Glycaemic variability is associated with coronary artery calcium in men with Type 1 diabetes: the Coronary Artery Calcification in Type 1 Diabetes study.

AIMS: We investigated coronary artery calcium in association with glucose levels and variability measured using continuous glucose monitoring in adults with Type 1 diabetes in the Coronary Artery Calcification in Type 1 Diabetes study. METHODS: Coronary artery calcium was measured by electron beam tomography. The presence of any coronary artery calcium was analysed with respect to glucose levels [mean(T) (mean glucose), % of values < 3.9 mmol/l, > 10 mmol/l and either < 3.9 or > 10 mmol/l] and glycaemic variability [sd(T) (sd of all glucose values); sd(dm) (sd of the daily mean glucose levels) and sd(hh:mm) (glucose sd for a specified time of day, over all days)] using 3-5 days of continuous glucose monitoring from 75 subjects (45 women, 30 men), age 42 ± 9 years (mean ± sd) and diabetes duration of 29 ± 8 years using logistic regression. RESULTS: We observed significant associations between coronary artery calcium and mean(T) (OR = 4.4, 95% CI 1.1-18.6), % of values > 10 mmol/l (OR = 5.5, 95% CI 1.3-22.6), % of measures < 3.9 or > 10 mmol/l (OR = 5.7, 95% CI 1.3-24.9), sd(T) (OR = 4.7, 95% CI 1.1-19.7), sd(dm) (OR = 6.0, 95% CI 1.2-30.4) and sd(hh:mm) (OR = 4.0, 95% CI 1.1-15.4), among men, but none of these variables were associated with the presence of coronary artery calcium in women. CONCLUSIONS: We report the novel finding that subclinical atherosclerosis is associated with glucose levels and variability in men with Type 1 diabetes. The relationship of coronary artery calcium and glucose variability in Type 1 diabetes, and potential gender differences in this association, deserve further study.

Polyacrylamide gel electrophoresis.

Polyacrylamide gel electrophoresis (PAGE) provides a versatile, gentle, high resolution method for fractionation and physical-chemical characterization of molecules on the basis of size, conformation, and net charge. The polymerization reaction can be rigorously controlled to provide uniform gels of reproducible, measurable pore size over a wide range. This makes it possible to obtain reproducible relative mobility (Rf) values as physical-chemical constants. Application and extension of Ogston's (random fiber) model for a gel allows for calculation of molecular volume, surface area, or radius, free mobility, and valence from RJ measurements at several gel concentrations, to calculate gel concentration for optimal resolution, and to predict behavior of macromolecules on gel gradients by computerized methods. Extension of classical moving boundary theory has been used to generate multiphasic buffer systems (providing selective stacking, unstacking, restacking, and preparative steady-state-stacking) with known operating characteristics for any pH at 0 degrees and 25 degrees C. A general strategy for isolation of macromolecules and for macromolecular mapping has been developed. Preparative scale PAGE is operational for milligram loads and feasible for gram quantities.

Transketolase abnormality in cultured fibroblasts from familial chronic alcoholic men and their male offspring.

We have investigated a thiamine-dependent enzyme, transketolase, in cultured fibroblasts from 41 human subjects, including patients with alcoholism-associated Wernicke-Korsakoff syndrome (n = 3), familial chronic alcoholic males (n = 7), their sons (n = 7), nonalcoholic men (n = 7), their male offspring (n = 7), and three generations of an Amish family (n = 10) without any history of alcoholism. This study was undertaken to delineate whether transketolase abnormality (i.e., high Michaelis Menton constant (Km) for thiamine pyrophosphate), previously reported in patients with Wernicke-Korsakoff syndrome is prevalent among familial chronic alcoholic men and their sons without prior history of alcohol abuse but who are at high risk for alcoholism. Our data suggest that an inborn error (i.e., high Km of transketolase for thiamine pyrophosphate) predisposing to thiamine deficiency diseases similar to those reported in Wernicke-Korsakoff syndrome may occur in the general population. However, for some as yet unexplained reason(s) this variant seems to occur more frequently among familial chronic alcoholic men and their male offspring without any history of alcoholism. The inheritance pattern of this enzyme variant as revealed from an Amish pedigree study may be autosomal recessive as previously suggested.

