RESUMO
Amino-acid-induced lysosomal mechanistic target of rapamycin complex 1 (mTORC1) localization through the Rag GTPases is a critical step for its activation by Rheb GTPase. However, how the mTORC1 interacts with Rheb on the lysosome remains elusive. We report that amino acids enhance the polyubiquitination of Rheb (Ub-Rheb), which shows a strong binding preference for mTORC1 and supports its activation, while the Ub-Rheb is subjected to subsequent degradation. Mechanistically, we identified ATXN3 as a Ub-Rheb deubiquitinase whose lysosomal localization is blocked by active Rag heterodimer in response to amino acid stimulation. Consistently, cells lacking functional Rag heterodimer on the lysosome accumulate Ub-Rheb, and blockade of its degradation instigates robust lysosomal mTORC1 localization and its activation without the Ragulator-Rag system. Thus, polyubiquitination of Rheb is an important post-translational modification, which facilitates the binding of mTORC1 to Rheb on the lysosome and is another crosstalk between the amino acid and growth factor signaling for mTORC1 activation.
Assuntos
Ataxina-3/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/fisiologia , Proteína Enriquecida em Homólogo de Ras do Encéfalo/metabolismo , Aminoácidos/metabolismo , Animais , Ataxina-3/fisiologia , Linhagem Celular , Enzimas Desubiquitinantes/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Complexos Multiproteicos/metabolismo , Ligação Proteica/fisiologia , Proteína Enriquecida em Homólogo de Ras do Encéfalo/fisiologia , Proteínas Repressoras/metabolismo , Transdução de Sinais/fisiologia , UbiquitinaçãoRESUMO
Although visceral adipocytes located within the body's central core are maintained at approximately 37°C, adipocytes within bone marrow, subcutaneous, and dermal depots are found primarily within the peripheral shell and generally exist at cooler temperatures. Responses of brown and beige/brite adipocytes to cold stress are well studied; however, comparatively little is known about mechanisms by which white adipocytes adapt to temperatures below 37°C. Here, we report that adaptation of cultured adipocytes to 31°C, the temperature at which distal marrow adipose tissues and subcutaneous adipose tissues often reside, increases anabolic and catabolic lipid metabolism, and elevates oxygen consumption. Cool adipocytes rely less on glucose and more on pyruvate, glutamine, and, especially, fatty acids as energy sources. Exposure of cultured adipocytes and gluteal white adipose tissue (WAT) to cool temperatures activates a shared program of gene expression. Cool temperatures induce stearoyl-CoA desaturase-1 (SCD1) expression and monounsaturated lipid levels in cultured adipocytes and distal bone marrow adipose tissues (BMATs), and SCD1 activity is required for acquisition of maximal oxygen consumption at 31°C.
Assuntos
Adipócitos Brancos/metabolismo , Regulação da Temperatura Corporal/fisiologia , Adaptação Fisiológica , Adipócitos/metabolismo , Adipócitos/fisiologia , Adipócitos Marrons/metabolismo , Adipócitos Brancos/fisiologia , Tecido Adiposo/metabolismo , Tecido Adiposo Branco/metabolismo , Animais , Temperatura Baixa , Ácidos Graxos/metabolismo , Feminino , Metabolismo dos Lipídeos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Consumo de Oxigênio , Ratos , Ratos Sprague-Dawley , Estearoil-CoA Dessaturase/metabolismoRESUMO
KEY POINTS: Several distinct strategies produce and conserve heat to maintain the body temperature of mammals, each associated with unique physiologies, with consequences for wellness and disease susceptibility Highly regulated properties of skin offset the total requirement for heat production We hypothesize that the adipose component of skin is primarily responsible for modulating heat flux; here we evaluate the relative regulation of adipose depots in mouse and human, to test their recruitment to heat production and conservation We found that insulating mouse dermal white adipose tissue accumulates in response to environmentally and genetically induced cool stress; this layer is one of two adipose depots closely apposed to mouse skin, where the subcutaneous mammary gland fat pads are actively recruited to heat production In contrast, the body-wide adipose depot associated with human skin produces heat directly, potentially creating an alternative to the centrally regulated brown adipose tissue ABSTRACT: Mammalian skin impacts metabolic efficiency system-wide, controlling the rate of heat loss and consequent heat production. Here we compare the unique fat depots associated with mouse and human skin, to determine whether they have corresponding functions and regulation. For humans, we assay a skin-associated fat (SAF) body-wide depot to distinguish it from the subcutaneous fat pads characteristic of the abdomen and upper limbs. We show that the thickness of SAF is not related to general adiposity; it is much thicker (1.6-fold) in women than men, and highly subject-specific. We used molecular and cellular assays of ß-adrenergic-induced lipolysis and found that dermal white adipose tissue (dWAT) in mice is resistant to lipolysis; in contrast, the body-wide human SAF depot becomes lipolytic, generating heat in response to ß-adrenergic stimulation. In mice challenged to make more heat to maintain body temperature (either environmentally or genetically), there is a compensatory increase in thickness of dWAT: a corresponding ß-adrenergic stimulation of human skin adipose (in vivo or in explant) depletes adipocyte lipid content. We summarize the regulation of skin-associated adipocytes by age, sex and adiposity, for both species. We conclude that the body-wide dWAT depot of mice shows unique regulation that enables it to be deployed for heat preservation; combined with the actively lipolytic subcutaneous mammary fat pads they enable thermal defence. The adipose tissue that covers human subjects produces heat directly, providing an alternative to the brown adipose tissues.
