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1.
Am J Physiol Endocrinol Metab ; 317(2): E374-E387, 2019 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-31211616

RESUMEN

Mitochondria are dynamic organelles with diverse functions in tissues such as liver and skeletal muscle. To unravel the mitochondrial contribution to tissue-specific physiology, we performed a systematic comparison of the mitochondrial proteome and lipidome of mice and assessed the consequences hereof for respiration. Liver and skeletal muscle mitochondrial protein composition was studied by data-independent ultra-high-performance (UHP)LC-MS/MS-proteomics, and lipid profiles were compared by UHPLC-MS/MS lipidomics. Mitochondrial function was investigated by high-resolution respirometry in samples from mice and humans. Enzymes of pyruvate oxidation as well as several subunits of complex I, III, and ATP synthase were more abundant in muscle mitochondria. Muscle mitochondria were enriched in cardiolipins associated with higher oxidative phosphorylation capacity and flexibility, in particular CL(18:2)4 and 22:6-containing cardiolipins. In contrast, protein equipment of liver mitochondria indicated a shuttling of complex I substrates toward gluconeogenesis and ketogenesis and a higher preference for electron transfer via the flavoprotein quinone oxidoreductase pathway. Concordantly, muscle and liver mitochondria showed distinct respiratory substrate preferences. Muscle respired significantly more on the complex I substrates pyruvate and glutamate, whereas in liver maximal respiration was supported by complex II substrate succinate. This was a consistent finding in mouse liver and skeletal muscle mitochondria and human samples. Muscle mitochondria are tailored to produce ATP with a high capacity for complex I-linked substrates. Liver mitochondria are more connected to biosynthetic pathways, preferring fatty acids and succinate for oxidation. The physiologic diversity of mitochondria may help to understand tissue-specific disease pathologies and to develop therapies targeting mitochondrial function.


Asunto(s)
Metabolismo Energético/fisiología , Hígado/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Músculo Esquelético/metabolismo , Proteoma/metabolismo , Animales , Femenino , Humanos , Hígado/química , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias Hepáticas/metabolismo , Mitocondrias Musculares/metabolismo , Proteínas Mitocondriales/análisis , Músculo Esquelético/química , Especificidad de Órganos , Mapeo Peptídico/métodos , Proteoma/análisis
2.
Adv Exp Med Biol ; 1158: 143-182, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31452140

RESUMEN

Mitochondrial dysfunction is discussed as a key player in the pathogenesis of type 2 diabetes mellitus (T2Dm), a highly prevalent disease rapidly developing as one of the greatest global health challenges of this century. Data however about the involvement of mitochondria, central hubs in bioenergetic processes, in the disease development are still controversial. Lipid and protein homeostasis are under intense discussion to be crucial for proper mitochondrial function. Consequently proteomics and lipidomics analyses might help to understand how molecular changes in mitochondria translate to alterations in energy transduction as observed in the healthy and metabolic diseases such as T2Dm and other related disorders. Mitochondrial lipids integrated in a tool covering proteomic and functional analyses were up to now rarely investigated, although mitochondrial lipids might provide a possible lynchpin in the understanding of type 2 diabetes development and thereby prevention. In this chapter state-of-the-art analytical strategies, pre-analytical aspects, potential pitfalls as well as current proteomics and lipidomics-based knowledge about the pathophysiological role of mitochondria in the pathogenesis of type 2 diabetes will be discussed.


Asunto(s)
Biología Computacional , Diabetes Mellitus Tipo 2 , Hígado , Mitocondrias , Músculo Esquelético , Proteómica , Diabetes Mellitus Tipo 2/fisiopatología , Humanos , Metabolismo de los Lípidos , Hígado/fisiopatología , Mitocondrias/metabolismo , Músculo Esquelético/fisiopatología
3.
Mol Metab ; 54: 101359, 2021 12.
Artículo en Inglés | MEDLINE | ID: mdl-34695608

RESUMEN

OBJECTIVE: Liver mitochondria adapt to high-calorie intake. We investigated how exercise alters the early compensatory response of mitochondria, thus preventing fatty liver disease as a long-term consequence of overnutrition. METHODS: We compared the effects of a steatogenic high-energy diet (HED) for six weeks on mitochondrial metabolism of sedentary and treadmill-trained C57BL/6N mice. We applied multi-OMICs analyses to study the alterations in the proteome, transcriptome, and lipids in isolated mitochondria of liver and skeletal muscle as well as in whole tissue and examined the functional consequences by high-resolution respirometry. RESULTS: HED increased the respiratory capacity of isolated liver mitochondria, both in sedentary and in trained mice. However, proteomics analysis of the mitochondria and transcriptomics indicated that training modified the adaptation of the hepatic metabolism to HED on the level of respiratory complex I, glucose oxidation, pyruvate and acetyl-CoA metabolism, and lipogenesis. Training also counteracted the HED-induced glucose intolerance, the increase in fasting insulin, and in liver fat by lowering diacylglycerol species and c-Jun N-terminal kinase (JNK) phosphorylation in the livers of trained HED-fed mice, two mechanisms that can reverse hepatic insulin resistance. In skeletal muscle, the combination of HED and training improved the oxidative capacity to a greater extent than training alone by increasing respiration of isolated mitochondria and total mitochondrial protein content. CONCLUSION: We provide a comprehensive insight into the early adaptations of mitochondria in the liver and skeletal muscle to HED and endurance training. Our results suggest that exercise disconnects the HED-induced increase in mitochondrial substrate oxidation from pyruvate and acetyl-CoA-driven lipid synthesis. This could contribute to the prevention of deleterious long-term effects of high fat and sugar intake on hepatic mitochondrial function and insulin sensitivity.


