RESUMEN
BACKGROUND: Older adults exposed to periods of inactivity during hospitalization, illness, or injury lose muscle mass and strength. This, in turn, predisposes poor recovery of physical function upon reambulation and represents a significant health risk for older adults. Bed rest (BR) results in altered skeletal muscle fuel metabolism and loss of oxidative capacity that have recently been linked to the muscle atrophy program. Our primary objective was to explore the effects of BR on mitochondrial energetics in muscle from older adults. A secondary objective was to examine the effect of ß-hydroxy-ß-methylbuturate (HMB) supplementation on mitochondrial energetics. METHODS: We studied 20 older adults before and after a 10-day BR intervention, who consumed a complete oral nutritional supplement (ONS) with HMB (3.0 g/d HMB, n = 11) or without HMB (CON, n = 9). Percutaneous biopsies of the vastus lateralis were obtained to determine mitochondrial respiration and H2O2 emission in permeabilized muscle fibers along with markers of content. RNA sequencing and lipidomics analyses were also conducted. RESULTS: We found a significant up-regulation of collagen synthesis and down-regulation of ribosome, oxidative metabolism and mitochondrial gene transcripts following BR in the CON group. Alterations to these gene transcripts were significantly blunted in the HMB group. Mitochondrial respiration and markers of content were both reduced and H2O2 emission was elevated in both groups following BR. CONCLUSIONS: In summary, 10 days of BR in older adults causes a significant deterioration in mitochondrial energetics, while transcriptomic profiling revealed that some of these negative effects may be attenuated by an ONS containing HMB.
Asunto(s)
Reposo en Cama/efectos adversos , Metabolismo Energético , Mitocondrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Anciano , Biopsia , Suplementos Dietéticos , Metabolismo Energético/efectos de los fármacos , Humanos , Lipidómica , Masculino , Persona de Mediana Edad , Mitocondrias Musculares/efectos de los fármacos , Músculo Esquelético/patología , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Valeratos/uso terapéuticoRESUMEN
An increasing body of evidence indicates that a local islet immune response is not only limited to type 1 diabetes, but also is associated with islet dysfunction in type 2 diabetes. Recently, the presence of pancreatic CD68+ macrophages within islet tissues was demonstrated by RT-PCR and immunohistochemical methods. However, the precise profile and activation status of intraislet leukocytes, which are present in both murine and human islets, are poorly defined. Here, we describe a detailed flow cytometry protocol designed to analyze both human and murine islets for intraislet leukocytes and leukocyte subsets. This approach permits the simultaneous identification of multiple intraislet leukocyte subsets, as well as their activation statuses. The use of flow cytometry-based approaches will advance the field of islet biology and help to identify unique changes in the immune cell composition that accompanies pathological islet inflammation and dysfunction in type 2 diabetes.
Asunto(s)
Citometría de Flujo , Islotes Pancreáticos/citología , Leucocitos/citología , Leucocitos/metabolismo , Animales , Biomarcadores , Recuento de Células , Separación Celular , Diabetes Mellitus Tipo 2 , Citometría de Flujo/métodos , Humanos , Inmunofenotipificación , RatonesRESUMEN
Skeletal muscle atrophy is a clinically important outcome of disuse because of injury, immobilization, or bed rest. Disuse atrophy is accompanied by mitochondrial dysfunction, which likely contributes to activation of the muscle atrophy program. However, the linkage of muscle mass and mitochondrial energetics during disuse atrophy and its recovery is incompletely understood. Transcriptomic analysis of muscle biopsies from healthy older adults subject to complete bed rest revealed marked inhibition of mitochondrial energy metabolic pathways. To determine the temporal sequence of muscle atrophy and changes in intramyocellular lipid and mitochondrial energetics, we conducted a time course of hind limb unloading-induced atrophy in adult mice. Mitochondrial respiration and calcium retention capacity were diminished, whereas H2O2 emission was increased within 3 days of unloading before significant muscle atrophy. These changes were associated with a decrease in total cardiolipin and profound changes in remodeled cardiolipin species. Hind limb unloading performed in muscle-specific peroxisome proliferator-activated receptor-γ coactivator-1α/ß knockout mice, a model of mitochondrial dysfunction, did not affect muscle atrophy but impacted muscle function. These data suggest early mitochondrial remodeling affects muscle function but not mass during disuse atrophy. Early alterations in mitochondrial energetics and lipid remodeling may represent novel targets to prevent muscle functional impairment caused by disuse and to enhance recovery from periods of muscle atrophy.
