Insulin processing and signal transduction in rat adipocytes

A glycine-HCl buffer (glycine, 50 mM/NaCl, 0.15 M/HCl, pH 3.5) was used to strip insulin bound to adipocyte cell surfaces. Adipocytes retained their integrity in the glycine buffer and their binding capacity for [125I]iodoinsulin could be completely recovered on transfer of the cells to physiological media. At 37 degrees C, [125I]iodoinsulin binds rapidly to plasma membrane receptors; maximal binding occurs within 10 min. At this temperature, the initial binding is followed by rapid internalization, degradation of the hormone and subsequent loss of label. Insulin treatment, at 37 degrees C, induced internalization of 37% of the plasma membrane insulin receptors. Phenylarsine oxide (PAO), a confirmed inhibitor of protein internalization, allowed insulin binding but completely inhibited degradation of the hormone. Monensin, a carboxylic ionophore which impairs uncoupling hormone-receptor complexes, effectively restricted insulin degradation over short time periods (less than 30 min). Addition of monensin to insulin-stimulated cells did not impair D-glucose uptake. It has previously been reported that PAO inhibits hexose transport through the direct interaction with the glucose transporters and low concentrations of PAO (1 microM) transiently inhibit insulin-stimulated glucose uptake. This recovery phenomenon was again observed when PAO was added to insulin-stimulated, monensin-treated adipocytes. The data suggests that lysosomal degradation of insulin is not requisite for signal transduction.     

Concentrations of signal transduction proteins exercise and insulin responses in rat extensor digitorum longus and soleus muscles

Differences in the concentrations of signal transduction proteins often alter cellular function and phenotype, as is evident from numerous, heterozygous knockout mouse models for signal transduction proteins. Here, we measured signal transduction proteins involved in the adaptation to exercise and insulin signalling in fast rat extensor digitorum longus (EDL; 3% type I fibres) and the slow soleus muscles (84% type I fibres). The EDL and soleus were excised from four rats, the proteins extracted and subjected to Western blots for various signal transduction proteins. Our results show major differences in signal transduction protein concentrations between EDL and soleus. The EDL to soleus concentration ratios were: Calcineurin: 1.43 +/- 0.10; ERK1: 0.38 +/- 0.18; ERK2: 0.61 +/- 0.16; p38alpha, beta: 1.36 +/- 0.15; p38gamma/ERK6: 0.95 +/- 0.11; PKB/AKT: 1.44 +/- 0.08; p70S6k: 6.86 +/- 3.58; GSK3beta: 0.69 +/- 0.03; myostatin: 1.95 +/- 0.43; NF-kappaB: 0.32 +/- 0.10 (values >1 indicate higher expression in the EDL, and values < 1 indicate higher expression in the soleus). With the exception of p38gamma/ERK6, the concentration of each signal transduction protein was uniformly higher in one muscle than in the other in all four animals. These experiments show that signal transduction protein concentrations vary between fast and slow muscles, presumably reflecting a concentration difference on a fibre level. Proteins that promote particular functions such as growth or slow phenotype are not necessarily higher in muscles with that particular trait (e.g. higher in larger fibres or slow muscle). Interindividual differences in fibre composition might explain variable responses to training and insulin.     

Action of insulin modulated by pertussis toxin in rat adipocytes

We studied the effect of pertussis toxin (PT) treatment on the ability of insulin to inhibit lipolysis and to stimulate glucose oxidation in isolated rat adipocytes. In cells maximally modified by PT (100% ADP ribosylation of a 41-kdalton protein in membranes), the ability of insulin to inhibit lipolysis stimulated either by PT alone or in combination with a catecholamine was abolished. In cells wherein ADP ribosylation was submaximal (about 67% modification), a small but variable antilipolytic action of insulin could still be detected. In cells maximally modified by PT, both basal and insulin-stimulated glucose oxidation were markedly reduced (to 10-15% of control levels). However, relative to the basal oxidation level, the fold stimulation by insulin in PT-treated cells was equivalent to the fold stimulation in control cells. Nonetheless, PT treatment caused a rightward shift in the dose-response curve for insulin-stimulated glucose oxidation as well as a small reduction in insulin binding. Our results point strongly not only to a link between the inhibitory guanine nucleotide regulatory complex (Gi) and the antilipolytic action of insulin but also to a link between the Gi complex and the overall regulation of glucose metabolism in adipocytes.     

