A novel crosstalk between Alk7 and cGMP signaling differentially regulates brown adipocyte function.
ABSTRACT: 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 ? (TGF?) 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 PPAR? 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.
Project description:Adaptation to nutrient availability is crucial for survival. Upon nutritional stress, such as during prolonged fasting or cold exposure, organisms need to balance the feeding of tissues and the maintenance of body temperature. The mechanisms that regulate the adaptation of brown adipose tissue (BAT), a key organ for non-shivering thermogenesis, to variations in nutritional state are not known. Here we report that specific deletion of the activin receptor ALK7 in BAT resulted in fasting-induced hypothermia due to exaggerated catabolic activity in brown adipocytes. After overnight fasting, BAT lacking ALK7 showed increased expression of genes responsive to nutrient stress, including the upstream regulator KLF15, aminoacid catabolizing enzymes, notably proline dehydrogenase (POX), and adipose triglyceride lipase (ATGL), as well as markedly reduced lipid droplet size. In agreement with this, ligand stimulation of ALK7 suppressed POX and KLF15 expression in both mouse and human brown adipocytes. Treatment of mutant mice with the glucocorticoid receptor antagonist RU486 restored KLF15 and POX expression levels in mutant BAT, suggesting that loss of BAT ALK7 results in excessive activation of glucocorticoid signaling upon fasting. These results reveal a novel signaling pathway downstream of ALK7 which regulates the adaptation of BAT to nutrient availability by limiting nutrient stress-induced overactivation of catabolic responses in brown adipocytes.
Project description:We previously identified a quantitative trait locus for adiposity, non-insulin-dependent diabetes 5 (Nidd5), on mouse chromosome 2. In the current study, we identified the actual genetic alteration at Nidd5 as a nonsense mutation of the Acvr1c gene encoding activin receptor-like kinase 7 (ALK7), one of the type I transforming growth factor-? receptors, which results in a COOH-terminal deletion of the kinase domain. We further showed that the ALK7 dysfunction causes increased lipolysis in adipocytes and leads to decreased fat accumulation. Conversely, ALK7 activation inhibits lipolysis by suppressing the expression of adipose lipases. ALK7 and activated Smads repress those lipases by downregulating peroxisome proliferator-activated receptor ? (PPAR?) and CCAAT/enhancer binding protein (C/EBP) ?. Although PPAR? and C/EBP? act as adipogenic transcription factors during adipocyte differentiation, they are lipolytic in sum in differentiated adipocytes and are downregulated by ALK7 in obesity to accumulate fat. Under the obese state, ALK7 deficiency improves glucose tolerance and insulin sensitivity by preferentially increasing fat combustion in mice. These findings have uncovered a net lipolytic function of PPAR? and C/EBP? in differentiated adipocytes and point to the ALK7-signaling pathway that is activated in obesity as a potential target of medical intervention.
Project description:Obesity is associated with blunted ?-adrenoreceptor (?-AR)-mediated lipolysis and lipid oxidation in adipose tissue, but the mechanisms linking nutrient overload to catecholamine resistance are poorly understood. We report that targeted disruption of TGF-? superfamily receptor ALK7 alleviates diet-induced catecholamine resistance in adipose tissue, thereby reducing obesity in mice. Global and fat-specific Alk7 knock-out enhanced adipose ?-AR expression, ?-adrenergic signaling, mitochondrial biogenesis, lipid oxidation, and lipolysis under a high fat diet, leading to elevated energy expenditure, decreased fat mass, and resistance to diet-induced obesity. Conversely, activation of ALK7 reduced ?-AR-mediated signaling and lipolysis cell-autonomously in both mouse and human adipocytes. Acute inhibition of ALK7 in adult mice by a chemical-genetic approach reduced diet-induced weight gain, fat accumulation, and adipocyte size, and enhanced adipocyte lipolysis and ?-adrenergic signaling. We propose that ALK7 signaling contributes to diet-induced catecholamine resistance in adipose tissue, and suggest that ALK7 inhibitors may have therapeutic value in human obesity.
