Elevated autophagy gene expression in adipose tissue of obese humans: A potential non-cell-cycle-dependent function of E2F1

Abstract

Autophagy genes' expression is upregulated in visceral fat in human obesity, associating with obesity-related cardio-metabolic risk. E2F1 (E2F transcription factor 1) was shown in cancer cells to transcriptionally regulate autophagy. We hypothesize that E2F1 regulates adipocyte autophagy in obesity, associating with endocrine/metabolic dysfunction, thereby, representing non-cell-cycle function of this transcription factor. E2F1 protein (N=69) and mRNA (N=437) were elevated in visceral fat of obese humans, correlating with increased expression of ATG5 (autophagy-related 5), MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 β), but not with proliferation/cell-cycle markers. Elevated E2F1 mainly characterized the adipocyte fraction, whereas MKI67 (marker of proliferation Ki-67) was elevated in the stromal-vascular fraction of adipose tissue. In human visceral fat explants, chromatin-immunoprecipitation revealed body mass index (BMI)-correlated increase in E2F1 binding to the promoter of MAP1LC3B, but not to the classical cell cycle E2F1 target, CCND1 (cyclin D1). Clinically, omental fat E2F1 expression correlated with insulin resistance, circulating free-fatty-acids (FFA), and with decreased circulating ADIPOQ/adiponectin, associations attenuated by adjustment for autophagy genes. Overexpression of E2F1 in HEK293 cells enhanced promoter activity of several autophagy genes and autophagic flux, and sensitized to further activation of autophagy by TNF. Conversely, mouse embryonic fibroblast (MEF)-derived adipocytes from e2f1 knockout mice (e2f1−/−) exhibited lower autophagy gene expression and flux, were more insulin sensitive, and secreted more ADIPOQ. Furthermore, e2f1−/− MEF-derived adipocytes, and autophagy-deficient (by Atg7 siRNA) adipocytes were resistant to cytokines-induced decrease in ADIPOQ secretion. Jointly, upregulated E2F1 sensitizes adipose tissue autophagy to inflammatory stimuli, linking visceral obesity to adipose and systemic metabolic-endocrine dysfunction.

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

“…602,608 In the clinical setting, tissue expression of ATG5, LC3A and LC3B and their respective proteins accompanies elevated autophagy flux in human adipose tissue in obesity. 217,609 Thus, assessing the mRNA levels of LC3 and other autophagy-related genes by northern blot or qRT-PCR may provide correlative data relating to the induction of autophagy. Downregulation of autophagy-related mRNAs has been observed in human islets under conditions of lipotoxicity 409 that impair autophagic flux.…”

Adipose tissue inflammation: a cause or consequence of obesity-related insulin resistance?

“…Regulation of autophagy has been studied in the context of aging, cellular development and cellular defense against pathogens, and is implicated in the pathogenesis of a growing number of human diseases [15]. In independent study settings and cohorts, we found generally higher markers of autophagy in visceral compared with SC adipose tissue and significant correlations with obesity, insulin resistance and adipose tissue inflammation [49,50,81]. However, since these associations have not been consistently found across different cohorts the dynamic changes due to autophagic flux need to be considered in the discussion of whether autophagy protects against obesity associated adipocyte dysfunction or could contribute to adipose tissue inflammation [82].…”

“…Individuals with hypertrophic obesity displayed lower improvement of insulin resistance after bariatric surgery [48]. Larger adipocytes are also more prone to store lipophilic toxins, which may increase intracellular stress, autophagy and apoptosis [49,50]. Adipocyte hypertrophy most likely results from a low capacity of de novo adipogenesis and an inability to respond to the energy surplus by adequately increasing adipocyte number (hyperplasia) [51].…”

ASK1 (MAP3K5) is transcriptionally upregulated by E2F1 in adipose tissue in obesity, molecularly defining a human dys-metabolic obese phenotype

“…Under these conditions, TNFα-induced increase in Ask1 mRNA was significantly inhibited by ∼50% (Figure 3D). We previously demonstrated that despite the role of E2F1 in adipogenesis [35], under in-vitro conditions, MEFs from E2F1 −/− mice can differentiate into adipocyte-like cells comparably to MEFs from wild-type (WT) mice, as assessed morphologically and by the similar induction of adipocyte-specific genes (Ppar γ , Cebp4α , Fabp4 , and Glut4 ) [30]. Utilizing these cells, we observed that in the absence of E2F1, “basal” ASK1 expression was markedly attenuated (Figure 3E–G).…”