An OLTAM system for analysis of brown/beige fat thermogenic activity

Abstract

BACKGROUND/OBJECTIVESThermogenic fat is present in humans and emerging evidence indicates that increasing the content and activity of these adipocytes may lead to weight loss and improved metabolic health. Multiple reporter systems have been developed to assay thermogenic fat activity based on the transcriptional and translational activation of Ucp1, the key molecule that mediates nonshivering thermogenesis. Our study aims to develop a much-needed tool to monitor thermogenic fat activity through a mechanism independent of Ucp1 regulation, therefore effectively assaying not only canonical β-adrenergic activation but also various non-UCP1-mediated thermogenic pathways that have been increasingly appreciated.METHODSWe detected increased luciferase activity upon thermogenic activation in interscapular brown and inguinal subcutaneous fat in ODD-Luc mice, a hypoxia reporter mouse model. We then developed an OLTAM (ODD-Luc based Thermogenic Activity Measurement) system to assay thermogenic fat cell activity.RESULTSIn both primary murine and human adipocytes and an immortalized adipose cell line that were transduced with the OLTAM system, luciferase activity can be readily measured and visualized by bioluminescence imaging in response to a variety of stimuli, including UCP1-independent thermogenic signaling. This system can offer a convenient method to assay thermogenic activity for both basic and translational research.CONCLUSIONSThe OLTAM system offers a convenient way to measure of the activation of thermogenic fat and presents opportunities to discover novel signaling pathways and unknown compounds targeting metabolically active adipocytes to counteract human obesity.

A dual Ucp1 reporter mouse model for imaging and quantitation of brown and brite fat recruitment

“…We wish to emphasize that all Ucp1 reporter mouse models published so far, including our new model, are suitable to monitor Ucp1 gene expression but do not report on thermogenic activity of UCP1 [44], [45], [46]. The real thermogenic activity and total capacity can be assessed with indirect calorimetry [47], [48] and new emerging technologies [49], [50], [51].…”

Hepatic hepcidin/intestinal HIF-2α axis maintains iron absorption during iron deficiency and overload

“…Knockout cells were verified by sequencing. The PHD enzyme activity luciferase reporter was previously described (46). Briefly, FPN GFP cells were infected at 10 MOI overnight and treated the next day with 250 ng/ml doxycycline, 100 μM FG4592 (Selleckchem), 200 μM ferric ammonium citrate (FAC) (Sigma-Aldrich), and/or 1 mg/ml human recombinant hepcidin (Bachem) or 200 μM DFO (Sigma-Aldrich).…”

TC‐E 5003, a protein methyltransferase 1 inhibitor, activates the PKA‐dependent thermogenic pathway in primary murine and human subcutaneous adipocytes

“…1E). Given the increased Ucp1 expression, we examined if TC‐E treatment increases thermogenesis of the cells using OLTAM [ODD‐Luc (luciferase fused with the oxygen‐dependent degradation domain)‐based Thermogenic Activity Measurement] system that we recently developed and reported to measure thermogenic activity of brown or beige adipocytes [14]. The result showed that TC‐E treatment increased ODD‐Luc activity to a level comparable with that observed following ISO treatment, suggesting that TC‐E enhances thermogenesis (Fig.…”

“…Human adipose precursor cells isolated from the SQ fat were obtained as previously described [14]. All specimens were collected under the protocols reviewed and approved by the University of Michigan Medical School Institutional Review Board (IRBMED).…”