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.

Abnormal regulation of protein tyrosine phosphatase activities in skeletal muscle of insulin-resistant humans.

Insulin resistance in skeletal muscle may be an expression of the genetic basis of a common form of non-insulin-dependent diabetes mellitus (NIDDM) in humans. Impaired insulin action results from an apparent postreceptor defect in insulin signal transduction that limits the influence of the hormone on various protein serine/threonine kinases and phosphatases that are thought to contribute to the mechanism by which insulin affects intracellular events. The fact that numerous responses to insulin are affected suggests that the cause of insulin resistance involves an early step in insulin action. Therefore, we examined the influence of insulin on protein tyrosine phosphatase (PTPase) activities, which may counteract the protein tyrosine kinase activity of the insulin receptor in skeletal muscle of insulin-sensitive and insulin-resistant humans. Insulin infusion in vivo produced a rapid 25% suppression of soluble-PTPase activity in muscle of insulin-sensitive subjects, but this response was severely impaired in subjects who were insulin resistant. Insulin did not affect PTPase activity in the particulate fraction of muscle from either group, but basal particulate activity was 33% higher in resistant subjects than in sensitive subjects. Either or both of these abnormal characteristics of PTPase activities could be central to the causes of insulin resistance and NIDDM.

Insulin stimulation of glycogen synthase in cultured human diploid fibroblasts.

The effect of insulin on glycogen synthase activity in human diploid fibroblasts has been studied. As little as 2 X 10(-10) M insulin increased the glycogen synthase / activity without changing the total activity. Stimulation occurred within 5 min and became maximal in 30 min. A half-maximal increase of / activity was achieved at 3 X 10(-9) M insulin. Glucose starvation increased the magnitude of response of glycogen synthase to insulin but did not change the insulin concentration necessary to give a half-maximal stimulation. Glucose increased the basal level of / activity in human diploid fibroblasts; the effect of insulin was additive. During in vitro senescence the total glycogen synthase activity declined, but the concentration of insulin that produced a half-maximal stimulation remained unchanged. These data indicate that regulation of glycogen synthase activity in human diploid fibroblasts is responsive to physiologic insulin levels and that the system provides a useful model for the in vitro study of insulin sensitivity.

Effects of NIDDM on very-low-density lipoprotein triglyceride and apolipoprotein B metabolism. Studies before and after sulfonylurea therapy.

To evaluate mechanisms of diabetes-induced changes in very-low-density lipoprotein (VLDL), VLDL triglyceride (TG) and VLDL apolipoprotein B (apoB) metabolism were studied in 12 obese Pima Indian control subjects and in 15 Pima Indian obese non-insulin-dependent diabetics. Eleven of the diabetics were restudied after reduction of hyperglycemia with oral sulfonylurea therapy. In addition, adipose, muscle, and postheparin lipoprotein lipase and postheparin hepatic lipase activities were measured in all subjects. Obese diabetics as compared with obese controls showed a trend toward increased production of VLDL TG (46 +/- 4 vs. 35 +/- 6 g/day, P = .10) but not of VLDL apoB (1595 +/- 106 vs. 1597 +/- 164 mg/day, NS); production of VLDL TG declined to control levels (33 +/- 4 g/day, P less than .05) during therapy, whereas there was no change in production of VLDL apoB. Diabetics had a clearance defect for VLDL, indicated by significantly lower fractional catabolic rates for both VLDL TG (10.6 +/- .9 vs. 13.1 +/- .9 pools/day, P less than .05) and VLDL apoB (5.6 +/- .4 vs. 7.5 +/- 0.7, P less than .05) as compared with controls; fractional catabolic rates increased after therapy (to 13.3 +/- 1.5, P less than .05, and 6.7 +/- .4, P less than .05, respectively). In the diabetics, this decrease in clearance was accompanied by a lower adipose lipoprotein lipase (.30 +/- .09 vs. .92 +/- .25 mumol X g-1 X h-1, P less than .01), which increased during therapy (to .61 +/- .17, P less than .05). Hepatic lipase also decreased significantly after therapy (27.4 +/- 3.6 to 26.4 +/- 3.2, P less than .01). Composition of VLDL in diabetics was also abnormal, indicated by a higher TG/apoB ratio (14.7 +/- .6 vs. 11.7 +/- .8, P less than .01); this ratio fell during therapy (to 12.5 +/- .8, P less than .05). The data indicate there are multiple abnormalities in structure and metabolism of VLDL in non-insulin-dependent diabetics. Control of hyperglycemia with sulfonylureas has the capability of reversing some of these abnormalities.

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.

Increased receptor binding of low-density lipoprotein from individuals consuming a high-carbohydrate, low-saturated-fat diet.

