RESUMO
A substantial body of evidence has established the contributions of both mitochondrial dynamics and lipid metabolism to the pathogenesis of diabetic kidney disease (DKD). However, the precise interplay between these two key metabolic regulators of DKD is not fully understood. Here, we uncover a link between mitochondrial dynamics and lipid metabolism by investigating the role of carbohydrate-response element-binding protein (ChREBP), a glucose-responsive transcription factor and a master regulator of lipogenesis, in kidney podocytes. We find that inducible podocyte-specific knockdown of ChREBP in diabetic db/db mice improves key biochemical and histological features of DKD in addition to significantly reducing mitochondrial fragmentation. Because of the critical role of ChREBP in lipid metabolism, we interrogated whether and how mitochondrial lipidomes play a role in ChREBP-mediated mitochondrial fission. Our findings suggest a key role for a family of ether phospholipids in ChREBP-induced mitochondrial remodeling. We find that overexpression of glyceronephosphate O-acyltransferase, a critical enzyme in the biosynthesis of plasmalogens, reverses the protective phenotype of ChREBP deficiency on mitochondrial fragmentation. Finally, our data also points to Gnpat as a direct transcriptional target of ChREBP. Taken together, our results uncover a distinct mitochondrial lipid signature as the link between ChREBP-induced mitochondrial dynamics and progression of DKD.
Assuntos
Diabetes Mellitus , Nefropatias Diabéticas , Animais , Camundongos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Diabetes Mellitus/metabolismo , Nefropatias Diabéticas/metabolismo , Regulação da Expressão Gênica , Rim/metabolismo , Lipidômica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Long noncoding RNAs (lncRNAs) have been shown to play key roles in a variety of biological activities of the cell. However, less is known about how lncRNAs respond to environmental cues and what transcriptional mechanisms regulate their expression. Studies from our laboratory have shown that the lncRNA Tug1 (taurine upregulated gene 1) is crucial for the progression of diabetic kidney disease, a major microvascular complication of diabetes. Using a combination of proximity labeling with the engineered soybean ascorbate peroxidase (APEX2), ChIP-qPCR, biotin-labeled oligonucleotide pulldown, and classical promoter luciferase assays in kidney podocytes, we extend our initial observations in the current study and now provide a detailed analysis on a how high-glucose milieu downregulates Tug1 expression in podocytes. Our results revealed an essential role for the transcription factor carbohydrate response element binding protein (ChREBP) in controlling Tug1 transcription in the podocytes in response to increased glucose levels. Along with ChREBP, other coregulators, including MAX dimerization protein (MLX), MAX dimerization protein 1 (MXD1), and histone deacetylase 1 (HDAC1), were enriched at the Tug1 promoter under high-glucose conditions. These observations provide the first characterization of the mouse Tug1 promoter's response to the high-glucose milieu. Our findings illustrate a molecular mechanism by which ChREBP can coordinate glucose homeostasis with the expression of the lncRNA Tug1 and further our understanding of dynamic transcriptional regulation of lncRNAs in a disease state.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Regulação da Expressão Gênica , Glucose/metabolismo , Podócitos/metabolismo , RNA Longo não Codificante/biossíntese , Transcrição Gênica , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Linhagem Celular Tumoral , Glucose/genética , Histona Desacetilase 1/genética , Histona Desacetilase 1/metabolismo , Humanos , Camundongos , RNA Longo não Codificante/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismoRESUMO
Dendritic cells (DCs) have the striking ability to cross-present exogenous antigens in association with major histocompatibility complex (MHC) class I to CD8(+) T cells. However, the intracellular pathways underlying cross-presentation remain ill defined. Current models involve cytosolic proteolysis of antigens by the proteasome and peptide import into endoplasmic reticulum (ER) or phagosomal lumen by the transporters associated with antigen processing (TAP1 and TAP2). Here, we show that DCs expressed an ER-resident 47 kDa immune-related GTPase, Igtp (Irgm3). Igtp resides on ER and lipid body (LB) membranes where it binds the LB coat component ADFP. Inactivation of genes encoding for either Igtp or ADFP led to defects in LB formation in DCs and severely impaired cross-presentation of phagocytosed antigens to CD8(+) T cells but not antigen presentation to CD4(+) T cells. We thus define a new role for LB organelles in regulating cross-presentation of exogenous antigens to CD8(+) T lymphocytes in DCs.
