Diabetes Metab J.  2024 Mar;48(2):215-230. 10.4093/dmj.2022.0332.

Extracellular Vimentin Alters Energy Metabolism And Induces Adipocyte Hypertrophy

• Park JH
• Kwon S
• Park YM

• KMID: 2553593
• DOI: http://doi.org/10.4093/dmj.2022.0332

Abstract

Background
Previous studies have reported that oxidative stress contributes to obesity characterized by adipocyte hypertrophy. However, mechanism has not been studied extensively. In the current study, we evaluated role of extracellular vimentin secreted by oxidized low-density lipoprotein (oxLDL) in energy metabolism in adipocytes.
Methods
We treated 3T3-L1-derived adipocytes with oxLDL and measured vimentin which was secreted in the media. We evaluated changes in uptake of glucose and free fatty acid, expression of molecules functioning in energy metabolism, synthesis of adenosine triphosphate (ATP) and lactate, markers for endoplasmic reticulum (ER) stress and autophagy in adipocytes treated with recombinant vimentin.
Results
Adipocytes secreted vimentin in response to oxLDL. Microscopic evaluation revealed that vimentin treatment induced increase in adipocyte size and increase in sizes of intracellular lipid droplets with increased intracellular triglyceride. Adipocytes treated with vimentin showed increased uptake of glucose and free fatty acid with increased expression of plasma membrane glucose transporter type 1 (GLUT1), GLUT4, and CD36. Vimentin treatment increased transcription of GLUT1 and hypoxia-inducible factor 1α (Hif-1α) but decreased GLUT4 transcription. Adipose triglyceride lipase (ATGL), peroxisome proliferator-activated receptor γ (PPARγ), sterol regulatory element-binding protein 1 (SREBP1), diacylglycerol O-acyltransferase 1 (DGAT1) and 2 were decreased by vimentin treatment. Markers for ER stress were increased and autophagy was impaired in vimentin-treated adipocytes. No change was observed in synthesis of ATP and lactate in the adipocytes treated with vimentin.
Conclusion
We concluded that extracellular vimentin regulates expression of molecules in energy metabolism and promotes adipocyte hypertrophy. Our results show that vimentin functions in the interplay between oxidative stress and metabolism, suggesting a mechanism by which adipocyte hypertrophy is induced in oxidative stress.

Keyword

Adipocytes; Fatty acids, nonesterified; Glucose; Glucose transporter type 1; Hypertrophy; Hypoxia-inducible factor 1; Oxidized low density lipoprotein; Triglycerides; Vimentin

Figure

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  Fig. 1. Oxidized low-density lipoprotein (oxLDL) induces vimentin secretion in 3T3-L1-derived adipocytes. (A) 3T3-L1-derived adipocytes were treated with or without oxLDL (50 µg/mL) for 8, 16, 24 hours and the concentrations of extracellular vimentin in the media were measured using enzyme-linked immunosorbent assay (ELISA). Data were normalized to the protein amount (mg) of the adipocytes used in the assay (n=3). (B) Detection of vimentin in the concentrated media of 3T3-L1-adipocytes cultured with LDL (50 µg/mL) or different concentrations of oxLDL (25, 50 µg/mL) for 24 hours. In the Western blot, His (molecular weight, 1.6 kDa)-tagged recombinant vimentin was used for a positive control. Data were normalized to the protein amount (mg) of the adipocytes obtained from the individual culture dish (n=3). (C) Detection of vimentin in the concentrated media of 3T3-L1-adipocytes cultured with LDL (50 µg/mL for 24 hours) or oxLDL (50 µg/mL for 8, 16, and 24 hours) using Western blotting. Data were normalized to the cellular protein (mg) (n=3). (D) 3T3-L1-adipocytes were pretreated with anti-CD36 antibody (2 µg/mL) before incubating with LDL (50 µg/mL for 24 hours) or oxLDL (50 µg/mL for 24 hours). Detection of vimentin in the concentrated media of the adipocytes using Western blotting and data were normalized to the cellular protein (mg) (n=3). (E) 3T3-L1-adipocytes were pretreated with or without H-89 (10 µM) before incubating with LDL (50 µg/mL for 24 hours) or oxLDL (50 µg/mL for 24 hours). Detection of vimentin in the concentrated media of adipocytes using Western blotting and data were normalized to the cellular protein (mg) (n=3). All experiments were done more than three times. One-way analysis of variance (ANOVA) with Bonferroni post hoc test was done. SF, serum free. aP<0.05, bP<0.01, cP<0.001.

