THE EFFECT OF SALBUTAMOL ON PGC-1 α AND GLUT4 mRNA EXPRESSION IN THE LIVER AND MUSCLE OF ELDERLY DIABETIC MICE.

BACKGROUND: Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) plays an important role in the regulation of cellular energy metabolism, and it is involved in obesity and type 2 diabetes mellitus (T2DM). Its expression is elevated in the liver of T2DM mouse models. Literature reports show that chronic ß2 stimulation improved insulin sensitivity in T2DM. OBJECTIVES: We aimed to test the hypotheses that chronic ß2 stimulation-induced improvement in insulin sensitivity involves changes in the expression of PGC-1α and glucose transporter 4 (GLUT4). ANIMALS AND METHODS: We fed a locally inbred, 8 months old mice, a high fat diet (HFD) to induce diabetes. These mice gained weight and became insulin resistant. The ß2 agonist salbutamol had a beneficial effect on both glucose tolerance and insulin sensitivity after 4 weeks. RESULTS: Salbutamol beneficial effect persisted after 4 weeks of its discontinuation. HFD caused an up regulation of the hepatic PGC-1 α expression by 5.23 folds (P< 0.041) and salbutamol reversed this effect and caused a down regulation by 30.3 folds (P< 0.0001). PGC-1 α and GLUT4 expression in the muscle was not affected by salbutamol (P> 0.05). CONCLUSION: Down regulation of the liver's PGC-1 α contributes to the beneficial effect of the chronic ß2 stimulation on glucose tolerance and insulin sensitivity in T2DM mice.

The bioequivalence study of folifer-Z: a new formulation of sustained-release iron and zinc.

The bioequivalence of Folifer-Z tablets, a new sustained-release iron and zinc formulation was evaluated and compared to that of Fefol-Z capsules in 30 healthy male subjects. Each subject received a single oral dose of either product according to a randomized two-way crossover design. A washout period of 1 week was allowed after each treatment. Blood samples were obtained over a 24-h period, and iron and zinc concentrations were measured. The pharmacokinetic parameters of Folifer-Z were Cmax (103 +/- 46.2 micrograms/dl), Tmax (5.93 +/- 2.94 h) and AUC0-24 h (1937 +/- 706 micrograms/dl per h), whereas the corresponding Fefol-Z values were Cmax (109 +/- 41.5 micrograms/dl), Tmax (6.64 +/- 2.54) and AUC0-24 h (1865 +/- 699 micrograms/h per dl). Analysis of variance on log-transformed data for Cmax and AUC0-24 h revealed lack of significant differences among the two formulations. The mean relative bioavailability of AUCtest/AUCreference was 1.07 (90% confidence interval range: 99-115%) and for Cmax test/Cmax reference was 0.96 (90% confidence interval range: 88-105%). Regarding the zinc results, the pharmacokinetic parameters of Folifer-Z values were Cmax (101 +/- 20.7 micrograms/dl), Tmax (4.86 +/- 1.53 h) and AUC0-24 h (1944 +/- 202 micrograms/h per dl), while the corresponding Fefol-Z values were Cmax (102 +/- 20.7), Tmax (4.93 +/- 1.51) and AUC0-24 h (1953 +/- 200). Analysis of variance on log-transformed zinc data for Cmax, Tmax and AUC0-24 h revealed lack of significant difference among the two formulations. The mean relative bioavailability of AUCtest/AUCreference was 0.98 (90% confidence interval range; 95-101%) and for Cmax test/Cmax reference was 0.92 (90% confidence interval range: 89-96%). The results also indicate a possible inhibition of zinc absorption by iron content of both formulations. It is concluded that Folifer-Z product is bioequivalent to Fefol-Z product.

Glucocorticoid elevation of mRNA encoding epinephrine-forming enzyme in lung.