Assuntos
Apresentação de Antígeno/imunologia , Apresentação Cruzada , Células Dendríticas/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Lipídeos/imunologia , Fagocitose , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Retículo Endoplasmático/imunologia , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/imunologia , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Perilipina-2RESUMO
While increased mitochondrial reactive oxygen species have been commonly implicated in a variety of disease states, their in vivo role in the pathogenesis of diabetic nephropathy remains controversial. Using a two-photon imaging approach with a genetically encoded redox biosensor, we monitored mitochondrial redox state in the kidneys of experimental models of diabetes in real-time in vivo. Diabetic (db/db) mice that express a redox-sensitive Green Fluorescent Protein biosensor (roGFP) specifically in the mitochondrial matrix (db/dbmt-roGFP) were generated, allowing dynamic monitoring of redox changes in the kidneys. These db/dbmt-roGFP mice exhibited a marked increase in mitochondrial reactive oxygen species in the kidneys. Yeast NADH-dehydrogenase, a mammalian Complex I homolog, was ectopically expressed in cultured podocytes, and this forced expression in roGFP-expressing podocytes prevented high glucose-induced increases in mitochondrial reactive oxygen species. Thus, in vivo monitoring of mitochondrial roGFP in diabetic mice confirms increased production of mitochondrial reactive oxygen species in the kidneys.
Assuntos
Diabetes Mellitus Tipo 2/patologia , Nefropatias Diabéticas/patologia , Rim/patologia , Mitocôndrias/patologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Técnicas Biossensoriais , Células Cultivadas , Diabetes Mellitus Tipo 2/complicações , Nefropatias Diabéticas/etiologia , Modelos Animais de Doenças , Proteínas de Fluorescência Verde/genética , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Microscopia de Fluorescência por Excitação Multifotônica , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Oxirredução , PodócitosRESUMO
Mitochondrial fission has been linked to the pathogenesis of diabetic nephropathy (DN). However, how mitochondrial fission affects progression of DN in vivo is unknown. Here, we report the effect of conditional podocyte-specific deletion of dynamin-related protein 1 (Drp1), an essential component of mitochondrial fission, on the pathogenesis and progression of DN. Inducible podocyte-specific deletion of Drp1 in diabetic mice decreased albuminuria and improved mesangial matrix expansion and podocyte morphology. Ultrastructure analysis revealed a significant increase in fragmented mitochondria in the podocytes of wild-type diabetic mice but a marked improvement in mitochondrial structure in Drp1-null podocytes of diabetic mice. When isolated from diabetic mice and cultured in high glucose, Drp1-null podocytes had more elongated mitochondria and better mitochondrial fitness associated with enhanced oxygen consumption and ATP production than wild-type podocytes. Furthermore, administration of a pharmacologic inhibitor of Drp1, Mdivi1, significantly blunted mitochondrial fission and rescued key pathologic features of DN in mice. Taken together, these results provide novel correlations between mitochondrial morphology and the progression of DN and point to Drp1 as a potential therapeutic target in DN.
Assuntos
Nefropatias Diabéticas/etiologia , Dinaminas/deficiência , Dinâmica Mitocondrial , Animais , Nefropatias Diabéticas/prevenção & controle , Progressão da Doença , Dinaminas/antagonistas & inibidores , Masculino , Camundongos , Camundongos Endogâmicos C57BL , PodócitosRESUMO
BACKGROUND: The resistance profiles for patients on first-line antiretroviral therapy (ART) regimens after viremia have not been well studied in community clinic settings in the modern treatment era. OBJECTIVE: To determine time to viremia and the ART resistance profiles of viremic patients. METHODS: HIV-positive patients aged ≥16 years initiating a three-drug regimen were retrospectively identified from 01/01/06 to 12/31/12. The regimens were a backbone of two nucleoside reverse transcriptase inhibitors (NRTIs) and a third agent: a protease inhibitor (PI), non-nucleoside reverse transcriptase inhibitor (NNRTI), or an integrase inhibitor (II). Time to viremia was compared using a proportional hazards model, adjusting for demographic and clinical factors. Resistance profiles were described in those with baseline