1. Volume 23, Issue 4, Pages 483-493 (April 2016)
Exogenous Fatty Acids Are the Preferred Source of Membrane Lipids in Proliferating Fibroblasts 
Cong-Hui Yao, Ronald Fowle-Grider, Nathanial G. Mahieu, Gao-Yuan Liu, Ying-Jr Chen, Rencheng Wang, Manmilan Singh, Gregory S. Potter, Richard W. Gross, Jacob Schaefer, Stephen L. Johnson, Gary J. Patti 
Cell Chemical Biology 
Volume 23, Issue 4, Pages (April 2016)
DOI: /j.chembiol
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2. Cell Chemical Biology 2016 23, 483-493DOI: (10. 1016/j. chembiol. 2016
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3. Figure 1 Establishing the Quiescent and Proliferative States
(A) Plot of 3T3-L1 fibroblast density as a function of culture time. Cells were harvested for comparative analysis at the times indicated by the arrows.
(B) Cell-cycle analysis at the times indicated by the arrows in (A).
(C) Rate of glucose consumption in quiescent and proliferating fibroblasts cultured in standard DMEM with 10% FBS.
Data are presented as mean ± SEM (n = 3). **p < 0.01; ***p <
Cell Chemical Biology  , DOI: ( /j.chembiol )
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4. Figure 2
[U-13C]Glucose Labeling of Quiescent and Proliferating Fibroblasts
(A) After 5 min of [U-13C]glucose labeling, glycolytic intermediates have increased isotopic enrichment percentages in proliferating fibroblasts relative to quiescent fibroblasts.
(B) After 6 hr of [U-13C]glucose labeling, TCA cycle intermediates and palmitate have decreased labeling percentages in proliferating fibroblasts relative to quiescent fibroblasts.
(C) The pool size of palmitate is ∼25% larger in proliferating fibroblasts. Data shown are from the 6-hr time point, but data from other time points are consistent.
(D) 13C CPMAS of intact quiescent fibroblasts (black) and intact proliferating fibroblasts (red) labeled with [U-13C]glucose for 12 hr. Spectra were normalized by scan number and dry sample mass. Natural-abundance contributions (measured experimentally) have been subtracted from the spectra. The narrow peak at 30 ppm corresponds to the aliphatic carbons of lipid chains. The data show that [U-13C]glucose labels more lipids in proliferating fibroblasts relative to quiescent fibroblasts.
Data are presented as mean ± SEM (n = 3). *p < 0.05; **p < 0.01; ***p < Star indicates that the difference in integrated intensities between black and red peaks is larger than the integrated red and black baselines by a value of p < 0.01.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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5. Figure 3
[U-13C]Glutamine Labeling of Quiescent and Proliferating Fibroblasts
(A) Proliferating fibroblasts take up more glutamine than quiescent fibroblasts when cultured in standard DMEM with 10% FBS.
(B) After 6 hr of [U-13C]glutamine labeling, TCA cycle intermediates have increased labeling percentages in proliferating fibroblasts relative to quiescent fibroblasts. The enrichment of palmitate is not statistically different between proliferating and quiescent fibroblasts.
(C) Citrate labeling in quiescent and proliferating fibroblasts from [U-13C]glutamine after 6 hr.
(D) Relative contributions of glucose and glutamine labels to de novo synthesis of palmitate at 6 hr in proliferating and quiescent fibroblasts cultured in standard DMEM with 10% FBS. Distributions were determined by using ISA and [U-13C]glucose and [U-13C]glutamine data at the 6-hr labeling time point.
(E) 13C CPMAS of intact quiescent fibroblasts (black) and intact proliferating fibroblasts (red) labeled with [U-13C]glutamine for 12 hr. Spectra were normalized by scan number and dry sample mass. Natural-abundance contributions (measured experimentally) have been subtracted from the spectra. The narrow peak at 30 ppm corresponds to the aliphatic carbons of lipid chains. The relative contribution of the 30 ppm peak, determined by experimental deconvolution (see Figure S2), is indicated by arrows. The data show that labeling of lipids by [U-13C]glutamine is the same between cell populations. In contrast, labeling of the peptide peak at 175 ppm increases in proliferating fibroblasts and indicates that the increased uptake of glutamine in proliferating cultures supports protein synthesis.
Data are presented as mean ± SEM (n = 3). *p < 0.05; **p < 0.01; ***p < Star indicates that the difference in integrated intensities between black and red peaks is larger than the integrated red and black baselines by a value of p < 0.01.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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6. Figure 4
Proliferating Fibroblasts Have Increased Palmitate Uptake and Deceased Palmitate Degradation Relative to Quiescent Fibroblasts
(A) Proliferating fibroblasts consume more palmitate than quiescent fibroblasts in media having two different palmitate concentrations.
(B) After 6 hr of [U-13C]palmitate labeling, TCA cycle intermediates have increased labeling percentages in quiescent fibroblasts relative to proliferating fibroblasts.
(C) The relative pool sizes of acyl carnitines decrease in proliferating fibroblasts.
(D) Labeling pattern of intracellular palmitate after cells were labeled with [U-13C]palmitate for 6 hr (corrected for natural abundance). The labeling pattern of M2-M14 is enlarged to show futile cycling between palmitate degradation and resynthesis.
(E) Labeling of complex lipids from [U-13C]palmitate after 6 hr. LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; LPS, lysophosphatidylserine; LPG, lysophosphoglycerol; PI, phosphatidylinositol.
Data are presented as mean ± SEM (n = 3). *p < 0.05; **p < 0.01; ***p <
Cell Chemical Biology  , DOI: ( /j.chembiol )
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7. Figure 5
Alterations in Palmitate Metabolism in Proliferating Fibroblasts
(A) Treatment of proliferating fibroblasts with SSO decreases palmitate uptake.
(B) Treatment of proliferating fibroblasts (3T3-P), H460, and HeLa cells with SSO decreases proliferation. Treatment of quiescent fibroblasts (3T3-Q) with SSO has no statistically significant effect.
(C) Proliferating fibroblasts have increased synthesis of palmitate from glucose carbon, increased palmitate uptake, and decreased palmitate degradation. Synthesis of complex lipids from palmitate is increased. The contribution of glutamine carbon to lipid synthesis has minimal change. Bigger changes in proliferating fibroblasts relative to quiescent fibroblasts are indicated with larger plus and minus signs.
Data are presented as mean ± SEM (n = 3). *p < 0.05; **p < 0.01; ***p <
Cell Chemical Biology  , DOI: ( /j.chembiol )
Copyright © 2016 Elsevier Ltd Terms and Conditions