Project description:All major cell types in pancreatic islets express the transforming growth factor (TGF)-beta superfamily receptor ALK7, but the physiological function of this receptor has been unknown. Mutant mice lacking ALK7 showed normal pancreas organogenesis but developed an age-dependent syndrome involving progressive hyperinsulinemia, reduced insulin sensitivity, liver steatosis, impaired glucose tolerance, and islet enlargement. Hyperinsulinemia preceded the development of any other defect, indicating that this may be one primary consequence of the lack of ALK7. In agreement with this, mutant islets showed enhanced insulin secretion under sustained glucose stimulation, indicating that ALK7 negatively regulates glucose-stimulated insulin release in beta-cells. Glucose increased expression of ALK7 and its ligand activin B in islets, but decreased that of activin A, which does not signal through ALK7. The two activins had opposite effects on Ca(2+) signaling in islet cells, with activin A increasing, but activin B decreasing, glucose-stimulated Ca(2+) influx. On its own, activin B had no effect on WT cells, but stimulated Ca(2+) influx in cells lacking ALK7. In accordance with this, mutant mice lacking activin B showed hyperinsulinemia comparable with that of Alk7(-/-) mice, but double mutants showed no additive effects, suggesting that ALK7 and activin B function in a common pathway to regulate insulin secretion. These findings uncover an unexpected antagonism between activins A and B in the control of Ca(2+) signaling in beta-cells. We propose that ALK7 plays an important role in regulating the functional plasticity of pancreatic islets, negatively affecting beta-cell function by mediating the effects of activin B on Ca(2+) signaling.
Project description:Ski-related oncogene SnoN (SnoN or SKIL) regulates multiple signaling pathways in a tissue- and developmental stage-dependent manner and has broad functions in embryonic angiogenesis, mammary gland alveologenesis, cancer, and aging. Here, we report that SnoN also plays a critical role in white adipose tissue (WAT) development by regulating mesenchymal stem cell (MSC) self-renewal and differentiation. We found that SnoN promotes MSC differentiation in the adipocyte lineage by antagonizing activin A/Smad2, but not TGFβ/Smad3 signaling. Mice lacking SnoN or expressing a mutant SnoN defective in binding to the Smads were protected from high-fat diet-induced obesity and insulin resistance, and MSCs lacking a functional SnoN exhibited defective differentiation. We further demonstrated that activin, via Smad2, appears to be the major regulator of WAT development in vivo We also noted that activin A is abundantly expressed in WAT and adipocytes through an autocrine mechanism and promotes MSC self-renewal and inhibits adipogenic differentiation by inducing expression of the gene encoding the homeobox transcription factor Nanog. Of note, SnoN repressed activin/Smad2 signaling and activin A expression, enabling expression of adipocyte-specific transcription factors and promoting adipogenic differentiation. In conclusion, our study has revealed that SnoN plays an important in vivo role in adipocyte differentiation and WAT development in vivo by decreasing activity in the activin/Smad2 signaling pathway.
Project description:OBJECTIVE:Recruitment of brown adipose tissue (BAT) is a potential new strategy for increasing energy expenditure (EE) to treat obesity. G protein-coupled receptors (GPCRs) represent promising targets to activate BAT, as they are the major regulators of BAT biological function. To identify new regulators of GPCR signaling in BAT, we studied the role of Regulator of G protein Signaling 2 (RGS2) in brown adipocytes and BAT. METHODS:We combined pharmacological and genetic tools to investigate the role of RGS2 in BAT in vitro and in vivo. Adipocyte progenitors were isolated from wild-type (WT) and RGS2 knockout (RGS2-/-) BAT and differentiated to brown adipocytes. This approach was complemented with knockdown of RGS2 using lentiviral shRNAs (shRGS2). Adipogenesis was analyzed by Oil Red O staining and by determining the expression of adipogenic and thermogenic markers. Pharmacological modulators and fluorescence staining of F-acting stress fibers were employed to identify the underlying signaling pathways. In vivo, the activity of BAT was assessed by ex vivo lipolysis and by measuring whole-body EE by indirect calorimetry in metabolic cages. RESULTS:RGS2 is highly expressed in BAT, and treatment with cGMP-an important enhancer of brown adipocyte differentiation-further increased RGS2 expression. Loss of RGS2 strongly suppressed adipogenesis and the expression of thermogenic genes in brown adipocytes. Mechanistically, we found increased Gq/Rho/Rho kinase (ROCK) signaling in the absence of RGS2. Surprisingly, in vivo analysis revealed elevated BAT activity in RGS2-deficient mice that was caused by enhanced Gs/cAMP signaling. CONCLUSION:Overall, RGS2 regulates two major signaling pathways in BAT: Gq and Gs. On the one hand, RGS2 promotes brown adipogenesis by counteracting the inhibitory action of Gq/Rho/ROCK signaling. On the other hand, RGS2 decreases the activity of BAT through the inhibition of Gs signaling and cAMP production. Thus, RGS2 might represent a stress modulator that protects BAT from overstimulation.