and follow-up genotypes. RESULTS: For 653 patients, distribution of third-agent use and viremia was: 244 (37%) on PIs with 80 viremia, 364 (56%) on NNRTIs with 84 viremia, and 45 (7%) on II with 11 viremia. Only for NNRTIs, time to viremia was longer than PIs (p = 0.04) for patients with a CD4 count ≥200 cells/mm(3). Of the 175 with viremia, 143 (82%) had baseline and 37 (21%) had follow-up genotype. Upon viremia, emerging ART resistance was rare. One new NNRTI (Y181C) mutation was identified and three patients taking PI-based regimens developed NRTI mutations (M184 V, M184I, and T215Y). CONCLUSIONS: Time to viremia for NNRTIs was longer than PIs. With viremia, ART resistance rarely developed without PI or II mutations, but with a few NRTI mutations in those taking PI-based regimens, and NNRTI mutations in those taking NNRTI-based regimens.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Farmacorresistência Viral , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Viremia , Adulto , Fármacos Anti-HIV/administração & dosagem , Feminino , Infecções por HIV/sangue , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Carga ViralRESUMO
MondoA is a basic helix-loop-helix (bHLH)/leucine zipper (ZIP) transcription factor that is expressed predominantly in skeletal muscle. Studies in vitro suggest that the Max-like protein X (MondoA:Mlx) heterodimer senses the intracellular energy status and directly targets the promoter region of thioredoxin interacting protein (Txnip) and possibly glycolytic enzymes. We generated MondoA-inactivated (MondoA-/-) mice by gene targeting. MondoA-/- mice had normal body weight at birth, exhibited normal growth and appeared to be healthy. However, they exhibited unique metabolic characteristics. MondoA-/- mice built up serum lactate and alanine levels and utilized fatty acids for fuel during exercise. Gene expression and promoter analysis suggested that MondoA functionally represses peroxisome-proliferator-activated receptor γ co-activator-1α (PGC-1α)-mediated activation of pyruvate dehydrogenase kinase 4 (PDK-4) transcription. PDK4 normally down-regulates the activity of pyruvate dehydrogenase, an enzyme complex that catalyses the decarboxylation of pyruvate to acetyl-CoA for entry into the Krebs cycle; in the absence of MondoA, pyruvate is diverted towards lactate and alanine, both products of glycolysis. Dynamic testing revealed that MondoA-/- mice excel in sprinting as their skeletal muscles display an enhanced glycolytic capacity. Our studies uncover a hitherto unappreciated function of MondoA in fuel selection in vivo. Lack of MondoA results in enhanced exercise capacity with sprinting.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/deficiência , Músculo Esquelético/metabolismo , Condicionamento Físico Animal/fisiologia , Resistência Física/fisiologia , Animais , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Técnicas de Cultura de Órgãos , Condicionamento Físico Animal/métodosRESUMO
Accumulating evidence suggests that microRNAs (miRNAs) contribute to a myriad of kidney diseases. However, the regulatory role of miRNAs on the key molecules implicated in kidney fibrosis remains poorly understood. Bone morphogenetic protein-7 (BMP-7) and its related BMP-6 have recently emerged as key regulators of kidney fibrosis. Using the established unilateral ureteral obstruction (UUO) model of kidney fibrosis as our experimental model, we examined the regulatory role of miRNAs on BMP-7/6 signaling. By analyzing the potential miRNAs that target BMP-7/6 in silica, we identified miR-22 as a potent miRNA targeting BMP-7/6. We found that expression levels of BMP-7/6 were significantly elevated in the kidneys of the miR-22 null mouse. Importantly, mice with targeted deletion of miR-22 exhibited attenuated renal fibrosis in the UUO model. Consistent with these in vivo observations, primary renal fibroblast isolated from miR-22-deficient UUO mice demonstrated a significant increase in BMP-7/6 expression and their downstream targets. This phenotype could be rescued when cells were transfected with miR-22 mimics. Interestingly, we found that miR-22 and BMP-7/6 are in a regulatory feedback circuit, whereby not only miR-22 inhibits BMP-7/6, but miR-22 by itself is induced by BMP-7/6. Finally, we identified two BMP-responsive elements in the proximal region of miR-22 promoter. These findings identify miR-22 as a critical miRNA that contributes to renal fibrosis on the basis of its pivotal role on BMP signaling cascade.
