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. 2018 Nov;50(5):557-564.
doi: 10.1097/SHK.0000000000001076.

Sepsis Induces Adipose Tissue Browning in Nonobese Mice But Not in Obese Mice

Itay Ayalon  1 , Hui Shen  1 , Lauren Williamson  1 , Keith Stringer  2 , Basilia Zingarelli  1 , Jennifer M Kaplan  1
Affiliations

Sepsis Induces Adipose Tissue Browning in Nonobese Mice But Not in Obese Mice

Itay Ayalon et al. Shock. 2018 Nov.

Abstract

Severe sepsis and septic shock are the biggest cause of mortality in critically ill patients. Obesity today is one of the world's greatest health challenges. Little is known about the extent of involvement of the white adipose tissue (WAT) in sepsis and how it is being modified by obesity. We sought to explore the involvement of the WAT in sepsis. We hypothesize that sepsis induces browning of the WAT and that obesity alters the response of WAT to sepsis. Six-week-old C57BL/6 mice were randomized to a high-fat diet to induce obesity (obese group) or control diet (nonobese group). After 6 to 11 weeks of feeding, polymicrobial sepsis was induced by cecal ligation and puncture (CLP). Mice were sacrificed at 0, 18, and 72 h after CLP and epididymal WAT (eWAT), inguinal WAT, and brown adipose tissue (BAT) harvested. Both types of WAT were processed for light microscopy and transmission electron microscopy to assess for morphological changes in both obese and nonobese mice. Tissues were processed for immunohistochemistry, image analyses, and molecular analyses. BATs were used as a positive control. Nonobese mice have an extensive breakdown of the unilocular lipid droplet and smaller adipocytes in WAT compared with obese mice after sepsis. Neutrophil infiltration increases in eWAT in nonobese mice after sepsis but not in obese mice. Nonobese septic mice have an increase in mitochondrial density compared with obese septic mice. Furthermore, nonobese septic mice have an increase in uncoupling protein-1 expression. Although the WAT of nonobese mice have multiple changes characteristic of browning during sepsis, these changes are markedly blunted in obesity.

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Figures

Figure 1
Figure 1. Weight of mice at time of CLP by cohort
At six weeks of age, mice were randomized to a high-fat diet or standard control diet for six (age of 12 weeks at CLP) or eleven weeks (age of 17 weeks at CLP) as indicated. *p≤0.001 vs. non-obese mice as analyzed by t-test.
Figure 2
Figure 2. WAT H&E staining from epididymal and inguinal WAT
Epididymal WAT in non-obese mice at 0h (e1,3), 18h (e5,7), and 72h (e9,11) and in obese mice at 0h (e2,4), 18h (e6,8), and 72h (e10,12) after sepsis at 10× and 40× magnification respectively. Inguinal WAT in non-obese mice at 0h (i1,3), 18h (i5,7), and 72h (i9,11) and in obese mice at 0h (i2,4), 18h (i6,8), and 72h (i10,12) after sepsis at 10× and 40× magnification respectively. Representative sections are illustrated. A similar pattern was seen in n=2-4 different samples in each experimental group. All mice were 12 weeks of age at the time of harvest.
Figure 3
Figure 3. Lipid droplet surface area in non-obese and obese mice at 0 and 18h after CLP by tissue type
Frequency of lipid droplet surface area in (A) eWAT and (B) iWAT from non-obese and obese non-septic and septic (CLP 18h) mice. (C) Lipid droplet surface area in epididymal and inguinal WAT at 0 and 18h after CLP. *p≤0.05 vs time 0h within diet, #p≤0.05 vs non-obese mice by 2-way ANOVA. n= 4-5 mice in each group. All mice were 17 weeks of age at the time of harvest.
Figure 4
Figure 4. Transmission electron microscopy of epididymal WAT in a non-obese mouse 18 hours after CLP
(A) and (B) demonstrate the budding process in which lipid droplets breakdown into 1st and 2nd generation lipid droplets. (C), (D) and (E) show the close proximity between the newly formed small lipid droplets and mitochondria at 1,500×, 3,000×, 8,000× respectively. Mice were 12 weeks of age at the time of harvest. Representative sections are illustrated. A similar pattern was seen in n=2-4 different samples. L=lipid droplets, B=budding of the lipid droplets, LBD=lipid breakdown, M=mitochondria.
Figure 5
Figure 5. Morphological changes of WAT by Transmission Electron Microscopy in non-obese and obese mice at timepoints after CLP
Epididymal and inguinal WAT in non-obese and obese mice at 0h, 18h, and 72h after sepsis at 200× and 2000× magnification. All mice were 12 weeks of age at the time of harvest. L=lipid droplet, N=nucleus, MX=extracellular matrix, LBD=lipid breakdown, B=budding, M=mitochondria, V=blood vessel.
Figure 6
Figure 6. Mitochondrial density in epididymal and inguinal WAT at various timepoints after CLP
Mitochondrial density as calculated by dividing the total number of mitochondria in a predefined magnification (×3000, TEM) by the total measured surface area. All mice were 17 weeks of age at the time of harvest. *p≤0.05 vs time 0h within diet, #p≤0.05 vs non-obese mice by 2-way ANOVA. n= 4-5 mice in each group.
Figure 7
Figure 7. UCP1 protein expression
(A) Mitochondrial UCP1 and VDAC1 (voltage-dependent anion-selective channel-1) expression in iWAT using Western blot analysis. BAT was used as a positive control. Expression of UCP1 in (B) epididymal WAT and (C) inguinal WAT in non-septic (CLP 0h) and septic (CLP 18h) mice. All mice were 17 weeks of age at the time of harvest. *p≤0.05 vs non-septic mice within diet, #p≤0.05 vs non-obese mice by 2-way ANOVA. n= 4-5 mice in each group.
Figure 8
Figure 8. WAT UCP1 expression by immunohistochemistry
(A) Representative images of epididymal WAT UCP1 IHC staining in non-obese mice at 0h (e1), 18h (e3) and in obese mice at 0h (e2), 18h (e4), after sepsis. Inguinal WAT UCP1 IHC staining in non-obese mice at 0h (i1), 18h (i3) and in obese mice at 0h (i2), 18h (i4) after sepsis. Quantification of (B) eWAT and (C) iWAT UCP1 staining using ImageScope software to determine positivity staining of UCP1 intensity in non-obese and obese mice at 0 and 18h after CLP. All mice were 17 weeks of age at the time of harvest. *p≤0.05 vs time 0h within diet, #p≤0.05 vs non-obese mice by 2-way ANOVA. n= 4-5 mice in each group.
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