Regulation of Lipolysis and Adipose Tissue Signaling during Acute Endotoxin-Induced Inflammation: A Human Randomized Crossover Trial

Abstract

Background: Lipolysis is accelerated during the acute phase of inflammation, a process being regulated by pro-inflammatory cytokines (e.g. TNF-α), stress-hormones, and insulin. The intracellular mechanisms remain elusive and we therefore measured pro- and anti-lipolytic signaling pathways in adipocytes after in vivo endotoxin exposure.

Methods: Eight healthy, lean, male subjects were investigated using a randomized cross over trial with two interventions: i) bolus injection of saline (Placebo) and ii) bolus injection of lipopolysaccharide endotoxin (LPS). A 3H-palmitate tracer was used to measure palmitate rate of appearance (Rapalmitate) and indirect calorimetry was performed to measure energy expenditures and lipid oxidation rates. A subcutaneous abdominal fat biopsy was obtained during both interventions and subjected to western blotting and qPCR quantifications.

Results: LPS caused a mean increase in serum free fatty acids (FFA) concentrations of 90% (CI-95%: 37-142, p = 0.005), a median increase in Rapalmitate of 117% (CI-95%: 77-166, p<0.001), a mean increase in lipid oxidation of 49% (CI-95%: 1-96, p = 0.047), and a median increase in energy expenditure of 28% (CI-95%: 16-42, p = 0.001) compared with Placebo. These effects were associated with increased phosphorylation of hormone sensitive lipase (pHSL) at ser650 in adipose tissue (p = 0.03), a trend towards elevated pHSL at ser552 (p = 0.09) and cAMP-dependent protein kinase A (PKA) phosphorylation of perilipin 1 (PLIN1) (p = 0.09). Phosphatase and tensin homolog (PTEN) also tended to increase (p = 0.08) while phosphorylation of Akt at Thr308 tended to decrease (p = 0.09) during LPS compared with Placebo. There was no difference between protein or mRNA expression of ATGL, G0S2, and CGI-58.

Conclusion: LPS stimulated lipolysis in adipose tissue and is associated with increased pHSL and signs of increased PLIN1 phosphorylation combined with a trend toward decreased insulin signaling. The combination of these mechanisms appear to be the driving forces behind the increased lipolysis observed in the early stages of acute inflammation and sepsis.

Trial registration: ClinicalTrials.gov NCT01705782.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1. CONSORT flowchart for the trial.

Fig 2. Flowchart showing the time course for the trial days.

At time = 0 min a bolus of isotonic saline (Placebo) or a bolus lipopolysaccharide (LPS) was given.

Fig 3. Metabolic measures.

Data are shown as dot-plots for each subject during the control conditions with saline administration (Placebo) and the day with lipopolysaccharide administration (LPS). A black horizontal bar indicates the median value for each group. A. Rapalmitate (n = 8), B. lipid oxidation rates from indirect calorimetry measurements (n = 7), and C. energy expenditure from indirect calorimtry measurements (n = 7). Paired sample t-tests were used to compare groups. Rapalmitate= rate of appearance of palmitate, FFA = free fatty acids.

Fig 4. Western blot analyses of subcutaneous abdominal fat tissue biopsies.

Representative western blots in abdominal adipose tissue during control conditions (Placebo) and during lipopolysaccharide (LPS) induced endotoxemia (n = 7). Data are presented as the ratio change compared to the median value for the Placebo condition. The black horizontal bars indicate the median value for each group (= 1 for Placebo in all graphs). Paired sample t-test was used to compare groups. A.G0S2 = G0/G1 switch protein 2, B.ATGL = adipose triglyceride lipase, C.CGI-58 = comparative gene identification-58, D.,E., and F.HSL = hormone sensitive lipase, andG.p-PKA Substrate = Phospho-PKA (protein kinase A) Substrate.

Fig 5. qPCR measurements of subcutaneous abdominal fat biopsies.

Quantitative PCR measurements (n = 6) of mRNA are shown for control conditions (Placebo) and during lipopolysaccharide (LPS): A.G0S2 = G0/G1 switch protein 2, B.ATGL = adipose triglyceride lipase, andC.CGI-58 = comparative gene identification-58.

Fig 6. Western blot analyses of subcutaneous abdominal fat tissue biopsies.

Representative western blots in abdominal adipose tissue during control conditions (Placebo) and during lipopolysaccharide (LPS) induced endotoxemia (n = 7). Data are presented as the ratio change compared to the median value for the Placebo condition. The black horizontal bars indicate the median value for each group (= 1 for Placebo in all graphs). Paired sample t-test was used to compare groups. A.PTEN = phosphatase and tensin homolog, B. and C.Akt, andD.AS160 = Akt substrate of 160 kDa.