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  Fig. 2. Extracellular vimentin induces adipocyte hypertrophy. (A) 3T3-L1-derived adipocytes treated with or without recombinant vimentin (20 µg/mL) for 24 hours, were stained with Oil-Red O and 4ʹ,6-diamidino-2-phenylindole (DAPI). Light microscopy (left panel) and fluorescence microscopy (right panel). Scale bar: 100 µm (left panel), 20 µm (right panel). (B) Comparison of adipocyte diameter (µm) between vimentin-treated (vimentin) and untreated (control) adipocytes. (C) Comparison of lipid droplet diameter (µm) between vimentin-treated (vimentin) and untreated (control) adipocytes. (D) Absorbance of the Oil-Red O dye extracted from the 3T3-L1-derived adipocytes in (A) (n=3). (E) Size distribution of 3T3-L1 adipocytes treated with or without vimentin (20 µg/mL) for 24 hours. (F) Size distribution of intracellular lipid droplets in the 3T3-L1 adipocytes treated with or without vimentin (20 µg/mL) for 24 hours. (G) Intracellular triglyceride was measured using 3T3-L1-derived adipocytes treated with or without recombinant vimentin (20 µg/mL) for 24 hours. Data were normalized to the amount (mg) of total protein from the adipocytes used in the assay (n=3). aP<0.05, bP<0.001.

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  Fig. 3. Extracellular vimentin promotes glucose uptake and free fatty acid (FFA) uptake via changes in expression of glucose transporters and fatty acid transporter. (A) 3T3-L1-derived adipocytes were treated with recombinant vimentin (20 µg/mL for 24 hours), insulin (1 µM for 30 minutes) or both vimentin (20 µg/mL for 24 hours) and insulin (1 µM for 30 minutes). 2-Deoxyglucose (2-DG) uptake was measured (n=4). (B) 3T3-L1-derived adipocytes were treated with or without recombinant vimentin (20 µg/mL) for 24 hours and FFA uptake was measured using fluorometric fatty acid dye-loading solution (TF2-C12 Fatty Acid) uptake assay (n=3). (C) Western blot analyses for CD36, glucose transporter type 4 (GLUT4), and GLUT1 were performed using fractionated lysates (plasma membrane and cytosol) of 3T3-L1-derived adipocytes treated with or without recombinant vimentin (20 µg/mL) for 24 hours. Band quantification was performed using either caveolin-1 (BD Biosciences) as a plasma membrane marker, or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a cytosol marker (n=3). (D) Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analyses for CD36, GLUT1, and GLUT4 were performed using RNA from 3T3-L1-derived adipocytes cultured with or without recombinant vimentin (20 µg/mL) for 24 hours. GAPDH was used as an internal control (n=5, n=6, n=4). (E) Western blot analyses for GLUT1 and GLUT4 using total lysates of 3T-3L1-derived adipocytes treated with or with recombinant vimentin (20 µg/mL) for 24 hours. Band quantification was performed using GAPDH, as an internal control (n=3). (F) Western blot analyses for GLUT1 and GLUT4 were performed using total lysates of 3T3-L1-derived adipocytes with or with recombinant vimentin (20 µg/mL) for 36 hours. Band quantification was performed using α-tubulin (AbFrontier), an internal control (n=3). (G) qRT-PCR analysis for hypoxia-inducible factor 1α (Hif-1α) was performed using RNA from 3T3-L1-derived adipocytes treated with or without recombinant vimentin (20 µg/mL) for 24 hours. GAPDH was used as an internal control (n=4). RFU, relative fluorescence unit. aP<0.05, bP<0.01, cP<0.001.

