A novel long-acting glucose-dependent insulinotropic peptide analogue: enhanced efficacy in normal and diabetic rodents.

AIM: Glucose-dependent insulinotropic peptide (GIP) is an incretin hormone that is released from intestinal K cells in response to nutrient ingestion. We aimed to investigate the therapeutic potential of the novel N- and C-terminally modified GIP analogue AC163794. METHODS: AC163794 was synthesized by solid-phase peptide synthesis. Design involved the substitution of the C-terminus tail region of the dipeptidyl peptidase IV (DPP-IV)-resistant GIP analogue [d-Ala(2) ]GIP(1-42) with the unique nine amino acid tail region of exenatide. The functional activity and binding of AC163794 to the GIP receptor were evaluated in RIN-m5F ß-cells. In vitro metabolic stability was tested in human plasma and kidney membrane preparations. Acute insulinotropic effects were investigated in isolated mouse islets and during an intravenous glucose tolerance test in normal and diabetic Zucker fatty diabetic (ZDF) rats. The biological actions of AC163794 were comprehensively assessed in normal, ob/ob and high-fat-fed streptozotocin (STZ)-induced diabetic mice. Acute glucoregulatory effects of AC163794 were tested in diet-induced obese mice treated subchronically with AC3174, the exendatide analogue [Leu(14) ] exenatide. Human GIP or [d-Ala(2) ]GIP(1-42) were used for comparison. RESULTS: AC163794 exhibited nanomolar functional GIP receptor potency in vitro similar to GIP and [d-Ala(2) ]GIP(1-42). AC163794 was metabolically more stable in vitro and displayed longer duration of insulinotropic action in vivo versus GIP and [d-Ala(2) ]GIP(1-42). In diabetic mice, AC163794 improved HbA1c through enhanced insulinotropic action, partial restoration of pancreatic insulin content and improved insulin sensitivity with no adverse effects on fat storage and metabolism. AC163794 provided additional baseline glucose-lowering when injected to mice treated with AC3174. CONCLUSIONS: These studies support the potential use of a novel GIP analogue AC163794 for the treatment of type 2 diabetes.

GLUT4 overexpression in db/db mice dose-dependently ameliorates diabetes but is not a lifelong cure.

We previously reported that overexpression of GLUT4 in lean, nondiabetic C57BL/KsJ-lepr(db/+) (db/+) mice resulted in improved glucose tolerance associated with increased basal and insulin-stimulated glucose transport in isolated skeletal muscle. We used the diabetic (db/db) litter mates of these mice to examine the effects of GLUT4 overexpression on in vivo glucose utilization and on in vitro glucose transport and GLUT4 translocation in diabetic mice. We examined in vivo glucose disposal by oral glucose challenge and hyperinsulinemic-hyperglycemic clamps. We also evaluated the in vitro relationship between glucose transport activity and cell surface GLUT4 levels as assessed by photolabeling with the membrane-impermeant reagent 2-N-(4-(1-azi-2,2,2-trifluoroethyl)benzoyl)-1,3-bis(D-mannose-4-yloxy)-2-propylamine in extensor digitorum longus (EDL) muscles. All parameters were examined as functions of animal age and the level of GLUT4 overexpression. In young mice (age 10-12 weeks), both lower (two- to threefold) and higher (four- to fivefold) levels of GLUT4 overexpression were associated with improved glucose tolerance compared to age-matched nontransgenic (NTG) mice. However, glucose tolerance deteriorated with age in db/db mice, although less rapidly in transgenic mice expressing the higher level of GLUT4. Glucose infusion rates during hyperinsulinemic-hyperglycemic clamps were increased with GLUT4 overexpression, compared with NTG mice in both lower and higher levels of GLUT4 overexpression, even in the older mice. Surprisingly, isolated EDL muscles from diabetic db/db mice did not exhibit alterations in either basal or insulin-stimulated glucose transport activity or cell surface GLUT4 compared to nondiabetic db/+ mice. Furthermore, both GLUT4 overexpression levels and animal age are associated with increased basal and insulin-stimulated glucose transport activities and cell surface GLUT4. However, the observed increased glucose transport activity in older db/db mice was not accompanied by an equivalent increase in cell surface GLUT4 compared to younger animals. Thus, although in vivo glucose tolerance is improved with GLUT4 overexpression in young animals, it deteriorates with age; in contrast, insulin responsiveness as assessed by the clamp technique remains improved with GLUT4 overexpression, as does in vitro insulin action. In summary, despite an impairment in whole-body glucose tolerance, skeletal muscle of the old transgenic GLUT4 db/db mice is still insulin responsive in vitro and in vivo.

Modulation of insulin secretion and glycemia by selective inhibition of cyclic AMP phosphodiesterase III.