Insulin resistance in Cushing's disease. Evaluation by studies of insulin binding to erythrocytes.

Studies of 125I -insulin binding to erythrocytes (RBC) from 5 patients with Cushing's disease were performed in an attempt to evaluate the insulin resistance in this disease. Five obese, nondiabetic patients and six normal subjects served as controls. Insulin resistance was present in both the obese, nondiabetic subjects and in the patients with Cushing's disease. Patients with Cushing's disease showed insulin resistance out of proportion to obesity, and of greater severity than in the obese subjects. As in previous studies, the insulin resistance of the obese subjects could be at least partially ascribed to a reduced number of receptors. In contrast, in our patients with Cushing's disease, no physiologically significant changes in the parameters of insulin-receptor interaction could be demonstrated. This suggests that the RBC insulin receptor is not involved in this type of insulin resistance.

Potential role of computers in clinical investigation and management of diabetes mellitus.

Computers are likely to play an increasingly important role in the clinical investigation and management of patients with diabetes. The applications fall primarily into four areas: 1) data-base systems in clinics, hospitals, and research centers to facilitate surveillance of patients and analysis of data; 2) personal microcomputers for use by physicians and patients in storing, analyzing, and transmitting data; 3) portable devices to provide recommendations regarding insulin dose for patient use; and 4) memory-equipped glucose meters that provide verified data and have associated software with the capacity for sophisticated graphic and statistical analysis and detection of trends. An important outgrowth of the memory-based meter and the personal microcomputer is the ambulatory glucose profile (AGP). This is a display of the average pattern of glucose throughout the day, usually based on data from a 2-wk period. The AGP shows the median blood glucose, range, and 25th and 75th percentiles for each hour or in relationship to meals. By virtue of signal averaging in the presence of large random variability, this display may reveal patterns that are not evident on individual days.

Computer simulation of plasma insulin and glucose dynamics after subcutaneous insulin injection.

We developed a computer program for the simulation of plasma insulin and glucose dynamics after subcutaneous injection of insulin. The program incorporates a pharmacokinetic model to calculate the time courses of plasma insulin for various combinations of popular preparations (regular, NPH, lente, and ultralente). With the use of a pharmacodynamic model describing the dependence of glucose dynamics on plasma insulin and glucose levels, the program can predict the expected time course of plasma glucose in response to a change in carbohydrate intake, insulin dose, timing, or regimen. A set of typical parameters has been obtained by analysis of data from the literature. The results of several computer simulations are presented showing the effect on a 24-h insulin and glucose profile of systematically changing insulin regimen, dose, timing of meals, or timing of preprandial insulin administration. The program can be used to explore on a theoretical basis the impact of various factors associated with glycemic control in insulin-dependent diabetes mellitus. As an educational tool, the program provides a realistic environment for demonstration of the combined or isolated effects of insulin and diet on glycemia.

Personal computer programs to assist with self-monitoring of blood glucose and self-adjustment of insulin dosage.

We have developed computer programs in compiled BASIC for the IBM-PC and compatible microcomputers for use by physicians, paramedical personnel, and/or patients to assist with self-monitoring of blood glucose (SMBG) and self-adjustment of insulin dosage. The programs can potentially assist with patient education and motivation, and provide: a customized "electronic notebook" for storage and retrieval of information on blood glucose, insulin dosage, hypoglycemic reactions, urinary ketones, diet, activity, weight, illness, apparent explanations for hypoglycemic reactions or glucose values outside target ranges, and comments; graphic displays of glucose and insulin versus date, and of a "glucose profile" versus time of day or versus day of the week; simple and detailed statistical analyses; a legible summary of data; a facility to permit the physician to prepare a "customized treatment plan" for each patient, involving a choice of six regimens, target levels for each of eight time periods, four supplement tables (when well or sick, before meals, or at bedtime), rules to reduce insulin in response to hypoglycemic reactions or documented hypoglycemia, rules to increase routine insulin doses in response to persistent unexplained hyperglycemia, and rules when the patient should call the physician; suggestions regarding compensatory supplements and adjustments of routine insulin dosage; explanations why various insulin dosages should or should not be altered, and why various glucose values should be tested; comparisons of the insulin dosage administered by the patient and the recommendations of the program, together with explanations for discrepancies offered by the patient, to help evaluate compliance. The program is "user-friendly," easy to learn, and easy to use.(ABSTRACT TRUNCATED AT 250 WORDS)

