Effects of the hepatocarcinogen 2-acetylaminofluorene on insulin binding to microsomal and Golgi fractions of rat liver cells.

Adult male Fischer rats fed the hepatocarcinogen 2-acetylaminofluorene [(2-AAF) CAS: 53-96-3; N-fluoren-2-yl-acetamide] at 0.02% showed a sharp decrease in the insulin binding to the Golgi fraction of the liver cells: from 10.0% specific binding per 0.1 mg protein in control animals to 5.9% after only 2 days and to 1.5% after 21 days of feeding 2-AAF. A less pronounced and slower decrease was observed in the microsomal fraction: from 19.1% specific binding per 0.5 mg protein in controls to a nadir of 10.8% after 46 days. The low binding of insulin to both fractions was observed for the 85 days of the experiment and persisted also in the animals fed 2-AAF for 90-107 days and then taken off the carcinogen for 30-75 days. The decrease in binding was due to the apparent decrease in the number of receptors. Neither 2-AAF nor its metabolites N-hydroxy-2-AAF (CAS: 53-95-2; N-fluoren-2-yl-acetohydroxamic acid) and N-acetoxy-2-AAF (CAS: 6098-44-8; N,O-diacetyl-N-fluoren-2-yl-hydroxylamine) influenced the insulin binding to the microsomes when added to the reaction mixture in vitro.

Effects of diethylnitrosamine on hepatic receptor binding and autophosphorylation of epidermal growth factor and insulin in rats.

Carcinogen-mediated alterations in hepatic membrane growth factor receptor activity were investigated with the use of the hepatocarcinogen diethylnitrosamine [(DENA) CAS: 55-18-5]. For the separate assessment of carcinogen-induced acute membrane receptor changes from changes associated with carcinogen-mediated alterations in growth, the receptor activity was measured both acutely after, and several months after, a single ip dose of DENA in male F344 rats. An acute dose-dependent decrease was observed in ligand binding and autophosphorylation of the epidermal growth factor (EGF) and insulin receptor. Binding decreased sharply by 36 hours and recovered by 30 days in Golgi fractions and more slowly in plasma membranes. Scatchard analysis revealed a decrease in receptor number but not affinity. Chronic DENA administration also decreased insulin and EGF binding. Hepatocellular carcinomas, induced by single DENA injections 1 year previously, had decreased EGF receptor binding and autophosphorylation compared to those seen in age-matched controls and also less extensive insulin receptor changes. Single DENA doses thus have two effects: an acute reversible receptor change and a delayed, apparently permanent change associated with altered growth.

Binding of epidermal growth factor and insulin and the autophosphorylation of their receptors in experimental primary hepatocellular carcinomas.

Experimental chemical hepatocellular carcinomas that were induced in male F344 rats using three different regimens of limited exposure to the carcinogens 2-acetylaminofluorene or diethylnitrosamine were characterized by very low (as compared to peritumorous or normal tissues) binding of epidermal growth factor and decreased autophosphorylation of the epidermal growth factor receptors. Similar changes were also found in the insulin receptors. We suggest that the carcinogens 2-acetylaminofluorene and diethylnitrosamine cause an initial chemical effect on the great majority of cells. Most of them with time recover their receptor function, and only a small minority become truly initiated and retain these changed characteristics up to the tumor stage. The observed changes appear to be associated with the altered growth state induced by chemical carcinogens. Simultaneous changes observed in the two receptors raise the possibility of a common underlying mechanism.

Insulin and epidermal growth factor receptors in rat liver after administration of the hepatocarcinogen 2-acetylaminofluorene: ligand binding and autophosphorylation.

