RESUMEN
Brown adipose tissue (BAT) dissipates nutritional energy as heat via the uncoupling protein-1 (UCP1) and BAT activity correlates with leanness in human adults. Here we profile G protein-coupled receptors (GPCRs) in brown adipocytes to identify druggable regulators of BAT. Twenty-one per cent of the GPCRs link to the Gq family, and inhibition of Gq signalling enhances differentiation of human and murine brown adipocytes. In contrast, activation of Gq signalling abrogates brown adipogenesis. We further identify the endothelin/Ednra pathway as an autocrine activator of Gq signalling in brown adipocytes. Expression of a constitutively active Gq protein in mice reduces UCP1 expression in BAT, whole-body energy expenditure and the number of brown-like/beige cells in white adipose tissue (WAT). Furthermore, expression of Gq in human WAT inversely correlates with UCP1 expression. Thus, our data indicate that Gq signalling regulates brown/beige adipocytes and inhibition of Gq signalling may be a novel therapeutic approach to combat obesity.
Asunto(s)
Tejido Adiposo Pardo/enzimología , Tejido Adiposo Blanco/enzimología , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Transducción de Señal , Adipocitos Marrones/citología , Adipocitos Marrones/enzimología , Adipocitos Blancos/citología , Adipocitos Blancos/enzimología , Adipogénesis , Animales , Diferenciación Celular , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Humanos , Canales Iónicos/genética , Canales Iónicos/metabolismo , Ratones , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Proteína Desacopladora 1RESUMEN
Brown adipose tissue is a special type of fat contributing to energy expenditure in human newborns and adults. Moreover, subcutaneous white adipose tissue has a high capacity to adapt an energy-consuming, brown-like/beige phenotype. Here, we developed an easy to handle and fast to accomplish method to efficiently transfer genes into brown and beige fat pads in vivo. Lentiviral vectors are directly injected into the target fat pad of anesthetized mice through a small incision using a modified, small needle connected to a microsyringe, which is well suited for infiltration of adipose tissues. Expression of the target gene can be detected in brown/beige fat one week after injection. The method can be applied within minutes to efficiently deliver transgenes into subcutaneous adipose tissues. Thus, this protocol allows for studying genes of interest in a timely manner in murine brown/beige fat and could potentially lead to new gene therapies for obesity.
RESUMEN
OBJECTIVE: Obesity is an enormous burden for patients and health systems world-wide. Brown adipose tissue dissipates energy in response to cold and has been shown to be metabolically active in human adults. The type I transforming growth factor ß (TGFß) receptor Activin receptor-like kinase 7 (Alk7) is highly expressed in adipose tissues and is down-regulated in obese patients. Here, we studied the function of Alk7 in brown adipocytes. METHODS: Using pharmacological and genetic tools, Alk7 signaling pathway and its effects were studied in murine brown adipocytes. Brown adipocyte differentiation and activation was analyzed. RESULTS: Alk7 is highly upregulated during differentiation of brown adipocytes. Interestingly, Alk7 expression is increased by cGMP/protein kinase G (PKG) signaling, which enhances brown adipocyte differentiation. Activin AB effectively activates Alk7 and SMAD3 signaling. Activation of Alk7 in brown preadipocytes suppresses the master adipogenic transcription factor PPARγ and differentiation. Stimulation of Alk7 during late differentiation of brown adipocytes reduces lipid content and adipogenic marker expression but enhances UCP1 expression. CONCLUSIONS: We found a so far unknown crosstalk between cGMP and Alk7 signaling pathways. Tight regulation of Alk7 is required for efficient differentiation of brown adipocytes. Alk7 has differential effects on adipogenic differentiation and the development of the thermogenic program in brown adipocytes.
RESUMEN
Activation of G protein-coupled receptors involves major conformational changes of the receptor protein ranging from the extracellular transmitter binding site to the intracellular G protein binding surface. GPCRs such as the muscarinic acetylcholine receptors are commonly probed with radioantagonists rather than radioagonists due to better physicochemical stability, higher affinity, and indifference towards receptor coupling states of the former. Here we introduce tritiated iperoxo, a superagonist at muscarinic M2 receptors with very high affinity. In membrane suspensions of transfected CHO-cells, [³H]iperoxo - unlike the common radioagonists [³H]acetylcholine and [³H]oxotremorine M - allowed labelling of each of the five muscarinic receptor subtypes in radioagonist displacement and saturation binding studies. [³H]iperoxo revealed considerable differences in affinity between the even- and the odd-numbered muscarinic receptor subtypes with affinities for the M2 and M4 receptor in the picomolar range. Probing ternary complex formation on the M2 receptor, [³H]iperoxo dissociation was not influenced by an archetypal allosteric inverse agonist, reflecting activation-related rearrangement of the extracellular loop region. At the inner side of M2, the preferred Gi protein acted as a positive allosteric modulator of [³H]iperoxo binding, whereas Gs and Gq were neutral in spite of their robust coupling to the activated receptor. In intact CHO-hM2 cells, endogenous guanylnucleotides promoted receptor/G protein-dissociation resulting in low-affinity agonist binding which, nevertheless, was still reported by [³H]iperoxo. Taken together, the muscarinic superagonist [³H]iperoxo is the best tool currently available for direct probing activation-related conformational transitions of muscarinic receptors.