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Biomedical subjects

B V Howard

Publications and source records attributed to B V Howard.

At least 181 records · Page 10Linked to original sources
PubMed1984

Effects of four-day fast on triglyceride mobilization in human adipocytes.

To examine the effect of a four-day fast on neutral glyceride mobilization in human adipocytes, subcutaneous abdominal fat tissue was obtained from ten human volunteers after an overnight fast and then after four days of fasting. Plasma insulin concentration decreased from 31 +/- 5 microU/ml before the four-day fast to 9 +/- 1 microU/ml after the fast (P less than 0.05). Plasma free fatty acid concentration increased from 13.9 +/- 1.3 to 23.6 +/- 3.2 mg/dl (P less than 0.05). Mean average cell size was similar before and after the four-day fast. Basal lipolysis increased threefold (P less than 0.005) regardless of cell size after four days of fasting. In contrast, isoproterenol (2 microM) stimulated maximum lipolysis was not increased after a four-day fast. The concentrations of isoproterenol required for a half maximum stimulation in the presence of 50 (7 microU/ml) and 8000 pM (1150 microU/ml) insulin were lower after a four-day fast (P less than 0.05, P less than 0.03, respectively) and were uninfluenced by average cell size. Basal and maximum insulin-stimulated glucose transport were similar after a four-day fast. However, glucose incorporation into neutral glycerides was reduced by 50 percent (P less than 0.02) following a four-day fast. The reduction was uninfluenced by average cell size. The data indicate that abdominal adipocytes after a four-day fast are metabolically adapted towards increased neutral glyceride mobilization by reducing glucose incorporation into neutral glycerides, increasing sensitivity of lipolysis to isoproterenol, and increasing basal lipolysis.

Adipose Tissue↗
PubMed1983

Effect of temperature on insulin binding and action in cultured human fibroblasts.

Insulin stimulation of glycogen synthase activity and insulin binding were measured in fibroblast monolayers at 24, 32, and 37 degrees C. Insulin stimulation of %I glycogen activity increased with increasing temperature. Maximum response was greater at 37 degrees C than at 32 degrees C, and half maximal stimulation required at 2.0 nM insulin at 37 degrees C vs. 10 nM at 32 degrees C. Insulin stimulation of glycogen synthase was greater and somewhat faster at 37 degrees C than at 32 degrees C. No insulin effect was observed at 24 degrees C. 125I-insulin binding to monolayers became maximal in 15 min at 37 degrees C, 60 min at 32 degrees C, and 120 min at 24 degrees C. However, insulin binding decreased with increasing temperature, and this decline was due to decreased numbers of receptors. Insulin binding and stimulation of glycogen synthase were comparable at 32 degrees C, with half maxima at 10 nM, indicating no evidence of "spare" receptors. The data indicate that temperature effects on insulin binding and action in fibroblasts are not directly related. The results also suggest that a rate limiting step(s) of insulin action is temperature sensitive, and that this step is not insulin binding.

Cells, Cultured↗
PubMed1983

Plasma and lipoprotein cholesterol and triglyceride concentrations in the Pima Indians: distributions differing from those of Caucasians.

Plasma and lipoprotein cholesterol and triglyceride concentrations were measured in Pima Indians, a genetically homogeneous population with a high prevalence of obesity and diabetes and a low frequency of coronary heart disease. The data for nondiabetic Pima Indians were compared with those of U.S. Caucasians measured during the Lipid Research Clinics Prevalence Study. Plasma total and low-density lipoprotein (LDL) cholesterol levels in Pima men older than 30 years of age and in women over 25 were lower than those of U.S. Caucasians, and in men the total and LDL cholesterol levels did not increase with age. In Pima male subjects of all ages high-density lipoprotein (HDL) cholesterol levels were lower than that in Caucasian male subjects, and in Pima female subjects HDL cholesterol levels averaged 11 mg/dl lower than those in female Caucasian subjects. On the other hand, plasma triglyceride levels in Pima male subjects up to 35 years of age and in female subjects up to 55 years old were higher than those of Caucasians. Comparison of plasma cholesterol and triglyceride levels in Pima male and female subjects showed the same relationships as those in Caucasians (i.e., higher in male subjects until the older age groups), but Pima male and female subjects had similar HDL cholesterol levels. There was a strong negative correlation between obesity and HDL cholesterol levels in Pimas. Obesity appeared to account for the lower HDL cholesterol levels in Pima male subjects compared with that in Caucasian male subjects, but obesity could not explain the lack of differences between the sexes in HDL levels in the Pimas. Triglyceride levels correlated positively with obesity in the Pimas, but the relationship was not as strong as that reported for Caucasians. The results indicate that there are differences in lipoprotein distributions between Pimas and U.S. Caucasians; the relationship between lipoprotein levels and the incidence of coronary disease in the Pimas must be determined.

