| 1. |
- Al-Khalili, Lubna, et al.
(författare)
-
Characterization of Human CD133+Cells in Biocompatible Poly(l-lactic acid) Electrospun Nano-Fiber Scaffolds
- 2016
-
Ingår i: Journal of Biomaterials and Tissue Engineering. - : American Scientific Publishers. - 2157-9083 .- 2157-9091. ; 6:12, s. 959-966
-
Tidskriftsartikel (refereegranskat)abstract
- CD133+ cells are potential myogenic progenitors for skeletal muscle regeneration to treat muscular dystrophies. The proliferation of human CD133+ stem cells was studied for 14 days in 3D biomimetic electrospun poly-L-lactic acid (PLLA) nano-fiber scaffolds. Additionally, the myogenic differentiation of the cells was studied during the last 7 days of the culture period. The cells were homogeneously distributed in the 3D scaffolds while colony formation and myotube formation occurred in 2D. After a lag phase due to lower initial cell attachment and an adaptation period, the cell growth rate in 3D was comparable to 2D after 7 and 14 days of culture. The expression of the stem cell (SC) marker PAX7 was 1.5-fold higher in 3D than 2D while the differentiation markers MyoG, Desmin and MyoD were only slightly changed (or remain unchanged) in 3D but strongly increased in 2D (12.6, 3.9, and 7.9-fold), and the myotube formation observed in 2D was absent in 3D. The marker expression during proliferation and differentiation, together with the absence of myotubes in 3D, indicates a better maintenance of stemness in 3D PLLA and stronger tendency for spontaneous differentiation in 2D culture. This makes 3D PLLA a promising biomaterial for the expansion of functional CD133+ cells.
|
|
| 2. |
- Al-Khalili, Lubna
(författare)
-
Gene regulation, intracellular signaling and membrane traffic : studies in primary human skeletal muscle cultures
- 2004
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Skeletal muscle is an insulin sensitive organ and plays a major role in whole body electrolyte and substrate homeostasis in the post prandial state. Impaired insulin action in skeletal muscle may lead to the pathological condition of insulin resistance. In this thesis primary human skeletal muscle cell culture (HSMC) has been used to investigate gene regulation, cellular signaling and membrane traffic. The expression of several myogenic and insulin responsive proteins, as well as insulin action, was followed in HSMC cultures as they differentiate from single cell myoblasts to mutlicellular myotubes. Insulin signaling to phosphatidyl inositol (PI) 3-kinase, extracellular regulated kinase (ERK) 1/2 mitogen activated protein kinase (MAPK) and protein kinase B (PKB) increased with differentiation. At the same time protein expression of PKBbeta, and ERK1/2 MAPK, and the insulin regulated glucose transported GLUT4, increased. In contrast, expression of GLUT1 decreased with differentiation. Compared to mature muscle, HSMC cultures express high levels of GLUT1. To differentiate between insulin effects on GLUT1 and GLUT4 membrane traffic we studied appearance of glucose transporter proteins at the plasma membrane using a specific photolabelling reagent. Using this technique we show that insulin increases plasma membrane content of GLUT4 but not GLUT1. On the other hand, GLUT1 content increases at the cell surface in response to serum stimulation. GLUT1 content in HSMC cells was specifically reduced using RNAi methodology. GLUT1 RNAi reduced GLUT1 content by 70%, and reduced serum-induced glucose uptake. The ability to specifically knock down proteins in primary human muscle cultures increases the utility of HSMC in the study of cellular signaling. Insulin also increases plasma membrane appearance of Na+, K+ATPase, as well as Na+, K+ATPase activity (determined by ouabain-suppressible 86Rb+ uptake) and specific [3H] ouabain binding in HSMC. We compared two different methods to study insulinmediated plasma membrane appearance of Na+, K+-ATPase alpha 1 and alpha 2 subunits. Using discontinuous sucrose gradients, insulin stimulation increased Na+, K+-ATPase alpha2 but not alpha 1 subunit in plasma membrane fraction. In contrast, when cell surface proteins were biotin labeled, we detected an insulinstimulated increase in both Na+, K+-ATPase a I a nd alpha2 s ubunits a t the c ell s urface. T hus insulin regulates membrane appearance of Na+, K+-ATPase alpha1 subunit, an effect which may previously have been overlooked due to technological limitations. Insulin-mediated regulation of Na+, K+-ATPase a subunits was determined in HSMC. In rodents a key regulatory role is played by protein kinase C phosphorylation of Ser23, however, this site is not present in human Na+, K+ATPase. We hypothesized insulin-mediated phosphorylation of Na+, K+-ATPase is dependent on ERK 1/2 signaling. Insulin-stimulated phosphorylation and translocation of Na+, K+-ATPase a was inhibited by the MEK1 inhibitor PD98059. Furthermore, purified ERK is able to directly phosphorylate Na+, K+-ATPase a subunits in vitro. Thus we conclude that insulin regulates Na+, K+ATPase via ERK dependent pathways. Insulin, AICAR, contraction and cellular stress increased the transcription factor myocyte enhancer factor 2 (MEF2) DNA binding activity in HSMC. Insulin-induced MEF2 DNA binding activity could be blocked using inhibitors against PI 3-kinase, PKC, p38- or MEK1. In contrast, AICAR mediated activation was only sensitive to the AMPK inhibitor compound C. Cellular stress-mediated activation of MEF2 DNA binding was sensitive to inhibition of p38 and MEK1, and partially sensitive to compound C. In isolated rat muscle, contraction-mediated activation of MEF2 DNA binding was sensitive to inhibition of p38 and MEK 1. In conclusion, GLUT4 mediates insulin-stimulated glucose uptake in primary cultures of human skeletal muscle, while GLUT1 primarily mediates serum induced glucose uptake. Differentiation of muscle cultures increases insulin-mediate activation of PI3-kinase, PKB, and ERK. We show that insulin activation of ERK is likely to be required for insulin induced activation of Na+, K+-ATPase and MEF2 DNA-binding. Thus although primary human skeletal muscle cell cultures differs in some aspects from mature skeletal muscle, it is a valuable and useful tool in the study of human cellular signaling and skeletal muscle research.
