CD28null T Cells from Polymyositis Patients Are Cytotoxic to Autologous Muscle Cells in Vitro Via... more CD28null T Cells from Polymyositis Patients Are Cytotoxic to Autologous Muscle Cells in Vitro Via Perforin-Dependent Mechanisms
CD28null T Cells from Polymyositis Patients Are Cytotoxic to Autologous Muscle Cells in Vitro Via... more CD28null T Cells from Polymyositis Patients Are Cytotoxic to Autologous Muscle Cells in Vitro Via Perforin-Dependent Mechanisms
Skeletal muscle is an insulin sensitive organ and plays a major role in whole body electrolyte an... more 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
Recombinant reverse transcriptase (RT) from HIV-1 subtype B was used to produce mouse anti-RT mon... more Recombinant reverse transcriptase (RT) from HIV-1 subtype B was used to produce mouse anti-RT monoclonal antibodies (MAbs). Immunization was done by mixing RT with the ISCOM matrix-forming adjuvant saponin (Quil A). Two different assays, both based on the interaction of native RT and antibodies, were used to monitor the immune response in mice and for screening, selection, and characterization of the MAbs. The first assay measures the capacity of antibodies to inhibit the polymerase activity of the RT and the second assay measures the ability of antibodies to capture enzymatically active RT. Twelve clones with the capacity to inhibit at least 50% of the RT activity and 34 clones with high RT-capturing capacity were found. The MAb panel was utilized to evaluate the immunological properties of 18 different RTs representing 9 different HIV1 subtypes. The RT-inhibitory MAbs could be divided into two groups based on their pattern of cross-reactivity toward the different HIV-1 RTs. The degree of diversity recorded among MAbs with RT-capturing capacity was larger. At least seven groups of MAbs with distinct cross-reactivity patterns were identified. Thus, the degree of isoenzyme specificity varied greatly, from MAbs that were quite specific for subtype B RT to one MAb that was able to capture the RTs from all HIV-1 isolates tested except one of the two group O isolates. In conclusion, our study revealed that there exist surprisingly large immunological differences between RTs from different HIV-1 subtypes as well as from the same subtype.
IgG binding to V3 peptides and serum neutralising responses were studied in four HIV-1 infected i... more IgG binding to V3 peptides and serum neutralising responses were studied in four HIV-1 infected individuals with progressive disease over a period of 31-70 months. The 18-20 mer peptides comprised residues 299-317 (numbering of HIV1 MN) in the N-terminal half of the V3 loop of the envelope glycoprotein gp120 and were derived from the sequences of autologous, as well as heterologous isolates. All four individuals studied lacked anti-V3 IgG binding to at least one autologous V3 sequence. V3 peptides to which autologous sera lacked binding IgG were all immunogenic in rabbits and induced antisera that were broadly cross-reactive by EIA and broadly cross-neutralising to primary HIV-1 isolates. This indicates that the peptides are immunogenic per se and that the respective human hosts have selective defects in recognising the corresponding V3 sequences. Despite the absence of antibody binding to autologous V3 peptides, the human sera had neutralising antibodies to autologous (three out of four cases), as well as heterologous isolates (all cases). Moreover, in vitro exposure of the patients' isolates to autologous neutralising serum or the homologous rabbit antiserum selected for variants with amino acid substitutions close to the crown of the V3 loop or in regions outside the sequence corresponding to peptides used for immunisation. The amino acid exchanges affected V3 positions known to be antigenic and which are also prone to change successively in infected persons. It is likely that neutralising antibodies recognise both linear and conformational epitopes in the V3 loop. Apparently, there are several, but restricted, numbers of ways for this structure to change its conformation and thereby give rise to neutralisation resistant viruses.
