Ageing and dexamethasone associated sarcopenia: peculiarities of regeneration

The purpose of this study was to assess the development of ageing- and glucocorticoid-related sarcopenia on the level of myofibrillar apparatus, paying attention to the synthesis (SR) and degradation rate (DR) of contractile proteins, muscle strength, and daily motor activity. We also wanted to test the effect of ageing and dexamethasone (Dex) excess on the regeneration peculiarities of skeletal muscle autografts. Four and 30-month-old male rats of the Wistar strain were used. Ageing associated sarcopenia was calculated from gastrocnemius muscle relative mass decrease (from 5.6 ± 0.08 to 3.35 ± 0.04; p < 0.001). The SR of MyHC in old rats was 30% and actin 23% lower than in young rats. Dex treatment decreased SR of two main contractile proteins significantly in both age groups (p < 0.001) and increased DR during ageing from 2.11 ± 0.15 to 4.09 ± 0.29%/day (p < 0.001). Hindlimb grip strength in young rats was 5.90 ± 0.35 N/100 g bw and 2.64 ± 0.2 N/100 g bw (p < 0.001) in old rats.

Autografts of old rats have a higher content of adipose tissue 14.9 ± 1.1% in comparison with young rats 6.8 ± 0.51% (p < 0.001) and less muscle tissue 39.8 ± 2.6% and 48.3 ± 2.8%, respectively (p < 0.05).

Both, ageing and dex-caused sarcopenic muscles have diminished capacity for regeneration.     

β3-Adrenergic Receptor Stimulation Induces E-Selectin-mediated Adipose Tissue Inflammation

Inflammation induced by wound healing or infection activates local vascular endothelial cells to mediate leukocyte rolling, adhesion, and extravasation by up-regulation of leukocyte adhesion molecules such as E-selectin and P-selectin. Obesity-associated adipose tissue inflammation has been suggested to cause insulin resistance, but weight loss and lipolysis also promote adipose tissue immune responses. While leukocyte-endothelial interactions are required for obesity-induced inflammation of adipose tissue, it is not known whether lipolysis-induced inflammation requires activation of endothelial cells. Here, we show that β₃-adrenergic receptor stimulation by CL 316,243 promotes adipose tissue neutrophil infiltration in wild type and P-selectin-null mice but not in E-selectin-null mice. Increased expression of adipose tissue cytokines IL-1β, CCL2, and TNF-α in response to CL 316,243 administration is also dependent upon E-selectin but not P-selectin. In contrast, fasting increases adipose-resident macrophages but not neutrophils, and does not activate adipose-resident endothelium. Thus, two models of lipolysis-induced inflammation induce distinct immune cell populations within adipose tissue and exhibit distinct dependences on endothelial activation. Importantly, our results indicate that β₃-adrenergic stimulation acts through up-regulation of E-selectin in adipose tissue endothelial cells to induce neutrophil infiltration.     

脂肪间质干细胞研究进展

脂肪间质干细胞,是脂肪组织中一类多能性干细胞。其在体外特定的培养条件下,可诱导分化形成脂肪、骨、软骨、肌肉等组织类型细胞。人体脂肪组织十分丰富,用其分离脂肪间质干细胞可避免分离胚胎干细胞所面临的道德伦理问题和获取极少量骨髓分离骨髓间质干细胞时给供者带来极大痛苦等。因此脂肪间质干细胞可作为组织再生工程的干细胞理想的替代资源。本文重点论述脂肪间质干细胞的研究进展,并探讨其临床应用前景。     

Activation of natural killer T cells promotes M2 Macrophage polarization in adipose tissue and improves systemic glucose tolerance via interleukin-4 (IL-4)/STAT6 protein signaling axis in obesity

Natural killer T (NKT) cells are important therapeutic targets in various disease models and are under clinical trials for cancer patients. However, their function in obesity and type 2 diabetes remains unclear. Our data show that adipose tissues of both mice and humans contain a population of type 1 NKT cells, whose abundance decreases with increased adiposity and insulin resistance. Although loss-of-function of NKT cells had no effect on glucose tolerance in animals with prolonged high fat diet feeding, activation of NKT cells by lipid agonist α-galactosylceramide enhances alternative macrophage polarization in adipose tissue and improves glucose homeostasis in animals at different stages of obesity. Furthermore, the effect of NKT cells is largely mediated by the IL-4/STAT6 signaling axis in obese adipose tissue. Thus, our data identify a novel therapeutic target for the treatment of obesity-associated inflammation and type 2 diabetes.     