The epinephrine-forming enzyme phenylethanolamine N-methyltransferase (PNMT) is present in lung and its activity is increased by the glucocorticoid dexamethasone. Chronic administration of dexamethasone (0.5 mg/kg twice daily) doubled levels of mRNA coding for PNMT in rat lung. Administration of the glucocorticoid antagonist RU 486 after 7 days of dexamethasone treated reduced PNMT mRNA by about two-thirds within 24 h. Lung epinephrine (E) levels correlated with lung PNMT activity in these dexamethasone-treated and control rats. In a separate experiment, lung PNMT mRNA levels were nearly tripled 6 h after dexamethasone (1 mg/kg sc). In a third experiment chronic administration of the PNMT inhibitor SKF 64139 (50 mg/kg twice daily) reduced in vitro lung PNMT activity in chronically dexamethasone-treated adrenalectomized rats by approximately 96% and reduced lung E levels by approximately 75%. We conclude that glucocorticoids increase lung PNMT activity by increasing levels of mRNA coding for this enzyme. The data also suggest that a substantial fraction of lung E is locally synthesized. We speculate that enhanced lung E synthesis may participate in glucocorticoid-induced dilation of the bronchioles.

Glucocorticoid induction of epinephrine synthesizing enzyme in rat skeletal muscle and insulin resistance.

Rat skeletal muscle contains two enzymes which can make epinephrine: phenylethanolamine N-methyltransferase (PNMT) and nonspecific N-methyltransferase. We studied the time-course and mechanism by which the glucocorticoid dexamethasone increases muscle PNMT activity. We also examined the hypothesis that increased muscle E synthesis may contribute to glucocorticoid-induced insulin resistance. Dexamethasone (1 mg/kg s.c. for 12 d) increased muscle PNMT activity seven-fold but did not change NMT activity. Immunotitration with an anti-PNMT antibody indicated that the PNMT elevation was due to increased numbers of PNMT molecules. Dexamethasone rapidly increased PNMT activity and this elevation was largely maintained 6 d after glucocorticoid treatment stopped. Muscle epinephrine levels were transiently elevated by dexamethasone. Dexamethasone-treated rats had elevated insulin levels after a glucose load, and chronic administration of the PNMT inhibitor SKF 64139 reversed this increase. Chronic SKF 64139 improved glucose tolerance in normal rats. Dexamethasone induced muscle synthesis of the epinephrine-forming enzyme PNMT. A PNMT inhibitor lowered insulin levels in glucocorticoid-treated rats and glucose levels in untreated rats. These findings are compatible with antagonism of insulin-mediated glucose uptake by epinephrine synthesized in skeletal muscle.

Glucocorticoid hypertension and nonadrenal phenylethanolamine N-methyltransferase.

Several drugs that block epinephrine synthesis by inhibiting phenylethanolamine N-methyltransferase (PNMT) lower blood pressure in hypertensive rats. We investigated the mechanism by which these drugs lower blood pressure in rats made hypertensive with the glucocorticoid dexamethasone. We performed adrenalectomy or sham operation on several rats and then gave them either dexamethasone chronically or vehicle. The dexamethasone-treated adrenalectomized rats also received either the centrally acting PNMT inhibitor SKF 64139 chronically or an equal dose of the primarily peripherally acting PNMT inhibitor SKF 29661. Both SKF 64139 and SKF 29661 reduced blood pressure by more than 25 mm Hg. SKF 64139 also reduced PNMT activity in hypothalamus, medulla oblongata, skeletal muscle, and cardiac atria and ventricles; SKF 29661 inhibited PNMT in muscle and heart tissue by 40-75%, did not inhibit PNMT in hypothalamus, and inhibited PNMT by only 29% in medulla oblongata. PNMT activity in peripheral tissues was also more highly correlated with blood pressure than was PNMT activity in the brain areas studied. Neither drug reduced tissue epinephrine levels, but SKF 64139 elevated dopamine or norepinephrine levels or both in several tissues. We conclude that the blood pressure-lowering action of PNMT-inhibiting drugs in glucocorticoid hypertensive rats may be due to inhibition of peripheral nonadrenal PNMT. We speculate that elevations in nonadrenal PNMT may mediate glucocorticoid hypertension.

Propranolol reduces rat dopamine-beta-hydroxylase activity and catecholamine levels.

