Quantitative estimation of beta cell sensitivity to glucose in the intact organism: a minimal model of insulin kinetics in the dog.

We propose an approach to quantifying the sensitivity of B cells to glucose in the intact organism, whereby we interpret the complex dynamic plasma insulin response to glucose injection in terms of a minimal mathematical model of posthepatic insulin delivery and insulin clearance. The best model for this purpose was chosen by comparing the ability of a series of proposed models to account precisely for plasma insulin dynamics. Intravenous glucose tolerance tests (IVGTT) (300 mg/kg) were performed on conscious dogs, and blood was sampled frequently until the basal steady state was reestablished. Glucose injection produced variable plasma insulin responses, which were characterized by an early peak (76 microU/ml above basal), a plateau with occasional additional peaks, and by an abrupt return of plasma insulin to basal by 37 min. A set of eight models was examined; one emerged as superior, in that it was able to account for insulin dynamics with the smallest number of physiologically meaningful parameters (N = 4). The chosen (minimal) model assumes that (1) clearance of insulin is of the first order, (2) the initial peak represents a bolus of insulin loaded into the plasma after the glucose injection, and (3) the rate of the secondary rise in insulin is determined by the concentration of glucose in plasma above a specific threshold value. The sensitivity of first phase insulin delivery to glucose (phi 1; 1.28 +/- 0.15 microU/ml per min per mg/dl), the sensitivity of the secondary phase to glucose concentration [phi 2; 0.038 +/- 0.005 (microU/mg) . min-2], and the threshold for glucose stimulation of second phase secretion (h; 125 +/- 8 mg/100 ml) were all precisely estimated from the dynamic insulin responses. These three parameters of insulin kinetics (phi 1, phi 2, and h) can be calculated from a single IVGTT, and they characterize the insulin responsiveness of a single individual. Estimating these characteristic parameters of insulin kinetics from IVGTT data has potential for quantitating the individual factors contributing to glucose-stimulated insulin secretion in intact animal models, and it may be applicable to man.

Inhibition of peripheral aromatization in the male cynomolgus monkey by a novel nonsteroidal aromatase inhibitor (R 76713).

R 76713 (6-[(4-chlorophenyl)(1-H-1,2,4-trizol-1-yl)methyl]1-H benzotriazole) is a highly potent and selective inhibitor of the aromatase enzyme both in vitro and in vivo. The ability of R 76713 to inhibit peripheral aromatization of androstenedione (A) to estrone (E1) in vivo was studied in male cynomolgus monkeys (Macaca fascicularis). Peripheral aromatization was measured using a primed constant infusion of [3H] A and [14C]E1 for 3.5 h. Blood samples, collected during the final hour of infusion, were analyzed for plasma radioactivity as infused and product steroids. MCRs, conversion ratios (CR), and percent conversion of A to E1 were calculated. R 76713 (0.03-10 microgram/kg) or vehicle (10% hydroxypropyl-beta-cyclodextrin) were administered iv 90 min before beginning the infusion of radiolabeled steroids. In vehicle-treated monkeys, the aromatization of A (mean +/- SEM, 1.35 +/- 0.11%) was similar to that previously reported for cynomolgus and rhesus monkeys, baboons, and humans. Aromatization of A, measured 4-5 h after injection of R 76713, was dose-dependently decreased from the control value by 87 +/- 3%, 85 +/- 2%, 61 +/- 5%, and 33 +/- 8% (all P less than 0.05) at doses of 10.0, 3.0, 0.3, and 0.03 micrograms/kg, respectively, with an ID50 of 0.13 microgram/kg, iv (95% confidence interval, 0.06-0.21). When measured 15-16 h after iv administration of 3.0 micrograms/kg R 76713, aromatization (0.55 +/- 0.13%) was significantly inhibited by 53 +/- 11% compared to that in control monkeys (1.16 +/- 0.18%). The CRs between androgens, the CRs between estrogens, and the MCRs of A and E1 were not significantly altered by R 76713 compared to those after vehicle treatment. R 76713 potently decreased peripheral conversion of androgen to estrogen in vivo in male cynomolgus monkeys and may be a useful therapeutic agent in treating estrogen-dependent diseases, including post-menopausal breast cancer.