Isolation of adipose tissue mesenchymal stem cells without tissue destruction: A non-enzymatic method

The conventional enzymatic method is widely used for mesenchymal stem cells (MSCs) isolation from adipose tissue. The method holds major drawbacks; it is costly, time-consuming and results in a heterogeneous cell population. Besides, digestion of extracellular matrix causes cell injury and compromise proliferation and differentiation of the cells. Also, because of over handling the samples are also prone to contamination. Here, we introduce a non-enzymatic method for MSCs isolation without disturbing the cells habitat. Small pieces of adipose tissue obtained from animal or human liposuction were explanted into a culture flask, immobilized by fetal bovine serum (FBS) and incubated overnight. The explants were then irrigated with DMEM containing FBS. Within few days, the fibroblast-like cells migrated from the tissue and proliferated rapidly. When subconfluent, the cells were harvested, expanded through 3 passages and used for immunophenotyping and differentiation assays. As judged by flow cytometric analysis of surface markers (CD44⁺, CD105⁺, CD34⁻, CD45⁻), Oil Red O and Alizarin Red staining, the MSCs isolated by our non-enzymatic method were pluripotent and exhibited the potential for differentiation into adipocyte and osteoblast. Great isolation yields, homogeneity of isolated cells, brief procedure, and high economy are the advantages of our method over the conventional protocol.     

Kinetic measures of restabilisation during volitional stepping reveal age-related alterations in the control of mediolateral dynamic stability

Research examining age-related changes in dynamic stability during stepping has recognised the importance of the restabilisation phase, subsequent to foot-contact. While regulation of the net ground reaction force (GRF_net) line of action is believed to influence dynamic stability during steady-state locomotion, such control during restabilisation remains unknown. This work explored the origins of age-related decline in mediolateral dynamic stability by examining the line of action of GRF_net relative to the centre of mass (COM) during restabilisation following voluntary stepping. Healthy younger and older adults (n=20 per group) performed three single-step tasks (varying speed and step placement), altering the challenge to stability control. Age-related differences in magnitude and intertrial variability of the angle of divergence of GRF_net line of action relative to the COM were quantified, along with the peak mediolateral and vertical GRF_net components. The angle of divergence was further examined at discrete points during restabilisation, to uncover events of potential importance to stability control. Older adults exhibited a reduced angle of divergence throughout restabilisation. Temporal and spatial constraints on stepping increased the magnitude and intertrial variability of the angle of divergence, although not differentially among the older adults. Analysis of the time-varying angle of divergence revealed age-related reductions in magnitude, with increases in timing and intertrial timing variability during the later phase of restabilisation. This work further supports the idea that age-related challenges in lateral stability control emerge during restabilisation. Age-related alterations during the later phase of restabilisation may signify challenges with reactive control.     

C1q/TNF-related protein-3 (CTRP3), a novel adipokine that regulates hepatic glucose output