Assuntos
Tecido Adiposo Marrom , Termogênese , Tecido Adiposo Marrom/fisiologia , Tecido Adiposo Branco/metabolismo , Animais , Feminino , Humanos , Lipólise , Gordura Subcutânea/metabolismo , Termogênese/fisiologiaRESUMO
CRISPR/Cas9 has enabled inducible gene knockout in numerous tissues; however, its use has not been reported in brown adipose tissue (BAT). Here, we developed the brown adipocyte CRISPR (BAd-CRISPR) methodology to rapidly interrogate the function of one or multiple genes. With BAd-CRISPR, an adeno-associated virus (AAV8) expressing a single guide RNA (sgRNA) is administered directly to BAT of mice expressing Cas9 in brown adipocytes. We show that the local administration of AAV8-sgRNA to interscapular BAT of adult mice robustly transduced brown adipocytes and ablated expression of adiponectin, adipose triglyceride lipase, fatty acid synthase, perilipin 1, or stearoyl-CoA desaturase 1 by >90%. Administration of multiple AAV8 sgRNAs led to simultaneous knockout of up to three genes. BAd-CRISPR induced frameshift mutations and suppressed target gene mRNA expression but did not lead to substantial accumulation of off-target mutations in BAT. We used BAd-CRISPR to create an inducible uncoupling protein 1 (Ucp1) knockout mouse to assess the effects of UCP1 loss on adaptive thermogenesis in adult mice. Inducible Ucp1 knockout did not alter core body temperature; however, BAd-CRISPR Ucp1 mice had elevated circulating concentrations of fibroblast growth factor 21 and changes in BAT gene expression consistent with heat production through increased peroxisomal lipid oxidation. Other molecular adaptations predict additional cellular inefficiencies with an increase in both protein synthesis and turnover, and mitochondria with reduced reliance on mitochondrial-encoded gene expression and increased expression of nuclear-encoded mitochondrial genes. These data suggest that BAd-CRISPR is an efficient tool to speed discoveries in adipose tissue biology.
Assuntos
Tecido Adiposo Marrom/metabolismo , Sistemas CRISPR-Cas , Animais , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Técnicas de Inativação de Genes , Camundongos , Camundongos Knockout , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismoRESUMO
The E3 ubiquitin ligase parkin is a critical regulator of mitophagy and has been identified as a susceptibility gene for type 2 diabetes (T2D). However, its role in metabolically active tissues that precipitate T2D development is unknown. Specifically, pancreatic ß cells and adipocytes both rely heavily on mitochondrial function in the regulation of optimal glycemic control to prevent T2D, but parkin's role in preserving quality control of ß cell or adipocyte mitochondria is unclear. Although parkin has been reported previously to control mitophagy, here we show that, surprisingly, parkin is dispensable for glucose homeostasis in both ß cells and adipocytes during diet-induced insulin resistance in mice. We observed that insulin secretion, ß cell formation, and islet architecture were preserved in parkin-deficient ß cells and islets, suggesting that parkin is not necessary for control of ß cell function and islet compensation for diet-induced obesity. Although transient parkin deficiency mildly impaired mitochondrial turnover in ß cell lines, parkin deletion in primary ß cells yielded no deficits in mitochondrial clearance. In adipocyte-specific deletion models, lipid uptake and ß-oxidation were increased in cultured cells, whereas adipose tissue morphology, glucose homeostasis, and beige-to-white adipocyte transition were unaffected in vivo In key metabolic tissues where mitochondrial dysfunction has been implicated in T2D development, our experiments unexpectedly revealed that parkin is not an essential regulator of glucose tolerance, whole-body energy metabolism, or mitochondrial quality control. These findings highlight that parkin-independent processes maintain ß cell and adipocyte mitochondrial quality control in diet-induced obesity.