|
|
| 3. |
- Al-Khalili, Lubna, et al.
(författare)
-
Profiling of human myotubes reveals an intrinsic proteomic signature associated with type 2 diabetes
- 2014
-
Ingår i: Translational Proteomics. - : Elsevier BV. - 2212-9634 .- 2212-9626. ; 2:1, s. 25-38
-
Tidskriftsartikel (refereegranskat)abstract
- The development of insulin resistance and type 2 diabetes (T2D) involves a complex array of metabolic defects in skeletal muscle. An in vitro cell culture system excludes the acute effects of external systemic factors existing in vivo. Thus, we aimed to determine whether intrinsic differences in the protein profile exist in cultured myotubes derived from T2D versus normal glucose tolerant (NGT) healthy people. Applying two dimensional difference gel electrophoresis technology (2-D DIGE), the abundance of 47 proteins differed in myotubes derived from T2D patients versus NGT donors. Proteins involved in fatty acid and amino acid metabolism, TCA cycle, mitochondrial function, mRNA processing, DNA repair and cell survival showed higher abundance, while proteins associated with redox signaling (PARK7; Parkinson disease 7), glutathione metabolism (glutathione S-transferase, GST, isoforms T1, P1 and M2), and protein dynamics (heat shock protein, HSP, isoform B1 and 90A) showed reduced abundance in myotubes derived from T2D versus NGT donors. Consistent with our proteome analysis results, the level of total glutathione was reduced in myotubes obtained from T2D versus NGT donors. Taken together, our data provide evidence for intrinsic differences in the profile of proteins involved in energy metabolism, cellular oxidative stress, protein dynamics and gene regulation in myotubes derived from T2D patients. These differences thereby suggest a genetic or epigenetic influence on protein content level, which can be further investigated to understand the molecular underpinnings of T2D progression and lead to new therapeutic approaches.
|
|
| 4. |
- Garrido, Pablo, et al.
(författare)
-
Negative regulation of glucose metabolism in human myotubes by supraphysiological doses of 17 beta-estradiol or testosterone
- 2014
-
Ingår i: Metabolism. - : Elsevier BV. - 0026-0495 .- 1532-8600. ; 63:9, s. 1178-1187
-
Tidskriftsartikel (refereegranskat)abstract
- Objective. Exposure of skeletal muscle to high levels of testosterone or estrogen induces insulin resistance, but evidence regarding the direct role of either sex hormone on metabolism is limited. Therefore, the aim of this study was to investigate the direct effect of acute sex hormone exposure on glucose metabolism in skeletal muscle. Materials/Methods. Differentiated human skeletal myotubes were exposed to either 17 beta-estradiol or testosterone and metabolic characteristics were assessed. Glucose incorporation into glycogen, glucose oxidation, palmitate oxidation, and phosphorylation of key signaling proteins were determined. Results. Treatment of myotubes with either 17 beta-estradiol or testosterone decreased glucose incorporation into glycogen. Exposure of myotubes to 17 beta-estradiol reduced glucose oxidation under basal and insulin-stimulated conditions. However, testosterone treatment enhanced basal palmitate oxidation and prevented insulin action on glucose and palmitate oxidation. Acute stimulation of myotubes with testosterone reduced phosphorylation of S6K1 and p38 MAPK. Exposure of myotubes to either 17 beta-estradiol or testosterone augmented phosphorylation GSK3 beta(ser9) and PKC delta(Thr505), two negative regulators of glycogen synthesis. Treatment of myotubes with a PKC specific inhibitor (GFX) restored the effect of either sex hormone on glycogen synthesis. PKC delta silencing restored glucose incorporation into glycogen to baseline in response to 17 beta-estradiol, but not testosterone treatment. Conclusion. An acute exposure to supraphysiological doses of either 17 beta-estradiol or testosterone regulates glucose metabolism, possibly via PKC signaling pathways. Furthermore, testosterone treatment elicits additional alterations in serine/threonine kinase signaling, including the ribosomal protein S6K1 and p38 MAPK.
|
|
| 5. |
- Krämer, David Kitz, et al.
