N-acetylcysteine inhibits lipid accumulation in mouse embryonic adipocytes.

Oxidative stress plays critical roles in the pathogenesis of diabetes, hypertension, and atherosclerosis; some authors reported that fat accumulation correlates to systemic oxidative stress in human and mice, but cellular redox environment effect on lipid accumulation is still unclear. In our laboratory we used mouse embryonic fibroblasts (undifferentiated cells: CC), which are capable of differentiating into mature adipocytes (differentiated cells: DC) and accumulate lipids, as obesity model. Here we analyzed the role of the well-known antioxidant and glutathione precursor N-acetylcysteine (NAC) in cellular MAPK modulation and lipid accumulation. We evaluated the effect of NAC on the adipogenic differentiation pathway using different doses: 0.01, 0.1, 1 and 5mM; no toxic doses in these cells. A dose of 5mM NAC [DCN-5] provoked a significant decrease in triglyceride accumulation (72±10 [DCN-5] vs 169±15 [DC], p<0.01), as well in Oil Red O stained neutral lipid content (120±2 [DCN-5] vs 139±12 [DC], p<0.01). Molecular mechanisms responsible for adipogenic differentiation involve increase of the expression of phosphoERK½ and phosphoJNK, 5mM NAC treatment inhibited both pERK½ and pJNK protein levels. We also evaluated the mitotic clonal expansion (MCE) which takes place during adipogenesis and observed an increase in DC at a rate of 1.5 cells number compared to CC at day 2, whereas the highest doses of NAC significantly inhibited MCE. Our results suggest that NAC inhibits lipid accumulation and the MAPK phosphorylation in mouse embryonic fibroblasts during adipogenic differentiation and further contribute to probe the importance of cellular redox environment in adipogenesis.

[Dietary interview: a useful tool for data collection]. / Entrevista dietética. Herramientas útiles para la recogida de datos.

Dietary interview is a very useful tool in the process of evaluating the nutritional state of a person and, of course, in the process of dietary counseling, particularly in the case of patients suffering from chronic conditions. The complexity of gathering information at individual or population level requires different models of questionnaires and tools to enhance the identification of the data collected. The current paper is based on the consensus document about dietary interview, made by a working group created during the 1st Meeting of the Spanish Dietitians' and Nutritionists' Association held in Barcelona 2002.

Isosorbide mononitrate in the prevention of first variceal bleed in patients who cannot receive beta-blockers.

BACKGROUND & AIMS: Nonselective beta-blockers (beta-blockers) are very effective in preventing first variceal bleeding (FVB) in patients with cirrhosis. However, 15%-25% of patients have contraindications or develop severe side effects precluding its use. The present study evaluates whether isosorbide-5-mononitrate (Is-MN) effectively prevents variceal bleeding in patients with contraindications or who could not tolerate beta-blockers. METHODS: One hundred thirty-three consecutive cirrhotic patients with gastro-esophageal varices and contraindications or intolerance to beta-blockers were included in a multicenter, prospective, double-blind randomized controlled trial. Sixty-seven were randomized to receive Is-MN, and 66 to receive placebo. RESULTS: There were no significant differences in the 1- and 2-year actuarial probability of experiencing a FVB between the 2 treatment groups. Presence of variceal red signs at endoscopy was the only variable independently associated with an increased risk of variceal bleeding on follow-up (relative risk 3.4; P < 0.01). Survival and adverse events were similar in the 2 groups. There were no significant differences in the incidence of ascites or changes in renal function. CONCLUSIONS: Is-MN does not reduce the incidence of FVB in patients with cirrhosis and esophageal varices who cannot be treated with beta-blockers because contraindications or intolerance to these drugs, suggesting that Is-MN has no place in the primary prophylaxis of variceal bleeding.

Mutation of CDH23, encoding a new member of the cadherin gene family, causes Usher syndrome type 1D.

