Satomi Itaya

DHEA improves glucose uptake via activations of protein kinase C and phosphatidylinositol 3-kinase

We have examined the effect of adrenal androgen, dehydroepiandrosterone (DHEA), on glucose uptake, phosphatidylinositol (PI) 3-kinase, and protein kinase C (PKC) activity in rat adipocytes. DHEA (1 μM) provoked a twofold increase in 2-[3H]deoxyglucose (DG) uptake for 30 min. Pretreatment with DHEA increased insulin-induced 2-[3H]DG uptake without alterations of insulin specific binding and autophosphorylation of insulin receptor. DHEA also stimulated PI 3-kinase activity. [3H]DHEA bound to purified PKC containing PKC-α, -β, and -γ. DHEA provoked the translocation of PKC-β and -ζ from the cytosol to the membrane in rat adipocytes. These results suggest that DHEA stimulates both PI 3-kinase and PKCs and subsequently stimulates glucose uptake. Moreover, to clarify the in vivo effect of DHEA on Goto-Kakizaki (GK) and Otsuka Long-Evans fatty (OLETF) rats, animal models of non-insulin-dependent diabetes mellitus (NIDDM) were treated with 0.4% DHEA for 2 wk. Insulin- and 12- O-tetradecanoyl phorbol-13-acetate-induced 2-[3H]DG uptakes of adipocytes were significantly increased, but there was no significant increase in the soleus muscles in DHEA-treated GK/Wistar or OLETF/Long-Evans Tokushima (LETO) rats when compared with untreated GK/Wistar or OLETF/LETO rats. These results indicate that in vivo DHEA treatment can result in increased insulin-induced glucose uptake in two different NIDDM rat models.We have examined the effect of adrenal androgen, dehydroepiandrosterone (DHEA), on glucose uptake, phosphatidylinositol (PI) 3-kinase, and protein kinase C (PKC) activity in rat adipocytes. DHEA (1 microM) provoked a twofold increase in 2-[3H]deoxyglucose (DG) uptake for 30 min. Pretreatment with DHEA increased insulin-induced 2-[3H]DG uptake without alterations of insulin specific binding and autophosphorylation of insulin receptor. DHEA also stimulated PI 3-kinase activity. [3H]DHEA bound to purified PKC containing PKC-alpha, -beta, and -gamma. DHEA provoked the translocation of PKC-beta and -zeta from the cytosol to the membrane in rat adipocytes. These results suggest that DHEA stimulates both PI 3-kinase and PKCs and subsequently stimulates glucose uptake. Moreover, to clarify the in vivo effect of DHEA on Goto-Kakizaki (GK) and Otsuka Long-Evans fatty (OLETF) rats, animal models of non-insulin-dependent diabetes mellitus (NIDDM) were treated with 0.4% DHEA for 2 wk. Insulin- and 12-O-tetradecanoyl phorbol-13-acetate-induced 2-[3H]DG uptakes of adipocytes were significantly increased, but there was no significant increase in the soleus muscles in DHEA-treated GK/Wistar or OLETF/Long-Evans Tokushima (LETO) rats when compared with untreated GK/Wistar or OLETF/LETO rats. These results indicate that in vivo DHEA treatment can result in increased insulin-induced glucose uptake in two different NIDDM rat models.

Effect of tumor necrosis factor-α on insulin signal transduction in rat adipocytes: relation to PKCβ and ζ 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.

Insulin regulates PKC isoform mRNA in rat adipocytes

Insulin and 12-O-tetradecanoyl phorbol-13-acetate (TPA) induce both glucose uptake and translocation of protein kinase C (PKC) from cytosol to membrane in insulin-sensitive tissues as previously reported by several investigators. We examined insulin-mediated PKC beta I, beta II, and epsilon translocation from cytosol to cytoskeleton, and expression of PKC alpha, beta I, beta II, gamma, and epsilon isoforms using the reverse transcription polymerase chain reaction (RT-PCR) method during treatment with insulin for 240 min in rat adipocytes. Insulin-induced increases in PKC beta I, beta II, and epsilon were greater in the cytoskeleton fraction than those in the membrane fraction. Insulin induced time-dependent increases in PKC alpha, gamma, epsilon and zeta mRNA levels for up to 240 min (555%, 117%, 236% and 138% increase, respectively). TPA also induced time-dependent increases in PKC alpha and gamma (34% and 500% increase, respectively) but not in PKC zeta. However, PKC beta I mRNA was decreased for up to 60 min and then maintained at under the basal level during stimulation with insulin and TPA. On the other hand, PKC beta II mRNA was markedly increased for up to 240 min. These results suggest that insulin-regulated PKC alpha, gamma and epsilon mRNA levels and PKC beta mRNA alternative splicing may occur in rat adipocytes.