(författare)
-
Direct activation of glucose transport in primary human myotubes after activation of peroxisome proliferator-activated receptor delta
- 2005
-
Ingår i: Diabetes. - Alexandria, USA : American Diabetes Association. - 0012-1797 .- 1939-327X. ; 54:4, s. 1157-1163
-
Tidskriftsartikel (refereegranskat)abstract
- Activators of peroxisome proliferator-activated receptor (PPAR)gamma have been studied intensively for their insulin-sensitizing properties and antidiabetic effects. Recently, a specific PPARdelta activator (GW501516) was reported to attenuate plasma glucose and insulin levels when administered to genetically obese ob/ob mice. This study was performed to determine whether specific activation of PPARdelta has direct effects on insulin action in skeletal muscle. Specific activation of PPARdelta using two pharmacological agonists (GW501516 and GW0742) increased glucose uptake independently of insulin in differentiated C2C12 myotubes. In cultured primary human skeletal myotubes, GW501516 increased glucose uptake independently of insulin and enhanced subsequent insulin stimulation. PPARdelta agonists increased the respective phosphorylation and expression of AMP-activated protein kinase 1.9-fold (P < 0.05) and 1.8-fold (P < 0.05), of extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase (MAPK) 2.2-fold (P < 0.05) and 1.7-fold (P < 0.05), and of p38 MAPK 1.2-fold (P < 0.05) and 1.4-fold (P < 0.05). Basal and insulin-stimulated protein kinase B/Akt was unaltered in cells preexposed to PPARdelta agonists. Preincubation of myotubes with the p38 MAPK inhibitor SB203580 reduced insulin- and PPARdelta-mediated increase in glucose uptake, whereas the mitogen-activated protein kinase kinase inhibitor PD98059 was without effect. PPARdelta agonists reduced mRNA expression of PPARdelta, sterol regulatory element binding protein (SREBP)-1a, and SREBP-1c (P < 0.05). In contrast, mRNA expression of PPARgamma, PPARgamma coactivator 1, GLUT1, and GLUT4 was unaltered. Our results provide evidence to suggest that PPARdelta agonists increase glucose metabolism and promote gene regulatory responses in cultured human skeletal muscle. Moreover, we provide biological validation of PPARdelta as a potential target for antidiabetic therapy.
|
|
| 6. |
|
|
| 7. |
|
|
| 8. |
- Pandya, Jayesh M., et al.
(författare)
-
CD4+and CD8+CD28(null) T Cells Are Cytotoxic to Autologous Muscle Cells in Patients With Polymyositis
- 2016
-
Ingår i: Arthritis & Rheumatology. - : John Wiley & Sons. - 2326-5191 .- 2326-5205. ; 68:8, s. 2016-2026
-
Tidskriftsartikel (refereegranskat)abstract
- Objective. Inflammatory T cell infiltrates in the skeletal muscle tissue of patients with polymyositis are dominated by CD28-negative effector (CD28(null)) T cells of both the CD4 and CD8 lineage. These cells are potentially cytotoxic, and the aim of the present study was to develop a fully autologous cell culture system in which to investigate the functional contribution of such CD28(null) T cells to myotoxicity. Methods. In vitro cocultures of autologous skeletal muscle cells and T cell subsets obtained from 5 polymyositis patients were performed. Myotoxicity of T cells was quantified by calcein release and flow cytometric analyses. T cell degranulation was blocked with concanamycin A. Specific blocking of perforin, cytokines, and HLA was performed using antibodies. Results. Both CD4+CD28(null) and CD8+CD28(null) T cells induced more muscle cell death than did their CD28+ counterparts. Differentiated muscle cells (myotubes) were more sensitive to T cell-mediated cell death than were their precursors (myoblasts). Both CD8+ and CD4+ CD28(null) T cells displayed perforin polarization toward muscle cells and secreted higher levels of granzyme B and interferon-gamma (IFN gamma) in coculture than did CD28+ T cells. The myotoxic effects of CD28(null) T cells were reduced upon the blocking of perforin, cytokines, and HLA. Addition of IFN gamma or tumor necrosis factor did not induce skeletal muscle cell death in the absence of T cells; however, it did up-regulate HLA expression on muscle cells. Conclusion. Myotoxicity of CD4+ and CD8+ CD28(null) T cells is mediated by directed perforin-dependent killing and can be further influenced by IFN gamma-induced HLA expression on muscle cells. The data suggest that CD28(null) T cells are key effector cells that contribute to the muscle cell damage in polymyositis.
|
|
| 9. |
|
|