Effects of pertussis toxin treatment on the metabolism of rat adipocytes

The protein toxin present in Bordetella pertussis vaccine blocks the inhibition of adenylate cyclase by prostaglandins and adenosine which may be secondary to ADP-ribosylation of an inhibitory guanine nucleotide-binding protein. The stimulatory effects of alpha 1-catecholamine agonists on 32P uptake into phosphatidic acid and phosphatidylinositol in isolated rat adipocytes were virtually abolished by pertussis toxin treatment. In contrast, the stimulatory effects of insulin were increased in adipocytes after pertussis toxin treatment. Pertussis toxin treatment did not alter insulin stimulation of glucose oxidation and actually increased glucose conversion to lipid. Basal lipolysis was elevated in adipocytes by pertussis toxin treatment but not basal cyclic AMP. However, the increases in cyclic AMP and lipolysis due to low concentrations of catecholamines and forskolin were markedly potentiated by pertussis toxin treatment. The inhibitory effects of adenosine on cyclic AMP stimulation due to catecholamines were abolished by pertussis toxin. These data indicate that pertussis toxin selectively interferes with inhibition of cyclic AMP accumulation in rat adipocytes by adenosine, potentiates the increases in cyclic AMP due to catecholamines, increases the stimulatory effects of insulin on adipocyte metabolism, and interferes with alpha 1-catecholamine stimulation of phosphatidylinositol turnover.     

Concentrations of signal transduction proteins mediating exercise and insulin responses in rat extensor digitorum longus and soleus muscles

Differences in the concentrations of signal transduction proteins often alter cellular function and phenotype, as is evident from numerous, heterozygous knockout mouse models for signal transduction proteins. Here, we measured signal transduction proteins involved in the adaptation to exercise and insulin signalling in fast rat extensor digitorum longus (EDL; 3% type I fibres) and the slow soleus muscles (84% type I fibres). The EDL and soleus were excised from four rats, the proteins extracted and subjected to Western blots for various signal transduction proteins. Our results show major differences in signal transduction protein concentrations between EDL and soleus. The EDL to soleus concentration ratios were: Calcineurin: 1.43 ± 0.10; ERK1: 0.38 ± 0.18; ERK2: 0.61 ± 0.16; p38, : 1.36 ± 0.15; p38/ERK6: 0.95 ± 0.11; PKB/AKT: 1.44 ± 0.08; p70S6k: 6.86 ± 3.58; GSK3: 0.69 ± 0.03; myostatin: 1.95 ± 0.43; NF-B: 0.32 ± 0.10 (values >1 indicate higher expression in the EDL, and values <1 indicate higher expression in the soleus). With the exception of p38/ERK6, the concentration of each signal transduction protein was uniformly higher in one muscle than in the other in all four animals. These experiments show that signal transduction protein concentrations vary between fast and slow muscles, presumably reflecting a concentration difference on a fibre level. Proteins that promote particular functions such as growth or slow phenotype are not necessarily higher in muscles with that particular trait (e.g. higher in larger fibres or slow muscle). Interindividual differences in fibre composition might explain variable responses to training and insulin. (Mol Cell Biochem 261: 111–116, 2004)     

Effect of pertussis toxin on water metabolism in the rat

Pertussis toxin administered to rats resulted in a polyuric state that was due to enhanced renal water excretion. Pertussis toxin also induced a defect in renal water conservation. These abnormalities in renal water excretion could not be attributed to polydipsia, impaired synthesis and/or release of arginine vasopressin or renal tubular dysfunction with solute diuresis. No evidence of pertussis toxin-induced renal tubular damage was present. These results indicate that pertussis toxin induces nephrogenic diabetes insipidus and this defect occurs at the level of the renal collecting tubule.     

Selective regulation by pertussis toxin of insulin-induced activation of particulate cAMP phosphodiesterase activity in 3T3-L1 adipocytes

Incubation of 3T3-L1 adipocytes with insulin or isoproterenol for 10 min increased particulate "low Km" cAMP phosphodiesterase activity by 42% and 50%, respectively. Pertussis toxin catalyzed the [32P]-ADP ribosylation of a 41,000 dalton protein in adipocyte particulate fractions; prior incubation of adipocytes with toxin markedly reduced incorporation of radiolabel. Exposure of adipocytes to pertussis toxin (0.3 microgram, 18 hr) increased glycerol production and inhibited activation of cAMP phosphodiesterase by insulin, but not by isoproterenol. These results suggest that pertussis toxin can interfere with receptor-mediated processes that stimulate cAMP hydrolysis as well as those that inhibit cAMP formation.     