Ambulatory glucose profile: representation of verified self-monitored blood glucose data.

Sixty-nine individuals with diabetes (23 with type I, 15 with pregestational, and 31 with gestational) used specially modified reflectance meters containing memory chips enabling the instruments to store 440 individual blood glucose values with corresponding time and date. These data were organized into 14-day periods and then collapsed into a graphic depiction, the Ambulatory Glucose Profile (AGP), which was represented as the pattern of the 25th, 50th, and 75th percentiles of blood glucose values. These three curves illustrate the median level of control and provide an index of variability in control at each hour of a "typical day." We observed distinctive AGPs related to the variability in metabolic control and the type of diabetes. Comparisons between diagnostic groups showed consistent differences between groups, independent of level of glycemic control. Review of serial AGPs obtained for sequential 2-wk periods for 23 non-pregnant individuals with type I diabetes and 10 women with gestational diabetes revealed changes in AGP corresponding to alterations in regimen. The AGP provides a new approach to the evaluation of glycemic control, with applications to patient and physician education, clinical investigation, and individual patient care.

Biosynthesis of proteoglycans by rat granuloma cells cultured in vitro: modulation by gonadotropins, steroid hormones, prostaglandins, and a cyclic nucleotide.

Rat ovarian granulosa cells were isolated from immature female rats 48 h after stimulation with 5 IU PMS gonadotropin and then maintained in culture. The effects of ovine (o)FSH, oLH, hCG, testosterone, 17 beta-estradiol, progesterone, prostaglandins E1, E2, F1 alpha, F2 alpha, N,O'-dibutyryl cAMP, and theophylline on proteoglycan synthesis by the granulosa cells in vitro were examined using [35S]sulfate as a precursor. oFSH, oLH, hCG, prostaglandins E1 and E2, N,O'-dibutyryl cAMP, theophylline and testosterone stimulated the production of 35S-labeled proteoglycans compared to control cultured. 17 beta-Estradiol, progesterone, and the prostaglandins F1 alpha, and F2 alpha, showed no stimulation. Two major species of proteoglycan are synthesized and secreted into the medium: 1) a population with large hydrodynamic size (Kav = 0.30 on Sepharose CL-2B); and 2) a population with relatively small size (Kav = 0.60 on Sepharose CL-2B). The stimulatory effect of the hormones was accounted for entirely by a net increase in synthesis of the smaller proteoglycan population. The hormones exert their stimulatory effects on proteoglycan synthesis at concentrations similar to those required for other in vitro biological responses of granulosa cells.

Aldosterone stimulated differentiation of mouse 3T3-L1 cells into adipocytes.

We find that 1-10 nM aldosterone can induce differentiation of 3T3-L1 cells into adipose cells as evaluated by microscopic accumulation of fat droplets and quantitative measurement of triglycerides and of glycerol-3-phosphate dehydrogenase, an enzyme specific for adipocyte differentiation. Moreover, the aldosterone antagonist ZK91587 inhibits aldosterone-but not glucocorticoid-mediated differentiation of 3T3-L1 cells. Steroid binding assays with 3T3-L1 cells indicate the presence of specific binding sites for aldosterone. We conclude that there is an aldosterone receptor-mediated pathway for terminal differentiation of 3T3-L1 cells into adipose cells. Receptors for aldosterone have also been found in a variety of cells that do not function to regulate sodium and potassium transport. The aldosterone receptor may have a role in regulation expression of genes involved in differentiation of these cells.

