Mitochondrial Activity in Human White Adipocytes Is Regulated by the Ubiquitin Carrier Protein 9/microRNA-30a Axis

Abstract

The acquisition of beige adipocyte features by white fat cells corresponds to protection against obesity-induced metabolic diseases in humans and animal models of type 2 diabetes. In adipose tissue, expression of the E2 small ubiquitin-like modifier ligase ubiquitin carrier protein 9 (Ubc9) is positively correlated with markers of insulin resistance and corresponds with impaired browning of human white adipocytes. However, the molecular regulation of Ubc9 expression in adipocytes and other cells remains unclear. In this study, we demonstrate that the mRNA and protein expression of Ubc9 are regulated by the microRNA miRNA-30a (miR-30a) in human subcutaneous adipocytes. Ubc9 and miR-30a exhibit inverse expression in adipose tissue, with miR-30a robustly elevated in brown fat. Depletion of Ubc9 by siRNA or enforced expression of a miR-30a mimic augments mitochondrial volume and respiration in human white adipocytes, reflecting features of brown fat cells. Furthermore, Ubc9 depletion induces a brown fat gene program in human subcutaneous adipocytes. Induction of the beige-selective gene program corresponds to stabilization of the PR domain-containing 16 (PRDM16) protein, an obligate transcriptional regulator of the brown/beige fat metabolic program in white adipocytes that interacts with Ubc9. Taken together, our data demonstrate a previously unappreciated molecular axis that controls browning of human white adipocytes.

Keywords: adipose tissue metabolism; bioenergetics; gene transcription; microRNA (miRNA); mitochondria; posttranscriptional regulation; respiration; tissue-specific transcription factor; transcription coregulator; transcription target gene.

FIGURE 1.

Ubc9 is a direct target of miR-30a. A, TargetScan and starBase predict that miR-30a binds the 3′ UTR of UBE2I (Ubc9). Plasmids with negative control (R01), PPIA, and Ubc9 3′ UTR luciferase fusions were co-transfected with a miR-30a mimic or control mimic in human adipocytes cells (n = 3; *, p < 0.05). RLU, relative light units; nt, non-targeting. B, the miR-30a putative binding site in the 3′ UTR of Ubc9 is conserved among species commonly queried in TargetScan, as shown by the sequence homology (box). con, control. C, human adipocytes were transfected with scrambled (scRNA) siRNA or Ubc9 siRNA and compared with expression of a control mimic or miR-30a mimic. qPCR was used to measure miR-30a-5p and Ubc9 relative (rel) gene expression normalized to TATA binding protein or sno412 RNA (n = 2; mean ± S.E.; *, p < 0.05). D, immunoblot (IB) analysis was used to measure the effects of siRNA and miRNA transient transfection on the protein expression of Ubc9.

FIGURE 2.

miR-30a inhibits Ubc9 expression in human white adipocytes. A–E, the gene expression levels of published miR-30a targets were analyzed in human adipocytes transfected with control or miR-30a mimics for 48 h. mRNA levels of Ubc9 (A), ATG5 (B), RIP140 (C), p53 (D), and BECN1 (E) were analyzed by qPCR. rel, relative. F, immunoblotting verified exclusive suppression of Ubc9 expression using samples analyzed by qPCR. *, p < 0.05 relative to cells transfected with control mimic (n ≥ 3 independent experiments). G, TargetScan cumulative weighted context++ scores of published miR-30a targets suggest that the miR-30/Ubc9 interaction is favorable relative to other targets. More negative values indicate a stronger interaction potential. nc, not-called.

FIGURE 3.

Reciprocal expression of Ubc9 and miR-30a in adipose tissues. A and B, qPCR was used to determine the expression of Ubc9, miR-30a, and marker genes from WAT and BAT (A) and skeletal muscle (SkM, B). Expression levels were normalized to TATA binding protein or U6 sno RNA (n ≥ 4 mice/group).

FIGURE 4.

The Ubc9/miR-30a axis remodels mitochondrial respiration in human white adipocytes. A, mitochondria (MitoTracker, red) and nuclei (DAPI, blue) were labeled in mature human adipocytes transfected with Ubc9 siRNA or a miR-30a mimic. Arrowheads indicate alterations in mitochondrial morphology between controls, Ubc9 siRNA, and miR-30a mimic transfections. B, mitochondrial DNA (ND6) was analyzed by qPCR from samples in A. *, p < 0.05 relative to control-transfected cells. C, basal respiration (as OCR) was measured in mature human adipocytes transfected with Ubc9 siRNA or a miR-30a mimic. D, respiration rate was measured after forskolin treatment to induce uncoupling. The percent change in OCR was normalized to baseline rates. E, percent uncoupling was calculated by subtracting the difference between oligomycin and rotenone. In this case, the OCR before oligomycin injection was set as 100%. Respiration data are presented as mean ± S.E.; n = 4 independent experiments; *, p < 0.05 relative to control transfections.

FIGURE 5.

PRDM16 stabilization and browning of human white adipocytes are regulated by the Ubc9/miR-30a axis. Ubc9 siRNA, miR-30a, or transfection controls were introduced into mature human adipocytes. A, the effects of Ubc9 siRNA and miR-30a overexpression in human adipocytes were characterized by qPCR analysis of PPARγ2, FABP4, ADIPOQ, PGC1α, UCP1, CIDEA, PRDM16, and Ubc9 mRNA levels (n ≥ 3 independent experiments; *, p < 0.05 relative to control transfections). B, expression levels of Ubc9, PRDM16, and UCP1 were analyzed by immunoblotting for human adipocytes transfected with Ubc9 siRNA or a miR-30a mimic. C, HEK293T cells were transfected with HA-Ubc9 and FLAG-PRDM16. Lysates were co-immunoprecipitated (IP) with anti-FLAG antibodies, and the precipitates were analyzed by immunoblotting (IB). The interaction between EHMT1 and PRDM16 was a positive control for PRDM16 binding partners. D, PRDM16 protein levels in a cycloheximide (CHX) chase experiment were analyzed by immunoblotting (top panel) and quantified by densitometric analysis of PRDM16 protein degradation rates normalized to HSP90 expression (bottom panel, n = 2 independent experiments). E, PPARγ ChIP was performed in human adipocytes transfected with Ubc9 siRNA, miR-30a, or transfection controls. qPCR was used to analyze genomic occupancy using primers flanking PPARγ binding sites in the UCP1 enhancer (enh), and CIDEA intron (intr) 1 regions. An intronic region of Cyclin D1 served as a negative control (n = 2 independent experiments; *, p < 0.05 relative to control transfections). All data are expressed as mean ± S.E.