Propranolol treatment (1 mg/kg i.p. twice daily for 8 days) reduced atrial dopamine-beta-hydroxylase (D beta H) activity by 60.5% and lung D beta H activity by 53.5% but did not alter ventricle D beta H activity. Propranolol also significantly reduced atrial noradrenaline (NA) by 66.3%, adrenaline (A) by 40%, dopamine (DA) by 72.4% and lung NA by 46.6% but did not change plasma or cardiac ventricle catecholamines. The addition of propranolol (10(-6) M) to cardiac and lung tissue homogenates in vitro did not inhibit tissue D beta H activities. The results suggest that chronic propranolol treatment inhibits NA synthesis and support the hypothesis of a centrally induced inhibition of sympathetic activity caused by beta-adrenoceptor blockade.

Epinephrine synthesis by rat arteries.

Carotid artery and aorta homogenates synthesized epinephrine (E) from norepinephrine (NE) in the presence of S-adenosylmethionine. Aorta synthesized epinine by the N-methylation of dopamine (DA) about 3 times as well as it synthesized E from NE. In contrast, adrenal homogenates which contain phenylethanolamine N-methyltransferase (PNMT) methylated DA only 1% as well as NE. The PNMT inhibitor SKF 29661 had no significant effect on methylation of NE by aorta but inhibited adrenal PNMT by 88%. N-Methylating activity in arterial homogenates was increased by dexamethasone and following catecholamine depletion by 6-hydroxydopamine (6-OHDA) and reserpine. Nine days after adrenal demedullation blood E levels collected at decapitation were less than 7% of levels found in sham operated controls but artery homogenate E was unchanged. Demedullated rats given 6-OHDA followed by reserpine for 4 days also had unchanged arterial E levels despite arterial NE levels that were less than 15% of controls. We conclude that arteries synthesize E in vitro and appear to synthesize E in vivo using an extraneuronal N-methyltransferase. This enzyme differs from adrenal PNMT in substrate and inhibitor specificity and its activity is enhanced by catecholamine depletion and by glucocorticoid treatment.

Sources of urinary catecholamines in renal denervated transplant recipients.

When a human kidney is transplanted, sympathetic nerves to that kidney are cut. We infused 3H-noradrenaline and then measured noradrenaline, dopamine and 3H-noradrenaline levels in the plasma and urine of renal transplant recipients and uninephrectomized control subjects. Less than 10% of 3H-noradrenaline cleared from the plasma appeared in the urine. Noradrenaline and dopamine appeared in the urine of transplant recipients at one-third the rate of control subjects, even though 3H-noradrenaline levels were slightly higher in the urine of transplant recipients. Transplant patients had a noradrenaline clearance of 128 +/- 50 ml/min, compatible with simple glomerular filtration, while controls had a higher calculated clearance of 229 +/- 41 ml/min. Plasma dopamine levels were very low compared with urinary dopamine. These results suggest that two-thirds of renal noradrenaline and dopamine depend on the presence of renal nerves. Almost all urinary dopamine comes from the kidney. For noradrenaline, urinary excretion is a very minor pathway for clearance from the plasma.

Epinephrine synthesis in the rat iris.

Epinephrine (E) alters blood flow, intraocular pressure and pupillary constriction. The rat iris contained E-forming activity that was moderately specific for a phenylethanolamine and was inhibited by the phenylethanolamine-N-methyltransferase (PNMT) inhibitor SKF 29661. Unilateral superior cervical ganglionectomy decreased iris norepinephrine (NE) 63%, but failed to lower PNMT activity or E in the iris. Removal of both adrenal medullae markedly lowered circulating E levels, but had no effect on iris E. Further treatment with 6-hydroxydopamine and reserpine greatly lowered iris NE levels, but failed to decrease either iris E or E forming activity. The rat iris has non-neuronal E-forming enzymes which appear to synthesize most of the E contained in the iris.

Lung epinephrine synthesis.

