Proinflammatory cytokines differentially regulate adipocyte mitochondrial metabolism, oxidative stress, and dynamics

Abstract

Proinflammatory cytokines differentially regulate adipocyte mitochondrial metabolism, oxidative stress, and dynamics. Macrophage infiltration of adipose tissue and the chronic low-grade production of inflammatory cytokines have been mechanistically linked to the development of insulin resistance, the forerunner of type 2 diabetes mellitus. In this study, we evaluated the chronic effects of TNFα, IL-6, and IL-1β on adipocyte mitochondrial metabolism and morphology using the 3T3-L1 model cell system. TNFα treatment of cultured adipocytes led to significant changes in mitochondrial bioenergetics, including increased proton leak, decreased ΔΨm, increased basal respiration, and decreased ATP turnover. In contrast, although IL-6 and IL-1β decreased maximal respiratory capacity, they had no effect on ΔΨm and varied effects on ATP turnover, proton leak, or basal respiration. Only TNFα treatment of 3T3-L1 cells led to an increase in oxidative stress (as measured by superoxide anion production and protein carbonylation) and C16 ceramide synthesis. Treatment of 3T3-L1 adipocytes with cytokines led to decreased mRNA expression of key transcription factors and control proteins implicated in mitochondrial biogenesis, including PGC-1α and eNOS as well as deceased expression of COX IV and Cyt C. Whereas each cytokine led to effects on expression of mitochondrial markers, TNFα exclusively led to mitochondrial fragmentation and decreased the total level of OPA1 while increasing OPA1 cleavage, without expression of levels of mitofusin 2, DRP-1, or mitofilin being affected. In summary, these results indicate that inflammatory cytokines have unique and specialized effects on adipocyte metabolism, but each leads to decreased mitochondrial function and a reprogramming of fat cell biology.

Keywords: adipocyte; cytokine; fusion; mitochondria; respiration.

Fig. 1.

Respiration profiling in chronic cytokine treatment of 3T3-L1 adipocytes. 3T3-L1 adipocytes were treated with 1 nM of the indicated cytokine in complete medium for 24–96 h beginning on day 6 of differentiation, and parameters linked to respiration and other biological processes were measured using the Seahorse XF24 Analyzer. A–D: values were used to calculate total oxygen consumption due to basal respiration (A), inner mitochondrial membrane proton leak (B), cellular ATP demand (C), and maximum respiratory capacity (D). E: extracellular acidification rate (ECAR) was compared in cytokine treatments of varying duration under basal respiration conditions. F: mitochondrial membrane potential (ΔΨ_m) was determined in 3T3-L1 adipocytes treated for 24 h with 1 nM cytokine (n = 3) by tetramethylrhodamine methyl ester (TMRM) staining and flow cytometry of 10,000 cells. All data are represented as means ± SE. **P < 0.005 and *P < 0.05, statistical significance determined by Student's t-test compared with untreated 0-h control; n = 5 unless noted. OCR, oxygen consumption rate.

Fig. 2.

Lipid and glucose metabolism following cytokine treatment of 3T3-L1 adipocytes. Nonesterified fatty acid (NEFA) efflux (A), perilipin abundance (B), NAD⁺/NADH (C), and 2-deoxyglucose uptake (D) were measured following 24-h treatment of adipocytes with 1 nM cytokine. In B, Western blot quantitation of perilipin protein signal was normalized to β-actin protein signal (n = 6). *P < 0.05 and **P < 0.01 compared with control.

Fig. 3.

Reactive oxygen species and protein carbonylation following cytokine treatment of 3T3-L1 adipocytes. A and B: on day 6, 3T3-L1 adipocytes were treated with the indicated cytokine for 24 h, and mitochondrial superoxide formation (A) and protein carbonylation (B) were evaluated; n = 3. C: protein carbonylation blot of TNFα treatment for 24–96 h was probed simultaneously with anti-manganese superoxide dismutase (MnSOD). The break in C represents a translocation of lanes from the same image and exposure purely for illustration purposes only and does not alter the image information. TPP, triphenylphosphonium hydroethidine. *P < 0.05; **P < 0.01.

Fig. 4.

Sphingolipid profiles following cytokine treatment. 3T3-L1 adipocytes were treated with 1 nM of the indicated cytokine for 0–96 h. At the indicated times, cellular extracts were subject to ultra performance liquid chromatography/mass spectrometry/mass spectrometry and the levels of sphigolipids determined. A–F: C16 ceramide (A), C24 ceramide (B), C18:1 ceramide (C), sphingosine-1 phosphate (S1P; D), sphingosine (E), and sphinganine (F). Each bar represents the mean ± SE; n = 3. *P < 0.05 by Student's t-test compared with 0-h control. **P < 0.01.

Fig. 5.

Expression of factors linked to mitochondrial biogenesis and function in response to cytokine treatment. 3T3-L1 adipocytes were treated with 1 nM of the indicated cytokine for 24 h, and the mRNA (A) and protein (B and C) expression of factors linked to mitochondrial biogenesis and function were evaluated. B: Western blot analysis of adipocytes. C: quantitation of each protein was normalized to β-actin and graphed relative to control (n = 6). QPCR results were normalized to reference mRNA transcription factor IIE. In A, the graph of endothelial nitric oxide synthase (eNOS) expression has a broken y-axis so that the TNFα treatment group is visible. Tfam, transcription factor A mitochondria; NRF1, nuclear respiratory factor 1; PGC-1α, PPARγ coactivator-1α; Con, control; T, TNFα; 1β, IL-1β; 6, IL-6; Cyt c, cytochrome c; COX IV, cytochrome c oxidase subunit IV. *P < 0.05.

Fig. 6.

Mitochondrial morphology in cytokine-treated 3T3-L1 adipocytes. A: live cell images of MitoTracker green-stained 3T3-L1 adipocytes after 24-h treatment with 1 nM cytokine. B and C: images of >15 cells were analyzed using Image J 3D software to determine average mitochondrial no. (B) and average volume per mitochondrion (C); n = 5–13. D: each of the cells sampled was scored for mitochondrial fragmentation, and the %cells exhibiting fission was calculated; n = 6–16 for each treatment. E: Western blot of whole cell extract detecting cleaved caspase-3 and β-actin. F: densitometric analysis of cleaved caspase-3 Western blots; n = 4–6. *P < 0.05 compared with control. **P < 0.01.

Fig. 7.

Expression of proteins linked to mitochondrial fission and fusion in response to cytokine treatment. A: extracts from 3T3-L1 adipocytes treated for 24 h with 1 nM cytokine in complete medium were immunoblotted for optic atrophy 1 (OPA1) or β-actin. B: densitometric analysis of OPA1 expression from 3 independent experiments. C and D: protein levels of dymanin-related protein-1 (DRP-1), mitofusin 2 (MFN2), mitofilin, and β-actin in cytosolic and mitochondrial cellular fractions measured by Western blot (C) and protein abundance in the mitochondrial fraction normalized to mitofilin (D). *P < 0.05.

Fig. 8.

Acute effects of TNFα on mitochondrial fragmentation. A: Western blot of whole cell extract detecting cleaved caspase-3 after no treatment control or 1 nM TNFα for 1, 2, 4, and 6 h. B: basal respiration measured by XF24 analyzer in adipocytes (Con) and after 1 nM TNFα for 1–2 h; n = 3–5. **P < 0.001. C: fluorescent microscopy and morphometric analysis of MitoTracker green-stained adipocytes treated or not (Con) for 2, 4, and 6 h with 1 nM TNFα. D: cells were scored for mitochondrial no. and mean mitochondrial volume, as reported in Fig. 6; n = 3.