Assuntos
Adipócitos/metabolismo , Homeostase , Células Secretoras de Insulina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Adipócitos/citologia , Adipócitos/enzimologia , Adiposidade , Animais , Peso Corporal , Diferenciação Celular , Diabetes Mellitus Tipo 2/metabolismo , Metabolismo Energético , Feminino , Teste de Tolerância a Glucose , Resistência à Insulina , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/enzimologia , Masculino , Camundongos , Mitocôndrias/metabolismo , OxirreduçãoRESUMO
Although adipogenesis is mainly controlled by a small number of master transcription factors, including CCAAT/enhancer-binding protein family members and peroxisome proliferator-activated receptor γ (PPARγ), other transcription factors also are involved in this process. Thyroid cancer cells expressing a paired box 8 (PAX8)-PPARγ fusion oncogene trans-differentiate into adipocyte-like cells in the presence of the PPARγ ligand pioglitazone, but this trans-differentiation is inhibited by the transcription factor NK2 homeobox 1 (NKX2-1). Here, we tested whether NKX family members may play a role also in normal adipogenesis. Using quantitative RT-PCR (RT-qPCR), we examined the expression of all 14 NKX family members during 3T3-L1 adipocyte differentiation. We found that most NKX members, including NKX2-1, are expressed at very low levels throughout differentiation. However, mRNA and protein expression of a related family member, NKX1-2, was induced during adipocyte differentiation. NKX1-2 also was up-regulated in cultured murine ear mesenchymal stem cells (EMSCs) during adipogenesis. Importantly, shRNA-mediated NKX1-2 knockdown in 3T3-L1 preadipocytes or EMSCs almost completely blocked adipocyte differentiation. Furthermore, NKX1-2 overexpression promoted differentiation of the ST2 bone marrow-derived mesenchymal precursor cell line into adipocytes. Additional findings suggested that NKX1-2 promotes adipogenesis by inhibiting expression of the antiadipogenic protein COUP transcription factor II. Bone marrow mesenchymal precursor cells can differentiate into adipocytes or osteoblasts, and we found that NKX1-2 both promotes ST2 cell adipogenesis and inhibits their osteoblastogenic differentiation. These results support a role for NKX1-2 in promoting adipogenesis and possibly in regulating the balance between adipocyte and osteoblast differentiation of bone marrow mesenchymal precursor cells.
Assuntos
Adipócitos/metabolismo , Adipogenia/genética , Diferenciação Celular/genética , Proteínas de Homeodomínio/genética , Proteínas Nucleares/genética , Osteoblastos/metabolismo , Fatores de Transcrição/genética , Células 3T3-L1 , Adipócitos/citologia , Animais , Linhagem Celular , Células Cultivadas , Regulação da Expressão Gênica , Células HEK293 , Proteínas de Homeodomínio/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Nucleares/metabolismo , Osteoblastos/citologia , Fator de Transcrição PAX8/genética , Fator de Transcrição PAX8/metabolismo , PPAR gama/genética , PPAR gama/metabolismo , Interferência de RNA , Fatores de Transcrição/metabolismoRESUMO
Maternal low-protein diet (LP) throughout gestation affects pancreatic ß-cell fraction of the offspring at birth, thus increasing their susceptibility to metabolic dysfunction and type 2 diabetes in adulthood. The present study sought to strictly examine the effects of LP during the last week of gestation (LP12.5) alone as a developmental window for ß-cell programming and metabolic dysfunction in adulthood. Islet morphology analysis revealed normal ß-cell fraction in LP12.5 newborns. Normal glucose tolerance was observed in 6- to 8-wk-old male and female LP12.5 offspring. However, male LP12.5 offspring displayed glucose intolerance and reduced insulin sensitivity associated with ß-cell dysfunction with aging. High-fat diet exposure of metabolically normal 12-wk-old male LP12.5 induced glucose intolerance due to increased body weight, insulin resistance, and insufficient ß-cell mass adaptation despite higher insulin secretion. Assessment of epigenetic mechanisms through microRNAs (miRs) by a real-time PCR-based microarray in islets revealed elevation in miRs that regulate insulin secretion (miRs 342, 143), insulin resistance (miR143), and obesity (miR219). In the islets, overexpression of miR143 reduced insulin secretion in response to glucose. In contrast to the model of LP exposure throughout pregnancy, islet protein levels of mTOR and pancreatic and duodenal homeobox 1 were normal in LP12.5 islets. Collectively, these data suggest that LP diet during the last week of pregnancy is critical and sufficient to induce specific and distinct developmental programming effects of tissues that control glucose homeostasis, thus causing permanent changes in specific set of microRNAs that may contribute to the overall vulnerability of the offspring to obesity, insulin resistance, and type 2 diabetes.