Identification and characterization of two classes of receptors for oxytocin and vasopressin in porcine tunica albuginea, epididymis, and vas deferens.

The neurohypophysial hormones oxytocin (OT) and vasopressin (VP) are involved in the regulation of the contractility of the male genital tract in several animal species. We investigated the presence of specific binding sites for [3H]OT and [3H]arginine VP (AVP) in membranes prepared from tunica albuginea, epididymis, and vas deferens from prepubertal pigs 2-16 weeks of age. Membranes were incubated with [3H]OT and [3H]AVP in the presence or absence of the corresponding unlabeled peptides. Binding equilibrium was reached in 60 min at 22 C. Millimolar concentrations of Mg2+ increased the specific binding of both ligands. Analysis of families of self- and cross-displacement curves using the computer program LIGAND clearly demonstrated that two classes of binding sites were present in all tissues investigated. The first class of sites, designated the OT site, shows high affinity for OT, AVP, lysine vasopressin, arginine vasotocin, the selective OT agonists [Thr4,Gly7]OT and [Asu1,6]OT, and the OT antagonists derived from ornithine vasotocin (OVT), namely d(CH2)5Tyr(Et)OVT and dEt2OVT. The second class of sites, designated the VP site, shows high affinity for AVP, lysine vasopressin, arginine vasotocin, and the selective V1 antagonist d(CH2)5Tyr(Me)AVP. The V2 agonist [1-deamino,4-valine]8-D-AVP shows low affinity for both sites. Isotocin, desglycinamide [Arg-8]AVP and tocinoic acid were ineffective in displacing [3H]AVP or [3H]OT. The highest density of OT receptors was found in tunica albuginea and epididymis, whereas the highest density of AVP receptors was found in vas deferens. Adenylate cyclase was not activated in any of the tissues studied by concentrations of AVP or OT up to 100-fold greater than their Kd values. This is the first demonstration and pharmacological characterization of specific OT and V1 VP receptors in the tunica albuginea, epididymis, and vas deferens. The recent demonstration of high local concentration of neurohypophysial hormones in the gonads of several mammals support a physiological role of these OT and VP receptors in regulation of the motility of the male genital tract.

Characterization of somatogenic and lactogenic binding sites in isolated rat hepatocytes.

Suspensions of rat hepatocytes isolated enzymatically by the method of Berry and Friend were used to study the binding of 125I-labeled human (hGH) and bovine (bGH) growth hormones and ovine prolactin (oPRL). Displacement of these labeled hormones by their unlabeled analogues was analyzed by means of Scatchard plots and affinity constants (K) and the number of binding sites per cell (q) were calculated. Specificity of binding was studied using hGH, bGH oPRL and rat growth hormone (rGH) and rat prolactin (rPRL). Rat hepatocytes contained two types of binding sites which bound hGH. The first, somatogenic, was specific for the growth-promoting hormones bGH and rGH. The second, lactogenic, was specific for lactogenic hormones, oPRL and rPRL. Human GH, which has both lactogenic and growth-promoting properties in rodents, bound to both sites. The somatogenic binding sites were present in both males and females, and the number of sites was similar in females and in males and was not affected by hypophysectomy. The lactogenic binding sites were present only in females, and the number of lactogenic and somatogenic sites was similar (40,000/cell). The affinity of hGH for the lactogenic binding sites was less than for the somatogenic (0.37 X 10(9) vs. 1 X 10(9)M-1). The lactogenic binding sites were lost when female rats were hypophysectomized and could not be restored by estrogen treatment.

Vasopressin and oxytocin receptors in vagina, myometrium, and oviduct of rabbits.