Effect of tumor necrosis factor-alpha on insulin signal transduction in rat adipocytes: relation to PKCbeta and zeta translocation

Although much evidence has been accumulated suggesting that tumor necrosis factor-alpha (TNF-alpha) is an important mediator of insulin resistance, the precise mechanism involved is still unclear. Recently, it has been reported that insulin-induced glucose uptake is mediated by activation of second messengers such as insulin receptor substrate 1 (IRS-1), phosphatidylinositol 3-kinase (PI3K), and diacylglycerol (DG)-protein kinase C (PKC). We have examined the effect of TNF-alpha on insulin-induced glucose uptake and activations of tyrosine kinase, IRS-1, PI3K and PKC in rat adipocytes. Pretreatment with 0.1-100 nM TNF-alpha for 60 min resulted in a significant decrease in 10 nM insulin- or 1 microM 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced [3H]2-deoxyglucose uptake without affecting basal glucose uptake. 10 nM insulin-stimulated activation of tyrosine kinase, IRS-1 and PI3K was suppressed by preincubation with 0.1-10 nM TNF-alpha for 60 min. 10 nM TNF-alpha pretreatment also suppressed 10 nM insulin- and 1 microM TPA-induced increases in membrane-associated PKCbeta and PKCzeta. Furthermore, 10 nM TNF-alpha, by itself, altered PKCbeta translocation from the membrane to cytosol. These results suggest that TNF-alpha inhibits insulin-stimulated activation of both the tyrosine kinase-IRS-1-PI3K-PKCzeta pathway and DG-PKC pathway. Finally, TNF-alpha contributes to insulin resistance in rat adipocytes.     

The effects of pertussis toxin and cholera toxin on mitogen-induced interleukin-2 production: evidence for G protein involvement in signal transduction

GTP-binding proteins, known as G proteins, play important roles in transducing signals generated by the binding of specific ligands to cell surface receptors. We examined the possibility that a G protein is involved in transducing the concanavalin A (Con A) signal for IL-2 production using a T-cell hybridoma, FS6-14.13, and the bacterial toxins, pertussis toxin (PTX) and cholera toxin (CTX). These toxins are known to interact with and modify the functions of G proteins. High concentrations of PTX (25-50 micrograms/ml) stimulated IL-2 production in the FS-6 cells in the absence of Con A, presumably due to the ability of its B subunit to crosslink membrane proteins. However, in the presence of Con A, PTX inhibited IL-2 production at concentrations ranging from 0.05 to 50 micrograms/ml. It is unlikely that this inhibition was due to a competitive interaction between Con A and PTX for binding sites at the cell surface, since high concentrations of PTX only minimally reduced Con A-FITC binding, evaluated by FACS analysis. In addition, concentrations of PTX which were not able to stimulate IL-2 production in the absence of Con A, retained their ability to inhibit IL-2 production in the presence of Con A. These data suggest the involvement of the PTX A subunit in this activity. In support of this possibility, PTX catalyzed ADP-ribosylation of a Mr = 41,000-Da protein in FS-6 membranes. This strongly suggests that a PTX substrate is involved in transducing the Con A signal for IL-2 production in FS-6 cells. CTX also inhibited Con A-induced IL-2 production, an effect mimicked by the addition of dibutyryl-cAMP. This suggests that a CTX substrate linked to the adenylyl cyclase-cAMP pathway is probably not involved in transducing the stimulatory Con A signal, but may play a role in downregulating T-cell activation.     

Effect of pertussis toxin on hormonal responsiveness of rat hepatocytes

The ureogenic action of epinephrine in hepatocytes from normal adult rats is mediated through activation of alpha 1-adrenoceptors. beta-Adrenoceptors in addition to alpha 1-adrenoceptors, became involved in mediating this effect in cells from animals treated with pertussis toxin. The accumulation of cyclic-AMP in response to epinephrine or isoproterenol was markedly increased in hepatocytes from pertussis-treated rats as compared to that observed in control cells. The accumulation of cyclic-AMP due to glucagon was also increased. It is suggested that pertussis toxin may release a constraint on adenylate cyclase activity by blocking the inhibitory coupling mechanism (Ni) or some other entity involved in the regulation of the activity of this enzyme.     

