Catalytically inactive lipoprotein lipase expression in muscle of transgenic mice increases very low density lipoprotein uptake: direct evidence that lipoprotein lipase bridging occurs in vivo

Lipoprotein lipase (LPL) is the central enzyme in plasma triglyceride hydrolysis. In vitro studies have shown that LPL also can enhance lipoprotein uptake into cells via pathways that are independent of catalytic activity but require LPL as a molecular bridge between lipoproteins and proteoglycans or receptors. To investigate whether this bridging function occurs in vivo, two transgenic mouse lines were established expressing a muscle creatine kinase promoter-driven human LPL (hLPL) minigene mutated in the catalytic triad (Asp156 to Asn). Mutated hLPL was expressed only in muscle and led to 3,100 and 3,500 ng/ml homodimeric hLPL protein in post-heparin plasma but no hLPL catalytic activity. Less than 5 ng/ml hLPL was found in preheparin plasma, indicating that proteoglycan binding of mutated LPL was not impaired. Expression of inactive LPL did not rescue LPL knock-out mice from neonatal death. On the wild-type (LPL2) background, inactive LPL decreased very low density lipoprotein (VLDL)-triglycerides. On the heterozygote LPL knock-out background (LPL1) background, plasma triglyceride levels were lowered 22 and 33% in the two transgenic lines. After injection of radiolabeled VLDL, increased muscle uptake was observed for triglyceride-derived fatty acids (LPL2, 1.7x; LPL1, 1.8x), core cholesteryl ether (LPL2, 2.3x; LPL1, 2.7x), and apolipoprotein (LPL1, 1.8x; significantly less than cholesteryl ether). Skeletal muscle from transgenic lines had a mitochondriopathy with glycogen accumulation similar to mice expressing active hLPL in muscle. In conclusion, it appears that inactive LPL can act in vivo to mediate VLDL removal from plasma and uptake into tissues in which it is expressed.     

Impaired glycolysis and protein catabolism induced by acid in L6 rat muscle cells

BACKGROUND: In skeletal muscle, metabolic acidosis stimulates protein degradation and oxidation of branched-chain amino acids. This could occur to compensate for impairment of glucose utilization induced by acid. METHODS: To test this hypothesis, glycolysis and protein degradation (release of [14C]-phenylalanine) were measured in L6 skeletal muscle cells cultured in Eagle's minimum essential medium at pH 7.1 or 7.5 for up to 3 days. RESULTS: No marked changes in total DNA or in cell viability were detected, nor was there any significant effect on intracellular pH or the water content of the cells (which is thought to be a key regulator of protein turnover, especially in liver). In spite of this, acid stimulated protein degradation, induced net protein loss from the cultures, inhibited glucose uptake and glycolysis (lactate output) and was associated with increased [1-14C]-leucine oxidation. Effects on protein degradation and glycolysis were gradual, reaching a maximum after 20-30 h. To investigate whether glycolytic flux itself can influence protein degradation, increased glycolysis was simulated by adding glucose (20 mmol L-1) or pyruvate (1 mmol L-1) to the medium. At pH 7.1, neither addition had any effect on protein degradation. CONCLUSION: Although acid-induced protein wasting is associated with impaired glycolysis, no obligatory coupling exists between glycolytic flux and protein degradation.     

The inhibition in vivo of lipoprotein lipase (clearing-factor lipase) activity by triton WR-1339

1. Lipoprotein lipase activity was measured in heart homogenates and in heparin-releasable and non-releasable fractions of isolated perfused rat hearts, after the intravenous injection of Triton WR-1339. 2. In homogenates of hearts from starved, rats, lipoprotein lipase activity was significantly inhibited (P less than 0.001) 2h after the injection of Triton. This inhibition was restricted exclusively to the heparin-releasable fraction. Maximum inhibition occurred 30 min after the injection and corresponded to about 60% of the lipoprotein lipase activity that could be released from the heart during 30 s perfusion with heparin. 3. Hearts of Triton-treated starved rats were unable to take up and utilize 14C-labelled chylomicron triacylglycerol fatty acids, even though about 40% of heparin-releasable activity remained in the hearts. 4. It is concluded that Triton selectively inhibits the functional lipoprotein lipase, i.e. the enzyme directly involved in the hydrolysis of circulating plasma triacylglycerols. 5. Lipoprotein lipase activities measured in homogenates of soleus muscle of starved rats and adipose tissue of fed rats were decreased by 25 and 39% respectively after Triton injection. It is concluded that, by analogy with the heart, these Triton-inhibitable activities correspond to the functional lipoprotein lipase.     

