Liver-Specific Nonviral Gene Delivery of Fibroblast Growth Factor 21 Protein Expression in Mice Regulates Body Mass and White/Brown Fat Respiration.

Viral-mediated in vivo gene delivery methods currently dominate among therapeutic strategies within the clinical and experimental settings, albeit with well documented limitations arising from immunologic constraints. In this study, we demonstrate the utility of nonviral hepatotropic in vivo gene delivery of unpackaged expression constructs, including one encoding fibroblast growth factor 21 (FGF21). FGF21 is an important hepatokine whose expression positively correlates with therapeutic outcomes across various animal models of obesity. Our data demonstrate that FGF21 expression can be restored into the livers of immunocompetent FGF21 knockout mice for at least 2 weeks after a single injection with an FGF21 expression plasmid. In wild-type C57BL6/J mice, in vivo transfection with an FGF21-expressing plasmid induced weight loss, decreased adiposity, and activated thermogenesis in white fat within 2 weeks. Furthermore, in vivo FGF21 gene delivery protected C57BL6/J mice against diet-induced obesity by decreasing adiposity and increasing uncoupling protein 1-dependent thermogenesis in brown fat and by boosting respiratory capacity in subcutaneous and perigonadal white fat. Together, the data illustrate a facile and effective methodology for delivering prolonged protein expression specifically to the liver. We contend that this method will find utility in basic science research as a practical means to enhance in vivo studies characterizing liver protein function. We further believe our data provide a rationale for further exploring the potential clinical utility of nonviral gene therapy in mouse models of disease. SIGNIFICANCE STATEMENT: This study presents a valuable method for nonviral gene delivery in mice that improves upon existing techniques. The data provide a rationale for further exploring the potential clinical utility of nonviral gene therapy in mouse models of disease and will likely enhance in vivo studies characterizing liver protein function.

The Aryl Hydrocarbon Receptor in Energy Balance: The Road from Dioxin-Induced Wasting Syndrome to Combating Obesity with Ahr Ligands.

The aryl hydrocarbon receptor (AHR) has been studied for over 40 years, yet our understanding of this ligand-activated transcription factor remains incomplete. Each year, novel findings continually force us to rethink the role of the AHR in mammalian biology. The AHR has historically been studied within the context of potent activation via AHR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), with a focus on how the AHR mediates TCDD toxicity. Research has subsequently revealed that the AHR is actively involved in distinct physiological processes ranging from the development of the liver and reproductive organs, to immune system function and wound healing. More recently, the AHR was implicated in the regulation of energy metabolism and is currently being investigated as a potential therapeutic target for obesity. In this review, we re-trace the steps through which the early toxicological studies of TCDD led to the conceptual framework for the AHR as a potential therapeutic target in metabolic disease. We additionally discuss the key discoveries that have been made concerning the role of the AHR in energy metabolism, as well as the current and future directions of the field.

Inducible Loss of the Aryl Hydrocarbon Receptor Activates Perigonadal White Fat Respiration and Brown Fat Thermogenesis via Fibroblast Growth Factor 21.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor highly expressed in hepatocytes. Researchers have employed global and liver-specific conditional Ahr knockout mouse models to characterize the physiological roles of the AHR; however, the gestational timing of AHR loss in these models can complicate efforts to distinguish the direct and indirect effects of post-gestational AHR deficiency. Utilizing a novel tamoxifen-inducible AHR knockout mouse model, we analyzed the effects of hepatocyte-targeted AHR loss in adult mice. The data demonstrate that AHR deficiency significantly reduces weight gain and adiposity, and increases multilocular lipid droplet formation within perigonadal white adipose tissue (gWAT). Protein and mRNA expression of fibroblast growth factor 21 (FGF21), an important hepatokine that activates thermogenesis in brown adipose tissue (BAT) and gWAT, significantly increases upon AHR loss and correlates with a significant increase of BAT and gWAT respiratory capacity. Confirming the role of FGF21 in mediating these effects, this phenotype is reversed in mice concomitantly lacking AHR and FGF21 expression. Chromatin immunoprecipitation analyses suggest that the AHR may constitutively suppress Fgf21 transcription through binding to a newly identified xenobiotic response element within the Fgf21 promoter. The data demonstrate an important AHR-FGF21 regulatory axis that influences adipose biology and may represent a "druggable" therapeutic target for obesity and its related metabolic disorders.

Hepatic Aryl Hydrocarbon Receptor Attenuates Fibroblast Growth Factor 21 Expression.

The Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor involved in many physiological processes. Several studies indicate that AHR is also involved in energy homeostasis. Fibroblast growth factor 21 (FGF21) is an important regulator of the fasting and feeding responses. When administered to various genetic and diet-induced mouse models of obesity, FGF21 can attenuate obesity-associated morbidities. Here, we explore the role of AHR in hepatic Fgf21 expression through the use of a conditional, hepatocyte-targeted AHR knock-out mouse model (Cre(Alb)Ahr(Fx/Fx)). Compared with the congenic parental strain (Ahr(Fx/Fx)), non-fasted Cre(Alb)Ahr(Fx/Fx) mice exhibit a 4-fold increase in hepatic Fgf21 expression, as well as elevated expression of the FGF21-target gene Igfbp1 Furthermore, in vivo agonist activation of AHR reduces hepatic Fgf21 expression during a fast. The Fgf21 promoter contains several putative dioxin response elements (DREs). Using EMSA, we demonstrate that the AHR-ARNT heterodimer binds to a specific DRE that overlaps binding sequences for peroxisome proliferator-activated receptor α (PPARα), carbohydrate response element-binding protein (ChREBP), and cAMP response element-binding protein, hepatocyte specific (CREBH). In addition, we reveal that agonist-activated AHR impairs PPARα-, ChREBP-, and CREBH-mediated promoter activity in Hepa-1 cells. Accordingly, agonist treatment in Hepa-1 cells ablates potent ER stress-driven Fgf21 expression, and pre-treatment with AHR antagonist blocks this effect. Finally, we show that pre-treatment of primary human hepatocytes with AHR agonist diminishes PPARα-, glucose-, and ER stress-driven induction of FGF21 expression, indicating the effect is not mouse-specific. Together, our data show that AHR contributes to hepatic energy homeostasis, partly through the regulation of FGF21 expression and signaling.