Changes in various plasma lipid components, glucose, and insulin in Spermophilus lateralis during hibernation.

This study evaluated fat mobilization as related to gluconeogenesis in two age groups of hibernating golden-mantled ground squirrels (Spermophilus lateralis). Our experimental group consisted of a total of 16 male and 15 female squirrels. Plasma samples were collected from selected animals being killed weekly from January to March, and the concentration of triglycerides, glycerol (GY), free fatty acids (FFA), total cholesterol, lipase activity, glucose, and insulin, were determined by biochemical assays and radioimmunoassay. Our results showed a mean FFA/GY ratio of five, which was higher than the predicted value of three, suggesting a significant depletion of GY and an enhanced rate of gluconeogenesis via GY to maintain glucose homeostasis in the hibernating animals. The factor of age did not significantly affect plasma lipid components. However, in the male group, plasma glucose levels were significantly higher for adults than for juveniles. Overall, females had significantly higher plasma glucose levels than males (150 +/- 11 vs. 110 +/- 8 mg%, P < 0.05). In the adult group, a gender influence was also seen on plasma insulin levels, with females' being higher than males' (66 +/- 13 vs. 25 +/- 3 microIU/ml, P < 0.01). We suggest that during hibernation, female squirrels may have a higher rate of lipolysis and gluconeogenesis along with a lower glucose utilization than their male counterparts. Additionally, adult females may exhibit a higher peripheral insulin resistance during hibernation than adult males, a possibility which merits further study.

Growth hormone treatment of short Chinese children with beta-thalassaemia major without GH deficiency.

OBJECTIVE: Despite regular transfusion and desferrioxamine treatment, growth failure is commonly seen in adolescent children with beta-thalassaemia major. The growth failure has been thought to be due to GH resistance rather than GH deficiency. We investigated the effect of GH on short non-GH deficient children with beta-thalassaemia. DESIGN: Recombinant human GH was given in a dose of 0.14 IU/kg/day subcutaneously in an open study. PATIENTS: Fifteen prepubertal Chinese children with beta-thalassaemia major (ranging from 7.16 to 14.7 years in age) with height -1.5 SD or more below the population mean for age and a growth velocity of less than 5 cm/year were treated with growth hormone for one year. All children had peak GH response > 15mlU/l to insulin induced hypoglycaemia and normal thyroid function and adrenal reserve. MEASUREMENTS: Anthropometric measurements were performed every 3 months. Morning urine was tested twice weekly for glycosuria. Blood count, renal and liver function tests, fasting blood glucose, IGF-I and fructosamine levels were assessed at entry and every 3 months during treatment. Fasting insulin was measured before and after 3 and 12 months of GH treatment. Skeletal maturity was assessed before and after one year of treatment. RESULTS: Treatment was stopped in two children after 6 months because of poor growth response and noncompliance with treatment and in one child at 9 months because of bone marrow transplantation. In the 13 children, the growth velocity increased from 3.6 +/- 0.7 cm/year to 8 +/- 1.2 cm/year after one year of GH treatment (P < 0.001). IGF-I was low before treatment (10.1 +/- 2.7 nmol/l), rising significantly to 15.8 +/- 4.8, 18.4 +/- 4.6, 19.3 +/- 6.4 and 21.9 +/- 7.5 nmol/l at 3, 6, 9 and 12 months of treatment (P < 0.005). The mean pretreatment bone age in the 13 children was 9.58 +/- 1.41 years and increased to 10.53 +/- 1.43 years after one year of treatment (delta BA/CA 0.95 +/- 0.3 years). None of the patients developed glycosuria or hypertension. There was no significant change in blood count, renal and liver function, thyroid function, fasting blood glucose or insulin concentrations during treatment. CONCLUSION: Growth failure in these children with normal GH reserve and low serum IGF-I concentrations would suggest GH insensitivity. Supraphysiological doses of exogenous GH can cause a significant increase in serum IGF-I levels and a significant improvement in short-term growth of short children with beta-thalassaemia major.

Plasma lipoprotein cholesterol concentrations in the golden-mantled ground squirrel (Spermophilus lateralis): a comparison between pre-hibernators and hibernators.