The substitution of saturated fat by complex carbohydrate, according to current dietary recommendations, results in a decrease of plasma and low-density lipoprotein (LDL) cholesterol levels. To determine whether this decrease might result from structural and thus functional changes in LDL particles, the binding internalization and degradation of 125I-LDL were measured using TR715-19 cells, a mutant CHO line into which has been transfected the human LDL receptor, and in which measurements of binding are highly reproducible. Eleven nondiabetic subjects (35 +/- 4 years, 27% +/- 3% body fat) were studied after they had 15% protein, and 560 mg cholesterol/d and the other containing 21% fat (6% saturated), 65% carbohydrate, 14% protein, and 524 mg cholesterol/d.LDL cholesterol levels decreased form 125 +/- 6 to 108 +/- 5 mg/dl (P < .01) on the high-carbohydrate diet. There was an increase in the binding affinity of LDL (Kd 6.6 +/- 2.6 v 7.3 +/- 2.7 micrograms/mL +/- SD; P < .02), and internalization (P < .10), and degradation (P < .05) were also higher. The data suggest that decreasing dietary saturated fat may cause alterations in LDL composition that result in increased receptor clearance; this may partially explain the LDL-decreasing effect of this dietary change.

Integrated study of low density lipoprotein metabolism and very low density lipoprotein metabolism in non-insulin-dependent diabetes.

The metabolisms of VLDL, IDL, and LDL and their interconversions have been studied in ten obese untreated male Pima Indian diabetics compared to 16 age-, sex-, and weight-matched nondiabetics. VLDL was elevated in the diabetics and had abnormal composition, as indicated by a significantly higher ratio of triglyceride/apo B. Fractional catabolic rates for both VLDL apoB and VLDL triglyceride were lower in diabetics, and diabetics had increased production of VLDL triglyceride but not VLDL apoB compared to obese nondiabetics. A higher proportion of VLDL apoB was removed without conversion to LDL in diabetics. LDL cholesterol and apoB were higher in diabetics, but production of LDL apoB was not different from nondiabetics. Fractional catabolic rate for LDL apoB, however, was significantly lower in the diabetics. The data indicate that the triglyceride-rich VLDL in non-insulin-dependent diabetics are less readily converted to LDL, whereas the elevated LDL in this group of diabetics is due to impaired clearance. Thus, decreased conversion of VLDL to LDL and impaired LDL clearance are two opposing phenomena which may influence the LDL concentration of diabetics in either direction. Thus, despite minimal changes in LDL concentration, there are multiple defects in the metabolism of LDL in non-insulin dependent diabetes which may contribute to the increased atherogenesis in this disorder.

Distinct deoxyribonucleic acid polymerase activities associated with Dane particles and naked Dane cores.

Two distinct deoxyribonucleic acid polymerase activities were found associated with liver fractions containing Dane particles and Dane cores.

Occurrence of a carrier state for Herpesvirus tamarinus in Marmosets.

The ecology of herpesviruses in marmosets and other nonhuman primates is important today, for colonies of these animals are being established for biomedical research. This paper presents the first reported isolations of Herpesvirus tamarinus from throat swabs of a healthy white-lipped marmoset carrier (Saguinus nigricollis) during a 2-month period. Infectivity studies with this virus in both white-moustached (S. mystax) and white-lipped marmosets demonstrated that the virus is not lethal to white-moustached marmosets (perhaps a more resistant species) at 1,000 TCID(50). Which environmental conditions trigger the unmasking of herpesviruses in marmosets is not known. Hoever, intermittent H. tamarinus shedding may help explain spontaneous infections in established colonies as well as suggest an additional mechanism for transmission of virus between marmosets under natural conditions.

Insulin stimulation of glucose entry in cultured human fibroblasts.

The effect of insulin on glucose entry has been studied in monolayer cultures of human diploid fibroblastic cells. Influence of insulin on total cell glucose incorporation was evaluated using [14C] glucose. Glucose incorporation was increased up to two-fold in the presence of insulin. Insulin action occurred within 30 minutes and could be observed with insulin concentrations as low as 10(-10) M (10 microU)ml). The action of insulin was enhanced by preincubation in glucose-free medium. After glucose starvation the cells converted glucose primarily to glycogen and nucleotides, and the stimulation by insulin was observed equally in both fractions. Influence of insulin on the kinetics of hexose transport was studied using 2-deoxyglucose and 3-0-methyl glucose. A large diffusion component was corrected using rho-chloromercuribenzoic acid or phloridzin. Km for facilitated diffusion averaged 1.9 mM for 2-deoxyglucose and 5.3 mM for 3-O-methyl glucose, and Vmax ranged from 10-24 nmoles/min/mg cell protein. Insulin resulted in a 150% increase in Vmax with no significant change in Km. The data suggest that human diploid fibroblasts can be a useful system for the study of insulin's glucoregulatory action.

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.

Cyclic excretion of hepatitis A virus in experimentally infected chimpanzees: biophysical characterization of the associated HAV particles.

Experimental infection of two chimpanzees with the Phoenix Antigen strain of HAV resulted in the cyclic excretion of virus particles on days 9-11, 14-15, and 20-21 postinoculation. Isopycnic banding in CsCl of stool suspensions prepared from 9-11; 14-15; and 17, 19, 21 dav stool pools revealed multiple buoyant densities for the associated HAV particles. Hollow HAV particles found in the 9-11 day pool banded primarily at a buoyant density of 1.30 g/cm3. HAV in the 14-15 day stool banded bimodally in a CsCl gradient, with antigen peaks at buoyant densities of 1.29 and 1.33 g/cm3. HAV in the days 17, 19, 21 stool pool also banded bimodally in a CsCl gradient; however, the antigen peaks occurred at buoyant densities of 1.33 and 1.40 g/cm3.