Differential regulation of glucose transport and glucose transporter (GLUT-1) gene expression by vanadate, phorbol ester and okadaic acid in L6 skeletal muscle cells

Vanadate, an inhibitor of protein tyrosine phosphatases (PTPases), elicited time-and-dose-dependent increases in glucose transport in rat muscle L6 cells in culture: the rate was increased by 150-175% over control in 24 h at 75-100 microM. In contrast, molybdate, another inhibitor of PTPases, failed to stimulate glucose transport. The effect of vanadate was not blocked by tyrosine kinase inhibitors, genistein or tyrphostin RG 50864, implying that tyrosine kinase activation may not mediate the action of vanadate. The ability of vanadate to stimulate glucose transport was preserved in cells whose protein kinase C (PKC) activity was down-regulated by prior exposure to phorbol esters (TPA), suggesting that the vanadate effect was unrelated to the TPA-sensitive PKC isoform(s). Okadaic acid, an inhibitor of protein phosphatases 1 and 2A, was a potent activator of glucose transport increasing the rate 7-fold in 24 h at a concentration of 50 nM. The increases in GLUT-1 mRNA level in response to vanadate and TPA were paralleled bh much smaller increases in immunoreactive GLUT-1 protein level, whereas okadaic acid treatment markedly elevated GLUT-1 protein without a concomitant change in GLUT-1 mRNA levels.     

Skeletal muscle cell-derived insulin-like growth factor (IGF) binding proteins inhibit IGF-I-induced myogenesis in rat L6E9 cells

The insulin-like growth factors (IGFs) stimulate the differentiation of skeletal muscle cells. IGF binding proteins (IGFBPs), which are expressed by skeletal muscle cells, may enhance or inhibit IGF actions. To explore the role of skeletal muscle-derived IGFBPs in IGF-induced myogenesis, we compared the differentiation-inducing effects of IGF-I and des(1-3)IGF-I in rat L6E9 skeletal myoblasts. Des(1-3)IGF-I is a naturally occurring IGF-I analog with markedly reduced affinity for IGFBPs but with an affinity for the IGF-I receptor that is comparable to that for native IGF-I. We find that rat L6E9 cells produce principally IGFBP-4 and BP-6, with a minor component of IGFBP-5. Both IGFBP-4 and BP-6 accumulate during differentiation and increase further in response to IGF-I or des(1-3)IGF-I treatment. We find that an IGF-I analog with reduced affinity for IGFBPs is significantly more potent than native IGF-I in stimulating myogenesis (as assessed by myogenin messenger RNA abundance and muscle creatine kinase activity), indicating that IGFBPs expressed by skeletal muscle cells inhibit differentiation induced by IGF-I. In view of the relative abundance of IGFBP-4, its relatively high affinity for IGF-I and the low affinity of IGFBP-6 for IGF-I, it is likely that the inhibitory effect of rat skeletal muscle-derived IGFBPs on IGF-I-induced myogenesis is mediated principally by IGFBP-4.     