The livers of male F344 rats which were fed 0.02% 2-acetylaminofluorene (2-AAF) for two days or more had decreased binding of insulin and epidermal growth factor (EGF) to their hepatic receptors in microsomal and Golgi fractions. Hepatic receptors which were partially purified from carcinogen-fed rats by Triton X-100 solubilization and wheat germ agglutinin affinity column chromatography also had decreased binding activity compared to receptors from normal rats. Scatchard analysis indicated that the decrease in insulin receptor binding was due to decreased receptor number whereas the change in EGF receptor binding was attributed to decreased receptor affinity. Insulin receptor phosphokinase activity was also decreased in 2-AAF-fed rats and correlated with the decrease in receptor binding. EGF receptor phosphokinase activity was unchanged in 2-AAF-fed rats when stimulated with a high concentration (1 microM) of EGF but was decreased when stimulated with low concentrations (0.01-0.1 microM) of EGF. No EGF or insulin competing activity for receptor binding was found using acid-ethanol extracts of 2-AAF-altered liver. These results suggest that 2-AAF causes different alterations in the insulin and EGF receptors of the rat liver.

Effect of 2-acetylaminofluorene on the binding of epidermal growth factor to microsomal and Golgi fractions of rat liver cells.

The livers of rats fed the hepatocarcinogen 2-acetylaminofluorene (0.02%) with chow showed a sharp decrease in the binding of epidermal growth factor to microsomes and Golgi fractions. The binding to the latter decreased from 15.3% specific binding per 0.1 mg protein in controls to 9.4% after 2 days and reached a nadir of 0.8% after 21 days. The binding to microsomes decreased from 26.3% specific binding per 0.5 mg protein in the controls to 17.4% after 4 days and reached a nadir of 7.5% after 46 days. The low binding which persisted until the end of the experiment (85 days) was due to the apparent decrease in the number of receptors without significant changes in their affinity. Also, there was only partial recovery in rats fed 2-acetylaminofluorene for 90 to 107 days and taken off the carcinogen for 30 to 75 days. In vitro, neither 2-acetylaminofluorene nor its metabolites hydroxy- and acetocy -2-acetylaminofluorene significantly decreased epidermal growth factor binding to the isolated microsomal fraction.

Chromium-associated 32P-labeling of proteins.

The incubation of proteins with chromium (Cr3+ or Cr6+) in the presence of 32P ([gamma-32P]ATP or H3(32)PO4) at room temperature for 10-30 min resulted in the labeling of these proteins with 32P. The 32P-labeled proteins could be separated by SDS-polyacrylamide gel electrophoresis and identified by exposure to X-ray film. The characteristics of this procedure included: the optimal chromium concentration was 100 microM; the minimum requirement of each protein was 1 microgram; the optimal pH value was between 6 and 8; metal ions such as V5+, Mn2+ and Fe3+ strongly inhibited the effect of chromium, whereas Ca2+ and Mg2+ had little effect. It was concluded that chromium binds to the proteins and forms a complex with 32P to achieve the 32P-labeling of the proteins. This technique can be applied for the rapid preparation of 32P labels on protein markers for gel electrophoresis and for the identification of unknown protein species.

Effect of low-dose prophylactic dopamine on high-dose cisplatin-induced electrolyte wasting, ototoxicity, and epidermal growth factor excretion: a randomized, placebo-controlled, double-blind trial.

PURPOSE: To evaluate the protective effect of low-dose dopamine given as continuous infusion in patients who undergo chemotherapy with the nephrotoxin cisplatin. PATIENTS AND METHODS: Forty-two patients who received high-dose cisplatin-containing chemotherapy entered a prospective, randomized, double-blind, placebo-controlled trial. Twenty-one patients received dopamine, and 21 received placebo. Patients were to receive either infusional dopamine 2 micrograms/kg/min over 48 hours or placebo. Cisplatin 125 mg/m2 was administered 12 hours after initiating dopamine (group D) or placebo (group P). This schedule was repeated twice, 1 week apart. Measurements of serum creatinine, urinary electrolytes and creatinine, urinary excretion of epidermal growth factor (EGF), ototoxicity, parameters of hematopoietic recovery, and duration of hospitalization were analyzed. RESULTS: We observed an increase in serum creatinine level to a peak of 1.9 mg/dL (range, 0.8 to 7.8) in the dopamine group, in comparison to 1.4 mg/dL (range, 0.9 to 3.3) in the placebo group (P = .04). Urinary magnesium excretion increased and EGF excretion decreased in both groups. Urinary sodium, chloride, and potassium excretion were increased in both groups, but more so in the placebo group. Dopamine had no measurable effect on hearing loss, duration of hospitalization, or hematopoietic recovery. CONCLUSION: The use of prophylactic dopamine increased peak serum creatinine levels relative to placebo and failed to prevent cisplatin-induced renal toxicity or ototoxicity. Determination of whether dopamine could reverse chemotherapy-induced renal damage would require a randomized prospective trial.

