RESUMO
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
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
Recently, lipid nanoparticles (LNPs) have attracted attention due to their emergent use for COVID-19 mRNA vaccines. The success of LNPs can be attributed to ionizable lipids, which enable functional intracellular delivery. Previously, the authors established an automated high-throughput platform to screen ionizable lipids and identified that the LNPs generated using this automated technique show comparable or increased mRNA functional delivery in vitro as compared to LNPs prepared using traditional microfluidics techniques. In this study, the authors choose one benchmark lipid, DLin-MC3-DMA (MC3), and investigate whether the automated formulation technique can enhance mRNA functional delivery in vivo. Interestingly, a 4.5-fold improvement in mRNA functional delivery in vivo by automated LNPs as compared to LNPs formulated by conventional microfluidics techniques, is observed. Mechanistic studies reveal that particles with large size accommodate more mRNA per LNP, possess more hydrophobic surface, are more hemolytic, bind a larger protein corona, and tend to accumulate more in macropinocytosomes, which may quantitatively benefit mRNA cytosolic delivery. These data suggest that mRNA loading per particle is a critical factor that accounts for the enhanced mRNA functional delivery of automated LNPs. These mechanistic findings provide valuable insight underlying the enhanced mRNA functional delivery to accelerate future mRNA LNP product development.
Assuntos
COVID-19 , Nanopartículas , Humanos , Lipossomos , Nanopartículas/química , RNA Mensageiro/química , SARS-CoV-2RESUMO
Peptide based nanomaterials have been gaining increased prominence due to their ability to form permeable scaffolds that promote growth and regeneration of new tissue. In this work for the first time a short hexapeptide motif VQIVYK, derived from the Tau protein family was conjugated with an organic polyamine linker, putrescine and utilized as a template for developing new materials for cartilage tissue regeneration. Our results showed that the conjugate formed extensive nanofibrous assemblies upon self-assembly under aqueous conditions. We then employed the layer-by-layer (LBL) approach to design the scaffold by first incorporating a short segment of the dentin sialophosphoprotein motif GDASYNSDESK followed by integration with the peptide sequence GSGAGAGSGAGAGSGAGA. This sequence mimics Ala, Gly, Ser repeats seen in the spider silk protein. We then incorporated the polysaccharide alginate which served as a hydrogel. To further enhance binding interactions with chondrocytes, and promote the formation of cartilage in vitro, the bionanocomposites were then attached to the chondrocyte binding peptide sequence HDSQLEALIKFM. The thermal properties as well as biodegradability of the scaffold was examined. To confirm biocompatibility, we examined cell viability, attachment and morphology in the presence of bovine chondrocytes. The cells were found to efficiently adhere to the scaffolds which formed an intricate mesh mimicking the extracellular matrix of cartilage tissue. To evaluate if differentiation occurred in the presence of the scaffolds, we examined in vitro deposition of proteoglycans. Thus, we have developed a new family of nanoscale scaffolds that may be utilized for cartilage tissue regeneration.
Assuntos
Alginatos/farmacologia , Cartilagem/efeitos dos fármacos , Peptídeos/farmacologia , Regeneração , Alginatos/química , Sequência de Aminoácidos , Animais , Cartilagem/fisiologia , Bovinos , Células Cultivadas , Ácido Glucurônico/química , Ácido Glucurônico/farmacologia , Ácidos Hexurônicos/química , Ácidos Hexurônicos/farmacologia , Microscopia Eletrônica de Varredura , Peptídeos/química , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
mRNA lipid nanoparticles (LNPs) are at the forefront of nucleic acid intracellular delivery, as exemplified by the recent emergency approval of two mRNA LNP-based COVID-19 vaccines. The success of an LNP product largely depends on the systematic optimisation of the four lipidic components, namely the ionisable lipid, PEG lipid, structural and helper lipids. However, the in vitro screening of novel lipidic components and LNP compositions is limited by the low-throughput of LNP preparation. To address these issues, we herein present an automated high-throughput screening platform to select novel ionisable lipids and corresponding LNPs encapsulating mRNA in vitro. This high-throughput platform employs a lab-based automated liquid handling system, amenable to high-throughput (up to 384 formulations per plate and several plates per run) and allows precise mixing and reproducible mRNA LNP preparation which ensures a direct head-to-head comparison of hundreds and even thousands of novel LNPs. Most importantly, the robotic process has been successfully applied to the screening of novel LNPs encapsulating mRNA and has identified the same novel mRNA LNP leads as those from microfluidics-mixing technology, with a correlation coefficient of 0.8751. This high-throughput platform can facilitate to narrow down the number of novel ionisable lipids to be evaluated in vivo. Moreover, this platform has been integrated into a fully-automated workflow for LNP property control, physicochemical characterisation and biological evaluation. The high-throughput platform may accelerate proprietary lipid development, mRNA LNP lead optimisation and candidate selection to advance preclinical mRNA LNP development to meet urgent global needs.
