Atg7 Knockdown Reduces Chemerin Secretion in Murine Adipocytes

Abstract

Context: In individuals with obesity, adipocyte endocrine function is affected by altered autophagy. Genetic variants in autophagy-related gene 7 (ATG7) correlated with serum chemerin (RARRES2) concentrations.

Objectives: To investigate a functional interplay between chemerin and ATG7, how it may relate to autophagy-mediated adipocyte dysfunction in obesity, and the relevance of genetic ATG7 variants in chemerin physiology.

Design: Adipose ATG7 mRNA expression and adiposity measures were available in two human study cohorts. The effect of a high-calorie diet on adipose Rarres2 and Atg7 expression was investigated in mice. In 3T3-L1 adipocytes, the effect of Atg7 knockdown on chemerin expression and secretion was studied. The influence of single nucleotide polymorphisms on ATG7 transcription and chemerin physiology was investigated using a luciferase assay.

Setting: Mouse model, clinical trials, in vitro studies.

Participants: Native American (n = 83) and white (n = 100) cohorts.

Main outcome measure: Adipocyte chemerin expression and secretion.

Results: In mice fed a high-calorie diet, adipose Atg7 mRNA expression did not parallel an increase in Rarres2 mRNA expression. ATG7 mRNA expression in human subcutaneous adipose tissue correlated with body mass index, fat mass (r > 0.27; P < 0.01), and adipocyte cell size (r > 0.24; P < 0.02). Atg7 knockdown in 3T3-L1 adipocytes decreased chemerin secretion by 22% (P < 0.04). Rs2606729 in ATG7 was predicted to alter ATG7 transcription and induced higher luciferase activity in vitro (P < 0.0001).

Conclusions: Human adipose ATG7 mRNA expression relates to measures of adiposity. Atg7 knockdown reduces chemerin secretion from adipocytes in vitro, supportive of a functional interplay between ATG7 and chemerin in autophagy-mediated adipocyte dysfunction.

Trial registration: ClinicalTrials.gov NCT00340132.

Figure 1.

Murine Rarres2 and Atg7 mRNA expression in response to dietary intervention. (A‒D) Response of Rarres2 and Atg7 mRNA expression in (A and B) VAT and (C and D) scAT to an HFD and an HSD. Data represented as means ± SEM. *P < 0.05; ****P < 0.001. ns, not significant; Ref, low-glycemic reference diet.

Figure 2.

Association analyses of Rarres2 and Atg7 mRNA expression in adipose tissue of mice receiving an HFD. In VAT (left panels) and scAT (right panels), Rarres2 mRNA expression correlated with (A and B) body weight as well as (C and D) fat mass. (E and F) Rarres2 mRNA expression related to Atg7 mRNA expression in these tissues as well. Pearson correlation is reported.

Figure 3.

Correlation of scAT ATG7 mRNA expression with measures of adiposity and expression of RARRES2 and CMKLR1 in humans. Correlation of scAT ATG7 mRNA expression (adjusted for age and sex) with (A) body weight, (B) BMI, (C) FM, (D) fat-free mass (FFM), (E) scAT RARRES2 mRNA expression, and (F) scAT CMKLR1 mRNA expression in 83 Native Americans. For mRNA expression, SD units are displayed, which account for sex- and batch-dependent differences in measurements (29).

Figure 4.

Correlation of ATG7 mRNA expression with scAT cell size in humans. (A and B) Partial correlation of scAT cell size (A, mean; B, maximal) and ATG7 mRNA expression in whites. Adjustments were made for age and sex. Variables were log₁₀-transformed to approach normal distribution and to reduce the influence of leveraged values on the model. For (A), sensitivity analyses showed an influence of highly leveraged values (n = 5) on the model (data not shown; r = 0.36; P = 0.0003 upon inclusion). Thus, these values were excluded from analyses, resulting in a sample size of n = 95. When correlation assessment was restricted to individuals with fasting glucose values below the diabetic range (≤7 mmol/L), mean scAT cell size tended to correlate with scAT ATG7 mRNA expression (n = 70; r = 0.21; P = 0.07). In this case, however, the association between maximal scAT cell size and scAT ATG7 mRNA expression remained significant (n = 75; r = 0.45; P < 0.0001).

Figure 5.

Functional assessment of alleles at rs2606729 and rs2606752 in ATG7 using a luciferase reporter enzyme assay. DNA fragments corresponding to each allele at rs2606729 and rs2606752 (±30 bases) were cloned into the firefly luciferase vector pGL4.23 in the 5′-3′- (forward) and 3′-5′- (reverse) orientation. Plasmids were transfected into HeLa-cells. Vector pGL4.23 was used as an internal control. Data represent mean ± SEM. For rs2606729 and rs2606752, n = 3 and n = 4 replicates, respectively. For pGL4.23, n = 5 replicates. Statistical analysis was done using one-way ANOVA followed by the Holm Sidak test as a post hoc test for multiple comparison of groups. Dotted line: Reference luciferase activity according to pGL4.23 normalization. ****P < 0.0001.

Figure 6.

Effect of siRNA-mediated knockdown of Atg7 on chemerin expression and secretion in 3T3-L1 adipocytes. In a separate experimental setup, the effect of siRNA-mediated knockdown on chemerin expression and secretion in 3T3-L1 adipocytes were assessed. (A‒C) In vitro effect of siRNA-mediated knockdown of Atg7 on (A) mRNA expression (n = 5 replicates) and (B and C) protein levels (n = 7 replicates) of Atg7 and autophagy markers. (C) Inhibition of autophagic activity was measured via assessment of reduction in relative LC3II protein levels upon siRNA-mediated knockdown of Atg7, comparing cells with and without bafilomycin A₁-treatment. (D) In this same experimental setup (i.e., in the setting of decreased autophagic activity), siRNA-mediated knockdown of Atg7 did not affect intracellular chemerin expression (n = 7 replicates). (E) However, compared with scrambled control (scr), lower levels of chemerin were measured in media of those cells transfected with Atg7 siRNA, indicating a decrease of chemerin secretion into media upon Atg7-mediated decrease of autophagic activity (n = 7 replicates). Statistical analysis was performed by the Student t test with a Holm-Sidak correction for multiple comparisons. Data are represented as mean ± SEM. Dashed line in (A) indicates relative mRNA expression of scrambled control. + or − indicates application of respective siRNA or Bafilomycin to in vitro setup. *P < 0.05; ***P < 0.001. ns, not significant.