocytes is regulated via various mechanisms (24). The breakdown of TGs requires the activation of adipose triglyceride lipase (ATGL), which is the first and most important step in lipolysis. However, the interplay between ATGL activity and mitochondrial function remains unknown. Although technical limitations prevented us from performing such analyses in the present study, future studies should clarify the relationship between mitochondrial function and lipolysis in adipocytes.

In conclusion, the present results revealed that impaired mitochondrial function can significantly reduce catecholamine-stimulated lipolysis in adipocytes. Furthermore, the reduction in lipolysis may be attributed to the utilization of glucose for ATP synthesis, thereby inhibiting its use in re-esterification.

Acknowledgements

Not applicable.

Funding

This work was supported by Grants-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science (grant nos. 20K11364 to HK and 19K11553 to NN).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

NT, KH and NN conceived and designed the research, performed the experiments, analyzed the data and interpreted the results of the experiments. NT and KH confirm the authenticity of all the raw data. NT and KH prepared the figures and drafted the manuscript. NT, KH and NN edited and revised, and read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References