Platelet protein kinase C isoform content in type 2 diabetes complicated with retinopathy and nephropathy

It has been reported that platelet aggregation in diabetic patients with microangiopathy is increased compared with healthy subjects. Chronic hyperglycemia is known to cause an increase in diacylglycerol level in various tissues. We examine whether protein kinase C (PKC) isoform content in platelets from diabetic patients is increased compared with healthy subjects, as previously described in the retina, aorta, and heart of diabetic rats. Platelet PKC α , β and ζ immunoreactivity in cytosol, membrane and cytoskeleton (CS) fractions were analyzed by immunoblotting in 20 type 2 diabetic patients (who had been treated with diet alone, sulphonylureas or insulin, and whose condition was complicated with retinopathy, nephropathy, neuropathy and/or macroangiopathy) and in five healthy subjects. PKC α , β and ζ immunoreactivity in cytosol, membrane and CS fractions in platelets from diabetic subjects were not significantly higher than those from healthy subjects. However, platelet PKC β immunoreactivity in cytosol fraction was significantly higher in diabetic patients with normal serum creatinine (Cr) level than in diabetic patients with abnormal Cr level (Cr S 1.5 mg/dl) or in healthy subjects. Moreover, significant negative correlation between PKC β immunoreactivity in cytosol fraction of platelets and serum Cr level was found in diabetic patients ( P < 0.05). To clarify the effect of treatment for diabetes, PKC isoform immunoreactivity in platelets was measured in type 2 diabetic patients treated with diet alone, sulphonylurea or insulin treatment. Serum creatinine level in diabetic patients with insulin treatment was significantly higher than in diabetic patients with sulphonylurea treatment and diet alone. In addition, PKC β immunoreactivity in diabetic patients with insulin treatment was significantly suppressed compared with that in patients treated by sulphonylurea treatment. These results suggest that chronic hyperglycemia may activate platelet PKC β isoform, and that insulin treatment may decrease platelet PKCβactivity. Finally, not only PKC β antagonists, but also glycemic control by insulin may prevent development of diabetic microangiopathy.

Phorbol ester and insulin stimulate protein kinase C isoforms in rat adipocytes.

We examined effect of insulin or 12-O-tetradecanoyl phorbol 13-acetate (TPA) on the subcellular redistribution of protein kinase C isoforms in rat adipocytes. Total Mono Q column-elutable novel PKCs (nPKCs) which are Ca(2+)-independent and phospholipid-dependent protein kinases, decreased in the cytosolic fraction and increased in the membrane fraction during treatment with insulin or phorbol ester for 10 min. Immunoblot analysis of novel PKCs, -epsilon, -delta and -zeta, showed that insulin stimulated the translocation of these PKC isoforms from cytosol to membrane, similar to the translocation of conventional Ca2+/phospholipid-dependent PKCs (cPKCs), -alpha, -beta, and -gamma. Phorbol esters stimulated the translocation of PKC-alpha, -beta, -gamma, -epsilon and -delta, but not PKC-zeta. These results suggest that (a) insulin and phorbol esters similarly stimulate the translocation of each PKC isoform except for PKC-zeta, and (b) the translocation of both nPKCs and cPKCs occurs during insulin and TPA actions in rat adipocytes.

Effect of glucocorticoid receptor antagonist RU 38486 on acute glucocorticoid-induced insulin resistance in rat adipocytes.