In view of the presence of distinct oxytocin (OT) and vasopressin (VP) receptors in the male genital tract (porcine) we have reexamined the receptors for OT and AVP in the classical OT target tissue, female genital tract (rabbit). Neurohypophysial hormone receptors have been investigated in vagina, myometrium, and oviduct using quantitative ligand binding, adenylate cyclase, and contractility studies. Our results clearly indicate the presence of distinct OT and V1 VP receptors in the myometrium, while only the latter was detected in vagina and oviduct. In myometrium, estrogen treatment increases the density of OT and AVP receptors, while progesterone administration inhibits the estrogen effect. At the time of spontaneous delivery a dramatic (17-fold) increase was observed for the OT sites, while the AVP sites were unchanged. AVP receptors in vagina were sensitive to sex steroid administration and were reduced during pregnancy and delivery. Isometric contractility studies suggest that not just OT, but AVP can stimulate uterine strips, an effect that is partially reversible by the V1 antagonist d(CH2)5TyrMeAVP. In vagina only AVP is effective in inducing contractions at nanomolar concentrations. These results suggest a role for AVP as well as OT in regulation of the motility of female genital tract.

Radioiodinated nondegradable gonadotropin-releasing hormone analogs: new probes for the investigation of pituitary gonadotropin-releasing hormone receptors.

Studies of pituitary plasma membrane gonadotropin-releasing hormone (GnRH) receptors using [125I]-iodo-GnRH suffer major disadvantages. Only a small (less than 25%) proportion of specific tracer binding is to high affinity sites, with more than 70% bound to low affinity sites (Ka = 1 x 10(6) M-1). [125I]Iodo-GnRH is also inactivated during incubation with pituitary plasma membrane preparations. Two superactive analongs of GnRH, substituted in positions 6 and 10, were used as the labeled ligand to overcome these problems. Both analogs bound to the same high affinity sites as GnRH on bovine pituitary plasma membranes, though the affinity of the analogs was higher than that of the natural decapeptide (Ka = 2.0 x 10(9), 6.0 x 10(9), and 3.0 x 10(8) M-1 for [D-Ser(TBu)6]des-Gly10-GnRH ethylamide, [D-Ala6]des-Gly10-GnRH ethylamide, and GnRH, respectively. The labeled analogs bound to a single class of high affinity sites with less than 15% of the specific binding being to low affinity sites (Ka approximately equal to 1 x 10(6) M-1). The labeled analogs were not inactivated during incubation with the pituitary membrane preparations. Using the analogs as tracer, a single class of high affinity sites (K1 = 4.0 x 10(9) M-1) was also demonstrated on crude 10,800 x g rat pituitary membrane preparations. Use of these analogs as both the labeled and unlabeled ligand offers substantial advantages over GnRH for investigation of GnRH receptors, allowing accurate determination of changes in their numbers and affinities under various physiological conditions.

Transport of steroid hormones: binding of 21 endogenous steroids to both testosterone-binding globulin and corticosteroid-binding globulin in human plasma.

This report describes a model of steroid transport in human plasma. The binding affinities of 21 endogenous steroids for both testosterone-binding globulin (TeBG) and corticosteroid-binding globulin (CBG) were determined under equilibrium conditions using a solid phase method at physiological pH and temperature. A computer program was used to solve the complex equilibrium interactions between these steroids and TeBG, CBG, and albumin. In this manner, we calculated the plasma distribution of each steroid into TeBG-bound, CBG-bound, albumin-bound, and unbound fractions in normal men, normal women during both the follicular and luteal phases of the ovarian cycle, and women during the third trimester of a normal pregnancy.

Improved diagnosis and management of hyper- and hypothyroidism by timing the arterial sounds.

"Sphygmo-Recording," a non-invasive method for timing the arterial pulse wave contour provides an objective measure of responses to medication in patients with hyper- and hypothyroidism. The QKd interval, i.e., the interval from the onset of the QRS complex (Q) to the onset of the Korotkoff sounds (K) at the brachial artery when the sphygmomanometer cuff is at diastolic pressure (d) is the QKd interval. QKd is normally 205 +/- 12 msec. In the hyperthyroidism the QKd interval may be shortened to 110 msec. In hypothyroidism the QKd interval may be prolonged to 320 msec. Changes in QKd parallel changes in clinical status and serum total T4 and T3, measured by radioimmunoassay. QKd can be used as an objective guide to antithyroid therapy in hyperthyroidism and replacement therapy with thyroid hormone in hypothyroid individuals.

