Sanford R. Sampson

Activation of Protein Kinase Cζ Induces Serine Phosphorylation of VAMP2 in the GLUT4 Compartment and Increases Glucose Transport in Skeletal Muscle

ABSTRACT Insulin stimulates glucose uptake into skeletal muscle tissue mainly through the translocation of glucose transporter 4 (GLUT4) to the plasma membrane. The precise mechanism involved in this process is presently unknown. In the cascade of events leading to insulin-induced glucose transport, insulin activates specific protein kinase C (PKC) isoforms. In this study we investigated the roles of PKCζ in insulin-stimulated glucose uptake and GLUT4 translocation in primary cultures of rat skeletal muscle. We found that insulin initially caused PKCζ to associate specifically with the GLUT4 compartments and that PKCζ together with the GLUT4 compartments were then translocated to the plasma membrane as a complex. PKCζ and GLUT4 recycled independently of one another. To further establish the importance of PKCζ in glucose transport, we used adenovirus constructs containing wild-type or kinase-inactive, dominant-negative PKCζ (DNPKCζ) cDNA to overexpress this isoform in skeletal muscle myotube cultures. We found that overexpression of PKCζ was associated with a marked increase in the activity of this isoform. The overexpressed, active PKCζ coprecipitated with the GLUT4 compartments. Moreover, overexpression of PKCζ caused GLUT4 translocation to the plasma membrane and increased glucose uptake in the absence of insulin. Finally, either insulin or overexpression of PKCζ induced serine phosphorylation of the GLUT4-compartment-associated vesicle-associated membrane protein 2. Furthermore, DNPKCζ disrupted the GLUT4 compartment integrity and abrogated insulin-induced GLUT4 translocation and glucose uptake. These results demonstrate that PKCζ regulates insulin-stimulated GLUT4 translocation and glucose transport through the unique colocalization of this isoform with the GLUT4 compartments.

Prodrugs of butyric acid. Novel derivatives possessing increased aqueous solubility and potential for treating cancer and blood diseases

The synthesis and biological activities of acidic, basic and neutral types of butyric acid (BA) prodrugs possessing increased aqueous solubility are described. The compounds are butyroyloxyalkyl derivatives of carboxylic acids, which possess functionalities suitable for aqueous solubilization. The anticancer activity of the prodrugs in vitro was evaluated by examining their effect on the growth of human colon, breast and pancreatic carcinoma cell lines, and their solubility in aqueous media was determined. The most promising compounds, with respect to activity and solubility, were found to be the butyroyloxymethyl esters of glutaric 2a and nicotinic acids 4a and phosphoric acid as its diethyl ester 10a, which displayed IC(50) values of 100 microM or lower. These prodrugs are expected to release formaldehyde upon metabolic hydrolysis. The corresponding butyroyloxyethyl esters (2b, 4b and 10b) that release acetaldehyde upon metabolism were significantly less potent. A similar correlation was observed for growth inhibition of the human prostate carcinoma cell lines PC-3 and LnCap and for induction of differentiation and apoptosis in the human myeloid leukemia cell line HL-60. The higher biological activity of the formaldehyde-releasing prodrugs 2a and 10a was further confirmed when induction of hemoglobin (Hb) synthesis in the human erythroleukemic cell line K562 was measured. Moreover, a therapeutic index (IC(50)/ED(50)) of ca. 5 was observed. The acute i.p. toxicity LD(50) in mice for 2a, 2b, 10a and 10b was similar and in the range of 400-600 mg kg(-1). The results obtained support the potential use of the butyric acid prodrugs for the treatment of neoplastic diseases and beta-globin disorders.

Patterns of Protein Kinase C Isoenzyme Expression in Transitional Cell Carcinoma of Bladder Relation to Degree of Malignancy

We determined the pattern of protein kinase C (PKC) isoform expression in human cell lines by Western blotting and immunofluorescent staining techniques. In addition, we examined PKC isoform expression in tissue samples of transitional cell carcinoma (TCC) of the bladder. PKC delta, PKC beta II, and PKC eta were found primarily in the RT4 cell line (low-grade tumor), and PKC zeta was expressed most strongly in the SUP cell line (invasive tumor). In tissue samples of urinary bladder cancer, PKC isoenzymes were expressed differentially as a function of tumor stage and grade; expression of PKC beta II and PKC delta was high in normal tissue and in low-grade tumors and decreased with increasing stage and grade of TCC. The opposite pattern was seen with PKC zeta. The differences in expression of specific isoenzymes as related to levels of malignancy of the cell lines and tissue samples suggest that the PKC family has an important role in normal and neoplastic urothelium.