Assuntos
Dieta Hiperlipídica , Resistência à Insulina/fisiologia , Células Secretoras de Insulina/metabolismo , Fenômenos Fisiológicos da Nutrição Materna/fisiologia , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Tecido Adiposo/metabolismo , Animais , Glicemia/metabolismo , Dieta com Restrição de Proteínas , Feminino , Teste de Tolerância a Glucose , Secreção de Insulina/fisiologia , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , GravidezRESUMO
Homeostatic temperature regulation is fundamental to mammalian physiology and is controlled by acute and chronic responses of local, endocrine and nervous regulators. Here, we report that loss of the heparan sulfate proteoglycan, syndecan-1, causes a profoundly depleted intradermal fat layer, which provides crucial thermogenic insulation for mammals. Mice without syndecan-1 enter torpor upon fasting and show multiple indicators of cold stress, including activation of the stress checkpoint p38α in brown adipose tissue, liver and lung. The metabolic phenotype in mutant mice, including reduced liver glycogen, is rescued by housing at thermoneutrality, suggesting that reduced insulation in cool temperatures underlies the observed phenotypes. We find that syndecan-1, which functions as a facultative lipoprotein uptake receptor, is required for adipocyte differentiation in vitro. Intradermal fat shows highly dynamic differentiation, continuously expanding and involuting in response to hair cycle and ambient temperature. This physiology probably confers a unique role for Sdc1 in this adipocyte sub-type. The PPARγ agonist rosiglitazone rescues Sdc1-/- intradermal adipose tissue, placing PPARγ downstream of Sdc1 in triggering adipocyte differentiation. Our study indicates that disruption of intradermal adipose tissue development results in cold stress and complex metabolic pathology.
Assuntos
Diferenciação Celular/genética , Proteína Quinase 14 Ativada por Mitógeno/genética , PPAR gama/genética , Estresse Fisiológico/genética , Sindecana-1/genética , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Tecido Adiposo Marrom/efeitos dos fármacos , Tecido Adiposo Marrom/metabolismo , Animais , Temperatura Baixa , Camundongos , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , PPAR gama/agonistas , PPAR gama/metabolismo , Rosiglitazona , Sindecana-1/metabolismo , Tiazolidinedionas/administração & dosagemRESUMO
In many skeletal diseases, including osteoporosis and disuse osteopenia, defective osteoblast differentiation is associated with increased marrow adipogenesis. The relative activity of two transcription factors, RUNX2 and PPARγ, controls whether a mesenchymal cell will differentiate into an osteoblast or adipocyte. Herein we show that the ERK/MAP kinase pathway, an important mediator of mechanical and hormonal signals in bone, stimulates osteoblastogenesis and inhibits adipogenesis via phosphorylation of RUNX2 and PPARγ. Induction of osteoblastogenesis in ST2 mesenchymal cells was associated with increased MAPK activity and RUNX2 phosphorylation. Under these conditions PPARγ phosphorylation also increased, but adipogenesis was inhibited. In contrast, during adipogenesis MAPK activity and phosphorylation of both transcription factors was reduced. RUNX2 phosphorylation and transcriptional activity were directly stimulated by MAPK, a response requiring phosphorylation at S301 and S319. MAPK also inhibited PPARγ-dependent transcription via S112 phosphorylation. Stimulation of MAPK increased osteoblastogenesis and inhibited adipogenesis, while dominant-negative suppression of activity had the opposite effect. In rescue experiments using Runx2(-/-) mouse embryo fibroblasts (MEFs), wild type or, to a greater extent, phosphomimetic mutant RUNX2 (S301E,S319E) stimulated osteoblastogenesis while suppressing adipogenesis. In contrast, a phosphorylation-deficient RUNX2 mutant (S301A,S319A) had reduced activity. Conversely, wild type or, to a greater extent, phosphorylation-resistant S112A mutant PPARγ strongly stimulated adipogenesis and inhibited osteoblastogenesis in Pparg(-/-) MEFs, while S112E mutant PPARγ was less active. Competition between RUNX2 and PPARγ was also observed at the transcriptional level. Together, these studies highlight the importance of MAP kinase signaling and RUNX2/PPARγ phosphorylation in the control of osteoblast and adipocyte lineages.
