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1.
Int J Mol Sci ; 22(14)2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-34299206

RESUMO

Despite the intensive investigation of the molecular mechanism of skeletal muscle hypertrophy, the underlying signaling processes are not completely understood. Therefore, we used an overload model, in which the main synergist muscles (gastrocnemius, soleus) of the plantaris muscle were surgically removed, to cause a significant overload in the remaining plantaris muscle of 8-month-old Wistar male rats. SIRT1-associated pro-anabolic, pro-catabolic molecular signaling pathways, NAD and H2S levels of this overload-induced hypertrophy were studied. Fourteen days of overload resulted in a significant 43% (p < 0.01) increase in the mass of plantaris muscle compared to sham operated animals. Cystathionine-ß-synthase (CBS) activities and bioavailable H2S levels were not modified by overload. On the other hand, overload-induced hypertrophy of skeletal muscle was associated with increased SIRT1 (p < 0.01), Akt (p < 0.01), mTOR, S6 (p < 0.01) and suppressed sestrin 2 levels (p < 0.01), which are mostly responsible for anabolic signaling. Decreased FOXO1 and SIRT3 signaling (p < 0.01) suggest downregulation of protein breakdown and mitophagy. Decreased levels of NAD+, sestrin2, OGG1 (p < 0.01) indicate that the redox milieu of skeletal muscle after 14 days of overloading is reduced. The present investigation revealed novel cellular interactions that regulate anabolic and catabolic processes in the hypertrophy of skeletal muscle.


Assuntos
Cistationina beta-Sintase/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/patologia , Animais , Hipertrofia/genética , Hipertrofia/metabolismo , Hipertrofia/patologia , Masculino , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Wistar , Proteínas Quinases S6 Ribossômicas/genética , Proteínas Quinases S6 Ribossômicas/metabolismo , Sirtuína 1/genética , Sirtuína 1/metabolismo , Sirtuínas/antagonistas & inibidores , Sirtuínas/genética , Sirtuínas/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo
2.
Biomed Opt Express ; 7(9): 3531-3542, 2016 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-27699118

RESUMO

A novel, Yb-fiber laser based, handheld 2PEF/SHG microscope imaging system is introduced. It is suitable for in vivo imaging of murine skin at an average power level as low as 5 mW at 200 kHz sampling rate. Amplified and compressed laser pulses having a spectral bandwidth of 8 to 12 nm at around 1030 nm excite the biological samples at a ~1.89 MHz repetition rate, which explains how the high quality two-photon excitation fluorescence (2PEF) and second harmonic generation (SHG) images are obtained at the average power level of a laser pointer. The scanning, imaging and detection head, which comprises a conventional microscope objective for beam focusing, has a physical length of ~180 mm owing to the custom designed imaging telescope system between the laser scanner mirrors and the entrance aperture of the microscope objective. Operation of the all-fiber, all-normal dispersion Yb-fiber ring laser oscillator is electronically controlled by a two-channel polarization controller for Q-switching free mode-locked operation. The whole nonlinear microscope imaging system has the main advantages of the low price of the fs laser applied, fiber optics flexibility, a relatively small, light-weight scanning and detection head, and a very low risk of thermal or photochemical damage of the skin samples.

3.
Microsc Res Tech ; 78(9): 823-30, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26208320

RESUMO

Nonlinear microscopy techniques are being increasingly used to perform in vivo studies in dermatology. These methods enable us to investigate the morphology and monitor the physiological process in the skin by the use of femtosecond lasers operating in the red, near-infrared spectral range (680-1,300 nm). In this work we used two different techniques that require no labeling: second harmonic generation (SHG) for collagen detection and coherent anti-Stokes Raman scattering (CARS) to assess lipid distribution in genetically obese murine skin. Obesity is one of the most serious public health problems due to its high and increasing prevalence and the associated risk of type 2 diabetes and cardiovascular diseases. Other than these diseases, nearly half of patients with diabetes mellitus suffer from dermatological complications such as delayed wound healing, foot ulcers and several other skin changes. In our experiment we investigated and followed the effects of obesity on dermal collagen alterations and adipocyte enlargement using a technique not reported in the literature so far. Our results indicate that the in vivo SHG and ex vivo CARS imaging technique might be an important tool for diagnosis of diabetes-related skin disorders in the near future.