Lipoprotein lipase transport in plasma: role of muscle and adipose tissues in regulation of plasma lipoprotein lipase concentrations

Lipoprotein lipase (LPL) is synthesized in tissues involved in fatty acid metabolism such as muscle and adipose tissue. LPL is also found in the circulation, but is mostly lipolytically inactive. The proportion of active circulating LPL increases after a fatty meal. We investigated the release of active and inactive LPL from adipose tissue and muscle in the fasting and postprandial states. Arteriovenous concentration gradients of LPL across adipose tissue and forearm muscle were measured in male subjects before and after a fat-rich meal (n = 7) and before and during infusion of a triacylglycerol emulsion (Intralipid) (n = 6). Plasma LPL activity rose after the meal and more so during Intralipid infusion. Plasma LPL mass (>95% inactive LPL) increased after the meal but decreased after Intralipid infusion. In the fasting state (n = 13) muscle efflux of LPL activity was 0.263 +/- 0.098 mU/min per 100 ml of muscle tissue whereas there was an influx of LPL activity to adipose tissue of 0.085 +/- 0.100 mU/min per 100 g of adipose tissue (P < 0. 02 muscle vs. adipose tissue). Similarly in the postprandial state only muscle released LPL activity. Both tissues released LPL mass. In the fasting state efflux was 17.8 +/- 8.8 ng/min per 100 ml muscle and 55.2 +/- 21.3 ng/min per 100 g of adipose tissue (P < 0. 05 muscle vs. adipose tissue). Release of LPL, either active or inactive, was not correlated with levels of non-esterified fatty acids or plasma triacylglycerol. In conclusion, there is a substantial release of LPL from adipose tissue and muscle, most of which is inactive. A small proportion of active LPL seems to be redistributed from muscle to adipose tissue.     

Expression of a voltage-dependent potassium current precedes fusion of human muscle satellite cells (myoblasts)

Using the whole-cell recording patch clamp technique in clonal cultures of human muscle satellite cells (SC), we studied a voltage-gated potassium current analogous to the delayed rectifier current (IKdr) described in adult human skeletal muscle. This current was absent in proliferating SC cultured in a growth medium containing 15% serum, except when the SC approached the end of their replicative life (between 77 and 124 days in culture); at that time, approximately 50% of the SC possessed IKdr. In contrast, IKdr was expressed within less than 4 days in approximately 70% of the SC cultured in a serum-free medium (SFM) and within 24 h in differentiating medium. We believe that IKdr may be a characteristic feature of fusion-component SC and that it may be involved in the fusion process for the following reasons: 1) after the transfer in differentiating medium, cultures of SC in which the expression of IKdr was previously promoted by exposure to SFM were found to fuse immediately, without the initial 24 h lag time observed in control sister cultures; 2) in the latter "naive" SC, IKdr was expressed during the first day in differentiating medium, before SC began to fuse; 3) most of the SC that did not fuse even after weeks of exposure to differentiating medium did not express IKdr; 4) TEA, at a concentration of 3 mM, reduces the amplitude of IKdr by 55% and the fusion index by 55-67%.     

Stimulation of glucose and amino acid transport and activation of the insulin signaling pathways by insulin lispro in L6 skeletal muscle cells

The monomeric insulin analogue insulin lispro (Lys B28, Pro B29) is a rapid-acting insulin with a shorter duration of activity than human regular insulin. This compound has the advantage of reducing early postprandial hyperglycemia and the accompanying late hypoglycemia, thereby improving overall blood glucose control. To date, all published studies of the functional properties of insulin lispro have been conducted in whole animals. This study aimed to characterize the cellular actions of insulin lispro and the signals it elicits in an insulin-sensitive muscle cell line, L6 cells. Comparing the cellular actions of insulin lispro with those of human regular insulin, a number of observations were made. (1) Insulin lispro stimulated glucose and amino acid transport into L6 myotubes with a dose dependency and time course virtually identical to those of human regular insulin. (2) Insulin lispro was as effective as human regular insulin in stimulating time-dependent phosphorylation of insulin receptor substrate 1 (IRS-1), p70 ribosomal S6 kinase, and two isoforms of mitogen-activated protein kinase (ERK1 and ERK2). (3) Insulin lispro's ability to induce the association of IRS-1 with the p85 subunit of phosphatidylinositol 3-kinase was similar to that of human regular insulin. (4) As with human regular insulin, 100 nmol of the fungal metabolite wortmannin completely inhibited insulin lispro stimulation of glucose uptake. We concluded that the cellular actions of insulin lispro are similar to those of human regular insulin with respect to glucose and amino acid uptake and that the biochemical signals elicited are also comparable.     