Characterization of the relation between sodium channels and electrical activity in cultured rat skeletal myotubes: regulatory aspects

The relation among sodium channel density, frequency of electrical activity and maximal rate of rise of the action potential was studied in developing and mature rat skeletal myotubes in culture. The number of tetrodotoxin (TTX)-sensitive Na-channels was determined by measurements of the amount of [3H]saxitoxin (STX) bound to the cultures, and electrical properties were recorded with intracellular microelectrodes. The EC50 for TTX-induced decreases in maximal STX-binding, frequency and rate of rise of action potentials was in the range 8-20 nM. The 3 variables increased in parallel with age in culture to reach peak values at age 7-8 days, and then decreased in parallel until 10-12 days in culture. The age-related increase in Na-channel density was decreased, but not abolished, by prevention of myoblast fusion. Treatment with the Ca2+ ionophore, A23187, down-regulated, and blockade of Ca-channels with verapamil up-regulated the number of Na-channels. Na-channel density was also increased by chronic treatment with TTX and elevated external [K+], which eliminated spontaneous electrical and contractile activity. Parallel effects were observed on frequency and rate of rise of action potentials. Up-regulation of Na-channels was prevented by simultaneous treatment of myotubes with inhibitors of protein synthesis. We conclude that electrical and mechanical activity of cultured myotubes regulate de novo synthesis of Na-channels through alterations in the level of cytosolic Ca2+.

Contribution of electrogenic sodium-potassium ATPase to resting membrane potential of cultured rat skeletal myotubes

The contribution of electrogenic Na+ -K+ ATPase to resting membrane potential (Em) of mature and developing rat skeletal myotubes in culture was determined by examining effects of inhibition of this enzyme on Em. Ouabain, a specific Na+-K+ ATPase inhibitor, caused resting Em to decrease within 30 s by 5-8 mV and reach a minimum value of about -60 mV after 5 min. The decrease in Em was not accompanied by a decrease in input resistance for up to 15 min after application. Resting Em was found to be dependent on the temperature of the recording medium with maximum values of Em ranging from -85 to -90 mV at a temperature of 35-37 degrees C and minimum values about -60 mV at 10-15 degrees C. Ouabain (1 mM), added to cultures at low temperature (10-15 degrees C) did not further decrease Em but did prevent the increase in Em that occurs with increasing temperature up to 37 degrees C. Resting Em of cultured myotubes was reduced to about -60 mV by reducing the supply of ATP either with 2,4 dinitrophenol (DNP), which inhibits oxidative phosphorylation or with fluorodinitrobenzene (FDNB), which inhibits creatine phosphokinase. Neither of these compounds, when added to cultures in the presence of ouabain, reduced resting Em to a value lower than that obtained with ouabain alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Sarcopoterium spinosum extract as an antidiabetic agent: In vitro and in vivo study

ETHNOPHARMACOLOGICAL RELEVANCE Sarcopoterium spinosum (L.) sp., a common plant in the Mediterranean region, is widely used as an antidiabetic drug by Bedouin healers. However, the antidiabetic properties of Sarcopoterium spinosum had not been fully validated using scientific tools. AIM OF THE STUDY To determine the effectiveness of Sarcopoterium spinosum extract as an antidiabetic agent in vitro and in vivo. MATERIALS AND METHODS RINm pancreatic beta-cells, L6 myotubes, 3T3-L1 adipocytes and AML-12 hepatocytes were treated with an aqueous Sarcopoterium spinosum extract (0.001-10mg/ml). The effect of the extract on specific physiological functions, including insulin secretion, pancreatic beta-cell viability, GSK3 beta phosphorylation, lipolysis and glucose uptake was measured. In vivo studies were performed using KK-A(y) mice, given the extract for several weeks. IPGTT was performed, and plasma insulin, FFA, food consumption and body weight were measured. In addition, diabetic KK-A(y) mice were given a single dose of the extract, and IPGTT was performed. RESULTS Sarcopoterium spinosum extract increased basal and glucose/forskolin-induced insulin secretion in RINm cells, and increased cell viability. The extract inhibited lipolysis in 3T3-L1 adipocytes, and induced glucose uptake in these cells as well as in AML-12 hepatocytes and L6 myotubes. GSK3 beta phosphorylation was also induced in L6 myotubes, suggesting increased glycogen synthesis. Sarcopoterium spinosum extract had a preventive effect on the progression of diabetes in KK-A(y) mice. Catechin and epicatechin were detected in Sarcopoterium spinosum extract using hyphenated LC-MS/MS. CONCLUSIONS Sarcopoterium spinosum extract has effects that mimic those of insulin and provide the basis for antidiabetic activity of the extract.

Influence of thyroid hormone on some electrophysiological properties of developing rat skeletal muscle cells in culture

Effects of thyroxine (T4) were examined on some electrophysiological properties of developing rat myotubes in culture. Thyroxine caused an increase in transmembrane resting potential (Em) of skeletal myotubes. Ouabain decreases Em in both control and T4-treated cells within 5-15 min of addition to the culture. Moreover, the effect of T4 is nearly eliminated by short-term ouabain treatment and by reduced extracellular K+ concentration. The change in Em in response to a 10-fold increase in extracellular K+ concentration is not altered by T4-treatment despite the higher Em of T4-treated cells. Studies on developmental effects of T4 showed that Em of T4-treated cells is higher than controls by as early as day 5. By this time, ouabain decreases Em of T4-treated cells but not that of untreated controls. Treatment with T4 also causes an increase in frequency of spontaneously-occurring action potentials generated by cultured myotubes. Chronic treatment with tetrodotoxin blocks this effect without affecting the T4-induced increase in Em. The findings demonstrate that activity of electrogenic Na,K-ATPase is increased by T4-treatment and that this effect is primarily responsible for the elevated Em in T4-treated cells. In addition, it appears that T4 causes the activity of this enzyme to be manifest earlier in development than might otherwise occur in cultured muscle. Finally, stimulation of electrogenic pump activity is not secondary to increased influx of Na+ ions associated with increased electrical activity and probably results from synthesis of new enzyme units in both developing and mature myotubes.