Assuntos
Proteína Morfogenética Óssea 6/metabolismo , Proteína Morfogenética Óssea 7/metabolismo , Rim/metabolismo , MicroRNAs/metabolismo , Animais , Sequência de Bases , Proteína Morfogenética Óssea 6/genética , Proteína Morfogenética Óssea 7/genética , Fibrose/metabolismo , Homeostase , Rim/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , MicroRNAs/genética , Dados de Sequência Molecular , Elementos de Resposta , Transdução de Sinais , Transcrição GênicaRESUMO
BACKGROUND: Lipid synthesis and storage are accomplished by lipid droplets (LDs). The perilipin family of LD-associated proteins, comprising 5 members (PLIN1-PLIN5), has been well characterized in adipocytes but not in sebocytes, epithelial cells in which LD formation is a key feature of the cellular differentiation. METHODS: Perilipin expression in the sebaceous gland cell line SZ95 and in human sebaceous glands was studied by qRT-PCR, Western blots, and immunohistochemistry. Lipid accumulation was evaluated by Nile red staining and mass spectrometry. RESULTS: PLIN2 and PLIN3 are the most abundant perilipins in undifferentiated sebocytes. Induction of lipogenesis by linoleic acid (LA) resulted in increased transcript levels of all perilipins except for PLIN3 and in a time-dependent increase of PLIN2 protein. Nile red staining revealed that siRNA-mediated downregulation of PLIN2 significantly impaired basal and LA-induced lipid accumulation. Mass spectrometry revealed PLIN2 deficiency to cause a reduction in the amount of several specific lipid fractions, including di- and triacyl-glycerol esters, phosphatidylcholine lipids, and ceramides in sebocytes under basal conditions. In contrast, PLIN2 downregulation exerted a statistically significant inhibitory effect only on the accumulation of specific LA-induced triglycerides. PLIN2-deficient mice showed normal morphology of sebaceous glands. However, their sebaceous glands were significantly reduced in size and showed less cell proliferation. CONCLUSIONS: PLIN2 is the major perilipin regulated during sebocyte differentiation in vitro. PLIN2 is also important for sebaceous lipid accumulation in vitro and regulates sebaceous gland size in vivo. GENERAL SIGNIFICANCE: Our study provides the first systematic analysis of LD-associated proteins in sebocytes.
Assuntos
Diferenciação Celular , Metabolismo dos Lipídeos , Proteínas de Membrana/fisiologia , Glândulas Sebáceas/citologia , Animais , Sequência de Bases , Linhagem Celular , Primers do DNA , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Perilipina-2 , Reação em Cadeia da Polimerase , Glândulas Sebáceas/metabolismoRESUMO
OBJECTIVES: The assembly and secretion of hepatic very low-density lipoprotein (VLDL) plays pivotal roles in hepatic and plasma lipid homeostasis. Protein disulfide isomerase A1 (PDIA1/P4HB) is a molecular chaperone whose functions are essential for protein folding in the endoplasmic reticulum. Here we investigated the physiological requirement in vivo for PDIA1 in maintaining VLDL assembly and secretion. METHODS: Pdia1/P4hb was conditionally deleted in adult mouse hepatocytes and the phenotypes characterized. Mechanistic analyses in primary hepatocytes determined how PDIA1 ablation alters MTTP synthesis and degradation as well as altering synthesis and secretion of Apolipoprotein B (APOB), along with complementary expression of intact PDIA1 vs a catalytically inactivated PDIA1 mutant. RESULTS: Hepatocyte-specific deletion of Pdia1/P4hb inhibited hepatic MTTP expression and dramatically reduced VLDL production, leading to severe hepatic steatosis and hypolipidemia. Pdia1-deletion did not affect mRNA expression or protein stability of MTTP but rather prevented Mttp mRNA translation. We demonstrate an essential role for PDIA1 in MTTP synthesis and function and show that PDIA1 interacts with APOB in an MTTP-independent manner via its molecular chaperone function to support APOB folding and secretion. CONCLUSIONS: PDIA1 plays indispensable roles in APOB folding, MTTP synthesis and activity to support VLDL assembly. Thus, like APOB and MTTP, PDIA1 is an obligatory component of hepatic VLDL production.
Assuntos
Hepatócitos , Lipoproteínas VLDL , Nucleotídeos de Timina , Animais , Camundongos , Apolipoproteínas B/genética , Apolipoproteínas B/metabolismo , Hepatócitos/metabolismo , Lipoproteínas VLDL/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Triglicerídeos/metabolismoRESUMO
The mitochondrial electron transport chain (ETC) is a highly adaptive process to meet metabolic demands of the cell, and its dysregulation has been associated with diverse clinical pathologies. However, the role and nature of impaired ETC in kidney diseases remains poorly understood. Here, we generate diabetic mice with podocyte-specific overexpression of Ndufs4, an accessory subunit of mitochondrial complex I, as a model investigate the role of ETC integrity in diabetic kidney disease (DKD). We find that conditional male mice with genetic overexpression of Ndufs4 exhibit significant improvements in cristae morphology, mitochondrial dynamics, and albuminuria. By coupling proximity labeling with super-resolution imaging, we also identify the role of cristae shaping protein STOML2 in linking NDUFS4 with improved cristae morphology. Together, we provide the evidence on the central role of NDUFS4 as a regulator of cristae remodeling and mitochondrial function in kidney podocytes. We propose that targeting NDUFS4 represents a promising approach to slow the progression of DKD.