Adolescent↗
PubMed1983

Altered insulin binding to monocytes and diploid fibroblasts from obese donors.

Insulin binding was studied in fibroblasts and monocytes from nondiabetic subjects with a range of body mass indices (BMI). Binding was compared in fibroblasts from extremely obese subjects and normal weight subjects. The [125I] insulin displacement curve for the obese subjects was shifted to the right. Scatchard analysis suggested that cells from the obese group have an increased number of receptors with decreased affinity. Significant correlations were observed between the cell donor's BMI and the concentration of insulin required to inhibit half of the [125I]insulin specifically bound to fibroblasts (r = 0.8; P less than 0.005), the high affinity dissociation constant (r = 0.8; P less than 0.005), and the number of receptors (r = 0.6; P less than 0.05). Insulin binding to monocytes was measured for nondiabetic Pima Indian subjects with a range of BMI values. Scatchard analysis of the data indicated that the high affinity dissociation constant was positively correlated with BMI (r = 0.6; P less than 0.02). The number of receptors was also positively correlated with BMI (r = 0.6; P less than 0.05). The observation that both cultured cells and monocytes exhibit changes in insulin binding that correlate with the obesity of the cell source suggests that the insulin resistance of obesity may be partially a reflection of genetic differences at the site of the insulin receptor.

Adolescent↗
PubMed1983

Very-low-density lipoprotein triglyceride metabolism in non-insulin-dependent diabetes mellitus. Relationship to plasma insulin and free fatty acids.

To investigate the mechanism of elevated plasma triglycerides in non-insulin-dependent (type II) diabetes, very-low-density lipoprotein triglyceride (VLDL-TG) metabolism was studied in 10 untreated male Pima Indian diabetics and compared with that of 15 weight-matched male nondiabetic controls. VLDL-TG metabolism was studied using tritiated glycerol as an endogenous precursor of VLDL-TG, and the resultant kinetic data were analyzed using a multicompartmental model, which includes two pathways for VLDL-TG synthesis and a stepwise delipidation process for VLDL catabolism. Pima diabetics had VLDL-TG concentrations approximately 150% those of nondiabetics. Rates of VLDL-TG production in the diabetics were not significantly different from those of controls. On the other hand, the fractional catabolic rate for VLDL-TG was significantly lower in the diabetics compared with the nondiabetics. Other catabolic parameters, such as the fraction of VLDL-TG delipidized at each step and the stepwise delipidation rate, were also decreased in the diabetics. To determine the relationships between the increased triglycerides and determinants of lipid metabolism that are altered in diabetes, insulin and free fatty acid concentrations were also assessed. Basal C-peptide levels in the diabetics during the metabolic study were slightly but not significantly higher than those of the nondiabetics. There was a highly significant correlation in the diabetics between plasma C-peptides and VLDL-TG production, whereas VLDL production in Pima nondiabetics was not related to insulin levels. Free fatty acid levels were not significantly elevated in the Pima diabetics. The data indicate that (1) the rise in VLDL-TG in Pima diabetics was a result of decreased capacity for clearance and (2) the absence of elevated VLDL-TG production may be attributed to the lack of increase in free fatty acids.

Adolescent↗
PubMed1983

Isopropanol precipitation method for the determination of apolipoprotein B specific activity and plasma concentrations during metabolic studies of very low density lipoprotein and low density lipoprotein apolipoprotein B.