IgG binding to V3 peptides and serum neutralising responses were studied in four HIV-1 infected i... more IgG binding to V3 peptides and serum neutralising responses were studied in four HIV-1 infected individuals with progressive disease over a period of 31-70 months. The 18-20 mer peptides comprised residues 299-317 (numbering of HIV1 MN) in the N-terminal half of the V3 loop of the envelope glycoprotein gp120 and were derived from the sequences of autologous, as well as heterologous isolates. All four individuals studied lacked anti-V3 IgG binding to at least one autologous V3 sequence. V3 peptides to which autologous sera lacked binding IgG were all immunogenic in rabbits and induced antisera that were broadly cross-reactive by EIA and broadly cross-neutralising to primary HIV-1 isolates. This indicates that the peptides are immunogenic per se and that the respective human hosts have selective defects in recognising the corresponding V3 sequences. Despite the absence of antibody binding to autologous V3 peptides, the human sera had neutralising antibodies to autologous (three out of four cases), as well as heterologous isolates (all cases). Moreover, in vitro exposure of the patients' isolates to autologous neutralising serum or the homologous rabbit antiserum selected for variants with amino acid substitutions close to the crown of the V3 loop or in regions outside the sequence corresponding to peptides used for immunisation. The amino acid exchanges affected V3 positions known to be antigenic and which are also prone to change successively in infected persons. It is likely that neutralising antibodies recognise both linear and conformational epitopes in the V3 loop. Apparently, there are several, but restricted, numbers of ways for this structure to change its conformation and thereby give rise to neutralisation resistant viruses.
Arthritis & rheumatology (Hoboken, N.J.), Jan 19, 2016
Objectives Inflammatory T cell infiltrates in the skeletal muscle tissue of polymyositis patients... more Objectives Inflammatory T cell infiltrates in the skeletal muscle tissue of polymyositis patients are dominated by CD28-negative effector (CD28null) T cells of both the CD4 and CD8 lineage. These cells are potentially cytotoxic and here we developed a fully autologous cell culture system to investigate the functional contribution of such CD28null T cells towards myotoxicity. Methods In vitro co-cultures of patient-derived autologous skeletal muscle cells and T cell subsets were performed (n=5). Myotoxicity of T cells was quantified by calcein release and flow-cytometry. T cell degranulation was blocked by Concanamycin-A and specific blocking of perforin, cytokines and HLA was performed using antibodies. Results Both CD4+CD28null and CD8+CD28null T cells induced more muscle cell-death compared to their CD28+ counterparts. Differentiated muscle cells (myotubes) were more sensitive to T cell-mediated cell-death compared to their precursors (myoblasts). Both CD8+ and CD4+CD28null T cell...
In skeletal muscle, insulin stimulation leads to phosphorylation of Na(+),K(+)-ATPase alpha-subun... more In skeletal muscle, insulin stimulation leads to phosphorylation of Na(+),K(+)-ATPase alpha-subunits on both serine/threonine and tyrosine residues, translocation of Na(+),K(+)-ATPase molecules to the plasma membrane, and increased Na(+),K(+)-ATPase activity. The molecular nature of the tyrosine kinase that phosphorylates Na(+),K(+)-ATPase is not yet identified. In vitro phosphorylation experiments show that the alpha-subunit of Na(+),K(+)-ATPase from skeletal muscle is a substrate for the tyrosine-specific protein kinase c-src. Tyrosine phosphorylation of the alpha-subunits of Na(+),K(+)-ATPase may be an important mechanism for insulin-mediated regulation of Na(+),K(+)-ATPase translocation and activity.
Biochemical and biophysical research communications, Jan 18, 2003
Using RNA interference (RNAi), we specifically down-regulate protein expression in differentiated... more Using RNA interference (RNAi), we specifically down-regulate protein expression in differentiated human skeletal myotube cultures. Serum stimulation of myotubes increases glucose uptake. Using a sensitive photolabeling technique, we demonstrate that this increase in glucose uptake is accompanied by increased cell-surface content of glucose transporter (GLUT) 1. Using RNAi, we specifically reduce GLUT1 mRNA and protein expression, leading to inhibition of serum-mediated increase in glucose transport. Thus, we demonstrate the utility of RNAi in a primary human differentiated cell system, and apply this methodology to demonstrate that serum-mediated increase in glucose transport in human skeletal muscle cells is dependent on GLUT1.
Exposure of skeletal muscle to high levels of testosterone or estrogen induces insulin resistance... more 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. Differentiated human skeletal myotubes were exposed to either 17β-estradiol or testosterone and metabolic characteristics were assessed. Glucose incorporation into glycogen, glucose oxidation, palmitate oxidation, and phosphorylation of key signaling proteins were determined. Treatment of myotubes with either 17β-estradiol or testosterone decreased glucose incorporation into glycogen. Exposure of myotubes to 17β-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 17β-estradiol or testosterone augmented phosphorylation GSK3β(Ser9) and PKCδ(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. PKCδ silencing restored glucose incorporation into glycogen to baseline in response to 17β-estradiol, but not testosterone treatment. An acute exposure to supraphysiological doses of either 17β-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.