1. The concentrations of total cholesterol (free cholesterol plus cholesteryl ester) in the plasma and in two lipoprotein fractions of golden-mantled ground squirrels (Spermophilus lateralis) were measured during pre-hibernation and compared to those values measured during hibernation. 2. Hibernating ground squirrels had significantly higher (P less than 0.005) very low density lipoprotein plus low density lipoprotein cholesterol (VLDL + LDL-C) concentrations than did pre-hibernating ground squirrels. 3. Hibernating squirrels additionally exhibited significantly higher (P less than 0.005) total plasma cholesterol concentration per high density lipoprotein cholesterol concentration (TPC/HDL-C) ratios than did pre-hibernating squirrels. 4. The significant differences in the lipoprotein cholesterol concentrations observed in this study suggest that lipoprotein metabolism in pre-hibernators was significantly different from that in hibernators and was a reflection of the marked biochemical and physiological adjustments these animals must undergo during their transition from pre-hibernation to hibernation.

Thyroid functions in patients treated with interleukin-2 and lymphokine-activated killer cells.

Treatment of malignant disease with interleukin-2 and lymphokine-activated killer cells activates autoreactive T lymphocytes, stimulates release of cytokines and induces expression of HLA-class II antigens by tumour cells. We studied eight patients with hepatocellular carcinoma treated with a total of 16 courses of recombinant human interleukin-2 and lymphokine-activated killer cells and observed them for features of autoimmune thyroid disease. During the course of treatment there were significant decreases in total serum T4 and T3 and free thyroxine levels, but no change in TSH levels when all patients were analysed as a group. This was due to a number of factors including suppression of thyroid hormone release, haemodilution during interleukin-2 infusion and actual removal of thyroid hormones from the circulation during leukapheresis. Thyroid hormones returned to normal levels during resting period. One patient subsequently developed compensated hypothyroidism (normal total T4, total T3 and free T4 but elevated TSH) and four patients had features of 'sick euthyroid syndrome' (low total T4, total T3 or free T4 but normal TSH). None of the patients studied developed antibodies to thyroglobulin or microsomes. In contrast, no abnormality of thyroid function was seen in any of the nine subjects who received no active treatment. In conclusion, thyroid dysfunction was associated with immunotherapy of malignant disease with interleukin-2 and lymphokine-activated killer cells. This may arise from direct hormonal effects of the cytokines on thyroid hormone production.

Circulating lipoprotein cholesterol concentrations in the summer-active ground squirrel (Spermophilus lateralis): a comparison with those in humans and rabbits.

1. The concentrations of total cholesterol (free cholesterol plus cholesteryl ester) in the sera and in two lipoprotein fractions of golden-mantled ground squirrels (Spermophilus lateralis) were measured and compared to those found in humans and New Zealand White rabbits (Oryctolagus cuniculus). 2. Squirrels showed significantly higher concentrations of total serum cholesterol (TSC; P less than 0.0005), high density lipoprotein cholesterol (HDL-C; P less than 0.0005), and very low density plus low density lipoprotein cholesterol (VLDL + LDL-C; P less than 0.0005) than those in rabbits. 3. Squirrels had significantly higher TSC (P less than 0.0005) and HDL-C (P less than 0.0005) concentrations than did humans. 4. Squirrels additionally exhibited significantly lower TSC/HDL-C ratios than did rabbits (P less than 0.005) or humans (P less than 0.0005). 5. The significant differences in lipoprotein metabolism observed in this study between the active hibernator and non-hibernators, may reflect the marked biochemical and physiological adjustments hibernating species make throughout their circannual cycle.

The use of glucagon challenge tests in the diagnostic evaluation of hypoglycemia due to hepatoma and insulinoma.

We previously found that patients with hypoglycemia due to chronic renal and liver disease had anomalous metabolic responses to glucose and glucagon stimulation. In this study we evaluated the use of glucagon (2 mg, iv) tests in the diagnosis of spontaneous hypoglycemia secondary to hepatocellular carcinoma (HCC) and insulinoma. Twenty-one normal subjects, 45 patients with HCC (11 with hypoglycemia), and 14 patients with insulinoma (all with hypoglycemia) were studied. The fasting blood glucose level was low in all patients with hypoglycemia. The fasting plasma insulin and C-peptide concentrations were high in patients with insulinoma and low in patients with HCC and hypoglycemia. The blood glucose responses to glucagon administration were less than normal in patients with HCC and hypoglycemia and within normal limits in patients with insulinoma. The insulinoma patients had increased plasma insulin and C-peptide responses to glucagon despite having low blood glucose levels. Compared with the HCC patients without hypoglycemia, HCC patients with hypoglycemia had impaired plasma insulin and C-peptide responses. The fasting hypoglycemia, hypoinsulinemia, and impaired insulin/C-peptide responses to glucagon in patients with hepatoma and hypoglycemia presumably reflect the production of insulin-like substances by the hepatoma. We conclude that glucagon administration results in characteristic responses in these groups of patients and can be of use in the diagnosis of spontaneous hypoglycemia secondary to hepatoma or insulinoma.