Asunto(s)
Hígado Graso/metabolismo , Mitocondrias/metabolismo , Condicionamiento Físico Animal/fisiología , Animales , Masculino , Ratones , Ratones Endogámicos C57BL
4.
J Clin Endocrinol Metab ; 105(11)2020 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-32827042

RESUMEN

CONTEXT: Exercise training improves glycemic control and increases mitochondrial content and respiration capacity in skeletal muscle. Rodent studies suggest that training increases mitochondrial respiration in adipose tissue. OBJECTIVE: To assess the effects of endurance training on respiratory capacities of human skeletal muscle and abdominal subcutaneous adipose tissue and to study the correlation with improvement in insulin sensitivity. DESIGN: Using high-resolution respirometry, we analyzed biopsies from 25 sedentary (VO2 peak 25.1 ± 4.0 VO2 mL/[kg*min]) subjects (16 female, 9 male; 29.8 ± 8.4 years) with obesity (body mass index [BMI] 31.5 ± 4.3 kg/m2), who did not have diabetes. They performed a supervised endurance training over 8 weeks (3 × 1 hour/week at 80% VO2 peak). RESULTS: Based on change in insulin sensitivity after intervention (using the Matsuda insulin sensitivity index [ISIMats]), subjects were grouped in subgroups as responders (>15% increase in ISIMats) and low-responders. The response in ISIMats was correlated to a reduction of subcutaneous and visceral adipose tissue volume. Both groups exhibited similar increases in fitness, respiratory capacity, and abundance of mitochondrial enzymes in skeletal muscle fibers. Respiratory capacities in subcutaneous adipose tissue were not altered by the intervention. Compared with muscle fibers, adipose tissue respiration showed a preference for ß-oxidation and complex II substrates. Respiratory capacities were higher in adipose tissue from female participants. CONCLUSION: Our data show that the improvement of peripheral insulin sensitivity after endurance training is not directly related to an increase in mitochondrial respiratory capacities in skeletal muscle and occurs without an increase in the respiratory capacity of subcutaneous adipose tissue.


Asunto(s)
Tejido Adiposo/metabolismo , Respiración de la Célula/fisiología , Entrenamiento Aeróbico , Ejercicio Físico/fisiología , Mitocondrias/metabolismo , Músculo Esquelético/metabolismo , Obesidad/metabolismo , Adulto , Índice de Masa Corporal , Femenino , Humanos , Resistencia a la Insulina/fisiología , Masculino , Mitocondrias Musculares/metabolismo , Resistencia Física/fisiología , Adulto Joven
5.
Sci Rep ; 8(1): 737, 2018 01 15.
Artículo en Inglés | MEDLINE | ID: mdl-29335583

RESUMEN

Measuring mitochondrial respiration in cultured cells is a valuable tool to investigate the influence of physiological and disease-related factors on cellular metabolism; however, the details of the experimental workflow greatly influence the informative value of the results. Working with primary cells and cell types capable of differentiation can be particularly challenging. We present a streamlined workflow optimised for investigation of primary human skeletal muscle cells. We applied the workflow to differentiated and undifferentiated cells and we investigated the effect of TGFß1 treatment. Differentiation of myoblasts to myotubes increased mitochondrial respiration and abundance of mitochondrial enzymes and mitochondrial marker proteins. Differentiation also induced qualitative changes in mitochondrial protein composition and respiration. TGFß1 reduced complex IV protein MTCO1 abundance in both myoblasts and myotubes. In myoblasts, spare electron transport system (ETS) capacity was reduced due to a reduction in maximal oxygen consumption. In TGFß1-treated myotubes, the reduction in spare ETS capacity is mainly a consequence of increased oxidative phosphorylation capacity and complex III protein UQCRC2. Taken together, our data shows that it is important to monitor muscle cell differentiation when mitochondrial function is studied. Our workflow is not only sensitive enough to detect physiological-sized differences, but also adequate to form mechanistic hypotheses.