Assuntos
Colágeno/análise , Fibroblastos/fisiologia , Processamento de Imagem Assistida por Computador , Lipídeos/análise , Microscopia/métodos , Obesidade/patologia , Pele/patologia , Animais , Fibroblastos/química , Humanos , Lasers , Camundongos Obesos , Pele/química , Análise Espectral Raman
4.
Exp Dermatol ; 23(8): 596-605, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24903756

RESUMO

Epidermal Langerhans cells (LCs) function as professional antigen-presenting cells of the skin. We investigated the LC-targeting properties of a special mannose-moiety-coated pathogen-like synthetic nanomedicine DermaVir (DV), which is capable to express antigens to induce immune responses and kill HIV-infected cells. Our aim was to use multiphoton laser microscopy (MLM) in vivo in order to visualize the uptake of Alexa-labelled DV (AF546-DV) by LCs. Knock-in mice expressing enhanced green fluorescent protein (eGFP) under the control of the langerin gene (CD207) were used to visualize LCs. After 1 h, AF546-DV penetrated the epidermis and entered the eGFP-LCs. The AF546-DV signal was equally distributed inside the LCs. After 9 h, we observed AF546-DV signal accumulation that occurred mainly at the cell body. We demonstrated in live animals that LCs picked up and accumulated the nanoparticles in the cell body.


Assuntos
Vacinas contra a AIDS/farmacocinética , Células de Langerhans/metabolismo , Nanomedicina/métodos , Nanopartículas , Animais , Transporte Biológico , Proteínas de Fluorescência Verde/genética , Células de Langerhans/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Microscopia Confocal , Microscopia de Fluorescência por Excitação Multifotônica , Modelos Animais
5.
Diabetes ; 63(6): 1881-94, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24430435

RESUMO

Induction of heat shock protein (HSP)72 protects against obesity-induced insulin resistance, but the underlying mechanisms are unknown. Here, we show that HSP72 plays a pivotal role in increasing skeletal muscle mitochondrial number and oxidative metabolism. Mice overexpressing HSP72 in skeletal muscle (HSP72Tg) and control wild-type (WT) mice were fed either a chow or high-fat diet (HFD). Despite a similar energy intake when HSP72Tg mice were compared with WT mice, the HFD increased body weight, intramuscular lipid accumulation (triacylglycerol and diacylglycerol but not ceramide), and severe glucose intolerance in WT mice alone. Whole-body VO2, fatty acid oxidation, and endurance running capacity were markedly increased in HSP72Tg mice. Moreover, HSP72Tg mice exhibited an increase in mitochondrial number. In addition, the HSP72 coinducer BGP-15, currently in human clinical trials for type 2 diabetes, also increased mitochondrial number and insulin sensitivity in a rat model of type 2 diabetes. Together, these data identify a novel role for activation of HSP72 in skeletal muscle. Thus, the increased oxidative metabolism associated with activation of HSP72 has potential clinical implications not only for type 2 diabetes but also for other disorders where mitochondrial function is compromised.