Delayed phytohemagglutinin-stimulated production of adenosine triphosphate by aged human lymphocytes: possible relation to mitochondrial dysfunction.

The decreased immune response associated with aging may, in part, reflect intrinsic age-related biochemical alterations in lymphocytes from older animals. We measured levels of lymphocyte adenosine triphosphate (ATP) and continuous [3H]thymidine incorporation in phytohemagglutinin-stimulated lymphocytes from young and old humans, and the effects thereon of inhibitors of mitochondrial oxidative phosphorylation and protein synthesis. No difference was found in adenine nucleotide content between young and old subjects. After 24 hours of culture there was a decrease in ATP, with recovery and 2--3-fold increase at 48 hours in young cells after phytohemagglutinin stimulation. We observed a clearcut delay in older lymphocytes of the increase in ATP and [3H]thymidine incorporation following phytohemagglutinin stimulation. We found no evidence for decreased viability or diminished number of responding units in aged cultures. The evidence suggests that mitochondrial dysfunction may play a role in the immunodeficiency of aging.

Alterations in cyclic nucleotides and cyclase-specific activities in T lymphocytes of aging normal humans and patients with Down's syndrome.

The levels of cyclic adenosine 3'5'-monophosphate (cAMP) in unstimulated (resting) peripheral blood thymus-derived lymphocytes (T cells) from normal old humans and young-adult Down's syndrome (DS) patients were markedly decreased when compared with those of young normal humans. By contrast, the cyclic guanosine 3'5'-monophosphate (cGMP) in resting T cells from normal old and young-adult DS patients were greatly increased. The cAMP/cGMP ratios for unstimulated T cells therefore declined in normal aged and DS subjects. The specific activity of adenylate cyclase (ATP pyrophosphate-lyase[cycling]E.C.4.6.1.1) was elevated in T cells from the aged and DS groups, whereas that of guanylate cyclase (GTP pyrophosphate-lyase[cycling]E.C.4.6.1.2) decreased with age and in DS. These results denote the existence of substantial age-related biochemical changes in peripheral T cells. An imbalance in resting cyclic nucleotide levels and their generating enzymes in T cells of normal aging and DS subjects might contribute to the immune dysfunction occurring both with aging and in DS.

Disulfide bonds and the structure of the chromatin complex in relation to aging.

Triton X-100 washed nuclei from livers of young-adult and old mice were digested with micrococcal nuclease and pelleted. Supernatants (1SF) were saved and the pellets lysed in a hypotonic EDTA buffer. A second supernatant (2SF) and a final pellet (P) were obtained by recentrifugation (7000 g, 7 minutes). The 1SF/2SF ratio, which has been shown to be an index of the transcriptionally active to inactive chromatin ratio, was lower in older mice. The fraction relatively resistant to solubilization by the nuclease (P) was found by isopycnic sucrose gradient centrifugation to be in a more compatc, condensed state when prepared from older mice. Higher amounts of heavy density chromatin were obtained from nuclei of old than young mice by hypotonic lysis plus minimal mechanical shearing. 2-Mercaptoethanol (2ME) treatment brought the density of the material of P from old mice back to the levels of young mice. In both age groups 2ME decreased the densities of mechanically sheared chromatin as well as of the whole Triton X-100 washed nuclei. In nuclease digestion experiments treatment of the nuclei from both age groups with S-S reducing agents increased the release of DNA from P into the supernatants. The results are consistent with S-S bonds being involved in the condensed structure of chromatin in young and old mice and in the shift of the chromatin complex towards a more compact, condensed state in old age.

Diamine oxidase activity in plasma and urine in uremia.