Synthesis, characterization, and anorectic testing of the four stereoisomers of cyclo(histidylproline).

All four possible stereoisomers of cyclo(histidylproline) were individually synthesized, purified, and characterized. They were each tested for anorectic activity in rats with a free feeding paradigm over 24 h. Contrary to literature reports, none significantly reduced food intake at any time over the test period.

A novel series of N-(1-aminoalkylidene)carboximidamides as potential hypoglycemic agents.

Nitrogen heterocyclic carboximidamides, such as linogliride, 1a, have been shown to possess significant hypoglycemic activity and have shown clinical efficacy as potential antidiabetic agents. We evaluated the biological significance of the heterocyclic ring A of general structure 1, which has always been maintained in this class of compounds, by preparing acyclic compounds of general structure 2. Preliminary in vivo biological testing, i.e., the glucose tolerance test in rats, indicates that a number of the specific acyclic carboximidamides prepared, 6a-kk, possessed significant hypoglycemic activity often comparable to, and in some cases better than, the activity noted for our model compound, 1a. These results suggest that the heterocyclic ring A of 1 is not essential for hypoglycemic activity for this class of compounds.

Carbohydrate biguanides as potential hypoglycemic agents.

A series of monosaccharides containing a biguanide functionality was prepared and evaluated for hypoglycemic activity. Among the analogues prepared were those involving D-glucose substituted on the 6- or 1-position (19 and 24), D-galactose substituted on the 6-position (7), and D-arabinose (31). The target compounds were evaluated in a modified rat glucose-tolerance test (oral glucose load/oral drug, 100 mg/kg). Compounds 8 [6-biguanidino-1,2:3,5-bis-O-(1-methylethylidene)-6-deoxy-al pha-D- glucofuranose] and 23 [methyl 6-biguanidino-6-deoxy-2,3,4-O-tribenzyl-alpha-D-glucopyra nos ide] were the most active, exhibiting nearly equivalent hypoglycemic activity to that of phenformin (1) and metformin (2), as measured by the inhibition of the rise of blood glucose. Compound 31 was somewhat less active with 26% inhibition, as compared to 64% inhibition with 1 and 41% inhibition with 2.

New non-steroidal aromatase inhibitors: focus on R76713.

R76713 is a novel triazole derivative which selectively blocks the cytochrome P450-dependent aromatase. In human placental microsomes, in FSH-stimulated rat and human granulosa cells and in human adipose stromal cells, 50% inhibition of estradiol biosynthesis was obtained at drug concentrations of 2-10 nM. In PMSG-injected female rats, R76713 lowered plasma estradiol levels by 50 and 90% 2 h after single oral doses of 0.005 and 0.05 mg/kg respectively. After 1 mg/kg, estradiol levels were suppressed by 90% for 16 h. In male cynomolgus monkeys, R76713 dose-dependently (0.03-10 micrograms/kg) inhibited peripheral aromatization with an ED50 of 0.13 microgram/kg without altering metabolic clearance rates and conversion ratios. In vitro R76713 had no effect on other P450-dependent steroidogenic enzymes up to 1000 nM at least. In rats, LHRH-, ACTH- and sodium-deprived diet stimulated plasma testosterone, corticosterone and aldosterone levels were not modified 2 h after single oral administrations of R76713 (up to 20 mg/kg). Furthermore, R76713 did not show any in vitro or in vivo estrogenic or antiestrogenic property. R76713 also induced regression of DMBA-induced mammary tumors after daily oral administration of 1 mg/kg b.i.d. In male volunteers (n = 4), a single oral dose of 5 and 10 mg lowered median plasma estradiol levels from 70 pM to the detection limit of the assay (40 pM) 4, 8 and 24 h after intake whereas no changes were detected after placebo administration. In premenopausal women (n = 15), receiving a single oral dose of 20 mg, median plasma estradiol levels decreased from 389 pM (before) to 168, 133 and 147 pM, 4, 8 and 24 h after intake whereas they remained above 420 pM after placebo (n = 7).

