Norman Kalant

Effect of diet restriction on glucose metabolism and insulin responsiveness in aging rats

The effect of age and of prolonged caloric restriction on glucose tolerance and insulin responsiveness has been studied in male Fischer 344 rats. Beginning at 1 month of age dietary intake of an experimental group (R) was limited to 60% of that of the control group (AL) which was allowed to eat ad libitum. Studies were carried out at intervals up to 24 months of age. In AL rats the oral glucose tolerance curve showed progressively higher peak levels of plasma glucose with age, and a decrease in the plasma insulin concentration at the time of the glucose peak. The R group did not show the increase in peak value with age and the corresponding insulin concentration was lower than that of the AL group. These results are compatible with a delay in the first phase of insulin secretion in aging AL rats. Insulin-stimulated glucose disposal was assessed by the method of Reaven et al. [Diabetes, 32 (1983) 175], at ages 4, 12, 18 and 24 months; using infusions of 2 mU of insulin and 1 mg of glucose/min per kg, the steady-state plasma glucose level (SSPG) was slightly lower in R than in AL rats, while the steady-state plasma insulin level was reduced by 40-60%. In rats aged 18-24 months the hepatic glucose output, measured with [3-3H]glucose, was the same for AL and R rats in the basal state and was reduced to the same extent by insulin. In the presence of epinephrine and propranolol, infusion of glucose and insulin at various rates demonstrated that the plasma glucose clearance rate increased linearly with increasing SSPI, and at comparable SSPI levels was lower in R than in AL rats. The ability of insulin to stimulate glycogenesis from glucose was measured in primary hepatocyte cultures. Insulin increased glycogenesis 3-fold in cells from AL rats and 4-6-fold in cells from R rats. There was no effect of age. The increased insulin responsiveness of R rats was not due to an increase in insulin binding or to a decrease in insulin degradation (measured with intact cells or as cytosolic insulinase activity).(ABSTRACT TRUNCATED AT 400 WORDS)

Interaction of native and cell-modified low density lipoprotein with collagen gel.

We have examined the binding of native and cell-modified low density lipoprotein (LDL) to gels of Type I collagen. Diffusion of native125I-LDL Into the collagen gel was slow, reaching equilibrium after 24 to 48 hours, while L-3H-glucose, a low molecular weight marker, equilibrated in 6 hours. Binding of125I-LDL was measured at 48 hours as the amount associated with the collagen after extensive washing. Binding was saturable with an Increasing concentration of LDL. Prior incubation with cell-free culture medium resulted in modest, but progressive, Increases In electrophoretlc mobility and binding to collagen. Incubation with cells produced a marked Increase In electrophoretlc mobility and a 5- to 10-fold Increase In collagen binding; the presence of butyiated hydroxytoluene during Incubation prevented both effects. These changes In LDL were Induced by porcine aortic endothellal cells, smooth muscle cells, human skin flbroblasts, and a variety of cell lines, as well as by acetylatlon. There was a curvilinear relationship between the amount of LDL protein bound and the net negative charge of the LDL; Increasing net charge was associated with progressively greater Increases In binding. These results suggest a potential role for collagen in trapping llpld In the extracellular matrix of arterial Intlma by slowing the diffusion of and by binding LDL. The data also demonstrate that binding of LDL to collagen Is enhanced by modifications that increase its net negative charge.

Effect of Age on Glucose Utilization and Responsiveness to Insulin in Forearm Muscle

To determine the effect of age on responsiveness to insulin, 34 healthy subjects (age range, 22–73 years) were studied. A glucose‐clamp technique was used to obtain a range of values for steady‐state arterial glucose and arterial insulin concentrations; total body glucose utilization was estimated from the rate of glucose infusion needed to maintain the steady state; and the uptake of glucose and insulin by forearm muscle was determined by a forearm perfusion procedure. The results were examined by multiple regression analyses. The rate of glucose utilization by the whole body as well as by forearm muscle was dependent upon the insulin concentration. Age had no apparent effect on body glucose utilization, the uptake of glucose or insulin by muscle, or the steady‐state insulin concentration in response to hyperglycemia. It is concluded that the abnormal glucose tolerance commonly associated with increased age is not due to a decrease in either insulin secretion or insulin stimulation of glucose uptake.