Diamine oxidase activity was measured in plasma or urine in 12 normal men, 4 men with chronic liver or heart disease, 13 men with chronic renal failure, and 12 men undergoing maintenance hemodialysis. Also in five studies in 4 patients, plasma diamine oxidase activity and total amine levels were measured at hourly intervals during a hemodialysis treatment. Plasma diamine oxidase activity was normal in patients with liver or heart disease and was at least three times normal in chronically uremic patients and in patients undergoing maintenance hemodialysis. Plasma diamine oxidase activities before and after a hemodialysis therapy were similar and did not change during dialysis until the 4th hour when they fell transiently; plasma total amine levels, which were elevated initially, tended to rise during the 4th hour of dialysis. Urinary diamine oxidase activity was reduced in the chronically uremic patients as compared to normal subjects. These observations are consistent with three alterations in diamine oxidase in patients with renal failure: activity (a) is increased in plasma of chronically uremic patients and those undergoing maintenance hemodialysis, (b) does not increase normally in response to heparin administration during dialysis therapy, and (c) is reduced in urine of chronically uremic patients.

Diamine oxidase in renal failure.

The enzyme, diamine oxidase, is present in many tissues and plays a role in the metabolism of certain amines, some of which may be toxic. In renal failure, plasma diamine oxidase activity was found to be increased in chronically uremic patients and before and after dialysis therapy in patients undergoing maintenance hemodialysis. Diamine oxidase activity was decreased in urine of the chronically uremic patients as compared to normal subjects. In chronically uremic rats, diamine oxidase activity was observed to be increased in plasma and reduced in urine as compared to sham-operated, pair-fed control rats. In the uremic rats diamine oxidase activity was also decreased in kidney and unchanged in liver and muscle. Total amine levels were elevated in plasma and reduced in urine of patients and rats with chronic renal failure. Although the clinical significance of abnormal diamine oxidase activity in renal failure is not clear, it is possible that this enzyme may have a pathophysiologic role in uremia.

Cyclic nucleotide levels in resting and mitogen-stimulated spleen cell suspensions from young and old mice.

The levels of cyclic adenosine 3', 5'-monophosphate (cAMP) in suspensions of unstimulated spleen cells from tumor-free 30-month old (C57BL/10Sn X C3H/HeDiSn)F1 hybrid mice averaged only 14% of that of 6-month old mice. By contrast, the level of cyclic guanosine 3', 5'-monophosphate (cGMP) in spleen cell suspensions from old mice was about 270% that of young mice. The cAMP/cGMP ratio for the unstimulated (resting) state showed a decline by 30 months to about 5% of its 6-month value. Cyclic nucleotide levels were also measured in cell suspensions from old and young mice at intervals over a two hour period following in vitro stimulation with the plant mitogens phytohemagglutinin, concanavalin-A and pokeweed mitogen. Quantitative and in some instances qualitative differences in responses were noted. These results might conceivably reflect either age-related changes in the splenic lymphoid cell subpopulations or intrinsic cellular alterations or both. It is unlikely that changes of this degree could be wholly explained by population shifts. An imbalance in cyclic nucleotide levels in both resting and stimulated lymphoid cells in older animals might contribute to the immune dysfunction known to occur with normal aging.

Amino acid metabolism in the chronically uremic rat.

The chronically uremic rat has been used as a model to study amino acid metabolism in uremia. Uremic rats fed low protein diets (6% casein) survived longer than uremic rats receiving either higher levels of dietary protein or a low protein diet supplemented with a mixture of nonessential amino acids. Alterations in plasma amino acid levels were observed in the uremic rats and were similar to those found in patients with renal failure. Plasma concentrations of citrulline, free tryptophan, glycine and the methylhistidines were increased and levels of serine, ornithine, lysine, total tryptophan, tyrosine, and the tyrosine-phenylalanine ratio were reduced. The metabolic basis of the altered tyrosine-phenylalanine ratio in plasma was studied. Tyrosine aminotransferase (TAT) and phenylalanine hydroxylase (PHL) activity were normal in the liver, but renal PHL activity of was decreased as compared to control rats. Tissue concentrations of citrulline were also found to be raised in liver and muscle of uremic rats. The activity of ornithine transcarbamoylase, was reduced in the liver and arginine synthetase activity was decreased in the kidneys of uremic rats. Thus elevated citrulline levels in uremic tissue appear to be caused by a decrease conversion of citrulline to arginine in the kidney. Preliminary studies of tryptophan metabolism in uremic rats have shown elevated brain levels of 5-hydroxyindoleacetic acid and increased hepatic tryptophan oxygenase activity. Increased plasma amine levels were associated with altered activities of monoamine oxidase and diamine oxidase in kidney and other tissues.