Assuntos
Vacinas contra COVID-19/administração & dosagem , COVID-19 , Nanopartículas , Vacinas Sintéticas/administração & dosagem , Vacinas de mRNA/administração & dosagem , COVID-19/prevenção & controle , Humanos , Lipossomos , RNA Interferente PequenoRESUMO
To investigate roles for bone marrow adipocyte (BMAd) lipolysis in bone homeostasis, we created a BMAd-specific Cre mouse model in which we knocked out adipose triglyceride lipase (ATGL, Pnpla2 gene). BMAd-Pnpla2-/- mice have impaired BMAd lipolysis, and increased size and number of BMAds at baseline. Although energy from BMAd lipid stores is largely dispensable when mice are fed ad libitum, BMAd lipolysis is necessary to maintain myelopoiesis and bone mass under caloric restriction. BMAd-specific Pnpla2 deficiency compounds the effects of caloric restriction on loss of trabecular bone in male mice, likely due to impaired osteoblast expression of collagen genes and reduced osteoid synthesis. RNA sequencing analysis of bone marrow adipose tissue reveals that caloric restriction induces dramatic elevations in extracellular matrix organization and skeletal development genes, and energy from BMAd is required for these adaptations. BMAd-derived energy supply is also required for bone regeneration upon injury, and maintenance of bone mass with cold exposure.
Assuntos
Medula Óssea , Lipólise , Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Animais , Medula Óssea/metabolismo , Lipase/metabolismo , Lipólise/genética , Masculino , CamundongosRESUMO
Gene transfer using viral or nonviral vectors enables the ability to manipulate specific cells and tissues for gene silencing, protein overexpression, or genome modification. Despite the widespread application of viral- and non-viral-mediated gene transfer to liver, heart, skeletal muscle, and the central nervous system, its use in adipose tissue has been limited. This is largely because adipose tissue is distributed throughout the body in distinct depots and adipocytes make up a minority of the cells within the tissue, making transduction difficult. Currently, there is no consensus methodology for efficient gene transfer to adipose tissue and many studies report conflicting information with regard to transduction efficiency and vector biodistribution. In this review, we summarize the challenges associated with gene transfer to adipose tissue and report on innovations that improve efficacy. We describe how vector and route of administration are the two key factors that influence transduction efficiency and outline a "gold standard" approach and experimental workflow for validating gene transfer to adipose tissue. Lastly, we speculate on how CRISPR/Cas9 can be integrated to improve adipose tissue research.
Assuntos
Tecido Adiposo/fisiologia , Técnicas de Transferência de Genes , Vetores Genéticos , Animais , Terapia Genética , Humanos , VírusRESUMO
OBJECTIVE: Obesity is a key risk factor for many secondary chronic illnesses, including type 2 diabetes and cardiovascular disease. Canonical Wnt/ß-catenin signaling is established as an important endogenous inhibitor of adipogenesis. This pathway is operative in mature adipocytes; however, its roles in this context remain unclear due to complexities of Wnt signaling and differences in experimental models. In this study, we used novel cultured cell and mouse models to investigate functional roles of Wnts secreted from adipocytes. METHODS: We generated adipocyte-specific Wntless (Wls) knockout mice and cultured cell models to investigate molecular and metabolic consequences of disrupting Wnt secretion from mature adipocytes. To characterize Wls-deficient cultured adipocytes, we evaluated the expression of Wnt target and lipogenic genes and the downstream functional effects on carbohydrate and lipid metabolism. We also investigated the impact of adipocyte-specific Wls deletion on adipose tissues and global glucose metabolism in mice fed normal chow or high-fat diets. RESULTS: Many aspects of the Wnt signaling apparatus are expressed and operative in mature adipocytes, including the Wnt chaperone Wntless. Deletion of Wntless in cultured adipocytes results in the inhibition of de novo lipogenesis and lipid monounsaturation, likely through repression of Srebf1 (SREBP1c) and Mlxipl (ChREBP) and impaired cleavage of immature SREBP1c into its active form. Adipocyte-specific Wls knockout mice (Wls-/-) have lipogenic gene expression in adipose tissues and isolated adipocytes similar to that of controls when fed a normal chow diet. However, closer investigation reveals that a subset of Wnts and downstream signaling targets are upregulated within stromal-vascular cells of Wls-/- mice, suggesting that adipose tissues defend loss of Wnt secretion from adipocytes. Interestingly, this compensation is lost with long-term high-fat diet challenges. Thus, after six months of a high-fat diet, Wls-/- mice are characterized by decreased adipocyte lipogenic gene expression, reduced visceral adiposity, and improved glucose homeostasis. CONCLUSIONS: Taken together, these studies demonstrate that adipocyte-derived Wnts regulate de novo lipogenesis and lipid desaturation and coordinate the expression of lipogenic genes in adipose tissues. In addition, we report that Wnt signaling within adipose tissues is defended, such that a loss of Wnt secretion from adipocytes is sensed and compensated for by neighboring stromal-vascular cells. With chronic overnutrition, this compensatory mechanism is lost, revealing that Wls-/- mice are resistant to diet-induced obesity, adipocyte hypertrophy, and metabolic dysfunction.