Assuntos
Adipogenia/fisiologia , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Osteogênese/fisiologia , PPAR gama/metabolismo , Animais , Osso e Ossos/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos , Osteoblastos/metabolismo , FosforilaçãoRESUMO
Recent literature suggests that the layer of adipocytes embedded in the skin below the dermis is far from being an inert spacer material. Instead, this layer of dermal white adipose tissue (dWAT) is a regulated lipid layer that comprises a crucial environmental defense. Among all the classes of biological molecules, lipids have the lowest thermal conductance and highest insulation potential. This property can be exploited by mammals to reduce heat loss, suppress brown adipose tissue activation, reduce the activation of thermogenic programs, and increase metabolic efficiency. Furthermore, this layer responds to bacterial challenge to provide a physical barrier and antimicrobial disinfection, and its expansion supports the growth of hair follicles and regenerating skin. In sum, this dWAT layer is a key defensive player with remarkable potential for modifying systemic metabolism, immune function, and physiology. In this review, we discuss the key literature illustrating the properties of this recently recognized adipose depot.
Assuntos
Gordura Subcutânea/fisiologia , Termogênese , Adipócitos Brancos/fisiologia , Adiposidade , Animais , Derme/fisiologia , Folículo Piloso/fisiologia , HumanosRESUMO
We have recently shown that the non-coding RNA, steroid receptor RNA activator (SRA), functions as a transcriptional coactivator of PPARγ and promotes adipocyte differentiation in vitro. To assess SRA function in vivo, we have generated a whole mouse Sra1 gene knock-out (SRA(-/-)). Here, we show that the Sra1 gene is an important regulator of adipose tissue mass and function. SRA is expressed at a higher level in adipose tissue than other organs in wild type mice. SRA(-/-) mice are resistant to high fat diet-induced obesity, with decreased fat mass and increased lean content. This lean phenotype of SRA(-/-) mice is associated with decreased expression of a subset of adipocyte marker genes and reduced plasma TNFα levels. The SRA(-/-) mice are more insulin sensitive, as evidenced by reduced fasting insulin, and lower blood glucoses in response to IP glucose and insulin. In addition, the livers of SRA(-/-) mice have fewer lipid droplets after high fat diet feeding, and the expression of lipogenesis-associated genes is decreased. To our knowledge, these data are the first to indicate a functional role for SRA in adipose tissue biology and glucose homeostasis in vivo.
Assuntos
Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Glicemia/metabolismo , Gorduras na Dieta/efeitos adversos , Obesidade/metabolismo , RNA Longo não Codificante/metabolismo , Adipócitos/patologia , Tecido Adiposo/patologia , Animais , Glicemia/genética , Gorduras na Dieta/farmacologia , Homeostase/genética , Camundongos , Camundongos Knockout , Obesidade/induzido quimicamente , Obesidade/genética , Obesidade/patologia , RNA Longo não Codificante/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Mammalian embryos have evolved to adjust their organ and tissue development in response to an atypical environment. This adaptation, called phenotypic plasticity, allows the organism to thrive in the anticipated environment in which the fetus will emerge. Barker and colleagues proposed that if the environment in which the fetus emerges differs from that in which it develops, phenotypic plasticity may provide an underlying mechanism for disease. Epidemiological studies have shown that humans born small- or large-for-gestational-age, have a higher likelihood of developing obesity as adults. The amount and quality of food that the mother consumes during gestation influences birth weight, and therefore susceptibility of progeny to disease in later life. Studies in experimental animals support these observations, and find that obesity occurs as a result of maternal nutrient-restriction during gestation, followed by rapid compensatory growth associated with ad libitum food consumption. Therefore, obesity associated with maternal nutritional restriction has a developmental origin. Based on this phenomenon, one might predict that gestational exposure to a westernized diet would protect against future obesity in offspring. However, evidence from experimental models indicates that, like maternal dietary restriction, maternal consumption of a westernized diet during gestation and lactation interacts with an adult obesogenic diet to induce further obesity. Mechanistically, restriction of nutrients or consumption of a high fat diet during gestation may promote obesity in progeny by altering hypothalamic neuropeptide production and thereby increasing hyperphagia in offspring. In addition to changes in food intake these animals may also direct energy from muscle toward storage in adipose tissue. Surprisingly, generational inheritance studies in rodents have further indicated that effects on body length, body weight, and glucose tolerance appear to be propagated to subsequent generations. Together, the findings discussed herein highlight the concept that maternal nutrition contributes to a legacy of obesity. Thus, ensuring adequate supplies of a complete and balanced diet during and after pregnancy should be a priority for public health worldwide. This article is part of a Special Issue entitled: Modulation of Adipose Tissue in Health and Disease.