Asunto(s)
Metabolismo Energético , Mitocondrias Musculares/metabolismo , Trastornos Musculares Atróficos/metabolismo , Anciano , Animales , Reposo en Cama , Calcio/metabolismo , Cardiolipinas/metabolismo , Femenino , Suspensión Trasera , Humanos , Peróxido de Hidrógeno/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Trastornos Musculares Atróficos/fisiopatología , Consumo de Oxígeno , Recuperación de la Función , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , TranscriptomaRESUMEN
The progression of age-related sarcopenia can be accelerated by impaired recovery of muscle mass following periods of disuse due to illness or immobilization. However, the mechanisms underlying poor recovery of aged muscle following disuse remain to be delineated. Recent evidence suggests that mitochondrial energetics play an important role in regulation of muscle mass. Here, we report that 22- to 24-month-old mice with low muscle mass and low glucose clearance rate also display poor early recovery of muscle mass following 10 days of hind limb unloading. We used unbiased and targeted approaches to identify changes in energy metabolism gene expression, metabolite pools and mitochondrial phenotype, and show for the first time that persistent mitochondrial dysfunction, dysregulated fatty acid ß-oxidation, and elevated H2O2 emission occur concomitantly with poor early recovery of muscle mass following a period of disuse in old mice. Importantly, this is linked to more severe whole-body insulin resistance, as determined by insulin tolerance test. The findings suggest that muscle fuel metabolism and mitochondrial energetics could be a focus for mining therapeutic targets to improve recovery of muscle mass following periods of disuse in older animals.
Asunto(s)
Envejecimiento/metabolismo , Envejecimiento/patología , Suspensión Trasera/efectos adversos , Mitocondrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Envejecimiento/genética , Animales , Metabolismo Energético , Ácidos Grasos/metabolismo , Suspensión Trasera/fisiología , Resistencia a la Insulina , Masculino , Metaboloma , Ratones , Ratones Endogámicos C57BL , Atrofia Muscular/genética , Atrofia Muscular/metabolismo , Atrofia Muscular/patología , Sarcopenia/genética , Sarcopenia/metabolismo , Sarcopenia/patología , TranscriptomaRESUMEN
Perilipin 5 (PLIN5) is a lipid droplet (LD) protein highly expressed in oxidative tissues, including the fasted liver. However, its expression also increases in nonalcoholic fatty liver. To determine whether PLIN5 regulates metabolic phenotypes of hepatosteatosis under nutritional excess, liver targeted overexpression of PLIN5 was achieved using adenoviral vector (Ad-PLIN5) in male C57BL/6J mice fed high-fat diet. Mice treated with adenovirus expressing green fluorescent protein (GFP) (Ad-GFP) served as control. Ad-PLIN5 livers increased LD in the liver section, and liquid chromatography with tandem mass spectrometry revealed increases in lipid classes associated with LD, including triacylglycerol, cholesterol ester, and phospholipid classes, compared with Ad-GFP liver. Lipids commonly associated with hepatic lipotoxicity, diacylglycerol, and ceramides, were also increased in Ad-PLIN5 liver. The expression of genes in lipid metabolism regulated by peroxisome proliferator-activated receptor-α was reduced suggestive of slower mobilization of stored lipids in Ad-PLIN5 mice. However, the increase of hepatosteatosis by PLIN5 overexpression did not worsen glucose homeostasis. Rather, serum insulin levels were decreased, indicating better insulin sensitivity in Ad-PLIN5 mice. Moreover, genes associated with liver injury were unaltered in Ad-PLIN5 steatotic liver compared with Ad-GFP control. Phosphorylation of protein kinase B was increased in Ad-PLIN5-transduced AML12 hepatocyte despite of the promotion of fatty acid incorporation to triacylglycerol as well. Collectively, our data indicates that the increase in liver PLIN5 during hepatosteatosis drives further lipid accumulation but does not adversely affect hepatic health or insulin sensitivity.