Caval occlusion technique for hepatic venous sampling: a new approach to estimating splanchnic substrate balance in conscious dogs.

We have proposed a new technique for sampling hepatic venous blood in conscious dogs. Sub-hepatic vena caval blood flow was temporarily occluded by a previously implanted inflatable snare so that all blood entering the inferior vena cava was hepatic venous effluent. Hepatic venous blood samples were collected from the inferior vena cava 8 seconds after beginning caval occlusion, with the total interval of flow occlusion lasting 12 to 15 seconds. No behavioral or metabolic alterations were observed when or metabolic alterations were observed when hepatic venous effluent was repetitively sampled using the caval occlusion technique. Net splanchnic glucose balance (NSGB) was measured in conscious dogs receiving saline, glucose or glucagon infusions. NSGB measurements made with the caval occlusion technique were in accord with previous results obtained via tracer methodology or arterio-venous difference techniques utilizing hepatic vein catheterization. The caval occlusion technique thus provides a method for collecting hepatic venous blood samples from conscious animals without the difficulties associated with hepatic vein catheterization.

Highly purified human growth hormone fails to stimulate lipolysis in rabbit adipocytes in vitro or in rabbits in vivo.

To determine whether the rapid lipolytic effect observed with human growth hormone (hGH) preparations in rabbits and rabbit adipose tissue is an intrinsic property of the hormone, we examined the lipolytic effects in vivo and in vitro of clinical grade preparations, hGH purified by DEAE cellulose chromatography, and hGH prepared by recombinant DNA techniques. Using isolated rabbit perirenal adipocytes, ACTH and clinical-grade hGH preparations both stimulated glycerol release to the same maximal rate with half-maximal hGH effects observed between 8 and 50 micrograms/mL. Purification of the most potent lipolytic preparation of clinical grade hGH by DEAE cellulose chromatography yielded a preparation (2 IU/mg of growth activity that retained insulinlike effects on rat fat pad [U-14C] glucose metabolism) that, at concentrations up to 0.2 mg/mL, failed to stimulate lipolysis by adipocytes incubated for one or four hours in the presence or absence of dexamethasone or trypsin inhibitor or when preincubated for three hours prior to addition of hGH. Recombinant DNA-derived hGH did not stimulate glycerol release at 0.1 mg/mL. While antiserum to purified hGH blocked the increase in glucose oxidation in rat fat pads produced by clinical grade hGH, it did not inhibit its lipolytic effect using rabbit adipocytes. Purified hGH (0.1 mg/kg IV) was also unable to elicit a rise in serum free fatty acid (FFA) levels of conscious rabbits while, at the same dose, clinical grade hGH increased FFA levels to 900 microEq/L over basal. Rapid lipolytic stimulation in rabbit adipocytes by hGH preparations could not be attributed to the 20,000 molecular weight variant of hGH (hGH20K) or the peptide corresponding to positions 32 to 46 of hGH (deletion peptide).(ABSTRACT TRUNCATED AT 250 WORDS)

Re-evaluation of histidyl-proline diketopiperazine [cyclo(His-Pro)] effects on food intake in the rat.