Rat skeletal muscle in culture expresses the ?1 but not the ?2 protein subunit isoform of the Na+/K+ pump

Studies from this laboratory have shown that the physiological expression of the Na+/K+ pump in primary cultures of rat skeletal muscle increases with development. The molecular mechanisms underlying these changes are not known. Therefore, we have examined the expression of α and β subunits of the Na+/K+ pump at both the protein and mRNA levels during myogenesis of primary skeletal muscle cell cultures obtained from newborn rats. Protein isoforms were identified by Western blotting techniques with specific monoclonal and polyclonal antibodies and subunit mRNA was studied with specific cDNA probes. Freshly isolated skeletal muscle from newborn rats expressed both α1 and α2 protein subunits. From day 1 after plating, primary cultures expressed only the α1 protein isoform. In contrast, both β1 and β2 isoforms were expressed in freshly isolated muscle and in primary cultures, with β1 expression being stronger in both preparations. Studies on RNA expression showed that mRNA for α1, α2, β1, and β2 isoforms was identified both in freshly isolated muscle and after plating of cells in culture. These findings indicate that the lack of α2 protein expression in primary muscle cell cultures reflects a form of posttranscriptional regulation. There did not appear to be a quantitative difference in isoform expression as a function of age or of fusion in spite of developmental increases in Na+/K+ pump activity and its dependence on cell fusion. The lack of expression of the α2 subunit isoform suggests that the developmental changes in physiological expression of the Na+/K+ pump in primary cultures of skeletal muscle may be attributable either to the changes in activity of the α1 subunit or to differential activities of αβ complexes involving either of the β subunits. J. Cell. Physiol. 180:236–244, 1999.

Maintenance of redox state and pancreatic beta‐cell function: Role of leptin and adiponectin

Whereas oxidative stress is linked to cellular damage, reactive oxygen species (ROS) are also believed to be involved in the propagation of signaling pathways. Studies on the role of ROS in pancreatic beta‐cell physiology, in contrast to pathophysiology, have not yet been reported. In this study we investigate the importance of maintaining cellular redox state on pancreatic beta‐cell function and viability, and the effects of leptin and adiponectin on this balance. Experiments were conducted on RINm and MIN6 pancreatic beta‐cells. Leptin (1–100 ng/ml) and adiponectin (1–100 nM) increased ROS accumulation, as was determined by DCFDA fluorescence. Using specific inhibitors, we found that the increase in ROS levels was mediated by NADPH oxidase (Nox), but not by AMP kinase (AMPK) or phosphatidyl inositol 3 kinase (PI3K). Leptin and adiponectin increased beta‐cell number as detected by the XTT method, but did not affect apoptosis, indicating that the increased cell number results from increased proliferation. The adipokines‐induced increase in viability is ROS dependent as this effect was abolished by N‐acetyl‐L‐cysteine (NAC) or PEG‐catalase. In addition, insulin secretion was found to be regulated by alterations in redox state, but not by adipokines. Finally, the effects of the various treatments on activity and mRNA expression of several antioxidant enzymes were determined. Both leptin and adiponectin reduced mRNA levels of superoxide dismutase (SOD)1. Adiponectin also decreased SOD activity and increased catalase and glutathione peroxidase (GPx) activities in the presence of H2O2. The results of this study show that leptin and adiponectin, by inducing a physiological increase in ROS levels, may be positive regulators of beta‐cell mass. J. Cell. Biochem. 113: 1966–1976, 2012.

Role of protein kinase Cα in melatonin signal transduction

Abstract Melatonin induces nuclear exclusion of the androgen receptor (AR) via activation of protein kinase C (PKC). The specific members of the PKC superfamily involved in AR nuclear exclusion were investigated in prostate cancer PC3 cells stably transfected with the wild-type androgen receptor (PC3-AR). PKCα was essentially cytoplasmic whereas PKCβ and PKCɛ were essentially membranal, suggesting their constitutive activity in the PC3-AR cells. Melatonin treatment induced membrane association of PKCα in a time and dose dependent manner. The PKCα and PKCβ1 specific inhibitor GO6976 and the PKCβ isoform-specific inhibitor hispidin had no effects on AR localization under basal conditions. However, GO6976 but not hispidin negated the melatonin-mediated nuclear exclusion of the AR. These data indicate that PKCα activation is a critical step in AR nuclear exclusion by melatonin. They also imply that PKCα-activation is a potentially effective way to control of the AR activity in prostate cancer cells.