Combined lipase deficiency (cld/cld) in mice affects differently post-translational processing of lipoprotein lipase, hepatic lipase and pancreatic lipase

A method is described for the measurement of 5,5-diphenylbarbituric acid in plasma using high-performance liquid chromatography with UV detection. Briefly, the compounds are separated on a C18 reversed-phase column using a mobile phase of 50 mM sodium acetate (pH 4.5) and methanol. The flow-rate is 1.0 ml/min and 25 microl are injected and detected at 215 nm. The method is specific and sensitive in the range of concentrations tested, with a limit of quantification of 0.25 microg/ml. The calibration curves are linear for concentrations between 0.25 and 10 microg/ml. Intra-day and inter-day coefficients of variation are less than 8.5 and 10.5%, respectively, over the linear range. Intra-day and inter-day bias are less than 7.0 and 8.0%, respectively. A pharmacokinetic study conducted in male Beagle dogs administered 10 mg/kg of 1,3-dimethoxymethyl-5,5-diphenylbarbituric acid or 8 mg/kg of 5,5-diphenylbarbituric acid intravenously demonstrates the utility of this method.     

Effect of zidovudine (AZT) on the structure and function of rat skeletal muscle

The role of the anti-HIV agent zidovudine (ZDV = AZT) in the generation of mitochondrial myopathies and subsequent skeletal muscle contractile deficiencies was evaluated in male rats given ZDV in drinking water (1 mg/mL). After 6 weeks, there was no difference in treadmill run time between experimental (n = 6) and control (n = 6) rats. ZDV did not affect tension output by the gastrocnemius-plantaris-soleus muscle group when stimulated in situ at frequencies of 15, 30, 45, and 75 tetani/min, nor did the drug affect the cytochrome oxidase activity of fast glycolytic, fast oxidative glycolytic, or slow oxidative fiber types after 6 or 15 weeks of treatment. A group of female rats, similarly evaluated after 6 weeks of ZDV at 1 (n = 4) or 2 (n = 4) mg/mL, also did not display any discernable deficiencies. However, when the data from all 10 control rats were compared with those of the 19 ZDV rats, the cytochrome oxidase activity of fast oxidative glycolytic muscle of the ZDV rats was significantly higher (35.0 +/- 1.36 versus 40.7 +/- 1.14 mumol.min-1.g-1; p < 0.05). No ultrastructural abnormalities were observed in 15-week ZDV-treated cardiac muscle or in any of the three skeletal muscle fiber types. These results suggest that ZDV-related myopathies observed in AIDS patients may be due to interactions between the drug and complications associated with HIV infection.     

Induction of c-myc and c-jun proto-oncogene expression in rat L6 myoblasts by cadmium is inhibited by zinc preinduction of the metallothionein gene

An outline of some procedures used for computer aided drug design has been given. The emphasis is on lead generation, both primary and secondary, and on current lead optimization processes which rely on an appreciation of ligand-receptor interactions. The areas covered include techniques for structure based drug design and lead optimization, ligand-receptor interaction studies using molecular surfaces and docking, extraction of pharmacophore pattern to obtain new leads, expert system application etc.     

Myosin heavy chain composition in young and old rat skeletal muscle: effects of endurance exercise

The objective of this study was to determine the effects of age and exercise on the myosin heavy chain (MHC) composition of skeletal muscle. Young (3 mo) and old (22 mo) female specific pathogen-free barrier-reared Fischer 344 rats were randomly assigned to young untrained or young trained and old untrained or old trained groups, respectively. Young trained and old trained animals performed endurance exercise training on a motorized treadmill for 8 wk. Succinate dehydrogenase activity and MHC isoforms were measured in the plantaris (Plan), lateral and medial gastrocnemius (Gast), and soleus (Sol) muscles. In sedentary animals, aging resulted in a decrease (P < 0.05) in type IIb MHC and an increase (P < 0.05) in type IIa MHC in both the Gast and Plan muscles. Also, aging resulted in a small but significant increase (approximately 4%; P < 0.05) in type I MHC in the Sol. Exercise training resulted in significant (P < 0.05) increases in Gast, Plan, and Sol succinate dehydrogenase activity in both young and old animals. Furthermore, exercise training resulted in a decrease (P < 0.05) in the percentage of type IIb MHC and an increase (P < 0.05) in the percentage of type IIa MHC in the Plan in both young and old animals. These data suggest that there is an age-related shift in locomotor muscle MHC isoforms from a faster to a slower isoform.     