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  Fig. 4. Extracellular vimentin-induced glucose transporter type 1 (GLUT1) expression depends on insulin-like growth factor 1 receptor (IGF1R) and activation of extracellular-signal-regulated kinase (ERK). (A) Western blot analyses for phospho-insulin receptor substrate 1 (P-IRS1; Tyrosine 612), P-Akt (Serine 473), and P-IGF1R (Tyrosine 1158+1162+1163) were performed using lysates of 3T3-L1-derived adipocytes treated with or without recombinant vimentin (20 µg/mL) for 30 minutes. Band quantification was performed using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control. Samples from three different batches of treatment were loaded onto a gel (n=3). (B) Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analyses for GLUT1, GLUT4, hypoxia-inducible factor 1α (Hif-1α) were performed with RNA from 3T3-L1-derived adipocytes treated or untreated with anti-IGF1R antibody (11 µg/mL) for 1 hour and then incubated with or without recombinant vimentin (20 µg/mL) for 24 hours. GAPDH was used as an internal control (GLUT1, n=3; GLUT4, n=3; Hif-1α, n=4). (C) Western blotting for P-ERK1/2 (Threonine 202/Tyrosine 204) was performed using lysates of 3T3-L1-derived adipocytes treated with or without recombinant vimentin (20 µg/mL) for 30 minutes. Band quantification was performed using GAPDH as an internal control (n=3). (D) qRT-PCR analyses for GLUT1, GLUT4, Hif-1α were performed with RNA from 3T3-L1-derived adipocytes treated or untreated with U0126 (ERK inhibitor, 25 µM) for 1 hour and then incubated with or without recombinant vimentin (20 µg/mL) for 24 hours. GAPDH was used as an internal control (n=3 for each). aP<0.05, bP<0.01, cP<0.001.

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  Fig. 5. Extracellular vimentin modulates expression of molecules for lipogenesis and lipolysis. (A) L-Lactate was measured in the culture media of 3T3-L1-derived adipocytes treated with or without recombinant vimentin (20 µg/mL) for 24 hours. Data were normalized to the protein amount (mg) of the adipocytes used in the assay (n=2). (B) Intracellular adenosine triphosphate (ATP) was measured in 3T3-L1-derived adipocytes treated with or without recombinant vimentin (20 µg/mL) for 24 hours. Data were normalized to the protein amount (mg) of the adipocytes used in the assay (n=4). (C) Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analyses for peroxisome proliferator-activated receptor γ 2 (PPARγ2), sterol regulatory element-binding protein 1 (SREBP1), diacylglycerol O-acyltransferase 1 (DGAT1), DGAT2, and Lipin1 were performed using RNA from 3T3-L1-derived adipocytes treated with or without recombinant vimentin (20 µg/mL) for 24 hours. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control (n=4, n=3, n=3, n=3). (D) Western blot analysis for PPARγ, fatty acid synthase (FASN), SREBP1, Lipin1, adipose triglyceride lipase (ATGL), and DGAT1 were performed using lysates of 3T3-L1-derived adipocytes treated with or without recombinant vimentin (20 µg/mL) for 24 hours. Band quantification was performed using GAPDH and α-tubulin as internal controls (n=3). (E) Western blot analysis for phosphor-hormone-sensitive lipase (P-HSL; Serine 564) was performed using lysates of 3T3-L1-derived adipocytes treated with or without recombinant vimentin (20 µg/mL) for 30 minutes. Band quantification was performed using total HSL and GAPDH as internal controls (n=3). aP<0.05, bP<0.01, cP<0.001.

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  Fig. 6. Extracellular vimentin induces endoplasmic reticulum (ER) stress and impairs autophagy. (A) Western blot analyses for protein kinase R-like ER kinase (PERK), inositol-requiring enzyme 1 α (IRE1α), and C/EBP homologous protein (CHOP) were performed using lysates of 3T3-L1-derived adipocytes treated with or without recombinant vimentin (20 µg/mL) for 24 hours. Band quantification was performed using β-actin and α-tubulin as internal controls (n=3). (B) Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analyses for IRE1α, CHOP, and binding immunoglobulin protein (BiP) were performed using RNA from 3T3-L1-derived adipocytes treated with or without vimentin (20 µg/mL) for 24 hours. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control (n=4). (C) Western blot analysis for p62, light chain 3 (LC3)-I, and LC3-II were performed using lysates of 3T3-L1-derived adipocytes treated with or without recombinant vimentin (20 µg/mL) for 24 hours. Band quantification was performed using α-tubulin and GAPDH as internal controls (n=3). (D) Autophagic vacuoles in 3T3-L1-derived adipocytes treated with or without recombinant vimentin (20 µg/mL) for 24 hours, were measured. Data were normalized to Hoechst 33342 staining in the adipocytes used in the assay (n=5). aP<0.05, bP<0.001.

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