Effects of Isorhamnetin on Adipocyte Mitochondrial Biogenesis and AMPK Activation

Abstract

Isorhamnetin (ISOR), 3-O-methylquercetin, is a naturally occurring flavonoid in many plants. It is a metabolite derived from quercetin and is known to exert beneficial effects on the prevention of obesity. However, the molecular mechanism of action involved in ISOR-mediated mitochondrial biogenesis, and AMP-activated protein kinase (AMPK) activation in 3T3-L1 cells remains unclear. The aim of this study was to determine whether ISOR affected mitochondrial biogenesis and AMPK activation, during 3T3-L1 adipocyte differentiation. Intracellular lipid and triglyceride accumulation, and glycerol-3-phosphate dehydrogenase (GPDH) activity decreased in ISOR-treated cells. The mRNA levels of adipogenic genes, such as the proliferator-activated receptor-γ (PPAR-γ), and adipocyte protein 2 (aP2), were inhibited by ISOR. In contrast, mRNA levels of mitochondrial genes, such as peroxisome proliferator-activated reporter gamma coactivator-1α (PGC-1α), nuclear respiratory factor (NRF)-1, transcription factor A (Tfam), and carnitine palmitoyl transferase-1α (CPT-1α), were all stimulated by ISOR treatment. Mitochondria DNA (mtDNA) copy number and AMPK activity were also stimulated by ISOR. The results suggested that the mitochondrial biogenic effect of ISOR in adipocytes might have been associated with stimulation of mitochondrial gene expression, mtDNA replication, and AMPK activation.

Keywords: 3T3-L1 adipocytes; AMPK; adipogenesis; isorhamnetin; mitochondrial biogenesis.

Figure 1

Chemical structures (a) and cell viability (b) of ISOR-treated 3T3-L1 cells. Cells were treated with 0 (control), 0.1, 0.5, 1, 10, 20, or 50 µM ISOR, and incubated for 1, 2, 5, or 7 days. Cell viability was determined using the water-soluble tetrazolium salt (WST)-8 assay. Values are expressed as means ± SE (n = 3) of three independent experiments. ISOR, isorhamnetin. * p < 0.05 vs. control.

Figure 2

Effects of ISOR on intracellular lipid and TG accumulation during adipocyte differentiation. 3T3-L1 cells were treated with 0 (control) or 20 µM ISOR, and incubated for 2, 5, or 7 days (d2 to d9). On day 7 (d9), change of adipocyte differentiation was presented with Oil Red O-staining. Intracellular lipid content (a) was stained with oil-red O dye, the stained oil droplets were dissolved with isopropanol and quantified by spectrophotometry. Representative cell images were captured at 200× magnification. Intracellular TG content (b) was treated with 0 (DM-treated control), 1, 10, and 20 µM ISOR, and incubated for 7 days, and determined using enzymatic colorimetric methods. Values are expressed as means ± SE (n = 3) of three independent experiments. TG, triglyceride; ISOR, isorhamnetin; DM, differentiation medium containing 3-isobutyl-1-methylxanthine, dexamethasone and insulin. * p < 0.05 and ** p < 0.01 vs. untreated control.

Figure 3

Effects of ISOR on GPDH activity in adipocytes. GPDH activity was determined using a GPDH assay kit. 3T3-L1 adipocytes were treated with 0 (DM-treated control), 1, 10, or 20 µM ISOR, and incubated for 7 days. Values are expressed as means ± SE (n = 3) of three independent experiments. GPDH, glycerol-3-phosphate dehydrogenase; ISOR, isorhamnetin; DM, differentiation medium containing 3-isobutyl-1-methylxanthine, dexamethasone, and insulin. * p < 0.05 and ** p < 0.01 vs. DM-treated control.

Figure 4

Effects of ISOR on the expression of genes involved in adipogenesis, and mitochondrial function in adipocytes. 3T3-L1 adipocytes were treated with 0 (control) or 20 μM ISOR, for 7 days. The mRNA levels were measured by qRT-PCR. Values are expressed as means ± SE (n = 3) of three independent experiments. ISOR, isorhamnetin. * p < 0.05, ** p < 0.01 vs. control.

Figure 5

Effects of ISOR on mtDNA content in in adipocytes. 3T3-L1 adipocytes were treated with 0 (control), 1, 10, and 20 μM ISOR, for 7 days. mtDNA copy number was measured by qRT-PCR. Values are expressed as means ± SE (n = 3) of three independent experiments. mtDNA, mitochondrial DNA; nDNA, nuclear DNA; ISOR, isorhamnetin. * p < 0.05 vs. control.

Figure 6

Effect of ISOR on AMPK activity in adipocytes. 3T3-L1 adipocytes were treated with 0 (control) or 20 μM ISOR, for 7 days and incubated with 1 μM of Comp C as an AMPK inhibitor for 16 h. AMPK activity was determined using an AMPK Kinase Assay Kit. Values are expressed as means ± SE (n = 3) of three independent experiments. AMPK, AMP-activated protein kinase; ISOR, isorhamnetin; Comp C, compound c. ** p < 0.01 vs. control (without or with Comp C.)

Figure 7

Schematic diagram showing the possible mechanisms of ISOR on adipogenesis, and mitochondrial biogenesis during 3T3-L1 adipocyte differentiation.

Figure 8

Experiment scheme and pattern of 3T3-L1 preadipocyte differentiation.