Asunto(s)
Dieta Alta en Grasa , Hígado Graso/metabolismo , Resistencia a la Insulina , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Hígado/metabolismo , Proteínas Musculares/metabolismo , Adenoviridae/metabolismo , Animales , Cromatografía Liquida , Hígado Graso/patología , Glucosa/metabolismo , Homeostasis , Gotas Lipídicas/metabolismo , Metabolismo de los Lípidos , Masculino , Ratones Endogámicos C57BL , Espectrometría de Masas en Tándem , Triglicéridos/metabolismo , Regulación hacia ArribaRESUMEN
Elevation of circulating fatty acids (FA) during fasting supports postprandial (PP) insulin secretion that is critical for glucose homeostasis and is impaired in diabetes. We tested our hypothesis that lipid droplet (LD) protein perilipin 5 (PLIN5) in ß-cells aids PP insulin secretion by regulating intracellular lipid metabolism. We demonstrated that PLIN5 serves as an LD protein in human islets. In vivo, Plin5 and triglycerides were increased by fasting in mouse islets. MIN6 cells expressing PLIN5 (adenovirus [Ad]-PLIN5) and those expressing perilipin 2 (PLIN2) (Ad-PLIN2) had higher [(3)H]FA incorporation into triglycerides than Ad-GFP control, which support their roles as LD proteins. However, Ad-PLIN5 cells had higher lipolysis than Ad-PLIN2 cells, which increased further by 8-Br-cAMP, indicating that PLIN5 facilitates FA mobilization upon cAMP stimulation as seen postprandially. Ad-PLIN5 in islets enhanced the augmentation of glucose-stimulated insulin secretion by FA and 8-Br-cAMP in G-protein-coupled receptor 40 (GPR40)- and cAMP-activated protein kinase-dependent manners, respectively. When PLIN5 was increased in mouse ß-cells in vivo, glucose tolerance after an acute exenatide challenge was improved. Therefore, the elevation of islet PLIN5 during fasting allows partitioning of FA into LD that is released upon refeeding to support PP insulin secretion in cAMP- and GPR40-dependent manners.
Asunto(s)
AMP Cíclico/metabolismo , Insulina/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Islotes Pancreáticos/metabolismo , Metabolismo de los Lípidos/fisiología , Proteínas Musculares/metabolismo , Periodo Posprandial/fisiología , Animales , Ayuno/metabolismo , Glucosa/metabolismo , Humanos , Secreción de Insulina , Péptidos y Proteínas de Señalización Intracelular/genética , Lipólisis/fisiología , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Proteínas Musculares/genética , Perilipina-2 , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
Neuropeptide Y (NPY) is highly expressed in the hypothalamus, where it regulates feeding and energy homeostasis. Interestingly, NPY and its receptors are also expressed in peripheral tissues with roles in metabolism, including pancreatic islets. In islets, NPY is known to suppress insulin secretion acutely. In addition, the role of NPY in ß-cell de-differentiation has been postulated recently. Therefore, we studied transgenic mice expressing NPY under rat insulin promoter (TG) to determine the effects of chronic up-regulation of NPY on islet morphology and function. NPY levels were 25 times higher in islets of TG mice compared with wild-type (WT) littermates, whereas no differences in NPY expression were noted in the brains of TG and WT mice. Islet NPY secretion was 2.3-fold higher in TG compared with WT mice. There were no significant changes in body weight, glucose tolerance, or insulin sensitivity in TG mice fed regular rodent diet or high-fat diet (HF). Islet ß-cell area was comparable between TG and WT mice both on regular rodent and HF diets, indicating that NPY overexpression is insufficient to alter ß-cell maturation or the compensatory increase of ß-cell area on HF. One abnormality noted was that the glucose-stimulated insulin secretion in islets isolated from TG was reduced compared with those from WT mice on HF diet. Overall, an increase in islet NPY level has little impact on islet function and is insufficient to affect glucose homeostasis in mice.
Asunto(s)
Células Secretoras de Insulina/fisiología , Neuropéptido Y/metabolismo , Adaptación Fisiológica , Animales , Glucemia/metabolismo , Dieta Alta en Grasa , Femenino , Homeostasis , Células Secretoras de Insulina/citología , Masculino , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
This study modified the degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR)-based whole genome amplification method for improvement of downstream genome-wide analysis of low copy number DNA samples (