Assuntos
Respiração Celular , Diabetes Mellitus Tipo 2/metabolismo , Proteínas de Choque Térmico HSP72/metabolismo , Resistência à Insulina , Mitocôndrias Musculares/metabolismo , Obesidade/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Glicemia , Western Blotting , Peso Corporal , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/fisiopatologia , Dieta Hiperlipídica , Metabolismo Energético , Ácidos Graxos/metabolismo , Leptina/metabolismo , Masculino , Camundongos , Músculo Esquelético/metabolismo , Obesidade/genética , Obesidade/fisiopatologia , Oxirredução , Fosforilação Oxidativa , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Sirtuína 1/metabolismo
6.
J Invest Dermatol ; 134(1): 105-111, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23884312

RESUMO

Recently, a transglutaminase 3 knockout (TGM3/KO) mouse was generated that showed impaired hair development, but no gross defects in the epidermal barrier, although increased fragility of isolated corneocytes was demonstrated. Here we investigated the functionality of skin barrier in vivo by percutaneous sensitization to FITC in TGM3/KO (n=64) and C57BL/6 wild-type (WT) mice (n=36). Cutaneous inflammation was evaluated by mouse ear swelling test (MEST), histology, serum IgE levels, and by flow cytometry from draining lymph nodes. Inflammation-induced significant MEST difference (P<0.0001) was detected between KO and WT mice and was supported also by histopathology. A significant increase of CD4+ CD25+-activated T cells (P<0.01) and elevated serum IgE levels (P<0.05) in KO mice indicated more the development of FITC sensitization than an irritative reaction. Propionibacter acnes-induced intracutaneous inflammation showed no difference (P=0.2254) between the reactivity of WT and KO immune system. As in vivo tracer, FITC penetration from skin surface followed by two-photon microscopy demonstrated a more invasive percutaneous penetration in KO mice. The clinically uninvolved skin in TGM3/KO mice showed impaired barrier function and higher susceptibility to FITC sensitization indicating that TGM3 has a significant contribution to the functionally intact cutaneous barrier.


Assuntos
Dermatite de Contato/imunologia , Dermatite de Contato/microbiologia , Infecções por Bactérias Gram-Positivas/imunologia , Propionibacterium acnes/imunologia , Transglutaminases/imunologia , Animais , Dermatite de Contato/etiologia , Edema/imunologia , Edema/metabolismo , Feminino , Citometria de Fluxo , Fluoresceína-5-Isotiocianato/toxicidade , Infecções por Bactérias Gram-Positivas/metabolismo , Imunoglobulina E/imunologia , Linfonodos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Propionibacterium acnes/metabolismo , Pele/imunologia , Pele/metabolismo , Pele/microbiologia , Transglutaminases/genética
7.
Pathol Oncol Res ; 18(4): 1071-6, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22743983

RESUMO

Atypical antipsychotic drugs (AAPD) are widely used to treat severe psychiatric disorders, have well documented metabolic side effects such as disturbances in glucose metabolism, insulin resistance and weight gain. It has been shown that BGP-15, a hydroxylamine derivative with insulin sensitizing activity can prevent AAPD provoked fat accumulation in adipocyte cultures, and insulin resistance in animal experiments and in healthy volunteers. The aim of this study was to compare the preventive effect of BGP-15 with conventional oral antidiabetics on metabolic side effects of AAPDs. We found that BGP-15 that does not belong to either conventional insulin sensitizers or oral antidiabetics, is able to counteract insulin resistance and weight gain provoked by antipsychotic agents in rats while rosiglitazone and metformin were not effective in the applied doses. Our results confirm that BGP-15 is a promising new drug candidate to control the metabolic side effects of atypical antipsychotics. Data indicate that this rat model is suitable to analyze the metabolic side effects of AAPDs and the protective mechanism of BGP-15.