Regulation of insulin binding and stimulation of sugar transport in cultured human fibroblasts by sugar levels in the culture medium

Studies were carried out on cultures of human skin fibroblasts to explore the effects of culture medium glucose levels on insulin binding and action. Cell cultures in 5.55 mM glucose-containing medium depleted their medium glucose within 3 days, and at that time exhibited elevated deoxy-D-glucose (2-DG) transport (84% greater than control cultures fed 22.2 mM glucose) and failure of insulin to stimulate 2-DG transport (an insulin:control transport ratio of 1.02). There was also a significant negative correlation between basal 2-DG transport and insulin binding (r = -0.621; n = 29; P less than 0.01), while insulin binding exhibited a significant positive correlation with insulin action (r = 0.816; n = 12; P less than 0.01). Glucose starvation of cultures for 18 h resulted in several changes: a 49% decrease in specific 125I-insulin binding due to a reduction in binding capacity; elevated basal 2-DG transport; and an absence of insulin stimulation of 2-DG transport. Exposure to increasing concentrations of glucose for 18 h led to a glucose concentration-dependent increase in specific insulin binding. Additionally, the various changes in the glucose-starved group were reversed after as little as 6 h of glucose refeeding. The results indicate that basal sugar transport, and insulin binding and action can be regulated by the amount of glucose in the medium.

Interrelationships of glucose and insulin uptake by muscle of normal and diabetic man. Evidence of a difference in metabolism of endogenous and exogenous insulin.

SummaryA forearm perfusion technique was used to study glucose and insulin uptake by muscle. In normal subjects at glycaemic levels above 130 mg/100 ml, glucose uptake was independent of glucose concentration; it was directly related to insulin concentration but not to insulin uptake. In non-obese maturity-onset diabetic subjects, glucose uptake was dependent on glucose concentration and insulin uptake, but not on insulin concentration. In both groups there was a strong correlation between insulin concentration and insulin uptake; diabetics had a normal insulin uptake in relation to concentration. For a given change in insulin concentration the increase in glucose uptake was as great in diabetics as in controls, but the effect of insulin was mediated through a mechanism involving its uptake. Thus in the non-obese maturity-onset diabetic, forearm muscle is not insulin resistant. The apparent uptake of insulin measured by a radioimmunoassay in relation to its arterial concentration was lower and more variable for heterologous than for endogenous insulin. With a receptor assay the venous insulin concentrations were lower than with the immunoassay and differences in uptake between endogenous and exogenous insulin disappeared. It is concluded that in muscle exogenous insulin was less severely degraded than endogenous insulin.

Inhibition by serum components of oxidation and collagen-binding of low-density lipoprotein

Low-density lipoprotein (LDL) is oxidized by cellular and noncellular mechanisms, both leading to an increased binding to collagen. We have investigated the effect of serum on lipid peroxidation, apoprotein oxidation and the binding of oxidized apoprotein to collagen. During noncellular oxidation, lipoprotein-deficient serum strongly inhibited all three processes. The serum fraction of M(r) > 100,000 was equally inhibitory; this effect was not due to alpha 1 or gamma globulins, alpha 2 macroglobulins, haptoglobins or ceruloplasmin. The serum fraction of M(r) 30,000-100,000 stimulated the binding of oxidized apoprotein but the albumin in this fraction inhibited lipid peroxidation and apoprotein oxidation. Serum ultrafiltrate (M(r) < 1000) inhibited lipid and protein oxidation, and binding; the inhibitory effect was abolished by deionization which removed histidine. The effects of lipoprotein-deficient serum and its fractions on cellular oxidation were similar but weaker than those on noncellular oxidation, HDL inhibited noncellular oxidation as well as binding of oxidized apoprotein. VLDL also inhibited oxidation; this could not be accounted for by its content of apo B. If present in vivo, these inhibitory effects would completely suppress both cellular and noncellular oxidation of LDL and its subsequent binding to collagen.