Brittle diabetes in pregnancy.

In eight brittle diabetics the insulin requirement increased during successful pregnancies from 35 to 64 U./day (in 29 stable severe diabetics from 53 to 78 U./day). The within-day glycemic excursions were calculated as the mean of the three differences between four time points: fasting, one hour after breakfast, two hours after breakfast, and two hours after lunch. This parameter was constant throughout the pregnancy in nondiabetics (34-46 mg./100 ml.) and stable severe diabetics (55-72 mg./100 ml.). In brittle diabetics it dropped from 147 mg./100 ml. before the pregnancy and 153 mg./ 100 ml. at eight weeks to 85 mg./100 ml. at 36 weeks. The between-day variability was calculated as the mean glycemic difference between two successive days at the four points of time noted above. It was very low in nondiabetics (49-53 mg./100 ml.). In brittle diabetics it decreased from 127 mg./100 ml. before pregnancy and 120 mg./100 ml. at eight weeks to 46-55 mg./100 ml. at 24-36 weeks. This shows that brittleness substantially decreased in the second half of pregnancy.

The diagnosis of postprandial hypoglycemia.

Our observation that hypoglycemia, often self-diagnosed by our patients, was seldom confirmed led the authors to establish norms for the glucose tolerance test. We first obtained values for 650 patients who were entirely free from symptoms before and during testing. The median nadir in this group was 64 mg/dl. Ten percent of the patients had plasma glucose nadirs of 47 mg/dl or below and 2.5% had values of 39 mg/dl or less. Utilizing these values in combination with clinical criteria, we confirmed hypoglycemia after glucose load in 16 (median nadir 39.5 mg/dl) of 118 patients presenting with this diagnosis, and only 5 of the 16 were hypoglycemic after their usual meals. The other 102 patients, whose many complaints were unrelated to measured plasma glucose levels, had nadirs similar to those of the control group. Placebo tests performed on 14 nonhypoglycemic patients provoked symptoms (recorded by the patients themselves) and they considered indicative of hypoglycemia. Some accepted other diagnoses after we demonstrated that their symptoms occurred when they were normoglycemic. Since nadirs of hypoglycemics and control subjects overlap, we conclude that accurate diagnosis of hypoglycemia requires that symptoms develop concurrently with low blood sugar and that they are absent at other times. Low plasma glucose must be considered only one of the criteria in diagnosing functional hypoglycemia along with a relationship between food intake, timing of symptoms, correlation of symptoms and low glucose levels, and reproducibility of test results.

Glycohemoglobin. Its use in the follow-up of diabetes and diagnosis of glucose intolerance.

In 140 persons with a normal glucose tolerance test (GTT), the median glycohemoglobin (GHb) level was 8.1% (95% limits, 6.5% to 9.0%). The GHb level was not significantly different in 30 patients with impaired GTT (8.6%). In all 14 patients with diabetic GTT and fasting plasma glucose (PG) level above 140 mg/dL, GHb level was above 10% (median, 11.3%). Of 25 patients with diabetic GTT but without fasting hyperglycemia, only two had GHb levels 10% or above. The GHb level correlated with the mean daily PG level in both 176 insulin-dependent (rs, +.530) and 107 non-insulin-dependent (rs, +.734) diabetics. Correlation was almost zero between GHb level and mean one-day PG level in 14 most brittle insulin-dependent diabetics. However, in them, there was a strong correlation (rs, +.763) between GHb level and the mean of 20 or more PG values for the preceding month. Follow-up of 100 diabetics showed strong correlation (rs, +.845) between the changes in GHb level and in the mea PG level. Decrease of the mean PG level of 100 mg/dL was accompanied by a drop of the GHb level of about 2%.