The effects of fibroblast growth factors in long-term primary culture of dystrophic (mdx) mouse muscle myoblasts

Neutralizing monoclonal antibodies specific for human interleukin-6 (IL-6) bind two distinct sites on the IL-6 protein (sites I and II). Their interference with IL-6 receptor binding suggested that site I is a receptor-binding site of IL-6, whereas site II is important for signal transduction. Mutagenesis of site II could therefore result in the isolation of IL-6 receptor antagonists. To test this hypothesis, a panel of IL-6 mutant proteins was constructed that did not bind to a site II-specific monoclonal antibody. One such site II mutant protein (with double substitution of Gln-160 with Glu and Thr-163 with Pro) was found to be an antagonist of human IL-6. It was inactive on human CESS cells, weakly active on human HepG2 cells, but active on mouse B9 cells. It could specifically antagonize the activity of wild-type IL-6 on CESS and HepG2 cells. The binding affinity of this variant for the 80-kDa IL-6 receptor was similar to that of wild-type IL-6. High affinity binding to CESS cells, however, was abolished, suggesting that the mutant protein is inactive because the complex of the 80-kDa IL-6 receptor and the mutant protein cannot associate with the signal transducer gp130. The human IL-6 antagonist protein may be potentially useful as a therapeutic agent.     

Decreased basal, noninsulin-stimulated glucose uptake and metabolism by skeletal soleus muscle isolated from obese-hyperglycemic (ob/ob) mice

Insulin resistance of diaphragms of ob/ob mice has been repeatedly demonstrated previously both in vitro and in vivo. In the present study, transport and metabolism of glucose with and without insulin stimulation were compared in a skeletal muscle more likely than diaphragm or heart to be representative of the overall striated muscle mass, i.e. isolated soleus muscle. Compared with soleus muscle from lean controls, unstimulated lactate release in the presence of exogenous glucose was depressed from 16.2 to 12.3 nmol/60 min per mg wet wt in soleus from ob/ob mutants; glycolysis was decreased from 6.6 to 3.7 and [14C]glucose oxidation to 14CO2 from 0.90 to 0.33 nmol glucose/60 min per mg wet wt. Uptake of 2-deoxyglucose (2-DOG), both with and without insulin, was very much less for soleus from ob/ob than from lean mice, at 2-DOG concentrations ranging from 0.1 to 10 mM, and in mice of 6-15 wk. When 2-DOG concentration was 1 mM, its basal uptake was 0.53 nmol/30 min per mg wet wt for soleus of ob/ob as against 0.96 for soleus of lean mice. The absolute increment due to 1 mU/ml insulin was 0.49 in muscle of ob/ob as against 1.21 in that of lean mice. When the resistance to insulin action was decreased by pretreatment in vivo by either streptozotocin injection or fasting, the decreased basal 2-DOG uptake of subsequently isolated soleus muscle was not improved. Inhibition of endogenous oxidation of fatty acids by 2-bromostearate, while greatly increasing 14CO2 production from [14C]glucose, did not affect basal [5-3H]glucose metabolism or 2-DOG uptake. It is suggested that transport and/or phosphorylation of glucose under basal, unstimulated conditions are depressed in soleus muscle of ob/ob mice, whether or not resistance to insulin and hyperinsulinemia are also present. Although the origin of the decreased basal glucose uptake remains unknown it might be related to a similar decrease in basal glucose uptake by ventromedial hypothalamic cells, an event presumably resulting in a tendency to hyperphagia. Decreased basal glucose uptake by soleus muscle of ob/ob mice might explain the hyperglycemia, and hence partly the hyperinsulinemia and excessive fat deposition of those animals.     