Different effects of two proteinase inhibitors on insulin-induced cellular responses in rat adipocytes

Among various proteinase inhibitors, N-acetyl-L-tyrosine ethyl ester (ATEE), a chymotrypsin substrate analog, and N alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), a trypsin inhibitor, showed significant inhibitory effects on insulin stimulated glucose transport in rat adipocytes. ATEE did not affect insulin binding, but inhibited insulin internalization. In intact adipocytes, ATEE inhibited tyrosine phosphorylation of the beta-subunit of the insulin receptor, a 170 kDa protein and a 60 kDa protein at almost the same concentration (ID50 = 0.24 +/- 0.05 mM, n = 4, mean +/- S.E.), but in a plasma membrane fraction, ATEE did not appreciably inhibit the tyrosine phosphorylation of the beta-subunit of the insulin receptor, TLCK did not inhibit insulin binding. At 0.25 mM, TLCK did not inhibit insulin internalization, but inhibited 70% of the insulin-stimulated glucose transport (ID50 = 0.19 +/- 0.02 mM, n = 7). TLCK inhibited insulin internalization at more than 0.25 mM. TLCK did not inhibit the tyrosine phosphorylation of the beta-subunit of the insulin receptor in intact cells or in the plasma membrane fraction. In intact cells, TLCK inhibited the phosphorylation of the 60 kDa protein and simultaneously it stimulated the phosphorylation of the 170 kDa protein more than 3-fold. These results indicate that there are at least two sites in the insulin-induced signal transduction pathway where proteinase inhibitors act to suppress the insulin signal transduction. A major ATEE site is very close to phosphorylation of the beta-subunit of the insulin receptor. On the other hand, TLCK inhibits a step(s) in the signal transduction pathway after the insulin receptor but before the glucose transporter.     

Sulfonylurea enhances insulin-induced acetyl coenzyme A carboxylase activity in rat adipocytes.

The effect of sulfonylurea on the activity of acetyl-coenzyme A carboxylase, a rate limiting enzyme of lipogenesis, was investigated using isolated rat adipocytes. Insulin significantly increased the enzyme activity by 170% of the control level, while glucagon and epinephrine decreased the activity of the enzyme by 53% and 64% of the control, respectively. In the presence of tolbutamide (10(-3) M) or glibenclamide (10(-6) M), a significant potentiation of insulin action was found in adipocytes. In addition, sulfonylurea restored the activity of acetyl-CoA carboxylase reduced by glucagon or epinephrine to the control level. Sulfonylurea enhancement of the acetyl-CoA carboxylase activity may offer one possible explanation for a mechanism of antilipolytic action of the drug in adipocytes.     

Effect of acetoacetate on glucose metabolism in the soleus and extensor digitorum longus muscles of the rat.

1. The effect of acetoacetate on glucose metabolism was compared in the soleus, a slow-twitch red muscle, and the extensor digitorum longus, a muscle composed of 50% fast-twitch red and 50% white fibres. 2. When incubated for 2h in a medium containing 5 mM-glucose and 0.1 unit of insulin/ml, rates of glucose uptake, lactate release and glucose oxidation in the soleus were 19.6, 18.6 and 1.47 micronmol/h per g respectively. Acetoacetate (1.7 mM) diminished all three rates by 25-50%; however, it increased glucose conversion into glycogen. In addition, it caused increases in tissue glucose, glucose 6-phosphate and fructose 6-phosphate, suggesting inhibition of phosphofructokinase. The concentrations of citrate, an inhibitor of phosphofructokinase, and of malate were also increased. 3. Rates of glucose uptake and lactate release in the extensor digitorum longus were 50-80% of those in the soleus. Acetoacetate caused moderate increases in tissue glucose 6-phosphate and possibly citrate, but it did not decrease glucose uptake or lactate release. 4. The rate of glycolysis in the soleus was approximately five times that previously observed in the perfused rat hindquarter, a muscle preparation in which acetoacetate inhibits glucose oxidation, but does not alter glucose uptake or glycolysis. A similar rate of glycolysis was observed when the soleus was incubated with a glucose-free medium. Under these conditions, tissue malate and the lactate/pyruvate ratio in the medium were decreased, and acetoacetate did not decrease lactate release or increase tissue citrate or glucose 6-phosphate. An intermediate rate of glycolysis, which was not decreased by acetoacetate, was observed when the soleus was incubated with glucose, but not insulin. 5. The data suggest that acetoacetate glucose inhibits uptake and glycolysis in red muscle under conditions that resemble mild to moderate exercise. They also suggest that the accumulation of citrate in these circumstances is linked to the rate of glycolysis, possibly through the generation of cytosolic NADH and malate formation.     