Clinical observation on brittle diabetes.

The brittleness of 100 severe diabetics was calculated as the mean of differences of blood glucose between two consecutive days at four time points (fasting, one and two hours after breakfast, and two hours after lunch). Mean daily difference (MDD) had a unimodal distribution; 15 patients with a MDD greater than 100 mg/100 ml were classified as most brittle. There was no correlation between MDD and insulin requirement. The brittle diabetics received 26 to 48 units of insulin/day. The insulin-resistant patients had low MDD values. No difference was found between seven patients with brittle diabetes and seven stable matched controls in insulin-binding capacity or total insulin. In two groups of six patients each with brittle diabetes, it was found that the stable dosage caused less brittleness than a sliding-scale regimen and that routine injection of 4 units of regular insulin before meals slightly decreased the mean diurnal glycemia level but increased the number of hypoglycemias. In two brittle diabetics, the blood glucose level was stabilized on intravenously administered insulin infusion, and in these patients, meals caused only a moderate hyperglycemia.

Nadirs of oral glucose tolerance tests are independent of age and sex.

We analyzed nadirs of 75-g oral glucose tolerance tests in 400 subjects who remained completely asymptomatic during the test. The median nadir was 63 mg/dl, the 10th percentile 48 mg/dl, and the 2.5th percentile 41 mg/dl. Seventy-five percent of the nadirs occurred at 3-4 h. There were no differences in the absolute level of nadirs between men and women, nor between the subjects 17-30 and 61-74 yr of age.

Different body-fat distributions in IDDM and NIDDM.

Stepwise logistic regression was used for the analysis of the following anthropometric parameters in 146 adult patients with diabetes: waist/hips ratio, waist/thighs ratio, ratio of the arms-to-thighs circumferences, and ratio of the subscapular-to-triceps skinfolds. After adjustment for age and body mass index, only the waist/thighs ratio was found to have an independent discriminating value between insulin-dependent (IDDM) and non-insulin-dependent (NIDDM) diabetic patients. The correlation of type of diabetes with the waist/thighs ratio was especially strong in women, exceeding even the role of age and body mass index. Other anthropometric parameters were related to type of diabetes through their correlation with waist/thighs ratio and had no independent discriminating value. The study shows that IDDM and NIDDM belong to different anthropometric types, which probably reflects their different pathogenesis.

C-peptide in NIDDM. Follow-up for 4-6 yr.

OBJECTIVE: To study whether fasting and 1-h postbreakfast C-peptide levels in NIDDM stabilize with time in individual patients. RESEARCH DESIGN AND METHODS: Within the period of 4-6 yr, 49 NIDDM patients had repeated tests of fasting and 1-h postprandial levels of plasma glucose and C-peptide with the aim of determining their individual qualitative patterns. Throughout the follow-up period, 13 patients were treated with insulin, 21 with oral sulfonylureas, and 15 were switched from oral drugs to insulin, with the tests done in both treatment periods. RESULTS: The group as a whole demonstrated no changes in mean fasting or postprandial C-peptide within 4-6 yr of observation, irrespective of the mode of therapy or its changes. Glycemic and C-peptide response to breakfast was qualitatively typical for each patient with the correlation between plasma glucose and C-peptide. However, the response was vastly different from patient to patient, and the cross-sectional data showed no correlation between postprandial changes in glycemia and C-peptide, nor between glycemic response to breakfast and fasting plasma glucose or C-peptide. In spite of high fasting glycemia, 25% of the patients showed remarkable tolerance to breakfast with only small increases in plasma glucose. In many other patients, however, in spite of similar increase in C-peptide, plasma glucose rose sharply after the meal. CONCLUSIONS: In our group, no deterioration of the insulin secretory function was observed within 4-6 yr of follow-up. Qualitative patterns of the glycemic and C-peptide responses to breakfast were typical for each patient but vastly different between patients. We see in NIDDM a syndrome with few common characteristics and recommend further work for its subclassification into forms with different pathogenesis.