Proliferative effects of oxidized low-density lipoprotein on vascular smooth muscle cells: role of dietary habits

The effects were studied of native, partially-oxidized and totally-oxidized human low-density lipoprotein (LDL) on the proliferation of cultured rat aortic smooth muscle cells (VSMC), measured as an altered DNA synthesis. The LDL was obtained from three different human long-term diet groups (a control diet rich in saturated fats, a vegetarian diet, and a fish diet). The oxidized LDLs were prepared by oxidizing the LDL with copper sulfate. The DNA synthesis was measured by [3H]-thymidine incorporation into the DNA. The partially-oxidized LDL was the most potent promoter of DNA synthesis compared to the native or totally-oxidized LDL of the same diet group. The partially-oxidized LDL had a true mitogenic effect in the absence of exogenous growth factors. The native and totally-oxidized LDL induced a significant increase in DNA synthesis, if they were obtained from the fish diet group. This study suggests an enhanced proliferative effect of partially-oxidized LDL on VSMC growth.     

Effects of membrane potential on just detectable movement in rat skeletal muscle: effects of denervation

The potential, Vt, at which a brief test depolarization first elicited movement was determined using two-microelectrode point voltage clamp. We expected that inactivation of excitation-contraction coupling at conditioning potentials between -60 and 0 mV would shift Vt to more positive potentials, and that fibers would become inactivatable with less conditioning depolarization in EDL than soleus. The curve relating Vt to conditioning potential had a negative slope (which was insensitive to addition of 1 mM cobalt or replacement of calcium with 20 mM CaEGTA) between -60 and -35 mV and a steep positive slope with further depolarization Unexpectedly, fibers became inactivatable with less conditioning depolarization in soleus than in EDL when Vt was measured with 50 msec test pulses. However, the positive shift in Vt became less steep as test pulse duration lengthened in soleus fibers. When Vt obtained with test pulses approaching rheobase (10 msec in EDL and 500 msec in soleus) was compared, EDL fibers became inactive with less conditioning depolarization than soleus fibers. The increase in Vt became steeper with 1 mM cobalt or 20 mM CaEGTA and was shifted to more positive potentials by denervation in soleus fibers. We conclude that inactivation (i) does not strongly influence threshold contractions at conditioning potentials between -60 and -40 mV and (ii) influences Vt between -40 and 0 mV in a manner that depends on test pulse duration.     

Rapid uptake and binding of estradiol-17beta-6-(O-carboxymethyl)oxime:125I-labeled BSA by female rat liver

To investigate potential membrane-mediated responses to estrogen, a membrane-impermeant, radioiodinated, steroid-BSA conjugate--estradiol-17beta-6-(O-carboxymethyl)oxime:125I-labeled BSA (17beta-E-6-125I-BSA)--or related steroid conjugates, or 125I-BSA was injected into female Sprague-Dawley rats, and tissues were collected at varying times postinjection. The liver, adrenal, and spleen displayed the most prominent uptake of 17beta-E-6-125I-BSA, reaching a maximum of 43 times blood levels in sonicated liver samples at 5 min postinjection, but no uptake of 125I-BSA. Isolation of liver membranes by differential centrifugation showed that over 50% of recovered radioactivity was in association with microsomes and plasmalemma (P3 fraction) at 30 sec postinjection. By 60 min postinjection, over 75% of recovered radioactivity was in association with mitochondrial and lysosomal membranes (P2 fraction), and less than 10% remained in the P3 fraction. In vitro competition assays demonstrated two binding sites in liver P3 fractions. The spleen and liver also showed saturable binding in vivo. These data suggest the presence of at least one membrane-binding protein for estrogen in liver, adrenal, and spleen. Initial studies of affinity-purified liver P3 fractions using ligand blots indicated the presence of two binding proteins. These potential membrane estrogen-binding proteins may be involved in a very rapid shuttling of estrogen from the plasmalemma to mitochondria and lysosomes.