Usher syndrome type I (USH1) is an autosomal recessive disorder characterized by congenital sensorineural hearing loss, vestibular dysfunction and visual impairment due to early onset retinitis pigmentosa (RP). So far, six loci (USH1A-USH1F) have been mapped, but only two USH1 genes have been identified: MYO7A for USH1B and the gene encoding harmonin for USH1C. We identified a Cuban pedigree linked to the locus for Usher syndrome type 1D (MIM 601067) within the q2 region of chromosome 10). Affected individuals present with congenital deafness and a highly variable degree of retinal degeneration. Using a positional candidate approach, we identified a new member of the cadherin gene superfamily, CDH23. It encodes a protein of 3,354 amino acids with a single transmembrane domain and 27 cadherin repeats. In the Cuban family, we detected two different mutations: a severe course of the retinal disease was observed in individuals homozygous for what is probably a truncating splice-site mutation (c.4488G-->C), whereas mild RP is present in individuals carrying the homozygous missense mutation R1746Q. A variable expression of the retinal phenotype was seen in patients with a combination of both mutations. In addition, we identified two mutations, Delta M1281 and IVS51+5G-->A, in a German USH1 patient. Our data show that different mutations in CDH23 result in USH1D with a variable retinal phenotype. In an accompanying paper, it is shown that mutations in the mouse ortholog cause disorganization of inner ear stereocilia and deafness in the waltzer mouse.

Total paracentesis in cirrhotic patients with tense ascites and dilutional hyponatremia.

OBJECTIVE: The safety of large-volume paracentesis with plasma expander infusion in ascitic cirrhotic patients with advanced liver disease, hyponatremia, or renal failure has not been elucidated. Our aim was to investigate the safety of total paracentesis in cirrhotic patients with ascites and severe hyponatremia. METHODS: Forty-five cirrhotic patients with tense ascites were treated with total paracentesis and infusion of plasma expanders. At inclusion, 20 patients showed severe hyponatremia (serum sodium <130 mEq/L). In the remaining 25 patients, serum sodium was >130 mEq/L (range, 133-146 mEq/L). RESULTS: Plasma renin activity (PRA) and plasma aldosterone concentration (PAC) were significantly higher in patients with hyponatremia (PRA: 19.7 +/- 5.8 ng/mL/h; PAC: 217 +/- 35 ng/dL) than in those patients without hyponatremia (PRA: 4.9 +/- 1.1 ng/mL/h; PAC: 95 +/- 31 ng/dL), indicating a more severe systemic hemodynamic deterioration. After paracentesis, PRA and PAC increased similarly in both groups of patients. Serum sodium levels remained unchanged after paracentesis in patients with hyponatremia (127 +/- 0.5 to 128 +/- 1.5 mEq/L) and decreased slightly in patients without hyponatremia (137 +/- 1 to 135 +/- 1 mEq/L; p < 0.005). The incidence of complications during the first hospitalization, the probability of readmission for complications of cirrhosis, and the probability of survival at 1 yr were similar in both groups of patients. CONCLUSIONS: These results indicate that therapeutic paracentesis is a safe treatment for tense ascites in cirrhotic patients with severe hyponatremia.

Risk of a first community-acquired spontaneous bacterial peritonitis in cirrhotics with low ascitic fluid protein levels.

BACKGROUND & AIMS: Long-term primary antibiotic prophylaxis of spontaneous bacterial peritonitis has been suggested to be useful in cirrhotic patients with low ascitic fluid protein levels. However, it is unlikely that all such patients need prophylactic treatment. The aim of this study was to identify the group of cirrhotic patients with low ascitic fluid protein levels at high risk of developing a first episode of spontaneous bacterial peritonitis during outpatient follow-up. METHODS: One hundred nine cirrhotic patients with low ascitic fluid protein levels and without previous episodes of spontaneous bacterial peritonitis were followed up in an outpatient clinic. RESULTS: Twenty-eight patients developed a first spontaneous bacterial peritonitis episode. In the multivariate analysis, serum bilirubin level (>3.2 mg /dL) and platelet count (<98.000/mm(3)) independently correlated with the risk of developing the first spontaneous bacterial peritonitis (P < 0.01 and P < 0.05, respectively). According to the median relative risk coefficient, a low-risk group (relative risk <1.09) and a high-risk group (relative risk >1.09) were established. The probability of developing a first spontaneous bacterial peritonitis episode at 1-year follow-up was significantly higher in the high risk-group (low-risk group, 23.6%; high-risk group, 55%; P < 0.01) as a consequence of a higher probability of the first community-acquired episode (13.7% vs. 47.6%, respectively, P < 0.01). One-year probability of survival was significantly lower in the high-risk group (low-risk group, 57.6%; high-risk group, 38%, P < 0.05). CONCLUSIONS: Cirrhotic patients with low ascitic fluid protein levels (

Infections caused by Escherichia coli resistant to norfloxacin in hospitalized cirrhotic patients.