Assuntos
Diabetes Mellitus Experimental , Nefropatias Diabéticas , Masculino , Animais , Camundongos , Nefropatias Diabéticas/genética , Diabetes Mellitus Experimental/genética , Membranas Mitocondriais , Rim , Mitocôndrias , Complexo I de Transporte de Elétrons/genéticaRESUMO
Plin4 is a lipid droplet protein (LDP) found predominantly in white adipose tissue (WAT). The Plin4 gene is immediately downstream of the Plin5 gene; the two genes exhibit distinct though overlapping tissue expression patterns. Plin4 is absent in brown adipose tissue (BAT) and liver and expressed at low levels in heart and skeletal muscle, whereas Plin5 is highly expressed in these oxidative tissues but at a low level in WAT. The physiological role of Plin4 remains unclear. We have generated Plin4(-/-) mice by gene targeting. Loss of Plin4 has no effect on body weight or composition or on adipose mass or development. However, the triacylglycerol (TAG) content in heart, but not other oxidative tissues such as BAT, soleus muscle, and liver, is markedly reduced in Plin4(-/-) mice. The heart of Plin4(-/-) mice displays reduced Plin5 mRNA and protein levels (by ~38 and 87%, respectively, vs. wild-type) but unchanged mRNA levels of other perilipin family genes (Plin2 and Plin3) or genes involved in glucose and lipid metabolism. Despite reduced cardiac TAG level, both young and aged Plin4(-/-) mice maintain normal heart function as wild-type mice, as measured by echocardiography. Interestingly, Plin4 deficiency prevents the lipid accumulation in the heart that normally occurs after a prolonged (48-h) fast. It also protects the heart from cardiac steatosis induced by high-fat diet or when Plin4(-/-) mice are bred into Lep(-/-) obese background. In conclusion, inactivation of Plin4 downregulates Plin5 and reduces cardiac lipid accumulation in mice.
Assuntos
Tecido Adiposo/metabolismo , Proteínas de Transporte/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Musculares/metabolismo , Miocárdio/metabolismo , RNA Mensageiro/análise , Triglicerídeos/metabolismo , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/metabolismo , Animais , Composição Corporal/genética , Composição Corporal/fisiologia , Peso Corporal/genética , Peso Corporal/fisiologia , Proteínas de Transporte/genética , Regulação para Baixo , Perfilação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Proteínas Musculares/genética , Perilipina-4 , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND & AIMS: Adult hepatocytes undergo cell cycle progression and proliferation in response to partial hepatectomy (PH). Transient lipid accumulation within hepatocytes preceding the peak proliferative phase is a characteristic feature of regenerating livers. However, the molecular mediators and mechanisms responsible for lipid accumulation in regenerating livers are not well understood. Adipose differentiation related protein (ADRP; Plin2) regulates hepatic triglyceride storage and Plin2-deficient (Plin2(-/-)) mice have significantly reduced triglyceride (TG) content in the liver. We sought to determine the functional significance of PLIN2 in liver regeneration in response to PH and toxic liver injury and examined whether absence of Plin2 expression modulates hepatocyte proliferation and liver regeneration. METHODS: We subjected wild-type (WT) and Plin2(-/-) mice to 70% PH or acute carbon tetrachloride (CCL4) treatment and examined the hepatic lipid content, the expression profile of lipid metabolism-related genes, the rate of cellular proliferation and the dynamics of liver regeneration in the treated animals. RESULTS: In response to PH, Plin2(-/-) mice showed decreased hepatic triglyceride accumulation and delayed cell cycle progression, which was associated with impaired liver regeneration. Fatty acid (FA) synthesis and lipid transfer gene expression profile were comparable between Plin2(-/-) and wild-type mice, while VLDL secretion rate was higher in the Plin2(-/-) mice. Downregulated ß-oxidation and reduced cytosolic FA level in Plin2(-/-) mice may have contributed to the attenuation of the liver regeneration capacity in these animals. In parallel experiments, we also observed attenuated hepatic lipid accumulation and proliferation in response to CCl4-mediated acute toxic liver injury in Plin2(-/-) mice. CONCLUSIONS: We conclude that PLIN2-mediated lipid accumulation and utilization by the liver is important for efficient liver regeneration in response to PH and toxic liver injury.