A method has been described for the measurement of apoB concentration and specific activity in very low density lipoprotein (VLDL) and low density lipoprotein (LDL) during metabolic studies. For measurement of specific activity, apoB was separated from other apolipoproteins and lipids by precipitation in, and subsequent washing with, isopropanol. For determination of apoB concentration, equal volumes of lipoprotein and isopropanol were mixed, and the protein content of the apoB precipitate was measured by the difference between total lipoprotein protein and the protein measured in the supernatant. Evidence that there was no apoB solubilization in isopropanol and that precipitated apoB was virtually free of soluble apolipoproteins was obtained by electrophoresis. Lipid contamination of the apoB precipitate was less than 1%. The methods were applicable to VLDL, intermediate density lipoprotein (IDL), and LDL from normolipemic patients with protein concentrations between 187 micrograms/ml and 1898 micrograms/ml. The data obtained using isopropanol were highly correlated with those using tetramethylurea, and recoveries of apoB were similar. Furthermore, the isopropanol method has been demonstrated to yield consistent data for apoB specific activities in a study of VLDL, IDL, and LDL metabolism.

1-Propanol↗
PubMed1982

Measurement of intracellular volume in monolayers of cultured cells.

Methods have been developed for measuring the intracellular water space (ICS) in cultured diploid fibroblast monolayers. The values obtained have been compared to similar measurements of ICS in suspended fibroblasts using an oil spin method. Markers commonly used for measurement of total water space (TWS) ([3H]H2O, [14C]urea) and extracellular space (ECS) ([3H]inulin, [14C]L-glucose, [3H]sucrose, [3H]D-mannitol) were investigated for use in cell monolayers. Monolayer incubations were terminated by rapidly rinsing the culture dishes three times with ice cold buffer. The distribution volume of the TWS marker [14C]urea plateaued at 10 to 15 min and was independent of urea concentration. [3H]H2O was not a suitable marker for measuring total water space in cell monolayers because of rapid loss of intracellular label during rinsing. Intracellular space was calculated by subtracting [3H]sucrose space (5 min) from [14C]urea space (20 min) after incubation of fibroblasts with both markers. Values obtained for ICS (mean +/- SE: microliter/10(6) cells) of fibroblasts from two cell lines measured in monolayer (1.74 +/- 0.11 and 1.60 +/- 0.10) and in suspension (1.88 +/- 0.07 and 1.78 +/- 0.11) was approximately 10% smaller than the values for cell size (2.01 and 2.22) obtained from Coulter Counter sizing. Thus, the methods developed for measurement of ICS in monolayer fibroblasts yield data comparable to those obtained with the more standard oil spin method. Furthermore, the methods can be applied to measurement of ICS in other types of adherent cells.

Body Fluids↗
PubMed1982

Characterization of lipase activities in obese Pima indians. Decreases with weight reduction.

Adipose tissue and muscle lipoprotein lipase and postheparin hepatic and lipoprotein lipase activities have been measured in a group of 21 Pima Indian males over a wide range of body weight to determine the relationship between obesity and these lipase activities. There was a significant positive correlation between adipose tissue lipoprotein lipase and obesity; muscle and postheparin lipoprotein lipase and hepatic lipase were not related to degree of obesity. Fasting insulin levels were not related to any of the measurements of lipase activity. There were racial differences in adipose and postheparin lipoprotein lipase activities; both were significantly lower in the Pimas as compared with a group of weight-matched Caucasian males. Lipase activities were remeasured in eight subjects after a period of weight reduction including several weeks of stabilization at the reduced weights. After the period of weight reduction adipose tissue lipoprotein lipase declined in all subjects. Hepatic lipase also declined in all but two patients. Muscle and postheparin lipolytic activities were not affected by weight loss. The data indicate that (a) there are racial differences in adipose tissue lipoprotein lipase; and (b) the elevated adipose lipoprotein lipase associated with obesity, like many other biochemical variables in the obese state, returns toward normal after weight reduction.

Adipose Tissue↗
PubMed1982

Glucose enhancement of insulin action: elevated glucose levels increase insulin stimulation of 2-deoxyglucose uptake in cultured human fibroblasts.