Effects of enalapril and nitrendipine on the excretion of epidermal growth factor and albumin in hypertensive NIDDM patients.

OBJECTIVE: To compare the effect of the antihypertensive drugs nitrendipine and enalapril on the excretion of epidermal growth factor (EGF) and albumin in hypertensive non-insulin-dependent diabetes mellitus (NIDDM) subjects. RESEARCH DESIGN AND METHODS: After a 4-week washout period, mildly hypertensive (systolic blood pressure [sBP] > or = 140 mmHg and/or diastolic blood pressure [dBP] > or = 90 mmHg) NIDDM patients with albuminuria (15-200 micrograms/min) were randomized into an 8-month-long therapy with either nitrendipine (n = 11) or enalapril (n = 10). Blood pressure, EGF, and microalbumin excretion were measured at baseline and throughout the treatment period. RESULTS: A significant fall in sBP was noticed in the enalapril group and in dBP in the nitrendipine group. In the enalapril group, EGF excretion progressively increased from 188 to 214 nmol/mmol creatinine after 6 weeks and to 274 after 8 months of therapy (P = 0.03). There was a significant fall in albumin excretion while patients were on enalapril, but in the nitrendipine group, neither albuminuria nor EGF excretion changed significantly. There was no correlation of improved EGF excretion with a decrease in albuminuria or BP. CONCLUSIONS: The angiotensin-converting enzyme inhibitor enalapril has been effective in decreasing albumin and increasing EGF excretion. Measurement of urinary EGF may provide a new valuable index of renal function.

Heart rate variability in diabetes: relationship to age and duration of the disease.

Heart rate variability (HRV) during deep breathing was studied with a neonatal heart monitor in 143 control subjects and 218 patients with diabetes (102 with IDDM and 116 with NIDDM). In the control group HRV decreased after age 20 by 4-5 beats per decade (from 29.7 +/- 5.8 beats at age 20-29 to 11.8 +/- 5.4 beats at age 60+). In all age groups HRV in IDDM was lower than in the controls, and both age and duration of diabetes played a role in the decrease of HRV (from 21.5 +/- 5.3 beats at age 20-29 to 6.3 +/- 5.4 at age 60+). In NIDDM aging seemed to play a less important role, and the influence of the duration of the disease was not statistically significant. In both groups of patients the frequency of HRV below the 2.5th percentile was 82% in those with symptoms and/or signs of autonomic neuropathy, 64% in patients with peripheral neuropathy only, and 36% in those who had no obvious signs or symptoms of neuropathy. Interindividual variability was pronounced, and age and duration of the disease together accounted for only 36% of the observed differences between IDDM and the controls. Determination of HRV with a standard neonatal heart monitor presents an easy, simple, and nonstressful test of cardiac autonomic neuropathy. The norms of the test are age related.

Effect of corticosterone on carbamylcholine-, leucine-, or calcium-induced insulin secretion by the isolated perfused rat pancreas.

Using the isolated pancreas of male Sprague-Dawley rats, three different concentrations of glucose were tested in the perfusion medium: 4.0, 6.7, and 9.4 mM. The insulinotropic effects of several potent secretagogues were examined with and without a 10(-7)-M corticosterone-21-acetate (CS) background. When the perfusion medium contained 4.0 mM glucose, there was no insulin secretion in the basal state, but 10(-6)M carbamylcholine chloride, 5 mM L-leucine, and 5 mM calcium chloride elicited moderate but sharp responses of insulin output. When the secretagogues were superimposed on CS infusion, their stimulatory effects were abolished. When the perfusion medium contained 6.7 mM glucose, a steady insulin secretion was observed. A 4- to 5-fold stimulation of insulin release was elicited by carbamylcholine, leucine, and calcium. With CS in the perfusate, the secretagogues were ineffective. However, at a higher glucose level (9.4 mM), the stimulants could partially escape the inhibitory effects of CS. It is concluded that CS may play a role in the regulation of insulin output by directly modulating the activities of some potent secretagogues.

