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.
Highlights
3-Iodothyronamine (T1AM) is a recently discovered endogenous hormone like molecule [1,2] structurally and metabolically related to thyroid hormone
Since our recent studies have shown that the thyronamine-like analog SG-2 represents a potent mimic of T1AM, with respect to TAAR1 agonist activity as well as metabolic and neurological effects [11,12], we explored the ability of this compound to modulate lipid metabolism in adipocytes (3T3-L1 cell) at concentrations comparable to our previous in vitro and in vivo studies [3,6,11,13]
We investigated the effects of SG-2 and T1AM on total lipid accumulation by using the lipid Oil Red O (ORO) staining kit, which is suitable for selective staining and detection of neutral lipids, including triglycerides and cholesterol esters, in cultured 3T3-L1 cells [20]
Summary
3-Iodothyronamine (T1AM) is a recently discovered endogenous hormone like molecule [1,2] structurally and metabolically related to thyroid hormone. Haviland et al demonstrated that chronic low dose administration of T1AM was able to induce significant weight loss in obese mice, and the effect was still maintained three weeks following cessation of the treatment [3] This weight loss was found to be due to a combination of increased lipolysis, and decreased lipid synthesis while maintaining glucose regulation. Our previous multidisciplinary in vivo studies provided compelling evidence that the weight loss incurred from T1AM treatment was due to both T1AM’s upregulation of fatty acid oxidation (i.e., increasing 3-hydroxybutyrate, a ketone body) as well as reduction in lipid synthesis [3,6] To examine this phenomenon more closely, we chose an in vitro adipocyte model, the 3T3-L1 mouse cell line, as a logical platform to gain insight into the cellular mechanisms of T1AM’s ability to affect macronutrient metabolism and contribute to weight loss. Blue: DAPI nuclear stain, Green: Mitochondria membrane stain, Red: FL-T1AM
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