Assuntos
Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Fenômenos Fisiológicos da Nutrição Materna , Obesidade/metabolismo , Adulto , Animais , Dieta Hiperlipídica , Embrião de Mamíferos/fisiologia , Feminino , Humanos , Obesidade/etiologia , Obesidade/patologia , GravidezRESUMO
Obesity has been associated with high bone mineral density (BMD) but a greater propensity to fracture. Some obese individuals have increased marrow adipose tissue (MAT), but the impact of MAT on bone turnover remains controversial, as do changes in BMD associated with a high fat diet (HFD). In this study we hypothesized that MAT volume would increase in response to HFD but would be independent of changes in BMD. Hence, we fed C57BL/6J (B6) male mice at 3 weeks of age either a high fat diet (60 kcal %) or regular diet (10 kcal %) for 12 weeks (n = 10/group). We measured MAT volume by osmium staining and micro-CT (µCT) as well as bone parameters by µCT, histomorphometry, and dual-energy X-ray absorptiometry. We also performed a short-term pilot study using 13-week-old B6 males and females fed a HFD (58 kcal %) for 2 weeks (n = 3/sex). Both long- and short-term HFD feedings were associated with high MAT volume, however, femoral trabecular bone volume fraction (BV/TV), bone formation rate and cortical bone mass were not altered in the long-term study. In the short-term pilot study, areal BMD was unchanged after 2 weeks of HFD. We conclude that, for B6 mice fed a HFD starting at wean or 13 weeks of age, MAT increases whereas bone mass is not altered. More studies are needed to define the mechanism responsible for the rapid storage of energy in the marrow and its distinction from other adipose depots.
Assuntos
Tecido Adiposo/crescimento & desenvolvimento , Medula Óssea/metabolismo , Dieta Hiperlipídica/efeitos adversos , Obesidade/metabolismo , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , Densidade Óssea/efeitos dos fármacos , Medula Óssea/efeitos dos fármacos , Medula Óssea/patologia , Metabolismo Energético , Feminino , Fêmur/efeitos dos fármacos , Fêmur/crescimento & desenvolvimento , Humanos , Leptina/metabolismo , Masculino , Camundongos , Obesidade/patologia , Osteogênese/efeitos dos fármacosRESUMO
G protein-coupled receptors mediate responses to a myriad of ligands, some of which regulate adipocyte differentiation and metabolism. The sweet taste receptors T1R2 and T1R3 are G protein-coupled receptors that function as carbohydrate sensors in taste buds, gut, and pancreas. Here we report that sweet taste receptors T1R2 and T1R3 are expressed throughout adipogenesis and in adipose tissues. Treatment of mouse and human precursor cells with artificial sweeteners, saccharin and acesulfame potassium, enhanced adipogenesis. Saccharin treatment of 3T3-L1 cells and primary mesenchymal stem cells rapidly stimulated phosphorylation of Akt and downstream targets with functions in adipogenesis such as cAMP-response element-binding protein and FOXO1; however, increased expression of peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding protein α was not observed until relatively late in differentiation. Saccharin-stimulated Akt phosphorylation at Thr-308 occurred within 5 min, was phosphatidylinositol 3-kinase-dependent, and occurred in the presence of high concentrations of insulin and dexamethasone; phosphorylation of Ser-473 occurred more gradually. Surprisingly, neither saccharin-stimulated adipogenesis nor Thr-308 phosphorylation was dependent on expression of T1R2 and/or T1R3, although Ser-473 phosphorylation was impaired in T1R2/T1R3 double knock-out precursors. In mature adipocytes, artificial sweetener treatment suppressed lipolysis even in the presence of forskolin, and lipolytic responses were correlated with phosphorylation of hormone-sensitive lipase. Suppression of lipolysis by saccharin in adipocytes was also independent of T1R2 and T1R3. These results suggest that some artificial sweeteners have previously uncharacterized metabolic effects on adipocyte differentiation and metabolism and that effects of artificial sweeteners on adipose tissue biology may be largely independent of the classical sweet taste receptors, T1R2 and T1R3.