Assuntos
Regeneração Hepática , Proteínas de Membrana/fisiologia , Animais , Ciclo Celular , Diferenciação Celular , Proliferação de Células , Hepatectomia , Hepatócitos/fisiologia , Lipogênese , Lipoproteínas VLDL/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Oxirredução , Perilipina-2RESUMO
Milk lipids originate by secretion of triglyceride-rich cytoplasmic lipid droplets (CLDs) from mammary epithelial cells. Adipophilin (ADPH)/Plin2, a member of the perilipin family of CLD binding proteins, is hypothesized to regulate CLD production in these cells during differentiation of the mammary gland into a secretory organ. We tested this hypothesis by comparing CLD accumulation in differentiating mammary glands of wild-type and ADPH-deficient mice. ADPH deficiency did not prevent CLD formation; however, it disrupted the increase in CLD size that normally occurs in differentiating mammary epithelial cells. Failure to form large CLDs in ADPH-deficient mice correlated with localization of adipose triglyceride lipase (ATGL) to the CLD surface, suggesting that ADPH promotes CLD growth by inhibiting lipolytic activity. Significantly, mammary alveoli also failed to mature in ADPH-deficient mice, and pups born to these mice failed to survive. The possibility that CLD accumulation and alveolar maturation defects in ADPH-deficient mice are functionally related was tested by in vivo rescue experiments. Transduction of mammary glands of pregnant ADPH-deficient mice with adenovirus encoding ADPH as an N-terminal GFP fusion protein prevented ATGL from localizing to CLDs and rescued CLD size and alveolar maturation defects. Collectively, these data provide direct in vivo evidence that ADPH inhibition of ATGL-dependent lipolysis is required for normal CLD accumulation and alveolar maturation during mammary gland differentiation. We speculate that impairing CLD accumulation interferes with alveolar maturation and lactation by disrupting triglyceride homeostasis in mammary epithelial cells.
Assuntos
Citoplasma/metabolismo , Metabolismo dos Lipídeos , Glândulas Mamárias Animais/fisiologia , Proteínas de Membrana/metabolismo , Animais , Caseínas/metabolismo , Feminino , Proteínas de Fluorescência Verde/metabolismo , Lactação/metabolismo , Lipase/metabolismo , Glândulas Mamárias Animais/metabolismo , Proteínas de Membrana/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Perilipina-2 , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The mitochondrial electron transport chain (ETC) is a highly adaptive process to meet metabolic demands of the cell, and its dysregulation has been associated with diverse clinical pathologies. However, the role and nature of impaired ETC in kidney diseases remains poorly understood. Here, we generated diabetic mice with podocyte-specific overexpression of Ndufs4, an accessory subunit of mitochondrial complex I, as a model to investigate the role of ETC integrity in diabetic kidney disease (DKD). We find that these conditional mice exhibit significant improvements in cristae morphology, mitochondrial dynamics, and albuminuria. By coupling proximity labeling with super-resolution imaging, we also identify the role of cristae shaping proteins in linking NDUFS4 with improved cristae morphology. Taken together, we discover the central role of NDUFS4 as a powerful regulator of cristae remodeling, respiratory supercomplexes assembly, and mitochondrial ultrastructure in vitro and in vivo. We propose that targeting NDUFS4 represents a promising approach to slow the progression of DKD.
RESUMO
Although several recent publications have suggested that microRNAs contribute to the pathogenesis of diabetic nephropathy, the role of miRNAs in vivo still remains poorly understood. Using an integrated in vitro and in vivo comparative miRNA expression array, we identified miR-29c as a signature miRNA in the diabetic environment. We validated our profiling array data by examining miR-29c expression in the kidney glomeruli obtained from db/db mice in vivo and in kidney microvascular endothelial cells and podocytes treated with high glucose in vitro. Functionally, we found that miR-29c induces cell apoptosis and increases extracellular matrix protein accumulation. Indeed, forced expression of miR-29c strongly induced podocyte apoptosis. Conversely, knockdown of miR-29c prevented high glucose-induced cell apoptosis. We also identified Sprouty homolog 1 (Spry1) as a direct target of miR-29c with a nearly perfect complementarity between miR-29c and the 3'-untranslated region (UTR) of mouse Spry1. Expression of miR-29c decreased the luciferase activity of Spry1 when co-transfected with the mouse Spry1 3'-UTR reporter construct. Overexpression of miR-29c decreased the levels of Spry1 protein and promoted activation of Rho kinase. Importantly, knockdown of miR-29c by a specific antisense oligonucleotide significantly reduced albuminuria and kidney mesangial matrix accumulation in the db/db mice model in vivo. These findings identify miR-29c as a novel target in diabetic nephropathy and provide new insights into the role of miR-29c in a previously unrecognized signaling cascade involving Spry1 and Rho kinase activation.