Selective intestinal decontamination with norfloxacin is useful to prevent bacterial infections in several groups of cirrhotic patients at high risk of infection. However, the emergence of infections caused by Escherichia coli resistant to quinolones has recently been observed in cirrhotic patients undergoing prophylactic norfloxacin. Our aim is to determine the characteristics of the infections caused by E. coli resistant to norfloxacin in hospitalized cirrhotic patients. One hundred and six infections caused by E. coli in 99 hospitalized cirrhotic patients were analyzed and distributed into two groups: group I (n = 67), infections caused by E. coli sensitive to norfloxacin, and group II (n = 39), infections caused by E. coli resistant to norfloxacin. The clinical and analytical characteristics at diagnosis of the infection were similar in both groups. Previous prophylaxis with norfloxacin was more frequent in group II (15/67, 22.4% vs. 32/39, 82%, P <.0001), as a result of a higher number of patients submitted to continuous long-term prophylaxis in this group, whereas previous short-term prophylaxis was similar in both groups. Infections were more frequently nosocomial-acquired in group II than in group I (17/67, 25.3% vs. 20/39, 51.2%, P =.01). The type of infections was similar in both groups: urinary tract infections 38 in group I and 24 in group II, spontaneous bacterial peritonitis 8 and 2, spontaneous bacteremia 4 and 4, and bacterascites 1 and 0, respectively (pNS). Mortality during hospitalization was similar in the two groups (4/67, 5.9% vs. 5/39, 12.8%, pNS). None of the E. coli resistant to norfloxacin were also resistant to cefotaxime and only one of them was resistant to amoxicillin-clavulanic acid. Prophylaxis with norfloxacin, usually continuous long-term prophylaxis, favors the development of infections caused by norfloxacin-resistant E. coli. Long-term antibiotic prophylaxis should therefore be restricted to highly selected groups of cirrhotic patients at high-risk of infection. Infections caused by E. coli resistant to norfloxacin show a severity similar to those caused by sensitive E. coli. No significant associated resistance between norfloxacin and the antibiotics most frequently used in the treatment of bacterial infections in cirrhotic patients has been observed.

Hemodynamic changes in patients developing effective hypovolemia after total paracentesis.

BACKGROUND/AIMS: In many centers paracentesis is considered the treatment of choice for tense ascites. However, the mechanism of effective hypovolemia after paracentesis, the main complication associated with this procedure, remains unknown. In the current study, systemic hemodynamics was sequentially studied before and after total paracentesis in 46 patients with cirrhosis and tense ascites. The aim of the study was to assess the mechanism of effective hypovolemia after paracentesis. METHODS: Plasma renin activity and aldosterone, mean arterial pressure, cardiac output (ECO-Doppler) and systemic vascular resistance were measured before, and 3 h, 6 h and 6 days after total paracentesis associated with plasma volume expansion. RESULTS: Effective hypovolemia after paracentesis (defined as 50% increase in plasma renin activity up to a level over 4 ng x m(-1) x h(-1) at the 6th day after paracentesis) occurred in 20 cases [plasma renin activity increased from 8+/-17 to 19+/-2.7 ng x m(-1) x h(-1)]. In the remaining 26 cases no changes in plasma renin activity [8.5+/-2.4 vs. 8.7+/-2.2 ng x m(-1) x h(-1)] were observed. The amounts of ascitic fluid volume removed were similar. Effective hypovolemia after paracentesis was associated with a significant decrease in mean arterial pressure (89+/-2 vs. 81+/-3 mmHg) and systemic vascular resistance [1263+/-67 vs. 1014+/-80 dyn x s(-1) x cm(-5)] 6 days after treatment. In contrast, no significant changes in these parameters were observed in patients not developing this complication. In the whole group of patients a significant inverse relation was observed between changes in plasma renin activity and in systemic vascular resistance (r=0.74;p< 0.001). CONCLUSIONS: These results indicate that effective hypovolemia after paracentesis in cirrhosis is predominantly due to an accentuation of the arteriolar vasodilation already present in these patients.

ACTH-mediated glucocorticoid and mineralocorticoid production is inhibited by an inositolphosphoglycan and a glycosylphosphatidylinositol-phospholipase C is activated by the hormone in mammalian adrenocortical cells.

