The incretin hormone glucagon-like peptide-1 (GLP-1) is an important insulin secretagogue and GLP-1 analogs are used for the treatment of type 2 diabetes. GLP-1 displays antiinflammatory and surfactant-releasing effects. Thus, we hypothesize that treatment with GLP-1 analogs will improve pulmonary function in a mouse model of obstructive lung disease. Female mice were sensitized with injected ovalbumin and treated with GLP-1 receptor (GLP-1R) agonists. Exacerbation was induced with inhalations of ovalbumin and lipopolysaccharide. Lung function was evaluated with a measurement of enhanced pause in a whole-body plethysmograph. mRNA levels of GLP-1R, surfactants (SFTPs), and a number of inflammatory markers were measured. GLP-1R was highly expressed in lung tissue. Mice treated with GLP-1R agonists had a noticeably better clinical appearance than the control group. Enhanced pause increased dramatically at day 17 in all control mice, but the increase was significantly less in the groups of GLP-1R agonist-treated mice (P < .001). Survival proportions were significantly increased in GLP-1R agonist-treated mice (P < .01). SFTPB and SFTPA were down-regulated and the expression of inflammatory cytokines were increased in mice with obstructive lung disease, but levels were largely unaffected by GLP-1R agonist treatment. These results show that GLP-1R agonists have potential therapeutic potential in the treatment of obstructive pulmonary diseases, such as chronic obstructive pulmonary disease, by decreasing the severity of acute exacerbations. The mechanism of action does not seem to be the modulation of inflammation and SFTP expression.

Summary Thermogenic adipocytes possess a therapeutically appealing, energy-expending capacity, which is canonically cold-induced by ligand-dependent activation of β-adrenergic G protein-coupled receptors (GPCRs). Here, we uncover an alternate paradigm of GPCR-mediated adipose thermogenesis through the constitutively active receptor, GPR3. We show that the N terminus of GPR3 confers intrinsic signaling activity, resulting in continuous Gs-coupling and cAMP production without an exogenous ligand. Thus, transcriptional induction of Gpr3 represents the regulatory parallel to ligand-binding of conventional GPCRs. Consequently, increasing Gpr3 expression in thermogenic adipocytes is alone sufficient to drive energy expenditure and counteract metabolic disease in mice. Gpr3 transcription is cold-stimulated by a lipolytic signal, and dietary fat potentiates GPR3-dependent thermogenesis to amplify the response to caloric excess. Moreover, we find GPR3 to be an essential, adrenergic-independent regulator of human brown adipocytes. Taken together, our findings reveal a noncanonical mechanism of GPCR control and thermogenic activation through the lipolysis-induced expression of constitutively active GPR3.

Brown adipose tissue takes up large amounts of glucose during cold exposure in mice and humans. Here we report an induction of glucose transporter 1 expression and increased expression of several glycolytic enzymes in brown adipose tissue from cold-exposed mice. Accordingly, these genes were also induced after β-adrenergic activation of cultured brown adipocytes, concomitant with accumulation of hypoxia inducible factor-1α (HIF-1α) protein levels. HIF-1α accumulation was dependent on uncoupling protein 1 and generation of mitochondrial reactive oxygen species. Expression of key glycolytic enzymes was reduced after knockdown of HIF-1α in mature brown adipocytes. Glucose consumption, lactate export and glycolytic capacity were reduced in brown adipocytes depleted of Hif-1α. Finally, we observed a decreased β-adrenergically induced oxygen consumption in Hif-1α knockdown adipocytes cultured in medium with glucose as the only exogenously added fuel. These data suggest that HIF-1α-dependent regulation of glycolysis is necessary for maximum glucose metabolism in brown adipocytes.

Brown adipose tissue is a promising therapeutic target for opposing obesity, glucose intolerance and insulin resistance. The ability to modulate gene expression in mature brown adipocytes is important to understand brown adipocyte function and delineate novel regulatory mechanisms of non-shivering thermogenesis. The aim of this study was to optimize a lipofection-based small interfering RNA (siRNA) transfection protocol for efficient silencing of gene expression in mature brown adipocytes. We determined that a critical parameter was to deliver the siRNA to mature adipocytes by reverse transfection, i.e. transfection of non-adherent cells. Using this protocol, we effectively knocked down both high- and low-abundance transcripts in a model of mature brown adipocytes (WT-1) as well as in primary mature mouse brown adipocytes. A functional consequence of the knockdown was confirmed by an attenuated increase in uncoupled respiration (thermogenesis) in response to β-adrenergic stimulation of mature WT-1 brown adipocytes transfected with uncoupling protein 1 siRNA. Efficient gene silencing was also obtained in various mouse and human white adipocyte models (3T3-L1, primary mouse white adipocytes, hMADS) with the ability to undergo “browning.” In summary, we report an easy and versatile reverse siRNA transfection protocol to achieve specific silencing of gene expression in various models of mature brown and browning-competent white adipocytes, including primary cells.

BackgroundIncreased adipose thermogenesis is being considered as a strategy aimed at preventing or reversing obesity. Thus, regulation of the uncoupling protein 1 (UCP1) gene in human adipocytes is of significant interest. Retinoic acid (RA), the carboxylic acid form of vitamin A, displays agonist activity toward several nuclear hormone receptors, including RA receptors (RARs) and peroxisome proliferator-activated receptor δ (PPARδ). Moreover, RA is a potent positive regulator of UCP1 expression in mouse adipocytes.ResultsThe effects of all-trans RA (ATRA) on UCP1 gene expression in models of mouse and human adipocyte differentiation were investigated. ATRA induced UCP1 expression in all mouse white and brown adipocytes, but inhibited or had no effect on UCP1 expression in human adipocyte cell lines and primary human white adipocytes. Experiments with various RAR agonists and a RAR antagonist in mouse cells demonstrated that the stimulatory effect of ATRA on UCP1 gene expression was indeed mediated by RARs. Consistently, a PPARδ agonist was without effect. Moreover, the ATRA-mediated induction of UCP1 expression in mouse adipocytes was independent of PPARγ coactivator-1α.ConclusionsUCP1 expression is differently affected by ATRA in mouse and human adipocytes. ATRA induces UCP1 expression in mouse adipocytes through activation of RARs, whereas expression of UCP1 in human adipocytes is not increased by exposure to ATRA.