Assuntos
Nefropatias Diabéticas/metabolismo , Regulação da Expressão Gênica , Glucose/metabolismo , Proteínas de Membrana/metabolismo , MicroRNAs/biossíntese , Fosfoproteínas/metabolismo , Podócitos/metabolismo , Regiões 3' não Traduzidas/genética , Proteínas Adaptadoras de Transdução de Sinal , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Transformada , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/patologia , Nefropatias Diabéticas/terapia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/biossíntese , Proteínas da Matriz Extracelular/genética , Técnicas de Silenciamento de Genes , Proteínas de Membrana/genética , Camundongos , MicroRNAs/genética , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Fosfoproteínas/genética , Podócitos/patologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismoRESUMO
Glucagon-like peptides (GLP-1/2) are cosecreted from endocrine L cells in the gut and preproglucagonergic neurons in the brain. Peripheral GLP-2 action is essential for maintaining intestinal homeostasis, improving absorption efficiency and blood flow, promoting immune defense, and producing efficacy in treatment of gastrointestinal diseases. However, it is unknown if CNS GLP-2 plays a physiological role in the control of energy homeostasis. Since GLP-1/2 are cotranslated from preproglucagongene and coproduced by prohormone convertase-1, it is challenging to knockout GLP-2 only. Instead, our laboratory has generated a Glp2r-floxed mouse line to dissect cell-specific GLP-2 receptor GLP-2R) action in the regulation of energy balance. Our objective was to determine if GLP-2R in the hypothalamus modulates feeding behavior and gastric emptying. We show that Glp2r mRNA and protein are highly expressed in the arcuate nucleus and dorsomedial nucleus of the mouse hypothalamus. Using the Cre-LoxP system, we generated mice that lack Glp2r expression in POMC neurons (KO; mainly in the hypothalamus). The KO mice showed hyperphagic behavior (such as increases in food intake and meal frequency), accelerated gastric emptying (assessed by [(13)C]octanoic acid breath test), and late-onset obesity, yet there was no decrease in basal metabolic rate. Infusion of GLP-2 (2.5 nmol into the 4th ventricle) suppressed food intake and gastric emptying, while GLP-2-mediated effects were abolished in the melanocortin receptor-4 (MC4R) KO mice. We conclude that Glp2r deletion in POMC neurons enhances feeding behavior and gastric motility, whereas icv GLP-2R activation suppresses food intake and gastric emptying through the MC4R signaling pathway. This study indicates that CNS GLP-2R plays a physiological role in the control of feeding behavior and gastric emptying and that this is mediated probably through the melanocortin system.
Assuntos
Comportamento Alimentar/fisiologia , Motilidade Gastrointestinal/fisiologia , Neurônios/fisiologia , Receptores de Glucagon/fisiologia , Animais , Núcleo Arqueado do Hipotálamo/fisiologia , Núcleo Hipotalâmico Dorsomedial/fisiologia , Feminino , Motilidade Gastrointestinal/genética , Peptídeo 2 Semelhante ao Glucagon/farmacologia , Receptor do Peptídeo Semelhante ao Glucagon 2 , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Pró-Opiomelanocortina/fisiologia , Receptor Tipo 4 de Melanocortina/genética , Receptor Tipo 4 de Melanocortina/fisiologia , Receptores de Glucagon/biossíntese , Receptores de Glucagon/genética , Transdução de Sinais/fisiologiaRESUMO
Stem cells may contribute to renal recovery following acute kidney injury, and this may occur through their secretion of cytokines, chemokines, and growth factors. Here, we developed an acellular, nanofiber-based preparation of self-assembled peptides to deliver the secretome of embryonic stem cells (ESCs). Using an integrated in vitro and in vivo approach, we found that nanofibers preconditioned with ESCs could reverse cell hyperpermeability and apoptosis in vitro and protect against lipopolysaccharide-induced acute kidney injury in vivo. The renoprotective effect of preconditioned nanofibers associated with an attenuation of Rho kinase activation. We also observed that the combined presence of follistatin, adiponectin, and secretory leukoprotease during preconditioning was essential to the renoprotective properties of the nanofibers. In summary, we developed a designer-peptide nanofiber that can serve as a delivery platform for the beneficial effects of stem cells without the problems of teratoma formation or limited cell engraftment and viability.