In the present paper, we report that an inositolphosphoglycan (IPG), derived from a Trypanosoma cruzi glycoinositolphosphoceramide (LPPG), is able to inhibit ACTH-mediated accumulation of a glucocorticoid, cortisol, in calf adrenocortical cells. This IPG is also able to inhibit the stimulation by ACTH of the production of the main glucocorticoid, corticosterone and the main mineralocorticoid, aldosterone, in rat adrenocortical cells. Nitrous acid deamination confirmed that IPG is responsible for this inhibition. In order to study the involvement of glycosylphosphatidylinositol (GPI) in ACTH response in rat adrenal cortex, the activation of a phospholipase that hydrolyzes GPI (GPI-PLC) was evaluated. It was found that the release of alkaline phosphatase, a GPI-anchored enzyme, to the extracellular medium is increased in rat adrenocortical cells by ACTH treatment. In addition, ACTH stimulates the release of ceramide from the glycoinositolphosphoceramide purified from T. cruzi. These data suggest that ACTH activates a GPI-PLC in rat adrenal cortex, which is in agreement with our previous data in calf adrenocortical cells; thus, the hydrolysis of GPI provoked by ACTH takes place in different mammals and the IPG released could inhibit ACTH-mediated synthesis of aldosterone, corticosterone and cortisol.

Continuous versus inpatient prophylaxis of the first episode of spontaneous bacterial peritonitis with norfloxacin.

Cirrhotic patients with ascites and low ascitic fluid total protein and/or high serum bilirubin levels are at high risk to develop the first episode of spontaneous bacterial peritonitis during long-term follow-up. The aim of the present study was to determine the efficacy of continuous long-term selective intestinal decontamination with norfloxacin in the prevention of this complication. One hundred nine cirrhotic patients with ascites and ascitic fluid total protein levels of < or = 1 g/dL or serum bilirubin levels of > 2.5 mg/dL without previous spontaneous bacterial peritonitis were prospectively randomized into two groups: group 1 (n = 56) received norfloxacin, 400 mg daily administered orally, and group 2 (n = 53) was the long-term control group, receiving norfloxacin only during hospitalization. During a mean follow-up of 43 +/- 3 weeks, there was one spontaneous bacterial peritonitis (1.8%) in group 1 and 9 (16.9%) in group 2 (P < .01). The incidence of community-acquired spontaneous bacterial peritonitis was lower in group 1 (1.8% vs. 13.2%, P < .05), whereas the incidence of nosocomial spontaneous bacterial peritonitis (0% vs. 3.7%) and the incidence of extraperitoneal infections (25% vs. 24.5%) were similar in both groups (P = NS). The actuarial probability of survival at 18 months was 75% in group 1 and 62% in group 2 (P = NS). Resistance to norfloxacin was observed in 9 of 10 (90%) Escherichia coli isolated in infections from group 1 and in 4 of 11 (36.3%) from group 2 (P < .05). The overall incidence of infections caused by norfloxacin-resistant bacteria was higher in group 1 (19.6% vs. 15%), but it did not reach statistical significance. Continuous long-term selective intestinal decontamination with norfloxacin is effective in preventing the first spontaneous bacterial peritonitis in cirrhotic patients at high risk. However, the emergence of infections caused by norfloxacin-resistant bacteria must be weighed carefully against the benefits of continuous long-term prophylaxis.

An inositol phosphoglycan from Trypanosoma cruzi inhibits ACTH action in calf adrenocortical cells.

We describe the effect of an inositol phosphoglycan (IPG) purified from Trypanosoma cruzi on the stimulation of aldosterone and cAMP production by ACTH in calf adrenocortical cells. T. cruzi IPG has two galactofuranose residues (Galf) which are not frequent in other IPGs. The effect of IPG with galactofuranose residues (IPG Galf) and IPG without these residues (IPG) was investigated. It was found that IPG Galf slightly decreased the stimulation of aldosterone and cAMP production by ACTH, whereas IPG significantly inhibited ACTH-mediated accumulation of both aldosterone and cAMP. The inhibition of aldosterone content in ACTH-treated cells by IPG was dose dependent. It was also found that the pretreatment of calf adrenocortical cells with IPG inhibited the accumulation of aldosterone provoked by ACTH and dibutyryladenosine-3',5'-cyclic monophosphate (db-cAMP). On the other hand, the activation of a GPI (glycosyl phosphatidylinositol)-phospholipase C by ACTH was evaluated. First it was found that the release of ceramide from a GPI-like molecule: a glycoinositol-phosphoceramide (LPPG) purified from T. cruzi is increased in ACTH-treated cells. Second, the release of alkaline phosphatase, a GPI-anchored enzyme, to the extracellular medium was increased in these cells by ACTH. These data suggest that ACTH activates a phospholipase C in calf adrenocortical cells, releasing IPG, which in turn may inhibit, or modulate ACTH action.