Adipose tissue-derived adipokines play important roles in controlling systemic insulin sensitivity and energy balance. Our recent efforts to identify novel metabolic mediators produced by adipose tissue have led to the discovery of a highly conserved family of secreted proteins, designated as C1q/TNF-related proteins 1-10 (CTRP1 to -10). However, physiological functions regulated by CTRPs are largely unknown. Here we provide the first in vivo functional characterization of CTRP3. We show that circulating levels of CTRP3 are inversely correlated with leptin levels; CTRP3 increases with fasting, decreases in diet-induced obese mice with high leptin levels, and increases in leptin-deficient ob/ob mice. A modest 3-fold elevation of plasma CTRP3 levels by recombinant protein administration is sufficient to lower glucose levels in normal and insulin-resistant ob/ob mice, without altering insulin or adiponectin levels. The glucose-lowering effect in mice is linked to activation of the Akt signaling pathway in liver and a marked suppression of hepatic gluconeogenic gene expression. Consistent with its effects in mice, CTRP3 acts directly and independently of insulin to regulate gluconeogenesis in cultured hepatocytes. In humans, alternative splicing generates two circulating CTRP3 isoforms differing in size and glycosylation pattern. The two human proteins form hetero-oligomers, an association that does not require interdisulfide bond formation and appears to protect the longer isoform from proteolytic cleavage. Recombinant human CTRP3 also reduces glucose output in hepatocytes by suppressing gluconeogenic enzyme expression. This study provides the first functional evidence linking CTRP3 to hepatic glucose metabolism and establishes CTRP3 as a novel adipokine.     

Heart-type fatty acid-binding protein is essential for efficient brown adipose tissue fatty acid oxidation and cold tolerance

Brown adipose tissue has a central role in thermogenesis to maintain body temperature through energy dissipation in small mammals and has recently been verified to function in adult humans as well. Here, we demonstrate that the heart-type fatty acid-binding protein, FABP3, is essential for cold tolerance and efficient fatty acid oxidation in mouse brown adipose tissue, despite the abundant expression of adipose-type fatty acid-binding protein, FABP4 (also known as aP2). Fabp3(-/-) mice exhibit extreme cold sensitivity despite induction of uncoupling and oxidative genes and hydrolysis of brown adipose tissue lipid stores. However, using FABP3 gain- and loss-of-function approaches in brown adipocytes, we detected a correlation between FABP3 levels and the utilization of exogenous fatty acids. Thus, Fabp3(-/-) brown adipocytes fail to oxidize exogenously supplied fatty acids, whereas enhanced Fabp3 expression promotes more efficient oxidation. These results suggest that FABP3 levels are a determinant of fatty acid oxidation efficiency by brown adipose tissue and that FABP3 represents a potential target for modulation of energy dissipation.     

Effects of plyometric and pneumatic explosive strength training on neuromuscular function and dynamic balance control in 60–70 year old males

The present study compared neuromuscular adaptations to 12 weeks of plyometric (PLY) or pneumatic (PNE) power training and their effects on dynamic balance control. Twenty-two older adults aged 60–70 (PLY n = 9, PNE n = 11) participated in the study. Measurements were conducted at Pre, 4, 8 and 12 weeks. Dynamic balance was assessed as anterior–posterior center of pressure (COP) displacement in response to sudden perturbations. Explosive isometric knee extension and plantar flexion maximal voluntary contractions (MVCs) were performed. Maximal drop jump performance from optimal dropping height was measured in a sledge ergometer. Increases in knee extensor and ankle plantar flexor torque and muscle activity were higher and occurred sooner in PNE, whereas in drop jumping, PLY showed a clearer increase in optimal drop height (24%, p < 0.01) after 8 weeks of training and soleus muscle activity after 12 weeks of training. In spite of these training mode specific adaptations, both groups showed similar improvements in dynamic balance control after 4 weeks of training (PLY 38%, p < 0.001; PNE 31%, p < 0.001) and no change thereafter. These results show that although power and plyometric training may involve different neural adaptation mechanisms, both training modes can produce similar improvements in dynamic balance control in older individuals. As COP displacement was negatively correlated with rapid knee extension torque in both groups (PLY r = −0.775, p < 0.05; PNE r = −0.734, p < 0.05) after training, the results also highlight the importance of targeting rapid force production when training older adults to improve dynamic balance.     

