Autophagy Ablation in Adipocytes Induces Insulin Resistance and Reveals Roles for Lipid Peroxide and Nrf2 Signaling in Adipose-Liver Crosstalk

Abstract

Autophagy is a homeostatic cellular process involved in the degradation of long-lived or damaged cellular components. The role of autophagy in adipogenesis is well recognized, but its role in mature adipocyte function is largely unknown. We show that the autophagy proteins Atg3 and Atg16L1 are required for proper mitochondrial function in mature adipocytes. In contrast to previous studies, we found that post-developmental ablation of autophagy causes peripheral insulin resistance independently of diet or adiposity. Finally, lack of adipocyte autophagy reveals cross talk between fat and liver, mediated by lipid peroxide-induced Nrf2 signaling. Our data reveal a role for autophagy in preventing lipid peroxide formation and its transfer in insulin-sensitive peripheral tissues.

Keywords: adipocytes; adiponectin; adipose tissue; autophagy; inflammation; insulin resistance; lipid peroxide; mitochondria.

Figure 1.. Post-Developmental Deletion of Adipocyte Autophagy Does Not Influence Adiposity but Causes Adipose Tissue Inflammation

(A and B) Western blot analysis of Atg3, LC3I, LC3II, p62, NBR1, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in eWAT and BAT, respectively, from NCD-fed mice with the indicated genotypes. (C and D) Densitometry of (A) and (B), respectively, normalized by GAPDH and expressed as fold change from wild-type (WT) controls. (E–H) WT and AdiAtg3KO mice were fed an NCD or high-fat diet (HFD) for 16 weeks. (E) Body weights. (F) Weight gain. (G) Percent lean mass. (H) Percent fat mass. (I and K) H&E staining of eWAT from NCD-fed mice with the indicated genotypes. (J) Morphometric analysis of adipocyte number and diameter in eWAT from NCD-fed mice with the indicated genotypes. (L) Morphometric quantification of crown-like structures in eWAT from NCD-fed mice with the indicated genotypes. (M) Relative mRNA levels of inflammatory genes expressed as fold change from WT in eWAT from NCD-fed mice with the indicated genotypes. Values are mean ± SEM (*p < 0.05, **p < 0.005 versus WT, ##p < 0.005 versus NCD of the same genotype); n = 5/group for (A)–(D) and n = 6–14/group for (E)–(M).

Figure 2.. Deletion of Autophagy in Mature Adipocytes Causes Peripheral Insulin Resistance in AdiAtg3 KO Mice Fed a NCD

(A–K) WT and AdiAtg3 KO mice were fed an NCD for 12 weeks. (A) Glucose tolerance tests (GTTs). (B) Area under the curve (AUC) for GTTs. (C) Insulin tolerance tests (ITTs). (D) AUC for ITTs. (E) Plasma insulin levels 0 and 20 min after glucose injection during GTTs. (F–I) Western blot analysis of phospho-Akt (Ser473) and total Akt levels in eWAT, BAT, liver, and skeletal muscle from NCD-fed mice with the indicated genotypes. (J) Densitometry of phospho-Akt to total Akt. (K) Insulin-stimulated 2-deoxyglucose uptake in eWAT explants of NCD-fedmice with the indicated genotypes. Values are mean ± SEM (*p < 0.05, **p < 0.005 versus WT, ##p < 0.005 versus basal). n = 8–10/group for (A)–(D), n = 6/group for (E), and n = 3/group for (F)–(K).

Figure 3.. Accumulation of Dysfunctional Mitochondria Autophagy-Deficient White and Brown Adipocytes

(A–D and N–S) WT and AdiAtg3 KO mice were fed an NCD for 12 weeks. (A and B) Western blot analysis of mitochondrial proteins (aconitase and HSP60) in eWAT and BAT, respectively. (C and D) Densitometry of aconitase and HSP60 normalized by GAPDH and expressed as fold change from WT controls in eWAT and BAT, respectively. (E–M) Primary brown preadipocytes were isolated from the interscapular BAT of Atg3^f/f mice and differentiated for 3 days before being infected with adenovirus-Cre-GFP or adenovirus-GFP control. Cells were analyzed 6 days post-infection. (E) Representative images of Ad-Cre-GFP- or Ad-GFP-infected cells 48 hr post-infection. (F) Relative mRNA expression of Atg3 and Atg7 in Ad-Cre-GFP- or Ad-GFP-infected cells 4 days post-infection. (G) Representative western blots of Atg3, LC3I, LC3II, p62, aconitase, HSP60, and tubulin in Ad-Cre-GFP- or Ad-GFP-infected cells 6 days post-infection. Cells were treated with saline or bafilomycin A1 (Baf-A1) for the last 24 hr. (H–M) Densitometry of (G) normalized by tubulin and expressed as fold change from Ad-GFP control cells. (N) Oxygen consumption rate (OCR) in BAT tissue sections. (O and P) OCR in eWAT and iWAT tissue sections, respectively. (Q and R) Extracellular acidification rate (ECAR) in eWAT and iWAT tissue sections, respectively. (S) Relative mRNA levels of genes involved in mitochondrial clearance in eWAT. Values are mean ± SEM (*p < 0.05, **p < 0.005 versus WT or Ad-GFP, #p < 0.05, ##p < 0.005 versus basal in (N) and versus without Baf-A1 in (I)–(K). n = 4–5/group for (A)–(D) and (N)–(S) and n = 3/condition for (E)–(M).

Figure 4.. Autophagy Deletion in Adipocytes Enhances Nrf2 and Keap1 Signaling in Adipose Tissue and Causes Adipose and Systemic Elevation of Lipid Peroxides

(A–O) WT and AdiAtg3 KO mice were fed an NCD for 12 weeks. (A) Heatmap showing hierarchical clustering of differentially expressed genes in AdiAtg3 KO mice. The FPKM (fragments per kilobase of exon per million reads mapped) values of individual samples were normalized to the average of WT controls. (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showing the most upregulated pathways. (C and D) Relative mRNA levels of the Nrf2 targets Nqo1 and Hmox1 in eWAT and BAT, respectively.