Histidyl-proline diketopiperazine [cyclo(His-Pro)], a metabolite of thyrotropin releasing hormone (TRH), has been reported to decrease food intake of rats in a variety of feeding models following intracerebroventricular (ICV) injection. We have re-evaluated the anorectic effects of cyclo(His-Pro) on food deprivation-induced and spontaneous feeding. When injected ICV at the end of the light period into ad lib fed rats, neither the naturally occurring cyclo(L-His-L-Pro) isomer (14 to 1000 nmole/rat) nor any of the four cyclo(D,L-His-D,L-Pro) stereoisomers (100 nmole/rat) significantly suppressed food intake at any hour for up to 12- or 24-hr post-injection. Bombesin (0.6 nmole/rat ICV) decreased food intake in the same model by up to 86% with anorexia still apparent 15-hr post-injection (71%, p less than 0.001). In two food deprivation-induced feeding models, cyclo(L-His-L-Pro) (100 and 1000 nmole/rat ICV) did not cause anorexia while TRH (10 and 1000 nmole/rat ICV) maximally suppressed food intake by 74% (p less than 0.02) and 50% (p less than 0.01). Occasional transient increases of food consumption were observed in cyclo(His-Pro)-treated rats during both spontaneous and induced feeding. Cyclo(L-His-L-Pro) was also without effect on food intake when intraperitoneally administered at 12.5 and 30 mumole/kg to schedule fed rats. TRH at 30 mumole/kg IP transiently suppressed food intake of schedule fed rats (p less than 0.005). These findings indicate that cyclo(His-Pro) does not exhibit anorectic activity in the rat and cast doubt on the concept that TRH-induced anorexia results from conversion of TRH to an active cyclo(His-Pro) metabolite.

Effects of nebivolol, atenolol and propranolol on in vivo cardiovascular and metabolic responses to isoproterenol in dogs.

Pentobarbital-anesthetized dogs were administered either dl-propranolol, atenolol or dl-nebivolol at cumulative doses of 0 (vehicle), 0.0025, 0.01, 0.04, 0.16 and 0.64 mg/kg i.v. After each dose, heart rate and diastolic blood pressure responses to isoproterenol (0.125 micrograms/kg/min i.v. for 5 min), as well as plasma glucose, insulin, lactate and free fatty acid responses, were measured. Heart rate and free fatty acid changes were taken as beta-1 adrenergic indices, with the other parameters taken as beta-2 adrenergic indices. The antagonist dose estimated to cause 50% inhibition of each isoproterenol response (ID50) was calculated. Nebivolol and atenolol had nearly identical cardiovascular profiles, which were much more beta-1 selective than that of propranolol [heart rate and blood pressure ID50 values, mg/kg: 0.034, 0.036 (propranolol); 0.058, 0.713 (nebivolol); 0.047, 0.506 (atenolol)]. Propranolol also potently inhibited isoproterenol-stimulated glucose, insulin and lactate increases (ID50S: 0.020, 0.078 and 0.007 mg/kg, respectively). Nebivolol and atenolol were much weaker inhibitors of these metabolic responses than propranolol (5-fold-35-fold and 8-fold-greater than 90-fold, respectively). Insulin responses were equivalently inhibited by both nebivolol and atenolol (ID50S greater than 0.4 mg/kg), whereas glucose and lactate ID50S for nebivolol were 0.183 and 0.243 mg/kg, respectively, with atenolol ID50S greater than 0.64 mg/kg. Free fatty acid responses were attenuated by all three antagonists with ID50 values of 0.103, 0.100 and 0.028 mg/kg for propranolol, nebivolol and atenolol, respectively. These in vivo studies demonstrate that dl-nebivolol significantly inhibited the beta-1 cardiac response at doses which did not produce either beta-2 cardiovascular or metabolic effects.

Cause of glucose oscillations during glucose infusion: periodic variation in glucose uptake.