Adipocyte Fetuin-A Contributes to Macrophage Migration into Adipose Tissue and Polarization of Macrophages

Macrophage infiltration into adipose tissue during obesity and their phenotypic conversion from anti-inflammatory M2 to proinflammatory M1 subtype significantly contributes to develop a link between inflammation and insulin resistance; signaling molecule(s) for these events, however, remains poorly understood. We demonstrate here that excess lipid in the adipose tissue environment may trigger one such signal. Adipose tissue from obese diabetic db/db mice, high fat diet-fed mice, and obese diabetic patients showed significantly elevated fetuin-A (FetA) levels in respect to their controls; partially hepatectomized high fat diet mice did not show noticeable alteration, indicating adipose tissue to be the source of this alteration. In adipocytes, fatty acid induces FetA gene and protein expressions, resulting in its copious release. We found that FetA could act as a chemoattractant for macrophages. To simulate lipid-induced inflammatory conditions when proinflammatory adipose tissue and macrophages create a niche of an altered microenvironment, we set up a transculture system of macrophages and adipocytes; the addition of fatty acid to adipocytes released FetA into the medium, which polarized M2 macrophages to M1. This was further confirmed by direct FetA addition to macrophages. Taken together, lipid-induced FetA from adipocytes is an efficient chemokine for macrophage migration and polarization. These findings open a new dimension for understanding obesity-induced inflammation.     

脂肪组织石蜡切片制作方法探讨

目的探讨脂肪组织石蜡切片制作方法,改善和提高其切片质量。方法将送检的脂肪组织切开固定于中性甲醛液中;取材后将标本放入脱水机中,调整脱水机设置程序,在常规方法的基础上适当增加组织再固定及脱水、透明时间;包埋时采取挤压组织等改进方法。结果通过改进和调整后制作的脂肪组织蜡块平整无塌陷,石蜡切片优良,厚薄均匀,完整,无空洞、无挤压、镜下组织结构完整清晰,细胞无挤压、重叠等现象。细胞形态清晰,着色均匀艳丽。结论通过上述方法的改进,能尽可能多地减少脂肪组织的脂滴,增加脂肪组织的硬度,使组织和石蜡能很好的融合在一起,有利于切出完整的厚薄均匀的石蜡切片,提高制片质量。     

Lecithin cholesterol acyltransferase null mice are protected from diet-induced obesity and insulin resistance in a gender-specific manner through multiple pathways

Complete lecithin cholesterol acyltransferase (LCAT) deficiency uniformly results in a profound HDL deficiency. We recently reported unexpected enhanced insulin sensitivity in LCAT knock-out mice in the LDL receptor knock-out background (Ldlr(-/-)×Lcat(-/-); double knock-out (DKO)), when compared with their Ldlr(-/-)×Lcat(+/+) (single knock-out (SKO)) controls. Here, we report that LCAT-deficient mice (DKO and Lcat(-/-)) are protected against high fat high sucrose (HFHS) diet-induced obesity without hypophagia in a gender-specific manner compared with their respective (SKO and WT) controls. The metabolic phenotypes are more pronounced in the females. Changes in endoplasmic reticulum stress were examined as a possible mechanism for the metabolic protection. The female DKO mice developed attenuated HFHS-induced endoplasmic reticulum stress as evidenced by a lack of increase in mRNA levels of the hepatic unfolded protein response (UPR) markers Grp78 and CHOP compared with SKO controls. The DKO female mice were also protected against diet-induced insulin resistance. In white adipose tissue of chow-fed DKO mice, we also observed a reduction in UPR, gene markers for adipogenesis, and markers for activation of Wnt signaling. In skeletal muscles of female DKO mice, we observed an unexpected increase in UCP1 in association with increase in phospho-AMPKα, PGC1α, and UCP3 expressions. This increase in UCP1 was associated with ectopic islands of brown adipocytes between skeletal muscle fibers. Our findings suggest that LCAT deficiency confers gender-specific protection against diet-induced obesity and insulin resistance at least in part through regulation in UPR, white adipose tissue adipogenesis, and brown adipocyte partitioning.     

