Naringenin and β-carotene convert human white adipocytes to a beige phenotype and elevate hormone- stimulated lipolysis

Abstract

Introduction: Naringenin, a peroxisome proliferator-activated receptor (PPAR) activator found in citrus fruits, upregulates markers of thermogenesis and insulin sensitivity in human adipose tissue. Our pharmacokinetics clinical trial demonstrated that naringenin is safe and bioavailable, and our case report showed that naringenin causes weight loss and improves insulin sensitivity. PPARs form heterodimers with retinoic-X-receptors (RXRs) at promoter elements of target genes. Retinoic acid is an RXR ligand metabolized from dietary carotenoids. The carotenoid β-carotene reduces adiposity and insulin resistance in clinical trials. Our goal was to examine if carotenoids strengthen the beneficial effects of naringenin on human adipocyte metabolism.

Methods: Human preadipocytes from donors with obesity were differentiated in culture and treated with 8µM naringenin + 2µM β-carotene (NRBC) for seven days. Candidate genes involved in thermogenesis and glucose metabolism were measured as well as hormone-stimulated lipolysis.

Results: We found that β-carotene acts synergistically with naringenin to boost UCP1 and glucose metabolism genes including GLUT4 and adiponectin, compared to naringenin alone. Protein levels of PPARα, PPARγ and PPARγ-coactivator-1α, key modulators of thermogenesis and insulin sensitivity, were also upregulated after treatment with NRBC. Transcriptome sequencing was conducted and the bioinformatics analyses of the data revealed that NRBC induced enzymes for several non-UCP1 pathways for energy expenditure including triglyceride cycling, creatine kinases, and Peptidase M20 Domain Containing 1 (PM20D1). A comprehensive analysis of changes in receptor expression showed that NRBC upregulated eight receptors that have been linked to lipolysis or thermogenesis including the β1-adrenergic receptor and the parathyroid hormone receptor. NRBC increased levels of triglyceride lipases and agonist-stimulated lipolysis in adipocytes. We observed that expression of RXRγ, an isoform of unknown function, was induced ten-fold after treatment with NRBC. We show that RXRγ is a coactivator bound to the immunoprecipitated PPARγ protein complex from white and beige human adipocytes.

Discussion: There is a need for obesity treatments that can be administered long-term without side effects. NRBC increases the abundance and lipolytic response of multiple receptors for hormones released after exercise and cold exposure. Lipolysis provides the fuel for thermogenesis, and these observations suggest that NRBC has therapeutic potential.

Keywords: PPARα; PPARγ; RXRγ; UCP1; adiponectin; carotenoid; lipolysis; naringenin.

Figure 1

Pro-vitamin A carotenoids and NR act synergistically to elevate levels of UCP1 mRNAs Adipocytes from 4 donors with obesity were treated for seven days with vehicle (control) or 8µM NR and 2µM carotenoids. (A) NR+BC (B) NR+Lutein (C) NR+Lycopene mRNA levels were measured using quantitative RTPCR. Data is expressed in least squares means ± standard error. Synergy for mRNA was calculated as: sum of differences ((NR- Control) + (BC - Control)) vs (NRBC - Control). *p < 0.001 for synergy, sum of differences versus NRBC. NR, naringenin; BC, beta carotene.

Figure 2

NR and BC synergistically induce metabolism genes (A) mRNA levels (B) Western Blots of Protein levels (C) Protein levels were measured with β-actin as loading control. Adipocytes from three or more donors with obesity were treated for seven days. mRNA data are expressed as least squares means ± standard error. Synergy for mRNA was calculated as: sum of differences ((NR- Control) + (BC - Control)) vs (NRBC - Control). *p < 0.05 for synergy. NR, Naringenin; BC, beta carotene; NRBC, naringenin and beta carotene.

Figure 3

NRBC upregulates a subset of key regulatory proteins without mRNA increases (A) PPARα; (B) PPARγ; (C) PGC-1α; and (D) NAMPT. Adipocytes from three to five donors with obesity were treated for seven days. Protein was measured by Western Blotting with β-actin used to adjust for loading. mRNA was quantified by real-time PCR. Data are expressed as least squares means ± standard error, *p < 0.001 NR, Naringenin; BC, beta carotene; NRBC, naringenin and beta carotene.