CD28null T Cells from Polymyositis Patients Are Cytotoxic to Autologous Muscle Cells in Vitro Via... more CD28null T Cells from Polymyositis Patients Are Cytotoxic to Autologous Muscle Cells in Vitro Via Perforin-Dependent Mechanisms
CD28null T Cells from Polymyositis Patients Are Cytotoxic to Autologous Muscle Cells in Vitro Via... more CD28null T Cells from Polymyositis Patients Are Cytotoxic to Autologous Muscle Cells in Vitro Via Perforin-Dependent Mechanisms
Skeletal muscle is an insulin sensitive organ and plays a major role in whole body electrolyte an... more 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
Recombinant reverse transcriptase (RT) from HIV-1 subtype B was used to produce mouse anti-RT mon... more Recombinant reverse transcriptase (RT) from HIV-1 subtype B was used to produce mouse anti-RT monoclonal antibodies (MAbs). Immunization was done by mixing RT with the ISCOM matrix-forming adjuvant saponin (Quil A). Two different assays, both based on the interaction of native RT and antibodies, were used to monitor the immune response in mice and for screening, selection, and characterization of the MAbs. The first assay measures the capacity of antibodies to inhibit the polymerase activity of the RT and the second assay measures the ability of antibodies to capture enzymatically active RT. Twelve clones with the capacity to inhibit at least 50% of the RT activity and 34 clones with high RT-capturing capacity were found. The MAb panel was utilized to evaluate the immunological properties of 18 different RTs representing 9 different HIV1 subtypes. The RT-inhibitory MAbs could be divided into two groups based on their pattern of cross-reactivity toward the different HIV-1 RTs. The degree of diversity recorded among MAbs with RT-capturing capacity was larger. At least seven groups of MAbs with distinct cross-reactivity patterns were identified. Thus, the degree of isoenzyme specificity varied greatly, from MAbs that were quite specific for subtype B RT to one MAb that was able to capture the RTs from all HIV-1 isolates tested except one of the two group O isolates. In conclusion, our study revealed that there exist surprisingly large immunological differences between RTs from different HIV-1 subtypes as well as from the same subtype.
IgG binding to V3 peptides and serum neutralising responses were studied in four HIV-1 infected i... more IgG binding to V3 peptides and serum neutralising responses were studied in four HIV-1 infected individuals with progressive disease over a period of 31-70 months. The 18-20 mer peptides comprised residues 299-317 (numbering of HIV1 MN) in the N-terminal half of the V3 loop of the envelope glycoprotein gp120 and were derived from the sequences of autologous, as well as heterologous isolates. All four individuals studied lacked anti-V3 IgG binding to at least one autologous V3 sequence. V3 peptides to which autologous sera lacked binding IgG were all immunogenic in rabbits and induced antisera that were broadly cross-reactive by EIA and broadly cross-neutralising to primary HIV-1 isolates. This indicates that the peptides are immunogenic per se and that the respective human hosts have selective defects in recognising the corresponding V3 sequences. Despite the absence of antibody binding to autologous V3 peptides, the human sera had neutralising antibodies to autologous (three out of four cases), as well as heterologous isolates (all cases). Moreover, in vitro exposure of the patients' isolates to autologous neutralising serum or the homologous rabbit antiserum selected for variants with amino acid substitutions close to the crown of the V3 loop or in regions outside the sequence corresponding to peptides used for immunisation. The amino acid exchanges affected V3 positions known to be antigenic and which are also prone to change successively in infected persons. It is likely that neutralising antibodies recognise both linear and conformational epitopes in the V3 loop. Apparently, there are several, but restricted, numbers of ways for this structure to change its conformation and thereby give rise to neutralisation resistant viruses.