Project description:Nodal proteins have crucial roles in mesendoderm formation and left-right patterning during vertebrate development. The molecular mechanisms of signal transduction by Nodal and related ligands, however, are not fully understood. In this paper, we present biochemical and functional evidence that the orphan type I serine/threonine kinase receptor ALK7 acts as a receptor for mouse Nodal and Xenopus Nodal-related 1 (Xnr1). Receptor reconstitution experiments indicate that ALK7 collaborates with ActRIIB to confer responsiveness to Xnr1 and Nodal. Both receptors can independently bind Xnr1. In addition, Cripto, an extracellular protein genetically implicated in Nodal signaling, can independently interact with both Xnr1 and ALK7, and its expression greatly enhances the ability of ALK7 and ActRIIB to respond to Nodal ligands. The Activin receptor ALK4 is also able to mediate Nodal signaling but only in the presence of Cripto, with which it can also interact directly. A constitutively activated form of ALK7 mimics the mesendoderm-inducing activity of Xnr1 in Xenopus embryos, whereas a dominant-negative ALK7 specifically blocks the activities of Nodal and Xnr1 but has little effect on other related ligands. In contrast, a dominant-negative ALK4 blocks all mesoderm-inducing ligands tested, including Nodal, Xnr1, Xnr2, Xnr4, and Activin. In agreement with a role in Nodal signaling, ALK7 mRNA is localized to the ectodermal and organizer regions of Xenopus gastrula embryos and is expressed during early stages of mouse embryonic development. Therefore, our results indicate that both ALK4 and ALK7 can mediate signal transduction by Nodal proteins, although ALK7 appears to be a receptor more specifically dedicated to Nodal signaling.
Project description:We investigated the ability of fetal mesenchymal stem cells (fMSCs) to differentiate into brown and white adipocytes and compared the expression of a number of marker genes and key regulatory factors. We showed that the expression of key adipocyte regulators and markers during differentiation is similar to that in other human and murine adipocyte models, including induction of PPARgamma2 and FABP4. Notably, we found that the preadipocyte marker, Pref-1, is induced early in differentiation and then declines markedly as the process continues, suggesting that fMSCs first acquire preadipocyte characteristics as they commit to the adipogenic lineage, prior to their differentiation into mature adipocytes. After adipogenic induction, some stem cell isolates differentiated into cells resembling brown adipocytes and others into white adipocytes. Detailed investigation of one isolate showed that the novel brown fat-determining factor PRDM16 is expressed both before and after differentiation. Importantly, these cells exhibited elevated basal UCP-1 expression, which was dependent on the activity of the orphan nuclear receptor ERRalpha, highlighting a novel role for ERRalpha in human brown fat. Thus fMSCs represent a useful in vitro model for human adipogenesis, and provide opportunities to study the stages prior to commitment to the adipocyte lineage. They also offer invaluable insights into the characteristics of human brown fat.
Project description:Transcriptional and epigenetic regulation is fundamentally involved in initiating and maintaining progression of cellular differentiation. The 2 types of thermogenic adipocytes, brown and beige, are thought to be of different origins but share functionally similar phenotypes. Here, we report that lysine-specific demethylase 2 (LSD2) regulates the expression of genes associated with lineage identity during the differentiation of brown and beige adipogenic progenitors in mice. In HB2 mouse brown preadipocytes, short hairpin RNA-mediated knockdown (KD) of LSD2 impaired formation of lipid droplet-containing adipocytes and down-regulated brown adipogenesis-associated genes. Transcriptomic analysis revealed that myogenesis-associated genes were up-regulated in LSD2-KD cells under adipogenic induction. In addition, loss of LSD2 during later phases of differentiation had no obvious influence on adipogenic traits, suggesting that LSD2 functions during earlier phases of brown adipocyte differentiation. Using adipogenic cells from the brown adipose tissues of LSD2-knockout (KO) mice, we found reduced expression of brown adipogenesis genes, whereas myogenesis genes were not affected. In contrast, when LSD2-KO cells from inguinal white adipose tissues were subjected to beige induction, these cells showed a dramatic rise in myogenic gene expression. Collectively, these results suggest that LSD2 regulates distinct sets of genes during brown and beige adipocyte formation.-Takase, R., Hino, S., Nagaoka, K., Anan, K., Kohrogi, K., Araki, H., Hino, Y., Sakamoto, A., Nicholson, T. B., Chen, T., Nakao, M. Lysine-specific demethylase-2 is distinctively involved in brown and beige adipogenic differentiation.
Project description:Human adipose tissue is a major site of expression of inhibin beta B (INHBB) which homodimerizes to form the novel adipokine activin B. Our aim was to determine if molecules needed for a local action of activin B are expressed in adipose tissue. Microarray analysis showed that adipose tissue expressed activin type I and II receptors and that the expression of activin receptor-like kinase 7 (ALK7) was adipose tissue specific. In obesity discordant siblings from the SOS Sib Pair study, adipose tissue ALK7 expression was higher in lean (n=90) compared to obese (n=90) subjects (p=4 x 10(-31)). Adipose tissue ALK7 expression correlated with several measures of body fat, carbohydrate metabolism and lipids. In addition, ALK7 and INHBB expression correlated but only in lean subjects and in subjects with normal glucose tolerance. We conclude that activin B may have local effects in adipose tissue and thereby influence obesity and its comorbidities.