We studied in vitro and in vivo epinephrine (E) synthesis by rat lung. Nine days after removal of the adrenal medullas, circulating E was reduced to 7% of levels found in sham-operated rats but 30% of lung E remained. Treatment of demedullated rats with 6 hydroxydopamine plus reserpine did not further reduce lung E. In the presence of S-[3H]adenosylmethionine lung homogenates readily N-methylated norepinephrine (NE) to form [3H]E. The rate of E synthesis by lung homogenates was progressively more rapid with increasing NE up to a concentration of 3 mM, above which it declined. The rate of E formation was optimal at an incubation pH of 8 and at temperatures of approximately 55 degrees C. We compared the E-forming enzyme(s) of lung homogenates with those of adrenal and cardiac ventricle. The adrenal contains mainly phenylethanolamine N-methyltransferase (PNMT), which is readily inhibited by SKF 29661 and methylates dopamine (DA) very poorly. Cardiac ventricles contain mainly nonspecific N-methyltransferase (NMT), which is poorly inhibited by SKF 29661 and readily methylates both DA and NE. Lung homogenates were inhibited by SKF 29661 about half as well as adrenal but more than ventricle. We used the rate of E formation from NE as an index of PNMT-like activity and deoxyepinephrine synthesis from DA as an index of NMT-like activity. PNMT and NMT activity in rat lung homogenates were not correlated with each other, displayed different responses to change in temperature, and were affected differently by glucocorticoids.(ABSTRACT TRUNCATED AT 250 WORDS)

Epinephrine synthesis by an N-methyltransferase in rat liver.

We investigated if liver can synthesize epinephrine in vitro and in vivo. Homogenates of rat liver readily synthesized [3H]epinephrine from [3H]S-adenosylmethionine and norepinephrine. Liver homogenates also N-methylated dopamine at more than twice the rate that they N-methylated norepinephrine. In contrast, adrenal homogenates, which N-methylate norepinephrine to form epinephrine using the enzyme phenylethanolamine-N-methyltransferase (PNMT), methylated dopamine only about 1% as well as norepinephrine. Synthesis of epinephrine by liver homogenates was not significantly inhibited by the PNMT inhibitor SKF 29661 at a concentration that inhibited adrenal homogenate epinephrine synthesis by nearly 90%. These findings indicate that liver can synthesize epinephrine in vitro using an enzyme other than PNMT. Adrenal demedullation of rats reduced plasma epinephrine levels to 7% of control values, but left liver epinephrine and epinephrine-forming enzyme levels unchanged. Treatment of demedullated rats with 6-hydroxydopamine plus reserpine also resulted in dramatically reduced plasma epinephrine levels but no change in hepatic epinephrine and N-methylating enzyme levels. We conclude that the liver synthesizes its own epinephrine.

Cardiac atria and ventricles contain different inducible adrenaline synthesising enzymes.

STUDY OBJECTIVE - The aim of the study was to investigate adrenaline synthesis in atrial and ventricular homogenates. DESIGN - The study involved the use of a new assay which measures the rate at which tissue homogenates convert noradrenaline into adrenaline, or dopamine into N-methyldopamine. This was coupled with a sensitive assay for tissue catecholamines in an investigation of ventricular and atrial homogenates from rats exposed to adrenal demedullation and chemical depletion of cardiac catecholamines. MEASUREMENTS and RESULTS - Atrial and ventricular homogenates from 12 male Sprague-Dawley rats were investigated. Atrial adrenaline forming activity resembled adrenal phenylethanolamine-N-methyltransferase (PNMT) in its relatively high affinity for noradrenaline, substrate specificity for noradrenaline over dopamine, and inhibition by the PNMT inhibitor SKF 29661. Ventricular tissue nonspecifically methylated both noradrenaline and dopamine, and was less inhibited by SKF 29661. Adrenal demedullation induced activity of ventricular adrenaline forming enzyme. CONCLUSIONS - The cardiac atria and ventricles contain different inducible adrenaline forming enzymes. About one third of cardiac adrenaline may be synthesised by the heart itself. The ventricular enzyme can synthesise adrenaline from noradrenaline, and N-methyldopamine from dopamine.

Epinephrine synthesis in rat skin by an N-methyltransferase.