Spontaneous bacterial peritonitis in cirrhotic patients treated using paracentesis or diuretics: results of a randomized study.

Diuretic treatment in cirrhotic patients with ascites increases ascitic fluid concentration of total protein and complement components, and opsonic activity. These changes are not observed in patients treated with paracentesis. Based on these data it has been suggested that therapeutic paracentesis may be associated with an increased risk of spontaneous bacterial peritonitis (SBP) development. To assess this possibility, 80 cirrhotic patients with tense ascites were randomly allocated in two therapeutic groups: group 1 (40 patients) was treated with total paracentesis associated with plasma volume expansion and group 2 was treated with diuretics. After mobilization of ascites, patients from both groups received diuretics to avoid reaccumulation of ascites; cases that developed tense ascites during follow-up (mean follow-up period, 60 +/- 6 and 55 +/- 4 weeks, respectively) were treated according to initial randomization. Patients from both groups had similar results regarding baseline clinical and standard laboratory data, ascitic fluid concentration of total protein, complement components, and opsonic activity. Sixteen patients (7 from group 1 and 9 from group 2) developed SBP during the study period. The 4-week and 1-year probability of SBP occurrence were 2.5% and 18.6%, respectively, in group 1 patients, and 11.9% and 24%, respectively, in group 2 patients. Therefore, our study indicates that therapeutic paracentesis does not increase the early- and long-term risk of SBP development in cirrhotic patients with tense ascites.

Total paracentesis with dextran 40 vs diuretics in the treatment of ascites in cirrhosis: a randomized controlled study.

The aim of the current study was to compare total paracentesis associated with dextran-40 infusion with diuretics in the treatment of tense ascites in patients with cirrhosis. Eighty patients were randomly allocated to two groups: 40 patients were treated with paracentesis plus dextran-40 infusion (8 g per liter of ascitic fluid removed), and 40 patients with diuretics. After treatment patients were discharged with diuretics, and patients developing tense ascites during follow up (54 +/- 4 weeks) were treated according to their initial schedule. Paracentesis was more effective than diuretics in mobilizing the ascitic fluid. The incidence of complications was significantly higher (p < 0.05) in the diuretic group (38%) than in the paracentesis group (15%). This difference was mainly due to a higher incidence of hepatic encephalopathy in the former group (30% vs. 2.5%). A significantly higher incidence of hepatic encephalopathy was also observed in the diuretic group during the follow-up readmissions for ascites recurrence. There were no significant differences between the two treatment groups in the probability of survival after inclusion. Plasma renin activity and plasma aldosterone concentration measured before and 2 and 6 days after paracentesis in 20 randomly selected patients increased significantly (p < 0.05) (baseline values: 5.3 +/- 1.4 ng.ml-1.h-1 and 63 +/- 21 ng/dl; 48 h after paracentesis: 11.7 +/- 3.9 ng.ml-1.h-1 and 99 +/- 31 ng/dl; 6 days after paracentesis: 10.9 +/- 3 ng.ml-1.h-1 and 110 +/- 27 ng/dl).(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of aldosterone production by hemin in calf adrenal glomerulosa cell cultures.

Aldosterone production from 11-deoxycorticosterone was stimulated by hemin in primary cultures and homogenates of calf adrenal zona glomerulosa, in a time- and dose-dependent fashion. The ferrochelatase inhibitor 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC) blocked the stimulation of aldosterone mediated by adrenocorticotropin (ACTH). Addition of hemin after treatment with DDC partially restored ACTH action. These results suggest that hemin may play a role in regulation of aldosterone production.

Adrenal receptors for natriuretic peptides and inhibition of aldosterone secretion in calf zona glomerulosa cells in culture.