Peripheral responses to thyroid hormone before and after L-thyroxine therapy in patients with subclinical hypothyroidism.

Twenty patients with serum levels of T4 and T3 within the normal range but with elevated serum concentrations of TSH were evaluated before and after treatment with L-T4. This therapy increased serum T4 (5.5 +/- 1.1 to 8.8 +/- 1.8 microgram/dl) and T3 (116 +/- 20 to 137 +/- 28 ng/dl) levels. Cardiac systolic time intervals (STI) were significantly (P less than 0.01) reduced by this therapy. The preejection period (123 +/- 18 to 114 +/- 14 msec; n = 12), the change in preejection period (+17 +/- 17 to +6 +/- 15 msec; n = 12), the ratio of preejection period to left ventricular ejection time (0.412 +/- 0.068 to 0.357 +/- 0.063 msec; n = 12), and the interval from the Q wave of the electrocardiogram to the pulse wave arrival time at the brachial artery (224 +/- 10 to 200 +/- 13 msec; n = 10) were consistently reduced. Cardiac STI were significantly correlated with serum TSH and T4 levels, but not with serum T3 levels. Normalization of serum TSH levels was associated with changes in QKd measurements even in those patients with minimal elevations in serum TSH. These studies demonstrate that patients having the combination of elevated TSH but T4 and T3 levels in the normal range have alterations in STI which can be changed significantly by L-T4 in doses which normalize TSH secretion. These data suggest that such patients have a mild form of primary hypothyroidism.

Naloxone administration does not affect gonadotropin secretion in agonadal men either basally or during testosterone treatment.

Naloxone administration has no effect on plasma gonadotropin levels of agonadal men. The present study was designed to evaluate whether testosterone replacement therapy could restore LH responsiveness to naloxone in such men. We measured plasma LH and FSH levels at 15-min intervals during naloxone infusion (8 mg in 1 min followed by 12 mg in 3 h) and for the following 3 h in a group of agonadal men both before and after at least 2 months of three different schedules of testosterone replacement therapy: 1) testosterone undecanoate, 40 mg three times a day by mouth; 2) testosterone enanthate 200 mg im every 2 weeks; and 3) testosterone enanthate 100 mg im once a week. Mean plasma gonadotropin levels as well as LH pulse frequency did not vary during naloxone infusion vs. placebo either basally or during each testosterone regimen. These results suggest that long term testosterone therapy does not affect the altered opioid modulation of gonadotropin secretion which is present in agonadal men.

Adrenal steroid responses to continuous intravenous adrenocorticotropin infusion compared to bolus injection in normal volunteers.

We compared the adrenal steroid responses after synthetic ACTH-(1-24) (Cosyntropin) administration given by either continuous iv infusion or bolus injection in 11 normal women and 6 normal men. Each subject received 250 micrograms Cosyntropin as a bolus iv injection on 1 occasion and as a continuous 2-h iv infusion on another occasion, in random order. There was a 1-week interval between the studies. We measured the plasma levels of cortisol, 11-deoxycortisol, 17-hydroxyprogesterone, progesterone, pregnenolone, 17-hydroxypregnenolone, dehydroepiandrosterone, dehydroepiandrosterone sulfate, delta 5-androstenediol, androstenedione, and testosterone by RIA 15 and 0 min before and 30, 45, 60, and 120 min after administering ACTH. The steroid concentrations and their increments, ratios, or areas above baseline did not differ significantly between the bolus injection and the continuous infusion. Thus, at the dose of 250 micrograms, a bolus ACTH injection stimulates adrenal steroid secretion as effectively as a 2-h continuous ACTH infusion.