Assuntos
Injúria Renal Aguda/terapia , Terapia Baseada em Transplante de Células e Tecidos/métodos , Células-Tronco Embrionárias/transplante , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/patologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Terapia Baseada em Transplante de Células e Tecidos/efeitos adversos , Células Cultivadas , Lipopolissacarídeos/efeitos adversos , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Nanofibras/uso terapêutico , Proteinúria/prevenção & controle , Quinases Associadas a rho/fisiologiaRESUMO
Vascular endothelial growth factor (VEGF) is a dimeric glycoprotein that plays a crucial role in microvascular complications of diabetes, including diabetic nephropathy. However, the precise regulatory mechanisms governing VEGF expression in the diabetic milieu are still poorly understood. Here, we provide evidence that microRNA-93 (miR-93) regulates VEGF expression in experimental models of diabetes both in vitro and in vivo. Comparative microRNA expression profile arrays identified miR-93 as a signature microRNA in hyperglycemic conditions. We identified VEGF-A as a putative target of miR-93 in the kidney with a perfect complementarity between miR-93 and the 3'-untranslated region of vegfa in several species. When cotransfected with a luciferase reporter construct containing the mouse vegfa 3'-untranslated region, expression of miR-93 markedly decreased the luciferase activity. We showed that forced expression of miR-93 in cells abrogated VEGF protein secretion. Conversely, anti-miR-93 inhibitors increased VEGF release. Transfection of miR-93 also prevented the effect of high glucose on VEGF downstream targets. Using transgenic mice containing VEGF-LacZ bicistronic transcripts, we found that inhibition of glomerular miR-93 by peptide-conjugated morpholino oligomers elicited increased expression of VEGF. Our findings also indicate that high glucose decreases miR-93 expression by down-regulating the promoter of the host MCM7 gene. Taken together, our findings provide new insights into the role of miR-93 in VEGF signaling pathway and offer a potentially novel target in preventing the progression of diabetic nephropathy.
Assuntos
Hiperglicemia/genética , Hiperglicemia/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Regiões 3' não Traduzidas/genética , Animais , Sequência de Bases , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/genética , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patologia , Diabetes Mellitus/fisiopatologia , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Glucose/farmacologia , Células HeLa , Humanos , Hiperglicemia/patologia , Hiperglicemia/fisiopatologia , Camundongos , Microvasos/citologia , Componente 7 do Complexo de Manutenção de Minicromossomo , Dados de Sequência Molecular , Morfolinas/química , Morfolinas/farmacologia , Proteínas Nucleares/genética , Podócitos/citologia , Podócitos/efeitos dos fármacos , Podócitos/metabolismo , Podócitos/patologia , Polímeros/química , Regiões Promotoras Genéticas/genética , Reprodutibilidade dos Testes , Transcrição Gênica , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
UNLABELLED: PNPLA3 (adiponutrin), a novel patatin-like phospholipase domain-containing enzyme, is expressed at high level in fat, but also in other tissues including liver. Polymorphisms in PNPLA3 have been linked to obesity and insulin sensitivity. Notably, a nonsynonymous variant rs738409(G) allele of the PNPLA3 gene was found to be strongly associated with both nonalcoholic and alcoholic fatty liver disease. We have generated Pnpla3(-/-) mice by gene targeting. Loss of Pnpla3 has no effect on body weight or composition, adipose mass, or development, whether the mice were fed regular chow or high-fat diet or bred into the genetic obese Lep(ob/ob) background. Plasma and liver triglyceride content and plasma aspartate aminotransferase and alanine aminotransferase levels were not different between Pnpla3(+/+) and Pnpla3(-/-) mice while they were on regular chow, fed three different fatty liver-inducing diets, or after they were bred into Lep(ob/ob) background. Hepatic Pnpla5 messenger RNA (mRNA) levels were similar in wild-type and Pnpla3(-/-) mice, although adipose Pnpla5 mRNA level was increased in Pnpla3(-/-) mice. A high-sucrose lipogenic diet stimulated hepatic Pnpla3 and Pnpla5 mRNA levels to a similar degree, but it did not affect adipose or liver triglyceride lipase (ATGL, known also as Pnpla2) mRNA in Pnpla3(+/+) and Pnpla3(-/-) mice. Finally, Pnpla3(+/+) and Pnpla3(-/-) mice displayed similar glucose tolerance and insulin tolerance tests while on regular chow or three different fatty liver-inducing diets. CONCLUSION: Loss of Pnpla3 does not cause fatty liver, liver enzyme elevation, or insulin resistance in mice.