Assuntos
Adipócitos/metabolismo , Regulação da Expressão Gênica , Lipogênese/genética , Doenças Metabólicas/etiologia , Doenças Metabólicas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Biomarcadores , Células Cultivadas , Dieta/efeitos adversos , Modelos Animais de Doenças , Suscetibilidade a Doenças , Glucose/metabolismo , Imuno-Histoquímica , Insulina/metabolismo , Metabolismo dos Lipídeos/genética , Doenças Metabólicas/diagnóstico , Camundongos , Camundongos Knockout , Receptores Acoplados a Proteínas G/genética , Via de Sinalização WntRESUMO
Bariatric surgeries are integral to the management of obesity and its metabolic complications. However, these surgeries cause bone loss and increase fracture risk through poorly understood mechanisms. In a mouse model, vertical sleeve gastrectomy (VSG) caused trabecular and cortical bone loss that was independent of sex, body weight, and diet, and this loss was characterized by impaired osteoid mineralization and bone formation. VSG had a profound effect on the bone marrow niche, with rapid loss of marrow adipose tissue, and expansion of myeloid cellularity, leading to increased circulating neutrophils. Following VSG, circulating granulocyte-colony stimulating factor (G-CSF) was increased in mice, and was transiently elevated in a longitudinal study of humans. Elevation of G-CSF was found to recapitulate many effects of VSG on bone and the marrow niche. In addition to stimulatory effects of G-CSF on myelopoiesis, endogenous G-CSF suppressed development of marrow adipocytes and hindered accrual of peak cortical and trabecular bone. Effects of VSG on induction of neutrophils and depletion of marrow adiposity were reduced in mice deficient for G-CSF; however, bone mass was not influenced. Although not a primary mechanism for bone loss with VSG, G-CSF plays an intermediary role for effects of VSG on the bone marrow niche.
Assuntos
Adipócitos/metabolismo , Células da Medula Óssea/metabolismo , Reabsorção Óssea/sangue , Gastroplastia , Fator Estimulador de Colônias de Granulócitos/sangue , Obesidade/sangue , Complicações Pós-Operatórias/sangue , Adipócitos/patologia , Adolescente , Adulto , Animais , Medula Óssea/patologia , Células da Medula Óssea/patologia , Reabsorção Óssea/etiologia , Reabsorção Óssea/genética , Reabsorção Óssea/patologia , Feminino , Gastrectomia , Humanos , Estudos Longitudinais , Camundongos , Camundongos Knockout , Obesidade/genética , Obesidade/patologia , Obesidade/cirurgia , Complicações Pós-Operatórias/genética , Complicações Pós-Operatórias/patologiaRESUMO
In this work we have developed a new family of biocomposite scaffolds for bone tissue regeneration by utilizing self-assembled fluorenylmethyloxycarbonyl protected Valyl-cetylamide (FVC) nanoassemblies as templates. To tailor the assemblies for enhanced osteoblast attachment and proliferation, we incorporated (a) Type I collagen, (b) a hydroxyapatite binding peptide sequence (EDPHNEVDGDK) derived from dentin sialophosphoprotein and (c) the osteoinductive bone morphogenetic protein-4 (BMP-4) to the templates by layer-by-layer assembly. The assemblies were then incubated with hydroxyapatite nanocrystals blended with varying mass percentages of TiO2 nanoparticles and coated with alginate to form three dimensional scaffolds for potential applications in bone tissue regeneration. The morphology was examined by TEM and SEM and the binding interactions were probed by FITR spectroscopy. The scaffolds were found to be non-cytotoxic, adhered to mouse preosteoblast MC3T3-E1 cells and promoted osteogenic differentiation as indicated by the results obtained by alkaline phosphatase assay. Furthermore, they were found to be biodegradable and possessed inherent antibacterial capability. Thus, we have developed a new family of tissue-engineered biocomposite scaffolds with potential applications in bone regeneration.