Assuntos
Adipócitos/metabolismo , Adipogenia/efeitos dos fármacos , Lipólise/efeitos dos fármacos , Receptores Acoplados a Proteínas G/metabolismo , Sacarina/farmacologia , Células-Tronco/metabolismo , Edulcorantes/efeitos adversos , Células 3T3-L1 , Adipogenia/genética , Adjuvantes Imunológicos/farmacologia , Animais , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Diferenciação Celular/efeitos dos fármacos , Colforsina/farmacologia , AMP Cíclico/genética , AMP Cíclico/metabolismo , Feminino , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Humanos , Lipólise/genética , Masculino , Camundongos , Pessoa de Meia-Idade , PPAR gama/genética , PPAR gama/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Acoplados a Proteínas G/genética , Esterol Esterase/genética , Esterol Esterase/metabolismo , Edulcorantes/farmacocinéticaRESUMO
Differentiation of adipocytes from preadipocytes contributes to adipose tissue expansion in obesity. Impaired adipogenesis may underlie the development of metabolic diseases such as insulin resistance and type 2 diabetes. Mechanistically, a well defined transcriptional network coordinates adipocyte differentiation. The family of paired-related homeobox transcription factors, which includes Prrx1a, Prrx1b, and Prrx2, is implicated with regulation of mesenchymal cell fate, including myogenesis and skeletogenesis; however, whether these proteins impact adipogenesis remains to be addressed. In this study, we identify Prrx1a and Prrx1b as negative regulators of adipogenesis. We show that Prrx1a and Prrx1b are down-regulated during adipogenesis in vitro and in vivo. Stable knockdown of Prrx1a/b enhances adipogenesis, with increased expression of peroxisome proliferator-activated receptor-γ, CCAAT/enhancer-binding protein-α and FABP4 and increased secretion of the adipokines adiponectin and chemerin. Although stable low-level expression of Prrx1a, Prrx1b, or Prrx2 does not affect 3T3-L1 adipogenesis, transient overexpression of Prrx1a or Prrx1b inhibits peroxisome proliferator-activated receptor-γ activity. Prrx1 knockdown decreases expression of Tgfb2 and Tgfb3, and inhibition of TGFß signaling during adipogenesis mimics the effects of Prrx1 knockdown. These data support the hypothesis that endogenous Prrx1 restrains adipogenesis by regulating expression of TGFß ligands and thereby activating TGFß signaling. Finally, we find that expression of Prrx1a or Prrx1b in adipose tissue increases during obesity and strongly correlates with Tgfb3 expression in BL6 mice. These observations suggest that increased Prrx1 expression may promote TGFß activity in adipose tissue and thereby contribute to aberrant adipocyte function during obesity.
Assuntos
Adipogenia , Proteínas de Homeodomínio/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Células 3T3-L1 , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Adipogenia/efeitos dos fármacos , Adipogenia/genética , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Técnicas de Silenciamento de Genes , Proteínas de Homeodomínio/genética , Humanos , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/genética , PPAR gama/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Especificidade da Espécie , Fatores de Transcrição/genética , Fator de Crescimento Transformador beta3/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Proteína Wnt3A/metabolismoRESUMO
Adipocyte differentiation consists of a complex series of events in which scores of cellular and extracellular factors interact to transform a fibroblast-like preadipocyte into a mature, lipid-filled adipocyte. Many of the pathways influencing this process have been identified using well-characterized preadipocyte culture systems and have subsequently been confirmed in animal models. Research conducted over the past decade has established the Wnt/beta-catenin signaling pathway as an important regulator of adipocyte differentiation. While initial reports implicated activators of Wnt/beta-catenin signaling as potent inhibitors of adipogenesis, recent investigations of mesenchymal cell fate, obesity, and type 2 diabetes highlight significant additional roles for Wnt signaling in metabolism and adipocyte biology.