Figure 4

Whole transcriptome sequencing analysis of pathways stimulated by naringenin and β-carotene (NRBC) Adipocytes from two donors with BMI of 27 and 36 kg/m² were treated with cell medium (vehicle control) or NRBC for seven days. cDNA libraries from expressed transcripts were constructed, sequenced and differential gene expression was analyzed. Gene-sets with false discovery rate (FDR) ≤ 5% were considered as significantly enriched, and the top pathways are shown.

Figure 5

RT-PCR validation of genes upregulated by naringenin and beta carotene (NRBC) in RNA sequencing analysis. Adipocytes from three donors with obesity were treated for seven days. mRNA levels were measured using quantitative RT-PCR. Data are expressed as mean ± standard error, *p < 0.001.

Figure 6

Relative receptor levels in white adipocytes (untreated) and NRBC-treated cells. (A) Low abundance (B) High abundance. Adipocytes from two donors with obesity were treated with cell medium (Control) or NRBC for seven days and transcript sequencing analysis was conducted. Data are expressed as mean normalized transcript counts (DEseq2). * indicates Padj< 0.002 for Control vs NRBC. β-adrenergic receptors (β1AR, β2AR, β3AR), G-protein coupled bile acid receptor (TGR5), Transient receptor potential cation channel subfamily M member 8 (TRPM8), Melanocortin-1 receptor (MC1R), Adenosine receptors A1 and A2B (ADORA1, ADORA2B), Natriuretic peptide receptors (NPR1, NPR3), G-protein coupled estrogen receptor 1 (GPER1), Parathyroid hormone receptor 1 (PTHR1), Growth hormone receptor (GHR).

Figure 7

Hormone-stimulated lipolysis in white adipocytes (untreated) and NRBC-pretreated cells. After 7d pretreatment with vehicle (untreated) or NRBC, adipocytes were exposed for 4 hours to receptor agonists in KRB buffer. Supernatants were removed for measurement of glycerol. Data are presented as least squares mean ± standard error from experiments using cells from four different donors with BMIs ranging from 27 to 36, each with at least 6 replicates. (*indicates a difference between untreated white adipocytes and NRBC-treated adipocytes p≤ 0.02) cAMP 8-Cpt-cAMP 200µM, ANP atrial natriuretic peptide 0.1 µM, PTH parathyroid hormone (1-34) 1 µM, isoprot isoproterenol 1µM, dobutam dobutamine 1µM, estradiol 1µM, GH growth hormone 250ng/ml, ACTH adrenocorticotropin hormone 1µM, CDCA chenodeoxycholic acid 30µM, adenosine 1µM, menthol 100µM.

Figure 8

Analysis of RXR isoforms bound to immunoprecipitated PPARγ complexes. Adipocytes were treated with vehicle or NRBC for seven days. RXRγ, RXRα and PPARγ protein levels were analyzed by Western blotting. For analysis of whole cell protein levels (input), 50 μg of protein lysate was loaded in each lane. PPARγ was immunoprecipitated from 600 μg of the same whole cell lysate for analysis of PPARγ cofactor binding. An anti-IgGκ light chain secondary antibody was used for detection. This experiment was repeated three times with adipocytes from obese donors.

Figure 9

Proposed paradigm for remodeling of white adipocytes by NRBC. NR and BC bind nuclear receptors and activate gene expression at PPRE motifs. UCP1 and other uncoupling compounds and mitochondrial proteins mediate multiple energy-wasting enzymatic cycles that generate heat. PM20D1 regulates synthesis and degradation of N-acyl amino acids (NAA), molecules that directly uncouple mitochondria and increase energy expenditure. The synthesis and breakdown of creatine phosphate by the mitochondrial creatine kinases CKMT1A, CKMT1B and CKMT2 facilitates ATP-coupled respiration and enhances oxygen consumption. PDK4 directs pyruvate into synthesis of glycerol, fatty acids, and TGs to promote futile TG recycling. Lipolytic receptors and PKA are upregulated, increasing responsiveness to hormones and lipolysis. Fatty acids are transferred into mitochondria to fuel thermogenesis. Genes are turned on for production of bioactive peptides and lipokines which have autocrine insulin sensitizing effects and are secreted into circulation. NR (Naringenin), BC (β-carotene) Created with Biorender.com.