Asunto(s)
Respiración de la Célula , Complejo IV de Transporte de Electrones/análisis , Mitocondrias/enzimología , Mitocondrias/metabolismo , Células Musculares/fisiología , Factor de Crecimiento Transformador beta/metabolismo , Diferenciación Celular , Células Cultivadas , Transporte de Electrón , Humanos , Masculino , Persona de Mediana Edad , Músculo Esquelético , Oxígeno/metabolismo
6.
Sci Rep ; 6: 21107, 2016 Feb 19.
Artículo en Inglés | MEDLINE | ID: mdl-26892142

RESUMEN

Subcellular lipidomics is a novel field of research that requires the careful combination of several pre-analytical and analytical steps. To define a reliable strategy for mitochondrial lipid profiling, we performed a systematic comparison of different mitochondria isolation procedures by western blot analyses and comprehensive high-resolution lipidomics. Using liver-derived HepG2 cells, we compared three common mitochondria isolation methods, differential centrifugation (DC), ultracentrifugation (UC) and a magnetic bead-assisted method (MACS). In total, 397 lipid species, including 32 cardiolipins, could be quantified in only 100 µg (by protein) of purified mitochondria. Mitochondria isolated by UC showed the highest enrichment in the mitochondria-specific cardiolipins as well as their precursors, phosphatidylglycerols. Mitochondrial fractions obtained by the commonly used DC and the more recent MACS method contained substantial contaminations by other organelles. Employing these isolation methods when performing lipidomics analyses from cell culture mitochondria may lead to inaccurate results. To conclude, we present a protocol how to obtain reliable mitochondria-specific lipid profiles from cell culture samples and show that quality controls are indispensable when performing mitochondria lipidomics.


Asunto(s)
Metabolismo de los Lípidos , Lípidos/aislamiento & purificación , Metabolómica/métodos , Mitocondrias/metabolismo , Cardiolipinas/aislamiento & purificación , Cardiolipinas/metabolismo , Técnicas de Cultivo de Célula , Centrifugación , Análisis por Conglomerados , Células Hep G2 , Humanos , Separación Inmunomagnética , Orgánulos/metabolismo , Flujo de Trabajo
7.
Mol Metab ; 4(9): 631-42, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-26413469

RESUMEN

OBJECTIVE: Several human and rodent obesity studies speculate on a causal link between altered white adipocyte mitochondria in the obese state and changes in glucose homeostasis. We here aimed to dissect whether alterations in white adipocyte mitochondrial respiratory function are a specific phenomenon of obesity or impaired glucose tolerance or both. METHODS: Mature white adipocytes were purified from posterior subcutaneous and intraabdominal epididymal fat of four murine obesity models characterized by either impaired or normal oral glucose tolerance. Bioenergetic profiles, including basal, leak, and maximal respiration, were generated using high-resolution respirometry. Cell respiratory control ratios were calculated to evaluate mitochondrial respiratory function. RESULTS: Maximal respiration capacity and cell respiratory control ratios were diminished in white adipocytes of each of the four murine obesity models, both in the absence and the presence of impaired glucose tolerance. Limitation was more pronounced in adipocytes of intraabdominal versus subcutaneous fat. CONCLUSION: Reduced mitochondrial respiratory capacity in white adipocytes is a hallmark of murine obesity irrespective of the glucose tolerance status. Impaired respiratory capacity in white adipocytes solely is not sufficient for the development of systemic glucose intolerance.

8.
Endocrinology ; 156(3): 923-33, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25549046

RESUMEN

Accumulation of visceral fat is associated with metabolic risk whereas excessive amounts of peripheral fat are considered less problematic. At the same time, altered white adipocyte mitochondrial bioenergetics has been implicated in the pathogenesis of insulin resistance and type 2 diabetes. We therefore investigated whether the metabolic risk of visceral vs peripheral fat coincides with a difference in mitochondrial capacity of white adipocytes. We assessed bioenergetic parameters of subcutaneous inguinal and visceral epididymal white adipocytes from male C57BL/6N mice employing a comprehensive respirometry setup of intact and permeabilized adipocytes as well as isolated mitochondria. Inguinal adipocytes clearly featured a higher respiratory capacity attributable to increased mitochondrial respiratory chain content compared with epididymal adipocytes. The lower capacity of mitochondria from epididymal adipocytes was accompanied by an increased generation of reactive oxygen species per oxygen consumed. Feeding a high-fat diet (HFD) for 1 week reduced white adipocyte mitochondrial capacity, with stronger effects in epididymal when compared with inguinal adipocytes. This was accompanied by impaired body glucose homeostasis. Therefore, the limited bioenergetic performance combined with the proportionally higher generation of reactive oxygen species of visceral adipocytes could be seen as a candidate mechanism mediating the elevated metabolic risk associated with this fat depot.


Asunto(s)
Adipocitos Blancos/metabolismo , Grasa Intraabdominal/citología , Mitocondrias/metabolismo , Grasa Subcutánea/citología , Animales , Grasas de la Dieta/administración & dosificación , Grasas de la Dieta/efectos adversos , Relación Dosis-Respuesta a Droga , Transporte de Electrón/fisiología , Glucosa/metabolismo , Homeostasis/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Consumo de Oxígeno , Fosforilación
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