Assuntos
Adipogenia/fisiologia , Tecido Adiposo/metabolismo , Transdução de Sinais/fisiologia , Proteínas Wnt/fisiologia , beta Catenina/fisiologia , Animais , Diferenciação Celular , HumanosRESUMO
Microfluidics has enabled new cell biology experiments. Incorporating chemical monitoring of cellular secretion into chips offers the potential to increase information content and utility of such systems. In this work, an integrated, multilayer polydimethylsiloxane microfluidic chip was developed to simultaneously measure fatty acids and glycerol secreted from cultured adipocytes on chip in near real time. Approximately 48,000 adipocytes were loaded into a cell chamber in a reversibly sealed chip. Cells were perfused at 0.75 µL/min. Cell perfusate was split and directed to separate, continuously operating fluorescent enzyme assay channel networks. The fluorescent assay products were detected simultaneously near the outlet of the chip. The fatty acid and glycerol assays had linear dynamic ranges of 150 and 110 µM and limit of detection (LOD) of 6 and 5 µM, respectively. Surface modifications including pretreatment with sodium dodecyl sulfate were utilized to prevent adsorption of fatty acids to the chip surface. Using the chip, basal fatty acid and glycerol concentrations ranged from 0.18 to 0.7 nmol × 10(6) cell(-1) min(-1) and from 0.23 to 0.85 nmol × 10(6) cell(-1) min(-1), respectively. Using valves built into the chip, the perfusion solution was switched to add 20 µM isoproterenol, a ß-adrenergic agonist, which stimulates the release of glycerol and fatty acids in adipocytes. This manipulation resulted in a rapid and stable 1.5- to 6.0-fold increase of non-esterified fatty acid (NEFA) and glycerol. The ratio of NEFA:glycerol released increased with adipocyte age. These experiments illustrate the potential for performing multiple real-time assays on cells in culture using microfluidic devices.
Assuntos
Adipócitos/metabolismo , Ensaios Enzimáticos/instrumentação , Ácidos Graxos/metabolismo , Glicerol/metabolismo , Lipólise/fisiologia , Técnicas Analíticas Microfluídicas/instrumentação , Células 3T3-L1 , Animais , Ensaios Enzimáticos/métodos , Fluorescência , Camundongos , Técnicas Analíticas Microfluídicas/métodosRESUMO
Skin has been shown to be a regulatory hub for energy expenditure and metabolism: mutations of skin lipid metabolism enzymes can change the rate of thermogenesis and susceptibility to diet-induced obesity. However, little is known about the physiological basis for this function. Here we show that the thermal properties of skin are highly reactive to diet: within three days, a high fat diet reduces heat transfer through skin. In contrast, a dietary manipulation that prevents obesity accelerates energy loss through skins. We found that skin was the largest target in a mouse body for dietary fat delivery, and that fat was assimilated both by epidermis and by dermal white adipose tissue. Dietary triglyceride acyl groups persist in skin for weeks after feeding. Using multi-modal lipid profiling, we have implicated both keratinocytes and sebocytes in the altered lipids which correlate with thermal function. In response to high fat feeding, wax diesters and ceramides accumulate, and triglycerides become more saturated. In contrast, in response to the dramatic loss of adipose tissue that accompanies restriction of the branched chain amino acid isoleucine, skin becomes highly heat-permeable: skins shows limited uptake of dietary lipids and editing of wax esters, and acquires a signature of depleted signaling lipids, which include the acyl carnitines and lipid ethers. We propose that skin should be routinely included in physiological studies of lipid metabolism, given the size of the skin lipid reservoir and its adaptable functionality.
RESUMO
Introduction: Unlike white adipose tissue depots, bone marrow adipose tissue (BMAT) expands during caloric restriction (CR). Although mechanisms for BMAT expansion remain unclear, prior research suggested an intermediary role for increased circulating glucocorticoids. Methods: In this study, we utilized a recently described mouse model (BMAd-Cre) to exclusively target bone marrow adipocytes (BMAds) for elimination of the glucocorticoid receptor (GR) (i.e. Nr3c1) whilst maintaining GR expression in other adipose depots. Results: Mice lacking GR in BMAds (BMAd-Nr3c1 -/-) and control mice (BMAd-Nr3c1 +/+) were fed ad libitum or placed on a 30% CR diet for six weeks. On a normal chow diet, tibiae of female BMAd-Nr3c1-/- mice had slightly elevated proximal trabecular metaphyseal bone volume fraction and thickness. Both control and BMAd-Nr3c1-/- mice had increased circulating glucocorticoids and elevated numbers of BMAds in the proximal tibia following CR. However, no significant differences in trabecular and cortical bone were observed, and quantification with osmium tetroxide and µCT revealed no difference in BMAT accumulation between control or BMAd-Nr3c1 -/- mice. Differences in BMAd size were not observed between BMAd-Nr3c1-/- and control mice. Interestingly, BMAd-Nr3c1-/- mice had decreased circulating white blood cell counts 4 h into the light cycle. Discussion: In conclusion, our data suggest that eliminating GR from BMAd has minor effects on bone and hematopoiesis, and does not impair BMAT accumulation during CR.