We examined the mechanism of acute glucocorticoid-induced insulin resistance in rat adipocytes using the glucocorticoid receptor antagonist RU 38486. Pretreatment with dexamethasone (DEX) and prednisolone for 60 minutes resulted in 50% inhibition of insulin-induced [3H]2-deoxyglucose (DOG) uptake at 10(-8) and 10(-7) mol/L, respectively, in rat adipocytes and 20% and 25% inhibition of insulin-induced [3H]2-DOG uptake, respectively, in soleus muscles. Our previous experiments indicated that DEX and prednisolone alone stimulate protein kinase C (PKC) in rat adipocytes. Accordingly, we examined [3H]DEX binding to PKC from MonoQ column-purified rat brain cytosol. Specific [3H]DEX binding to MonoQ column-purified PKC was observed (kd, 56.8 nmol/L; Bmax, 725 fmol/mg protein). Thus, insulin-induced PKC translocation from the cytosol to the membrane was suppressed by pretreatment with 10(-7) mol/L DEX and 10(-6) mol/L prednisolone for 80 minutes. During treatment with RU 38486 for 60 minutes, there was no change in the glucocorticoid-induced inhibitory effect on insulin-induced [3H]2-DOG uptake and PKC translocation from the cytosol to the membrane. Moreover, pretreatment with RU 38486 for 120 minutes slightly prevented the DEX-mediated inhibition of insulin-induced glucose uptake. These results suggest that acute glucocorticoid-induced insulin resistance may be mainly mediated through the other non-glucocorticoid receptor pathway.

Glucose‐ and phorbol ester‐induced insulin secretion in human insulinoma cells ‐ Association with protein kinase C activation‐

This study examined the effect of glucose and 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) on insulin secretion in isolated human insulinoma cells. In addition, we analyzed conventional PKCα and β activation in the membrane fractions, respectively. Treatment with 5 mM and 20 mM glucose for 5 min and 20 min resulted in 6∼7‐fold increases in insulin secretion, and treatment with 1 μM TPA for 5 min also resulted in 3‐fold increases in insulin secretion from the basal level. Immunoblot analysis of membrane fractions showed increases in PKCα and β immunoreactivities after treatment with 5 mM, 20 mM glucose and 1 μM TPA. Translocations of PKCα after treatment with glucose and TPA were greater than those of PKCβ in membrane fractions. These results suggest that TPA independently provokes insulin secretion via PKC activation and that PKCα and β activation may be involved in insulin secretion in human insulinoma cells.

Acute effects of phorbol ester and insulin on insulin-induced glucose uptake and protein kinase C activation in rat adipocytes

We examined the acute effect of pretreatment with phorbol ester and insulin on insulin-induced glucose uptake and protein kinase C (PKC) translocation from cytosol to the membrane in rat adipocytes. Adipocytes were preincubated with 1 microM tetradecanoylphorbol 13-acetate (TPA) and 10 nM insulin for 60 min and then stimulated with 10 nM insulin for 10 and 30 min to measure PKC activity in cytosol and membrane fractions using a Mono Q column connected onto an HPLC system and [3H]2-deoxyglucose (DOG) uptake, respectively. Pretreatment with 1 microM TPA and 10 nM insulin for 60 min resulted in the marked decreases of insulin-induced [3H]2-DOG uptake. Translocation of Mono Q column-purified cytosolic PKC enzyme activity and PKC beta immunoreactivity from cytosol to the membrane was suppressed by pretreatment with TPA and insulin for 60 min. These results indicate that acute treatment with TPA and insulin which are PKC activators suppress translocation/activation of PKC, and accordingly inhibit insulin-induced glucose uptake. We suggest that a decrease of cytosolic PKC activity may mainly-contribute to the impaired responsiveness of the glucose transport system after acute TPA and insulin treatment.

Effects of wortmannin on glucose uptake and protein kinase C activity in rat adipocytes

Wortmannin is known to be an inhibitor of myosin light chain kinase and phosphatidylinositol 3-kinase (PI 3-kinase) (J. Biol. Chem. 268, 25846, 1993). We studied the effects of wortmannin on insulin- and 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced glucose uptake, purified PKC activity and in vitro 80 kDa protein phosphorylation to elucidate the relationship between insulin-induced PI 3-kinase and PKC activations. Pretreatment with 10(-12)-10(-6) M wortmannin for 60 min resulted in a dose-responsive reduction of 10 nM insulin-stimulated glucose uptake in rat adipocytes. Pretreatment with 10(-6) M wortmannin resulted in 80% and 20% decreases of glucose uptake stimulated by insulin and TPA, respectively. Partially purified rat brain PKC activity and 80 kDa protein in vitro phosphorylation of rat adipocyte cytosol by addition of Ca2+ and phospholipid were dose-dependently decreased by 10(-8)-10(-6) M wortmannin; 20% decrease of PKC activity and 50% decrease of 80 kDa protein phosphorylation by 10(-6) M wortmannin were observed. These results suggest that wortmannin has a potent inhibitory effect on PI 3-kinase and a weak inhibitory effect on PKC activity, and both effects cause a significant inhibition of insulin-stimulated glucose uptake in rat adipocytes.