Constant infusion of glucose (10 mg . kg-1 . min-1) into conscious, intact dogs induced oscillations in the plasma concentrations of glucose and insulin. Glucose rose from basal (98 +/- 1 mg/dl) and, after 3 h, entered oscillations that persisted until the end of the 9-h glucose infusion. Between 240 and 540 min, glucose fluctuated by +/- 17 mg/dl about a mean value of 143 +/- 2 mg/dl; frequency of the glucose oscillation was 0.54 +/- 0.03 cycles/h. During the same time interval, insulin increased from basal 13 +/- 2 mu U/ml to mean 46 +/- 4 mu U/ml. Insulin oscillated at an amplitude (peak-to-peak) of 48 mu U/ml, with frequency not different from that of glucose (0.60 +/- 0.09 cycles/h). The oscillation in glucose "led" the insulin oscillation by 22 +/- 5 min. In three animals, [2-3H]glucose was infused along with unlabeled glucose during oscillations, and it was determined that almost all (98%) of the glucose appearance was from the exogenous infusion. Thus varying endogenous glucose production was ruled out as a contributory factor to the glucose oscillation. Total glucose uptake (Rd) fluctuated periodically at the same frequency as glucose and insulin (0.56 +/- 0.05 cycles/h) and with a large amplitude (Rd mean = 248 mg/min; peak-to-peak amplitude = 85 mg/min). Direct splanchnic balance measurements were made in the interval 240-540 min to determine the specific contributions of splanchnic (Rds) and peripheral glucose uptake (Rdp) to the oscillation in total Rd. Peripheral uptake oscillated in phase with plasma insulin and accounted for 80% (57.5 g) of total Rd. The splanchnic bed (presumably liver) sequestered 20% (14.1 g) of infused glucose, and Rds varied in phase with plasma glucose. The liver extracted 5.6 +/- 0.3% of the total amount of glucose presented to it in the 5-h interval of observation. It is concluded that a) large fluctuations in peripheral glucose utilization are responsible for the observed periodicities in glucose concentration; b) peripheral uptake fluctuations result from periodic bursts in insulin secretion; c) during glucose infusion, glucose is the primary moment-to-moment regulator of hepatic glucose uptake, whereas insulin is the principal regulator of peripheral glucose utilization.

Quantitative estimation of insulin sensitivity.

We have evaluated the feasibility of using a mathematical model of glucose disappearance to estimate insulin sensitivity. Glucose was injected into conscious dogs at 100, 200, or 300 mg/kg. The measured time course of insulin was regarded as the "input," and the falling glucose concentration as the "output" of the physiological system storing and using glucose. Seven mathematical models of glucose uptake were compared to identify the representation most capable of simulating glucose disappearance. One specific nonlinear model was superior in that it 1) predicted the time course of glucose after glucose injection, 2) had four parameters that could be precisely estimated, and 3) described individual experiments with similar parameter values. Insulin sensitivity index (SI), defined as the dependence of fractional glucose disappearance on plasma insulin, was the ratio of two parameters of the chosen model and could be estimated with good reproducibility from the 300 mg/kg injection experiments (SI = 7.00 X 10(-4) +/- 24% (coefficient of variation) min-1/(microU/ml) (n = 8)). Thus, from a single glucose injection it is possible to obtain a quantitative index of insulin sensitivity that may have clinical applicability.

Fuel partitioning and food intake: role for mitochondrial fatty acid transport.

Administration of methyl palmoxirate (MP; 10 mg/kg po), an inhibitor of carnitine palmitoyltransferase I (CPT I), increased the food intake of rats maintained on a diet high in triglycerides comprised of long-chain fatty acids, which require CPT I for mitochondrial uptake and oxidation. MP did not affect food intake in rats fed a comparable diet high in medium-chain fatty acids, which do not require CPT I for mitochondrial uptake and oxidation. The feeding response to MP was reduced more effectively by an intragastric preload of medium-chain triglyceride (MCT) oil than a preload of a long-chain triglyceride (LCT) oil. Food intake of MCT- and LCT-fed rats differed under control conditions (no MP), and this appeared to reflect differences in the diurnal distribution of feeding. Measurement of plasma ketone body concentrations indicated that the dietary manipulations and MP had their intended metabolic effects. The results strongly suggest that mitochondrial transport of fatty acids plays a role in the control of food intake. CPT I participates in that control by regulating the partitioning of long-chain fatty acids between pathways of storage and intramitochondrial oxidation.

Stimulation by risperidone of rat prolactin secretion in vivo and in cultured pituitary cells in vitro.