Atrial and brain natriuretic peptides specifically bind to primary cultures of calf adrenal glomerulosa cells. Binding of both natriuretic peptides to the same receptor has been proved by: a Dixon plot showing competitive effects for the binding of 125I-labeled brain natriuretic peptide in the presence of increasing concentrations of unlabeled atrial natriuretic peptide; a Scatchard plot showing a lower dissociation constant (Kd) for atrial natriuretic peptide than for brain natriuretic peptide binding, but the maximum binding (Bmax) values were the same; autoradiography of sodium dodecyl sulfate polyacrylamide gels after cross-linking of 125I-labeled atrial natriuretic peptide and 125I-labeled brain natriuretic peptide, showing the same molecular weights for both peptide receptors--a single 66-kD band in whole cells and a main band at 125 kD in membranes. C-Type atrial natriuretic peptide only slightly displaced atrial natriuretic peptide binding. Angiotensin II- and potassium-mediated stimulation of aldosterone production were inhibited strongly and to the same degree by atrial and brain natriuretic peptide but only slightly by C-type atrial natriuretic peptide. Stimulation of aldosterone production mediated by adrenocorticotropin was only partially inhibited by atrial and brain natriuretic peptide, while baseline aldosterone was not affected. These results suggest that atrial and brain natriuretic peptide bind to the same receptors and provoke the same effects on aldosterone production. The weak effects found with C-type atrial natriuretic peptide suggest that the primary culture of calf adrenal glomerulosa cells contain the guanylate cyclase A receptor.

Risk factors for spontaneous bacterial peritonitis in cirrhotic patients with ascites.

BACKGROUND: This study was performed to investigate the risk factors for a first episode of spontaneous bacterial peritonitis in cirrhotic patients. METHODS: One hundred ten cirrhotics with sterile ascites, without previous spontaneous bacterial peritonitis (SBP), were included from March 1988 to October 1989 and followed up until October 1990 (follow-up, 46 +/- 3.5 weeks; range, 4-120 weeks). RESULTS: Twenty-eight patients (25.45%) suffered SBP. In multivariate analysis (Cox's regression model) including only variables commonly used in clinical practice, ascitic fluid protein concentration and serum bilirubin level independently correlated with first SBP development. Using these two variables the relative risk of a first SBP episode was calculated for each patient. According to the median relative risk coefficient (1.2), a low-risk group (relative risk, < 1.2) and a high-risk group (relative risk, > 1.2) were established. Kaplan-Meier estimates of patients free of SBP were significantly higher in the low-risk group. CONCLUSIONS: The probability of a first SBP episode is significantly influenced by the antimicrobial capacity of ascitic fluid and hepatic function.

Insulin rapidly increases glycerol-3-phosphate-acyltransferase activity in rat adipocytes.

Glycerol-3-phosphate acyltransferase (G3PAT) was activated by insulin in intact rat adipocytes within 1 min: this activation persisted for 10 min, and was due to a decrease in the Km of the enzyme. The addition of insulin to control adipocyte membranes also increased G3PAT activity, and this effect was mimicked by phosphatidylinositol-specific phospholipase C. Cytosol fractions from insulin-treated adipocytes stimulated G3PAT activity of control membranes, suggesting that a soluble mediator is released during insulin action, possibly through activation of a PI-specific PLC.

Sulfonylurea-stimulated glucose transport association with diacylglycerollike activation of protein kinase C in BC3H1 myocytes.

The extrapancreatic effects of sulfonylurea drugs include increased glucose uptake by certain peripheral tissues. To study this effect, we used BC3H1 myocytes, which are reported to respond to these drugs. Within 30 min, tolbutamide and glyburide increased [3H]-2-deoxyglucose uptake in a dose-dependent manner. The inactive analogue carboxytolbutamide had no effect on glucose transport. Because increases in glucose transport may be mediated by activation of the diacylglycerol-protein kinase C signaling system, we examined the effects of these drugs on lipid metabolism and protein kinase C activity. Unlike insulin, tolbutamide and glyburide failed to increase [3H]glycerol labeling of diacylglycerol or labeling of phospholipids by 32P. After 30 min of treatment with tolbutamide or glyburide, however, membrane-associated and cytosolic protein kinase C activity were each increased. When cells were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) for 48 h to deplete certain isoforms of protein kinase C, glyburide, tolbutamide, and acute TPA treatment failed to increase glucose uptake, suggesting that TPA and sulfonylureas operate through activation of a common pathway. The effect of glyburide was additive to TPA in stimulating glucose uptake at low but not high TPA concentrations. As with insulin and TPA, extracellular Ca2+ was not essential for sulfonylurea-stimulated glucose uptake. Staurosporine, a protein kinase C inhibitor, blocked glyburide-, tolbutamide-, and insulin-stimulated glucose uptake. In intact cells, glyburide stimulated the phosphorylation of both 80,000-Mr and 40,000-Mr proteins, which are markers for protein kinase C activation. Addition of sulfonylureas directly to the protein kinase C assay system in vitro provoked dioleinlike effects, in that sensitivity of the enzyme to Ca2+ was increased.(ABSTRACT TRUNCATED AT 250 WORDS)