Insulin stimulation of 2-deoxyglucose uptake in cultured human skin fibroblasts has been examined in the presence of varying concentrations of extracellular glucose. When cell monolayers were preincubated in glucose-free medium, insulin stimulation of hexose transport was small. Increasing glucose levels from 2.7 to 16.7 mM in the preincubation medium enhanced insulin-stimulation 2-deoxyglucose uptake (18-65% of basal uptake). Insulin stimulation of hexose transport in the presence of glucose was the result of an increase in the Vmax without a significant change in the Km of transport. Maximum enhancement of insulin action was achieved 18-24 h after glucose exposure. After 24 h, the insulin response decreased. Glucose did not influence the concentration of insulin required for half-maximal stimulation. Growth conditions also altered the magnitude of insulin stimulation of 2-deoxyglucose uptake; the response was maximal in confluent cells (5-10 days of culture) and decreased during senescence. These data demonstrate that extracellular glucose levels influence insulin action in cultured fibroblasts. This implies that insulin responsiveness in vivo may be regulated in some systems by ambient glucose levels.

Adult↗
PubMed1981

Cell culture studies of a patient with congenital lipoatrophic diabetes--normal insulin binding with alterations in intracellular glucose metabolism and insulin action.

Insulin binding and the action of insulin on several aspects of glucose metabolism have been investigated in cultured fibroblasts derived from a patient with congenital lipoatrophic diabetes and compared to cultures from 9 nondiabetic controls. Incorporation of glucose was elevated in the patient's cells at glucose levels above 0.1 mM. When distribution of labelled glucose was examined, cell associated glycogen and acid soluble material were increased, but the greatest increment was in lactate production. Insulin binding, as indicated by maximum specific 125I-insulin binding and concentration of unlabelled insulin for 50% displacement, was normal, although insulin regulation of the insulin receptor was diminished. Insulin stimulation of total glucose incorporation was reduced in cells from the patient. When insulin stimulation of glycogen synthase was measured directly, the response to insulin was also attenuated. On the other hand, insulin stimulation of hexose transport appeared to be unimpaired. The data indicate alterations in both cell glucose metabolism and insulin response which may be related to the observed insulin resistance of this disorder.

Adult↗
PubMed1981

Alloxan action on glucose metabolism in cultured fibroblasts. I. Stimulation and inhibition of glucose utilization.

The influence of alloxan on mammalian cell glucose metabolism has been investigated using human diploid fibroblastic cells in culture. When cell monolayers were exposed to D-[14C]glucose, the presence of alloxan (0.31-1.87 mM) resulted initially in a dose-dependent enhancement of total cell glucose incorporation. This was observed within 2 min and declined by 6 min. After that time, alloxan inhibited glucose incorporation. When hexose transport was examined directly using glucose analogues, alloxan neither enhanced nor inhibited the uptake of 3-O-methylglucose or 2-deoxyglucose. Alloxan exerted no effect on cell permeability or cell viability. These results suggest that alloxan may directly influence cell glucose metabolism beyond the level of phosphorylation. The dual effect of alloxan on glucose incorporation may be related to the alloxan stimulation and subsequent inhibition of glucose-induced insulin release in pancreatic islets.

Alloxan↗
PubMed1981

Alloxan action on glucose metabolism in cultured fibroblasts. II. Effects on pentose-monophosphate shunt and tricarboxylic acid pathways.

The site of action of alloxan on glucose metabolism has been investigated using cultured human fibroblasts. Analysis of cell extracts after cell monolayers were exposed to D-[U-14C]glucose indicated that the initial stimulation of glucose incorporation by alloxan was observed primarily in the nucleotide fraction (ribose) with inhibition of lactate production. The subsequent inhibition of glucose incorporation was observed in the nucleotide fraction. Assay of 14CO2 production indicated that alloxan enhanced 14CO2 formation from D-[U-14C]glucose for approximately 10 min, followed by inhibition. To probe the site of alloxan action, rates of 14CO2 formation from 1- and 6-position labeled [14C]glucose, and [U-14C]pyruvate were compared. The initial stimulation was observed mainly in D-[1-14C]glucose oxidation, whereas inhibition was measurable with the 6-position tracer and [14C]pyruvate. The results suggest that alloxan initially stimulates the pentose-monophosphate shunt and then subsequently inhibits both the pentose-monophosphate shunt and tricarboxylic acid pathways.

Alloxan↗
PubMed1981

Characterization of the human insulin receptor solubilized from cultured fibroblast and erythrocyte cell membrane preparations.