Assuntos
Antipsicóticos/toxicidade , Oximas/farmacologia , Piperidinas/farmacologia , Substâncias Protetoras/farmacologia , Análise de Variância , Animais , Interações Medicamentosas , Feminino , Técnica Clamp de Glucose , Resistência à Insulina , Ratos , Ratos Wistar , Ganho de Peso/efeitos dos fármacos
8.
Cell Stress Chaperones ; 17(4): 517-21, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22322357

RESUMO

Weight gain and dysfunction of glucose and lipid metabolism are well-known side effects of atypical antipsychotic drugs (AAPD). Here, we address the question whether a heat-shock protein (HSP) co-inducer, insulin sensitizer drug candidate, BGP-15, can prevent AAPD-induced glucose, lipid, and weight changes. We also examined how an AAPD alters HSP expression and whether BGP-15 alters that expression. Four different experiments are reported on the AAPD BGP-15 interventions in a human trial of healthy men, a rodent animal model, and an in vitro adipocyte cell culture system. Olanzapine caused rapid insulin resistance in healthy volunteers and was associated with decreased level of HSP72 in peripheral mononuclear blood cells. Both changes were restored by the administration of BGP-15. In Wistar rats, weight gain and insulin resistance induced by clozapine were abolished by BGP-15. In 3T3L1 adipocytes, clozapine increased intracellular fat accumulation, and BGP-15 inhibited this process. Taken together, our results indicate that BGP-15 inhibits multiple metabolic side effects of atypical antipsychotics, and this effect is likely to be related to its HSP co-inducing ability.


Assuntos
Adipócitos/efeitos dos fármacos , Antipsicóticos/efeitos adversos , Proteínas de Choque Térmico HSP72/metabolismo , Hipoglicemiantes/farmacologia , Oximas/farmacologia , Piperidinas/farmacologia , Ganho de Peso/efeitos dos fármacos , Animais , Células Cultivadas , Feminino , Humanos , Masculino , Ratos , Ratos Wistar , Regulação para Cima/efeitos dos fármacos
9.
Cytometry ; 47(4): 207-16, 2002 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-11933010

RESUMO

BACKGROUND: In this study, the effect of antigen-presenting cells (APC), peptide concentration, and CD28 costimulation on calcium signaling, induced by antigen-specific T-cell activation, was studied by flow cytometry. METHODS: We used two experimental approaches, which differed in their time scale and in the duration of the T cell-APC interaction, to measure the increase of intracellular free calcium levels ([Ca(2+)](i)) in activated T cells: (1) Fluo-3-loaded T cells were activated by cocentrifugation with peptide-loaded APC and the kinetics of fluorescence intensity changes was monitored continuously and (2) peptide-loaded APC and T cells were mixed, cocultured, and the fluorescence intensity was measured at various time intervals. RESULTS: The calcium signal of T cells was dependent on the APC as demonstrated by the ratio of cells exhibiting high versus low fluorescence intensity and by the magnitude of the calcium signal in the activated population. Short-term interaction of T cells with less potent APC or with efficient APC in the presence of low antigen concentration resulted in decreased calcium signaling. CD28-mediated costimulation enhanced the magnitude and sustained the increase of intracellular calcium levels. In line with the strong and sustained calcium signals, the activation of the calcium-dependent transcription factors NF-AT, AP-1, and NF-kappaB was induced. CONCLUSIONS: Flow cytometric methods, feasible for the rapid and flexible analysis of calcium signaling upon antigen-specific T-cell activation, were established. Kinetics of the increase of mean fluorescence intensity reflected the calcium response of the total cell population whereas statistical analysis of fluorescence intensity at selected time points provided information on the activation state of single cells.


Assuntos
Células Apresentadoras de Antígenos/metabolismo , Antígenos/imunologia , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Citometria de Fluxo/métodos , Ativação Linfocitária/imunologia , Proteínas Nucleares , Linfócitos T/metabolismo , Animais , Células Apresentadoras de Antígenos/citologia , Células Apresentadoras de Antígenos/imunologia , Antígenos de Superfície/imunologia , Antígeno B7-1/imunologia , Antígenos CD28/imunologia , Comunicação Celular/imunologia , Proteínas de Ligação a DNA/imunologia , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/imunologia , Fatores de Transcrição NFATC , Linfócitos T/citologia , Linfócitos T/imunologia , Fator de Transcrição AP-1/imunologia , Fatores de Transcrição/imunologia , Células Tumorais Cultivadas
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