Homogenates of rat skin N-methylated norepinephrine to form epinephrine. In the brain and adrenal medulla the enzyme phenylethanolamine-N-methyltransferase synthesizes epinephrine, but the skin epinephrine forming enzyme was an N-methyltransferase distinct from phenylethanolamine-N-methyltransferase. Skin N-methyltransferase was not inhibited by the phenylethanolamine-N-methyltransferase inhibitor SKF 29661. Unlike phenylethanolamine-N-methyltransferase, skin readily methylated dopamine to form epinine. Sympathetic denervation by superior cervical ganglionectomy had no effect on skin N-methyltransferase levels. Procedures that reduced skin norepinephrine levels to 2% of control left skin epinephrine levels at 38% of control even when plasma epinephrine levels were very low. Skin contains an extraneuronal enzyme that synthesizes epinephrine in vitro and appears to synthesize part of the epinephrine normally present in skin. The enzyme can synthesize epinephrine and epinine, both of which can regulate epidermal proliferation, skin blood flow, and atopic responses.

Rat renal epinephrine synthesis.

Rats that underwent adrenal demedullation had a 93% decrease in plasma epinephrine (E) levels, but did not decrease their renal E. Even further treatment with 6-hydroxydopamine and reserpine failed to lower renal E levels. Similarly, urine E levels failed to decrease after adrenal demedullation and renal denervation. There is a renal E-synthesizing enzyme that differs from adrenal phenylethanolamine-N-methyltransferase (PNMT) in that it is only weakly inhibited by SKF 29661 and can synthesize epinine from dopamine, while adrenal PNMT does so poorly. When an adrenalectomized rat received intravenous [3H]methionine, its urine contained radioactivity that appeared to be [3H]E, with small amounts of [3H]epinine. However, after [3H]methionine was infused in the renal artery, the major product in urine appeared to be [3H]epinine, with a small amount of [3H]E. Adrenal demedullation induced renal E synthesis, but denervation returned the rate of renal E synthesis to control values. The combination of adrenal demedullation, 6-hydroxydopamine, and reserpine treatments increased renal E-forming activity to 350% of control. We conclude that appreciable portions of renal and urinary E are synthesized in the kidney by an enzyme distinct from PNMT. The enzyme is induced by some treatments that lower E and NE levels.

Extraadrenal adrenaline formation by two separate enzymes.

Adrenaline (A) is synthesized in the adrenal medullae by the enzyme phenylethanolamine-N-methyltransferase (PNMT). After surgical removal of the adrenal medullae tissue A levels ranged from 22% of control in the heart to 125% of control in the liver. Use of a novel assay to measure tissue A formation revealed that many tissues can synthesize A using PNMT and another enzyme that N-methylates both noradrenaline and dopamine. These enzymes are non-neuronal, inducible and synthesize a major fraction of tissue and urine A.

Hypoglycemic effects of Teucrium polium.

Teucrium polium has a folk reputation as a hypoglycemic agent. The hypoglycemic activity of an aqueous decoction of plant aerial parts was tested in normoglycemic and streptozotocin-hyperglycemic rats. Results indicate that this extract caused significant reductions in blood glucose concentration 4 h after intravenous administration and 24 h after intraperitoneal administration. This effect could be due to enhancement of peripheral metabolism of glucose rather than an increase in insulin release.

A sensitive radioenzymatic assay for epinephrine forming enzymes.

Epinephrine (E) is formed in the adrenal medulla by phenylethanolamine-N-methyltransferase (PNMT), and in other tissues. Enzymes other than PNMT may be able to synthesize E, but this has been difficult to investigate because most assays do not have E as their final product. This assay produces 3H-E from norepinephrine (NE) and 3H-S-adenosylmethionine. The 3H-E is isolated on alumina, 3H-S-adenosylmethionine is precipitated and the 3H-E is suspended in diethylhexyl phosphoric acid in toluene for scintillation counting. The assay is sensitive and linear over a wide range. E was formed by most tissues tested. Brain and adrenal contained an enzyme specific for NE, but cardiac ventricle contained an enzyme that methylated both NE and dopamine. Denervated tissues in adrenal medullectomized rats contained very little NE, but still had E and E forming enzyme present. This assay detects a non-neuronal E forming enzyme with activity in vitro and in vivo.