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. 2019 Jun 3;49(5):802-810.e6.
doi: 10.1016/j.devcel.2019.04.009. Epub 2019 May 9.

NF-κB Shapes Metabolic Adaptation by Attenuating Foxo-Mediated Lipolysis in Drosophila

Maral Molaei  1 , Crissie Vandehoef  2 , Jason Karpac  3
Affiliations

NF-κB Shapes Metabolic Adaptation by Attenuating Foxo-Mediated Lipolysis in Drosophila

Maral Molaei et al. Dev Cell. .

Abstract

Metabolic and innate immune signaling pathways have co-evolved to elicit coordinated responses. However, dissecting the integration of these ancient signaling mechanisms remains a challenge. Using Drosophila, we uncovered a role for the innate immune transcription factor nuclear factor κB (NF-κB)/Relish in governing lipid metabolism during metabolic adaptation to fasting. We found that Relish is required to restrain fasting-induced lipolysis, and thus conserve cellular triglyceride levels during metabolic adaptation, through specific repression of ATGL/Brummer lipase gene expression in adipose (fat body). Fasting-induced changes in Brummer expression and, consequently, triglyceride metabolism are adjusted by Relish-dependent attenuation of Foxo transcriptional activation function, a critical metabolic transcription factor. Relish limits Foxo function by influencing fasting-dependent histone deacetylation and subsequent chromatin modifications within the Bmm locus. These results highlight that the antagonism of Relish and Foxo functions are crucial in the regulation of lipid metabolism during metabolic adaptation, which may further influence the coordination of innate immune-metabolic responses.

Keywords: ATGL; Bmm; Drosophila; Foxo; NF-κB; Relish; histone acetylation; innate immune; lipid metabolism; metabolic adaptation.

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Conflict of interest statement

Declaration of Interests

The authors declare no competing interests.