Defective Adipose Lipolysis and Altered Global Energy Metabolism in Mice with Adipose Overexpression of the Lipolytic Inhibitor G0/G1 Switch Gene 2 (G0S2)

Biochemical and cell-based studies have identified the G0S2 (G₀/G₁ switch gene 2) as a selective inhibitor of the key intracellular triacylglycerol hydrolase, adipose triglyceride lipase. To better understand the physiological role of G0S2, we constructed an adipose tissue-specific G0S2 transgenic mouse model. In comparison with wild type animals, the transgenic mice exhibited a significant increase in overall fat mass and a decrease in peripheral triglyceride accumulation. Basal and adrenergically stimulated lipolysis was attenuated in adipose explants isolated from the transgenic mice. Following fasting or injection of a β3-adrenergic agonist, in vivo lipolysis and ketogenesis were decreased in G0S2 transgenic mice when compared with wild type animals. Consequently, adipose overexpression of G0S2 prevented the “switch” of energy substrate from carbohydrates to fatty acids during fasting. Moreover, G0S2 overexpression promoted accumulation of more and larger lipid droplets in brown adipocytes without impacting either mitochondrial morphology or expression of oxidative genes. This phenotypic change was accompanied by defective cold adaptation. Furthermore, feeding with a high fat diet caused a greater gain of both body weight and adiposity in the transgenic mice. The transgenic mice also displayed a decrease in fasting plasma levels of free fatty acid, triglyceride, and insulin as well as improved glucose and insulin tolerance. Cumulatively, these results indicate that fat-specific G0S2 overexpression uncouples adiposity from insulin sensitivity and overall metabolic health through inhibiting adipose lipolysis and decreasing circulating fatty acids.     

The Farnesoid X Receptor Regulates Adipocyte Differentiation and Function by Promoting Peroxisome Proliferator-activated Receptor-γ and Interfering with the Wnt/β-Catenin Pathways

The bile acid receptor farnesoid X receptor (FXR) is expressed in adipose tissue, but its function remains poorly defined. Peroxisome proliferator-activated receptor-γ (PPARγ) is a master regulator of adipocyte differentiation and function. The aim of this study was to analyze the role of FXR in adipocyte function and to assess whether it modulates PPARγ action. Therefore, we tested the responsiveness of FXR-deficient mice (FXR^−/−) and cells to the PPARγ activator rosiglitazone. Our results show that genetically obese FXR^−/−/ob/ob mice displayed a resistance to rosiglitazone treatment. In vitro, rosiglitazone treatment did not induce normal adipocyte differentiation and lipid droplet formation in FXR^−/− mouse embryonic fibroblasts (MEFs) and preadipocytes. Moreover, FXR^−/− MEFs displayed both an increased lipolysis and a decreased de novo lipogenesis, resulting in reduced intracellular triglyceride content, even upon PPARγ activation. Retroviral-mediated FXR re-expression in FXR^−/− MEFs restored the induction of adipogenic marker genes during rosiglitazone-forced adipocyte differentiation. The expression of Wnt/β-catenin pathway and target genes was increased in FXR^−/− adipose tissue and MEFs. Moreover, the expression of several endogenous inhibitors of this pathway was decreased early during the adipocyte differentiation of FXR^−/− MEFs. These findings demonstrate that FXR regulates adipocyte differentiation and function by regulating two counteracting pathways of adipocyte differentiation, the PPARγ and Wnt/β-catenin pathways.     

Fatty Acids Change the Conformation of Uncoupling Protein 1 (UCP1)

UCP1 catalyzes proton leak across the mitochondrial inner membrane to disengage substrate oxidation from ATP production. It is well established that UCP1 is activated by fatty acids and inhibited by purine nucleotides, but precisely how this regulation occurs remains unsettled. Although fatty acids can competitively overcome nucleotide inhibition in functional assays, fatty acids have little effect on purine nucleotide binding. Here, we present the first demonstration that fatty acids induce a conformational change in UCP1. Palmitate dramatically changed the binding kinetics of 2′/3′-O-(N-methylanthraniloyl)-GDP, a fluorescently labeled nucleotide analog, for UCP1. Furthermore, palmitate accelerated the rate of enzymatic proteolysis of UCP1. The altered kinetics of both processes indicate that fatty acids change the conformation of UCP1, reconciling the apparent discrepancy between existing functional and ligand binding data. Our results provide a framework for how fatty acids and nucleotides compete to regulate the activity of UCP1.