The effect of glucagon on metabolism of glycine-1-C14

Abstract Fasted intact rats were injected with glycine-1-C 14 and glucose-1-C 14 , and the effects of glucagon and epinephrine on incorporation of the C 14 into liver glycogen and expired carbon dioxide were studied. 1. 1. The rate of incorporation of C 14 from glycine into liver glycogen was increased by a previous injection of glucagon, and decreased by a previous injection of epinephrine. 2. 2. The rate of incorporation of glycine-1-C 14 into glycogen was decreased, while the incorporation into CO 2 was increased by concurrent injections of glucagon. 3. 3. The incorporation of C 14 from glucose into liver glycogen was increased by a previous injection of glucagon. 4. 4. Glucagon appears to cause a “rebound” accumulation of liver glycogen comparable to that produced by epinephrine.

Insulin Responsiveness of Superficial Forearm Tissues in Type 2 (Non-Insulin Dependent) Diabetes

SummaryForearm perfusion studies were carried out to determine the responsiveness to insulin of the superficial forearm tissues in non-obese Type 2 (non-insulin-dependent) diabetics, and the interrelationships among plasma concentrations of glucose, insulin and non-esterified fatty acids (NEFA), tissue uptake of glucose and insulin and tissue release of NEFA. It was found that: (1) in normal subjects, up-take of glucose was dependent on glucose concentration. It was also dependent on insulin concentration in the range of 0–30 mU/l, but not over a wider range of insulin concentration (< 66 mU/l), indicating that the insulin effect was maximal at approximately 30 mU/l. In contrast, glucose uptake in diabetics was independent of glucose concentration but dependent on insulin uptake over an insulin concentration range up to 140 mU/l; glucose uptake reached the same levels as in control subjects but only at higher concentration and higher uptake of insulin. (2) Insulin uptake was directly dependent on insulin concentration and the regression coefficients were very similar in the two groups. (3) NEFA concentration fell to comparable levels in the two groups of subjects in response to insulin. It is concluded that in Type 2 diabetes: (1) the superficial forearm tissues show decreased responsiveness to the stimulatory effect of both hyperglycaemia and hyperinsulinaemia on glucose utilization but the NEFA-lowering effect of insulin is undiminished, and (2) tissue uptake of insulin is normal, despite the decrease in receptor capacity that has been demonstrated by others.

Effects of porcine aortic smooth muscle cell conditioned medium on endothelial cell replication.

Previous studies suggested that arterial smooth muscle cells (SMC) may be Involved In regulating the growth of capillaries Into atherosclerotic plaques. In the present study, we determined the effect of SMC products on porcine aortic endothelial cell (EC) replication In vitro. Quiescent or slowly growing EC In medium without endothelial cell growth factor (ECGF) were stimulated to proliferate In the presence of porcine aortic SMC conditioned medium, while the same conditioned medium Inhibited the growth of rapidly dividing EC In high serum concentrations or with ECGF. The magnitude of both activities depended on SMC conditioned medium concentration. The dose-dependent increase In EC number stimulated by ECGF was completely Inhibited by SMC conditioned medium. This effect was not due to a direct Interaction of conditioned medium with ECGF because SMC conditioned medium Inhibited the growth of EC that were rapidly proliferating In 10% serum without ECGF. The Inhibitory activity was retained by an ultraflttratlon membrane with an exclusion limit of 1000 daltons; the stimulatory activity was recovered In the urtrafirtrate and remained stable after boiling, treatment with add or base and trypsin, and repeated freezing and thawing, but was removed by activated charcoal. The growth-promoting activity could not be accounted for by release of cell contents from lysed cells or of thymldlne Into the medium. Conditioned medium from SMC Incubated in the presence of serum contained less EC growth-stimulatory activity but more growth-Inhibitory activity than that from SMC In serum-free medium.

Stimulation of glycolysis by imidazole.

Abstract Imidazole stimulated glycolysis by rat diaphragm homogenates; this effect increased as the concentration of imidazole was raised. The simultaneous changes in concentration of some of the glycolytic intermediates suggested an action of imidazole at a step between triose phosphate and pyruvate. Addition of glyceraldehyde phosphate dehydrogenase increased glycolysis and abolished the stimulating effect of imidazole, thus leading to the conclusion that in the homogenate the activity of this enzyme was rate-limiting for glycolysis and was increased by imidazole.