Risperidone, a new antipsychotic agent which antagonizes both 5-hydroxytryptamine-2 (5-HT2) and dopamine-2 (D2) receptors, was evaluated for its effects on prolactin release. One hr after either p.o. or i.p. dosing, risperidone was 3 to 5 times more potent than the classical D2 receptor antagonist haloperidol in stimulating rat prolactin levels in vivo. This result was unexpected because haloperidol is a more potent D2 receptor antagonist than risperidone in vitro. Mechanisms which might explain these observations were investigated. Compared to haloperidol, risperidone was 0.34 (95% confidence interval: 0.23, 0.48) times as potent in reversing the suppression by dopamine of rat anterior pituitary cell prolactin release in vitro, which is consistent with the compounds' striatal D2 receptor binding potencies in vitro. Administration of ketanserin, a 5-HT2 receptor antagonist, along with haloperidol did not modify haloperidol's activity on either in vitro pituitary cell prolactin release (up to 1000 nM ketanserin) or in vivo prolactin concentrations (5 mg/kg ketanserin, i.p.). In vitro incubation of haloperidol and risperidone with a liver homogenate supernatant (S-9) led to extensive (greater than 86%) metabolism of each compound. S-9 treatment abolished haloperidol's effects on pituitary cell prolactin release. Risperidone's ability to increase prolactin release was unchanged after S-9 treatment, and 9-hydroxy-risperidone, identified as a major metabolite in the S-9 conditioned media, was equipotent to risperidone in modulating prolactin release in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of the fatty acid oxidation inhibitor methyl palmoxirate (methyl 2-tetradecylglycidate) on recovery from insulin-induced hypoglycemia in diabetic dogs.

Methyl palmoxirate, an effective hypoglycemic agent administered p.o., has been shown to decrease hepatic glucose production secondary to inhibition of mitochondrial fatty acid oxidation. Because the ability to increase hepatic glucose production is an important counter-regulatory defense against hypoglycemia, we compared the ability of streptozotocin/alloxan-induced diabetic dogs treated p.o. with vehicle or methyl palmoxirate (2.5 mg/kg/day X 7 days) to recover from insulin-induced hypoglycemia. Hepatic glucose production and glucose utilization were determined by isotope dilution before and after acute reduction of plasma glucose by i.v. insulin injection (0.10 or 0.13 U/kg). Diabetic dogs treated with methyl palmoxirate for 6 days had lower overnight fasting plasma glucose levels than vehicle-treated animals (158 +/- 7 vs. 171 +/- 11, respectively, P less than .05). Plasma glucose at 4 hr after the last dose of drug decreased to 115 +/- 5 mg/dl, whereas glucose in the vehicle-treated dogs was unchanged (172 +/- 8 mg/dl). Recovery from insulin-induced hypoglycemia (nadirs of 58 +/- 5 and 42 +/- 4 mg/dl in the vehicle- and methyl palmoxirate-treated groups, respectively) was not significantly different between the two groups of dogs. Restoration of plasma glucose was primarily due to increased hepatic glucose production in both treatment groups, as glucose utilization did not fall significantly below baseline levels. Plasma glucagon levels increased in both vehicle- and methyl palmoxirate-treated dogs in response to hypoglycemia, indicating that release of an important counter-regulatory hormone was not compromised by drug treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Aromatase inhibition by R 76713: experimental and clinical pharmacology.

R 76713 is a new non-steroidal compound which inhibits aromatase in vitro and in vivo with a potency of at least 1000-fold that of aminoglutethimide. In male cynomolgus monkeys peripheral conversion of labeled androstenedione to estrone is decreased by 85%, 4-5 h after a single intravenous dose of 0.003 mg/kg of R 76713, without altering steroid metabolic clearance rates. In rats fed a sodium-depleted diet for 3 weeks, plasma levels of aldosterone and plasma renin activity remain unchanged 2 h after a single oral dose of up to 20 mg/kg of R 76713. This confirms previous data on the selectivity of R 76713 for aromatase inhibition as compared to inhibition of other enzymes involved in steroid biosynthesis. In male volunteers, a single oral dose of 5 or 10 mg of R 76713 lowers median plasma estradiol levels from 70 pM to the detection limit of the assay (30 pM) 4 and 8 h after intake, whereas no important changes are detected after placebo administration. In 15 premenopausal female volunteers receiving a single oral dose of 20 mg of R 76713, mean plasma estradiol levels decrease from 415 pM (before) to 179, 149 and 185 pM respectively 4, 8 and 24 h after intake whereas they remain above 380 pM after placebo (n = 7).