Human insulin receptors have been partially purified from cultured diploid fibroblasts and from erythrocytes. Cell membrane fractions were prepared by differential centrifugation and solubilized with Triton X-100. Gel filtration chromatography of the 45,000 x g pellet yielded a single sharp peak of binding activity with an apparent molecular weight of 370,000. Solubilized receptor preparations showed binding affinity, pH optimum, and analog specificity similar to those for whole cells. These data indicate that insulin receptors may be isolated from readily obtainable normal human cells. In the case of the fibroblast, the system has the potential for the coordinated study of insulin receptor biochemistry and insulin action.

Centrifugation↗
PubMed1981

Effect of pH and 3-hydroxybutyrate on insulin binding and action in cultured human fibroblasts.

The influence of pH and 3-hydroxybutyrate on insulin binding and action has been studied in cultured human fibroblasts. When the pH of the incubation medium was decreased from 7.6 to 6.8, insulin stimulation of glycogen synthase and total glucose uptake was decreased. The decreased pH induced both an increase in the insulin concentration for half-maximal response, and a decrease in maximal responsiveness. When insulin binding was measured at lower pH, receptor affinity was decreased. The effect of 3-hydroxybutyrate on insulin binding and action was assessed at pH 7.4 and 6.9. In the presence of 3-hydroxybutyrate, insulin binding increased, but insulin action on glycogen synthase and total glucose uptake was unaffected. The data show that insulin action is impaired at lower pH. The decreased sensitivity is probably related to the decrease in insulin binding affinity at lower pH, but decreased maximal responsiveness implies that postreceptor components are also affected by lower pH. The results also suggest that 3-hydroxybutyrate induced a dissociation between binding and response, since an increase of insulin binding was not accompanied by changes in the regulation of glycogen synthase and total glucose uptake.

3-Hydroxybutyric Acid↗
PubMed1981

Type II diabetes and insulin resistance. Evidence of lack of inherent cellular defects in insulin sensitivity.

To determine if inherent cellular differences in insulin sensitivity account for the insulin resistance of non-insulin-dependent diabetes, the effect of insulin on several aspects of cell glucose metabolism was compared in fibroblasts from diabetics and matched nondiabetic controls. The response of total cell glucose metabolism to insulin was assessed by measurement of 14C-glucose uptake. Insulin stimulated cell glucose incorporation in nondiabetic cells up to twofold with half-maximal stimulation at approximately 3 X 10(-9) M insulin. This was similar to that observed in diabetic cells. Insulin stimulation of I glycogen synthase activity was also compared in the cells from diabetics and nondiabetics. Both groups demonstrated a threefold increase in %I activity in the presence of insulin with half-maximal stimulation at approximately 2 X 10(-9) M. There were no differences between diabetics and nondiabetics in either magnitude of response or insulin concentration for half-maximal stimulation. Finally, insulin stimulation of hexose transport was compared in the two cell types using 2-deoxyglucose. In both groups hexose transport was elevated approximately 40% over baseline in the fibroblast in the presence of insulin, with half-maximal stimulation at approximately 2 X 10(-9) M insulin. No differences were found in insulin action on glucose metabolism in fibroblasts form diabetics and nondiabetics; these results may indicate that there are no inherent differences in cell sensitivity to insulin's glucoregulatory action in non-insulin-dependent diabetics.

Adult↗
PubMed1980

Oral contraceptives raise the cholesterol saturation of bile by increasing biliary cholesterol secretion.

To discover the mechanism by which oral contraceptives increase the level of cholesterol saturation of human bile, we measured biliary lipid secretion rates, gallbladder and hepatic bile lipid composition, bile acid pool size, bile acid composition, and plasma lipoprotein levels in five healthy women during routine oral contraceptive treatment and again during normal menstrual cycles on no medication. The molar percent cholesterol in both gallbladder and hepatic bile was higher in every subject while taking oral contraceptives (p less than .02). Oral contraceptive usage was accompanied by a significant enhancement of biliary cholesterol secretion (65 versus 46 mg/hr, p less than .01), but there was no significant change in bile acid or phospholipid secretion, total bile acid pool size, or bile acid composition. These findings indicate that oral contraceptive usage increases biliary cholesterol secretion, thereby raising the level of cholesterol saturation of bile and predisposing to cholesterol precipitation and gallstone formation.

Adult↗