Figures

Figure 1:
Figure 1:. Relish Function in Fat body Directs Lipid Metabolism in Response to Metabolic Adaptation
(A-D) Relish-dependent changes in lipid metabolism and survival in response to fasting (A) Total triglyceride (TAG) levels of whole flies (OreR (WT-wild type) control, relE20/+ (heterozygote control), or relE20/relE20 (mutant) genotypes) before and after fasting (20 hours). n = 4–5 samples. (B) Starvation resistance of female flies. n = 5 cohorts (total 87–95 flies). The red arrow indicates time-point of fasting assays. (C) Oil Red O (ORO) and (D) Nile red stain of dissected carcass/ fat body before and after fasting (20 hours). Nile red (neutral lipids; red) and DAPI (DNA; blue) detected by fluorescent histochemistry. (E-G) Re-expressing Relish (UAS-Rel) in fat body (CGGal4) of Relish-deficient flies restores metabolic adaptation responses. (E) Starvation resistance of female flies (CGGal4/+; relE20/+ (control), CGGal4/+; relE20/relE20 (mutant), or CGGal4/UAS-Rel; relE20/relE20 (Rescue)). n = 5 cohorts (total 79–98 flies). The red arrow indicates time-point of fasting assays. (F) Total TAG levels of whole flies (n = 4–5 samples) and (G) ORO stain of dissected carcass/ fat body before and after fasting (20 hours). (H-J) Changes in lipid metabolism and survival upon Relish depletion (RNAi lines v108469-KK and v49413-GD) in fat body (CGGal4). (H) Starvation resistance of female flies. n = 6 cohorts (total 139–149 flies). The red arrow indicates time-point of fasting assays. (I) Total TAG levels of whole flies (n = 4–5 samples) and (J) ORO stain of dissected carcass/ fat body (only RNAi v108469-KK shown) before and after fasting (64 and/or 90 hours). Bars and line graph markers represent mean ± SE. All flies were 7 days old post-eclosion. See also Figures S1–S3.
Figure 2:
Figure 2:. Relish Controls Fasting-induced Triglyceride Lipase Bmm Transcription and Lipolysis
(A) Drosophila HSL, lip4, CG5966, and bmm transcription (measured by qRTPCR in whole flies, plotted as fold induction (20 hours fasted/fed) of relative expression). relE20/+ (heterozygote control), or relE20/relE20 (mutant) genotypes. n = 3 samples. (B-C) Changes in lip4, CG5966, and bmm transcription (measured by qRT-PCR in dissected carcass / fat body, plotted as fold induction (64 hours fasted/fed) of relative expression) upon Relish depletion (RNAi line v108469-KK) in fat body (CGGal4). (C) Relative expression values (from (B)) for bmm transcription. n = 3–4 samples. (D) Quantification of lipid breakdown. Incorporation of 14C-labeled glucose into total lipids (from whole flies) from labeled-glucose fed (1.5 hours or 16 hours) or fasted (20 hours) flies are shown. Percent change in loss of 14C-labeled lipids after fasting is also shown. n = 3–4 samples. (E) Free fatty acid (FFA) levels measured in whole flies before and after fasting (20 hours). n = 4 samples. (F-G) Attenuating Bmm (RNAi line v37877) in fat body (CGGal4) of Relish-deficient flies restores metabolic adaptation responses. (F) Total TAG levels of whole flies (n = 3–4 samples) and (G) Oil Red O stain of dissected carcass/ fat body before and after fasting (20 hours, CGGal4/+; relE20/+ (control), CGGal4/+; relE20/relE20 (mutant), or CGGal4/UAS-Bmm RNAi; relE20/relE20 (Rescue)). Bars represent mean ± SE. All flies were 7 days old post-eclosion. See also Figure S4.
Figure 3:
Figure 3:. Relish binds to a Regulatory Region in the Bmm Locus
(A) Schematic shows Bmm locus (focusing on first intron proximal to transcription start site) and putative NF-κB/Rel binding motifs (identified by Clover). R1 and R2 represent regional target sites (and corresponding primer sets) tested in ChIP-qPCR analysis. The histogram represents ChIP-qPCR analysis of Relish binding to the Bmm locus (compared to the Actin5c promoter (Act5cP) and the Diptericin promoter (DiptP) in fed or fasted (20 hours) conditions. ChIP-qPCR analysis with normal goat serum (NGS) is included as a control. Plotted as fold change (FC) of indicated PCR primer sets compared to a negative control (NC) primer set. n = 3 biological replicates. (B) Requirement of Bmm locus Rel binding site in limiting induced gene expression measured by RFP fluorescence in transgenic flies carrying indicated reporters (during fed and fasted (48 or 72 hours) conditions). (C) ChIP-PCR analysis of H3K9ac enrichment in R1/Relish-binding region of the Bmm locus in wild type (WT; OreR) and relE20/relE20 (mutant) genotypes before and after fasting (20 hours). n = 3 biological replicates. (D) Changes bmm transcription (measured by qRT-PCR in whole flies) before and after fasting (20 hours) upon Rpd3 depletion (RNAi line TRiP 36800) in fat body (CGGal4). Controls are a genetically matched RNAi targeting luciferase. n = 4–6 samples. Bars represent mean ± SE. All flies were 7 days old post-eclosion. See also Figure S3 and S4.
Figure 4:
Figure 4:. Foxo and Relish Antagonism Dictate Fasting-induced Bmm Transcription and Lipolysis
(A) Putative model highlighting the integration of Relish (Rel) and other fasting-induced transcription factors (TF). (B-C) Changes in bmm transcription (measured by qRT-PCR in whole flies or dissected fat body) before and after fasting (64 hours) in Foxo mutant (w1118;; foxo24/foxoΔ94) and control (w1118) genotypes, as well as upon Foxo depletion (RNAi line v106097) in fat body (CGGal4). n = 4 samples. (D) Percent eclosion of adult animals of indicated mutants / double mutants. n = 67–90 animals. (E) Changes in bmm transcription (measured by qRT-PCR in whole flies) before and after fasting (20 hours) in controls (relE20/+ and relE20, foxo24/+), mutant (relE20/relE20) and mutant with reduction in Foxo gene dose (relE20, foxo24/relE20) genotypes. n = 3 samples. (F) Starvation resistance of Relish-deficient female flies with reduction in Foxo gene dose (n = 4 cohorts (total 68–78 flies)), and (G) ORO stain of dissected carcass/ fat body before and after fasting (20 hours). (H-J) Attenuating Foxo (RNAi line v106097) in fat body (CGGal4) of Relish-deficient flies restores metabolic adaptation responses. (H) Starvation resistance of female flies (CGGal4/+; relE20/+ (control), CGGal4/+; relE20/relE20 (mutant), or CGGal4/UAS-Foxo RNAi; relE20/relE20 (Rescue)). n = 6 cohorts (total 117–140 flies). The red arrow indicates time-point of fasting assays. (I) Total TAG levels of whole flies (n = 5 samples) and (J) ORO stain of dissected carcass/ fat body before and after fasting (20 hours). Bars and line graph markers represent mean ± SE. All flies were 7 days old post-eclosion. See also Figure S4.
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