Binding of growth hormone to rat liver during experimental chemical hepatocarcinogenesis.

Male F-344 rats (180-200 g) received either a single injection of diethylnitrosamine (DEN) or continuous feeding with 2-acetylaminofluorene (AAF). DEN induced a decrease in the binding of human GH (hGH) to its hepatic Golgi receptors in a dose-dependent manner; the changes were due to a decrease in the number of hGH receptors without significant changes in their affinity. Thirty days after DEN, the binding of hGH returned to normal. After the administration of AAF, the binding of hGH increased owing to the greater number of binding sites, and this effect persisted for the 30-day period of the continuous AAF feeding. In three separate hepatocellular carcinomas, the hGH binding to the Golgi fraction of the tumors was only one quarter of the binding to the peritumorous tissues. We conclude that DEN and AAF, administered acutely for a short time, affect hepatic hGH receptors in a different way, but that hepatocellular carcinomas bind much less hGH than peritumorous or normal tissues. The results show that hepatocarcinogenesis encompasses changes in receptors belonging to different classes.

Mitogenic factors accelerate later-age diseases: insulin as a paradigm.

Some genes are expressed differently in earlier and later generations of most cell lines. Many diseases become clinically expressed only later in life, and show clustering of the age at onset in the affected siblings, which may be related to the changing expression with age of the genes involved. Because insulin and its receptor are extremely ancient and well preserved structures with almost universal mitogenic effects, insulin may serve a paradigm of this process. It is suggested that by stimulating cell proliferation, hyperinsulinemia speeds up the appearance of later generations of cells with different expression of the genes. Insulin resistance, accompanying any hyperinsulinemia and considered to be a pathogenetic factor of some common later-age diseases, involves only some biochemical, but not mitogenic effects of the hormone. In humans, high levels of insulin in blood are encountered both physiologically after meals and in many pathological conditions: insulin therapy inevitably causes peripheral hyperinsulinemia; in type 2 diabetes hyperinsulinemia precedes hyperglycemia by many years; hyperinsulinemia is an independent risk factor of atherosclerosis, of type 2 diabetes itself, of some forms of dementia and other diseases; obesity is an obligatory hyperinsulinemic condition. The opposite of hyperalimentation, i.e. calorie restriction (at least, in rodents) may exert its life-prolonging effects through decreasing insulinemia and therefore the rate of cell proliferation. Insulin is only one example, and different mitogens regulate proliferation of different cells. It is likely that growth factors in general accelerating the replication of cells, play a role in speeding up the appearance of later-age diseases involving these cells.

Homogeneity of the age at diagnosis in sibs with Type 2 diabetes: implications for sib-pair analysis.

Homogeneity of the age at diagnosis of Type 2 diabetes was studied in 1,228 sibs in 300 unrelated families: 100 consecutive single-affected and 200 consecutive multiple-affected ones. There were 635 diabetic sibs. The mean and median age at diagnosis in all affected individuals was 50 years (range, 19-75 years). The mean age at diagnosis in the multiply affected families was 49 years (median 50); the between-sibs range of age at diagnosis within multiple-affected families was (mean and median) 17 years (range, 0-55), with 42% of these diagnosed within a 5-year age span, 66% within 10 years, and 90% within 13 years. When one parent had diabetes, it was more often the mother (79% P = 0.0023). In order to examine this apparent tendency toward homogeneity of age at diagnosis within families, with full regard for and parsimonious to right-censored data, we employed a Cox proportional-hazards survival analysis, with family as the explanatory variable. Deviance residuals resulting from that model were analyzed in a variance components, random effects model ANOVA which indicated a significant (P << 0.001) effect of family on age of diagnosis, with an intraclass correlation of 0.29. In many families the clustering of age at diagnosis appeared very tight, with single outliers, and in 20 families with the longest history, diabetes was diagnosed in 68 sibs within the span of 8 +/- 7 years, whereas 38 unaffected sibs remain free of diabetes 25 +/- 8 years later. The wide differences of the age at diagnosis between families and its intrafamilial homogeneity should be considered in planning genetic analysis of Type 2 diabetes.