Effect of dilution rate on the release of pertussis toxin and lipopolysaccharide ofBordetella pertussis

Summary The kinetics ofBordetella pertussis growth was studied in a glutamate-limited continuous culture. Growth kinetics corresponded to Monod's model. The saturation constant and maximum specific growth rate were estimated as well as the energetic parameters, theoretical yield of cells and maintenance coefficient. Release of pertussis toxin (PT) and lipopolysaccharide (LPS) were growth-associated. In addition, they showed a linear relationship between them. Growth rate affected neither outer membrane proteins nor the cell-bound LPS pattern.Nomenclature X cell concentration (g L^–1) - specific growth rate (h^–1) - _m maximum specific growth rate (h^–1) - D dilution rate (h^–1) - S concentration of growth rate-limiting nutrient (glutamate) (mmol L^–1 or g L^–1) - Ks substrate saturation constant (mol L^–1) - m_s maintenance coefficient (g g^–1 h^–1) - Y_x/s theoretical yield of cells from glutamate (g g^–1) - Y_x/s yield of cells from glutamate (g g^–1) - Y_PT/s yield of soluble PT from glutamate (mg g^–1) - Y_KDO/s yield of cell-free KDO from glutamate (g g^–1) - Y_PT/x specific yield of soluble PT (mg g^–1) - Y_KDO/x specific yield of cell-free KDO (g g^–1) - q_PT specific soluble PT production rate (mg g^–1 h^–1) - q_KDO specific cell-free KDO production rate (g g^–1 h^–1)     

Inactivation of excitation-contraction coupling in rat extensor digitorum longus and soleus muscles

K contractures and two-microelectrode voltage-clamp techniques were used to measure inactivation of excitation-contraction coupling in small bundles of fibers from rat extensor digitorum longus (e.d.l.) and soleus muscles at 21 degrees C. The rate of spontaneous relaxation was faster in e.d.l. fibers: the time for 120 mM K contractures to decay to 50% of maximum tension was 9.8 +/- 0.5 s (mean +/- SEM) in e.d.l. and 16.8 +/- 1.7 s in soleus. The rate of decay depended on membrane potential: in e.d.l., the 50% decay time was 14.3 +/- 0.7 s for contractures in 80 mM K (Vm = 25 mV) and 4.9 +/- 0.4 s in 160 mM K (Vm = -3 mV). In contrast to activation, which occurred with less depolarization in soleus fibers, steady state inactivation required more depolarization: after 3 min at -40 mV in 40 mM K, the 200 mM K contracture amplitude in e.d.l. fell to 28 +/- 10% (n = 5) of control, but remained at 85 +/- 2% (n = 6) of control in soleus. These different inactivation properties in e.d.l. and soleus fibers were not influenced by the fact that the 200 mM K solution used to test for steady state inactivation produced contractures that were maximal in soleus fibers but submaximal in e.d.l.: a relatively similar depression was recorded in maximal (200 mM K) and submaximal (60 and 80 mM K) contracture tension. A steady state "pedestal" of tension was observed with maintained depolarization after K contracture relaxation and was larger in soleus than in e.d.l. fibers. The pedestal tension was attributed to the overlap between the activation and inactivation curves for tension vs. membrane potential, which was greater in soleus than in e.d.l. fibers. The K contracture results were confirmed with the two-microelectrode voltage clamp: the contraction threshold increased to more positive potentials at holding potentials of -50 mV in e.d.l. or -40 mV in soleus. At holding potentials of -30 mV in e.d.l. or 0 mV in soleus, contraction could not be evoked by 15-ms pulses to +20 mV. Both K contracture and voltage-clamp experiments revealed that activation in soleus fibers occurred with a smaller transient depolarization and was maintained with greater steady state depolarization than in e.d.l. fibers. The K contracture and voltage-clamp results are described by a model in which contraction depends on the formation of a threshold concentration of activator from a voltage-sensitive molecule that can exist in the precursor, activator, or inactive states.     

Peroxisomes of the rat cardiac and soleus muscles increase after starvation

Summary We have investigated the change of catalase activity in the homogenates of rat cardiac and skeletal muscles. After 7 days' starvation, the catalase activity of heart increased about 3-fold and that of soleus muscle enhanced 2-fold higher than that of control rats. Immunoblot analysis of catalase showed a single band in the homogenates of cardiac and soleus muscles and increase of catalase antigen after starvation. Light microscopic immunoenzyme staining showed that after starvation catalase positive granules markedly increased in both the cardiac and soleus muscle. Quantitative analysis of the staining showed that number of the granules per 100 m² of tissue section was about 1.4-fold in the soleus muscle and 1.7-fold in the cardiac muscle after starvation. By electron microscopy of alkaline DAB staining, we confirmed that the granules were peroxisomes, which increased in both number and size. Furthermore, we stained the peroxisomes for catalase by a protein A-gold technique. Labeling density (gold particles/m²) of the cardiac and soleus muscles from the starved rat increased approximately 1.4 times as much as that of normal animal. When the numerical density is multiplied by the labeling density, the values are largely consistent with the enhancement of catalase activity. These results show that increase in the catalase activity of the muscle tissue after starvation is caused by increase in number and size of peroxisomes.