Lipolytic Effects of 3-Iodothyronamine (T1AM) and a Novel Thyronamine-Like Analog SG-2 through the AMPK Pathway

Abstract

3-Iodothyronamine (T1AM) and its synthetic analog SG-2 are rapidly emerging as promising drivers of cellular metabolic reprogramming. Our recent research indicates that in obese mice a sub-chronic low dose T1AM treatment increased lipolysis, associated with significant weight loss independent of food consumption. The specific cellular mechanism of T1AM's lipolytic effect and its site of action remains unknown. First, to study the mechanism used by T1AM to gain entry into cells, we synthesized a fluoro-labeled version of T1AM (FL-T1AM) by conjugating it to rhodamine (TRITC) and analyzed its cellular uptake and localization in 3T3-L1 mouse adipocytes. Cell imaging using confocal microscopy revealed a rapid intercellular uptake of FL-T1AM into mitochondria without localization to the lipid droplet or nucleus of mature adipocytes. Treatment of 3T3-L1 adipocytes with T1AM and SG-2 resulted in decreased lipid accumulation, the latter showing a significantly higher potency than T1AM (10 µM vs. 20 µM, respectively). We further examined the effects of T1AM and SG-2 on liver HepG2 cells. A significant decrease in lipid accumulation was observed in HepG2 cells treated with T1AM or SG-2, due to increased lipolytic activity. This was confirmed by accumulation of glycerol in the culture media and through activation of the AMPK/ACC signaling pathways.

Keywords: 3-iodothyronamine (T1AM), thyroid hormone analogs; AMPK pathway; lipid metabolism; metabolic reprogramming; mitochondria; rhodamine (TRITC), cell imaging.

Figure 1

Synthesis of fluorescently labeled T1AM (FL-T1AM). One-step conjugation of Tetramethylrhodamine-6-isothiocyanate (6-TRITC) with T1AM efficiently produces fluorescently-labeled T1AM (FL-T1AM).

Figure 2

Fluorescent labeling of internalized T1AM in differentiated 3T3-L1 cells. (A) Internalization of T1AM is extremely rapid, with FL-T1AM being detected inside cells within seconds of addition to media. (B) Snapshots of a zoomed cell to show each individual staining (i.e., Green: Cell membrane stain, Blue: DAPI nuclear stain, Red: FL-T1AM), and composite picture of stains for ease of visualization. (C) FL-T1AM does not co-localize into the lipid droplet of mature adipocytes (i.e., lack of yellow signal resulting from red and green overlap). Blue: DAPI nuclear stain, Green: Lipid stain, Red: FL-T1AM. (D) FL-T1AM remains perinuclear, but not within the nucleus when internalized into adipocytes. Blue: DAPI nuclear stain, Green: Cell membrane stain, Red: FL-T1AM. (E) FL-T1AM co-localizes to mitochondria in 3T3-L1 adipocytes. Blue: DAPI nuclear stain, Green: Mitochondria membrane stain, Red: FL-T1AM.

Figure 3

Uptake rate of fluorescent labeled T1AM (FL-T1AM). 3T3-L1 cells were given FL-T1AM and their mean shift in fluorescent intensity was measured with 10,000 cells per time point. T1AM uptake appears to involve a three-phase pattern, consisting of a rapid exponential uptake, followed by a linear steady increase, and ending with a plateau. (A) Mean fluorescent shift of FL-T1AM signal at 100 nM ± SEM. (B) Fluorescent signal intensity over time at 10, 50, and 100 nM. (C) Fluorescent signal intensity over time at 200 nM and 500 nM.

Figure 4

T1AM and SG-2 treatments are associated with decreased lipid accumulation. 3T3-L1 adipocytes were treated with T1AM or SG-2 (1, 2, 5, 10 µM) at the beginning of differentiation (day 0) (A) Visible decreases in lipid accumulation were readily observable at day 5 with SG-2 treatment. Treatment with T1AM did not result in visibly detectible decreases in lipid accumulation (pictures not shown). (B) Fluorescent intensity of Nile Red lipid stain in 3T3-L1 differentiated adipocytes treated with 20 µM T1AM and 10 µM SG-2 and measured by flow cytometry. Nile red is a fluorescent stain that integrates into and fluoresces when in contact with lipid, allowing for measured fluorescent intensity to correspond to the degree of lipid present in cells. Flow cytometry data are presented as mean channel fluorescence, with +/− SEM. * denotes a significant difference from control at p < 0.05 (ANOVA Dunnett’s test).

Figure 5

Effects of T1AM and SG-2 on lipid accumulation in fully differentiated 3T3-L1 cells. Mature adipocytes were treated for 24 h with T1AM or SG-2 (1, 10, 25, 50 µM). 10 µM isoproterenol (ISO) was used as positive control. Oil red O stained intercellular oil droplets were eluted with isopropanol and quantified by spectrophotometry analysis at 510 nm. Values represent the mean ± SEM of 4–8 experiments. The groups were compared using the One-Way ANOVA followed by Tukey’s range test. * p < 0.05; ** p < 0.01; *** p < 0.005.

Figure 6

Effects of T1AM and SG-2 on lipids droplets in HepG2 cells. Cells were treated for 24 h with T1AM or SG-2 at two doses (10–25 µM). Cells without treatments labeled as CTRL. A total of 10 µM isoproterenol (ISO) and 25 µM cloroquine (CLO) were used as positive controls. (A) Lipid accumulation in HepG2 Cells. Oil Red O stained intercellular oil droplets were eluted with isopropanol and quantified by spectrophotometry analysis at OD = 510 nm. Values represent the mean ± SEM of 4–8 experiments. (B) Lipolysis in HepG2 cells. Glycerol released in the culture medium (0.5 mL) of HepG2 cells after 24 h treatment with T1AM or SG-2 at two doses (10 and 25 µM). Values represent the mean ± SEM of 3 to 6 experiments. (C) Lack of toxicity in HepG2 cells. Cell viability after 24 h after treatment with T1AM or SG-2. Cell viability was assessed by MTT assay and reported values shown on Y-axis at OD = 570nm. Values represent the mean ± SEM of three independent experiments (D) Regulation of metabolism in HepG2 via AMPK and ACC pathways, respectively. Representative immunoblotting images and the quantitative analysis of phosphorylation of AMPK and ACC are shown. The groups were compared using the one-way ANOVA followed by Tukey’s range test. * p < 0.05; ** p < 0.01.