IgG binding to V3 peptides and serum neutralising responses were studied in four HIV-1 infected i... more IgG binding to V3 peptides and serum neutralising responses were studied in four HIV-1 infected individuals with progressive disease over a period of 31-70 months. The 18-20 mer peptides comprised residues 299-317 (numbering of HIV1 MN) in the N-terminal half of the V3 loop of the envelope glycoprotein gp120 and were derived from the sequences of autologous, as well as heterologous isolates. All four individuals studied lacked anti-V3 IgG binding to at least one autologous V3 sequence. V3 peptides to which autologous sera lacked binding IgG were all immunogenic in rabbits and induced antisera that were broadly cross-reactive by EIA and broadly cross-neutralising to primary HIV-1 isolates. This indicates that the peptides are immunogenic per se and that the respective human hosts have selective defects in recognising the corresponding V3 sequences. Despite the absence of antibody binding to autologous V3 peptides, the human sera had neutralising antibodies to autologous (three out of four cases), as well as heterologous isolates (all cases). Moreover, in vitro exposure of the patients' isolates to autologous neutralising serum or the homologous rabbit antiserum selected for variants with amino acid substitutions close to the crown of the V3 loop or in regions outside the sequence corresponding to peptides used for immunisation. The amino acid exchanges affected V3 positions known to be antigenic and which are also prone to change successively in infected persons. It is likely that neutralising antibodies recognise both linear and conformational epitopes in the V3 loop. Apparently, there are several, but restricted, numbers of ways for this structure to change its conformation and thereby give rise to neutralisation resistant viruses.
Arthritis & rheumatology (Hoboken, N.J.), Jan 19, 2016
Objectives Inflammatory T cell infiltrates in the skeletal muscle tissue of polymyositis patients... more Objectives Inflammatory T cell infiltrates in the skeletal muscle tissue of polymyositis patients are dominated by CD28-negative effector (CD28null) T cells of both the CD4 and CD8 lineage. These cells are potentially cytotoxic and here we developed a fully autologous cell culture system to investigate the functional contribution of such CD28null T cells towards myotoxicity. Methods In vitro co-cultures of patient-derived autologous skeletal muscle cells and T cell subsets were performed (n=5). Myotoxicity of T cells was quantified by calcein release and flow-cytometry. T cell degranulation was blocked by Concanamycin-A and specific blocking of perforin, cytokines and HLA was performed using antibodies. Results Both CD4+CD28null and CD8+CD28null T cells induced more muscle cell-death compared to their CD28+ counterparts. Differentiated muscle cells (myotubes) were more sensitive to T cell-mediated cell-death compared to their precursors (myoblasts). Both CD8+ and CD4+CD28null T cell...
In skeletal muscle, insulin stimulation leads to phosphorylation of Na(+),K(+)-ATPase alpha-subun... more In skeletal muscle, insulin stimulation leads to phosphorylation of Na(+),K(+)-ATPase alpha-subunits on both serine/threonine and tyrosine residues, translocation of Na(+),K(+)-ATPase molecules to the plasma membrane, and increased Na(+),K(+)-ATPase activity. The molecular nature of the tyrosine kinase that phosphorylates Na(+),K(+)-ATPase is not yet identified. In vitro phosphorylation experiments show that the alpha-subunit of Na(+),K(+)-ATPase from skeletal muscle is a substrate for the tyrosine-specific protein kinase c-src. Tyrosine phosphorylation of the alpha-subunits of Na(+),K(+)-ATPase may be an important mechanism for insulin-mediated regulation of Na(+),K(+)-ATPase translocation and activity.
Biochemical and biophysical research communications, Jan 18, 2003
Using RNA interference (RNAi), we specifically down-regulate protein expression in differentiated... more Using RNA interference (RNAi), we specifically down-regulate protein expression in differentiated human skeletal myotube cultures. Serum stimulation of myotubes increases glucose uptake. Using a sensitive photolabeling technique, we demonstrate that this increase in glucose uptake is accompanied by increased cell-surface content of glucose transporter (GLUT) 1. Using RNAi, we specifically reduce GLUT1 mRNA and protein expression, leading to inhibition of serum-mediated increase in glucose transport. Thus, we demonstrate the utility of RNAi in a primary human differentiated cell system, and apply this methodology to demonstrate that serum-mediated increase in glucose transport in human skeletal muscle cells is dependent on GLUT1.
Exposure of skeletal muscle to high levels of testosterone or estrogen induces insulin resistance... more 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. Differentiated human skeletal myotubes were exposed to either 17β-estradiol or testosterone and metabolic characteristics were assessed. Glucose incorporation into glycogen, glucose oxidation, palmitate oxidation, and phosphorylation of key signaling proteins were determined. Treatment of myotubes with either 17β-estradiol or testosterone decreased glucose incorporation into glycogen. Exposure of myotubes to 17β-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 17β-estradiol or testosterone augmented phosphorylation GSK3β(Ser9) and PKCδ(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. PKCδ silencing restored glucose incorporation into glycogen to baseline in response to 17β-estradiol, but not testosterone treatment. An acute exposure to supraphysiological doses of either 17β-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.
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Papers by Lubna Al-khalili