The effects of selective inhibition of cyclic AMP phosphodiesterase type III on insulin and glucose levels during an oral glucose challenge were evaluated in obese, diabetic ob/ob mice and in lean, non-diabetic littermates using the selective inhibitor, milrinone. Oral administration of milrinone increased plasma insulin levels both in ob/ob and in lean mice. Glucose tolerance was improved in lean, but not in ob/ob mice, where glucose levels were increased by milrinone treatment. In isolated hepatocytes from normal rats incubation with 200 microM milrinone caused a 30% increase in glucose release with a corresponding depletion of glycogen stores. Stimulation of isolated rat adipocytes with 200 microM milrinone increased glycerol release 7-fold. We conclude that selective inhibitors of cyclic AMP phosphodiesterase III are effective insulin secretagogues, but their therapeutic utility may be limited by their concurrent stimulation of lipolysis and hepatic glucose output.

Glucose transport-enhancing and hypoglycemic activity of 2-methyl-2-phenoxy-3-phenylpropanoic acids.

A series of 2-phenoxy-3-phenylpropanoic acids has been prepared which contains many potent hypoglycemic agents as demonstrated by assessing glucose lowering in ob/ob mice. Some compounds (32, 33, 59) normalize plasma glucose in this diabetic model at doses of approximately 1 mg/kg. The mechanism of action of these drugs may involve enhanced glucose transport, especially in fat cells, but the compounds do not stimulate GLUT4 translocation and do not increase the levels of GLUT1 or GLUT4 in vivo. Thus, these compounds may enhance the intrinsic activity of the glucose transporter GLUT1 or GLUT4. Some compounds also modestly decrease hepatocyte gluconeogenesis in vitro, but this is not likely to be a major contributor to the hypoglycemic effect observed in vivo. Likewise, a modest decrease in food consumption observed with some of these compounds was shown by a pair-feeding experiment not to be the primary cause of the hypoglycemia observed.

Comparison of the glucose dependency of glucagon-like peptide-1 (7-37) and glyburide in vitro and in vivo.

The purpose of the present study was to compare the glucose dependency of the insulin secretagogue activity of the sulfonylurea, glyburide, versus that of glucagon-like peptide-1(7-37) [GLP-1(7-37)] in vitro and in vivo. In freshly isolated rat islets, maximally effective concentrations of glyburide (10 micromol/L) and GLP-1(7-37) (10 nmol/L) were equally effective in stimulating insulin secretion in the presence of 15 mmol/L glucose (2.4-fold increase relative to 15 nmol/L glucose alone). At 5 nmol/L glucose, both agents increased insulin secretion, but the effect for glyburide was threefold greater than for GLP-1(7-37) (122% and 41% increase in insulin secretion, respectively). In conscious catheterized rats infused with glucose at a variable rate to clamp plasma glucose concentration at 11 mmol/L, glyburide (1 mg/kg orally) and GLP -1(7-37) (infused intravenously [IV] at 5 pmol/min/kg) produced similar increase in insulin levels (1.8-fold relative to the respective vehicle controls) that were sustained through 60 minutes of measurement. These doses of GLP-1(7-37) and glyburide were then administered to fasted and fed rats (basal plasma glucose concentration, 5.8 and 7.3 mmol/L, respectively). Relative to the vehicle control group, GLP-1(7-37) infusion produced a transitory increase (30%) in plasma insulin concentration and a modest sustained decrease (10% to 20%) in glucose in both fasted and fed rats, whereas glyburide induced a sustained 2.4- and 1.7-fold increase in plasma insulin concentration in fasted and fed rats, respectively, and a 50% decrease in plasma glucose in both fasted and fed rats. Results of these studies demonstrate the higher glucose threshold for the insulin secretagogue activity of GLP-1(7-37) relative to glyburide in vitro and in vivo.

Glucose-dependent action of glucagon-like peptide-1 (7-37) in vivo during short- or long-term administration.

In vitro, truncated glucagon-like peptides [GLP-1(7-36)-amide and GLP-1(7-37)] increase insulin secretion in a glucose-dependent manner, and desensitization to the action of GLP-1(7-37) has been demonstrated acutely with high concentrations. The purpose of these studies was to evaluate the glucose dependency and threshold of GLP-1(7-37) action in normal rats and in a rat model of type II diabetes and to assess the effects of long-term administration in vivo. All studies were conducted in conscious catheterized rats. An intravenous (IV) infusion of GLP-1(7-37) at 0.5, 5, or 50 pmol/min/kg during the second hour of a 2-hour 11-mmol/L hyperglycemic clamp in Sprague-Dawley rats produced a dose-related enhancement of the glucose-induced increase in plasma insulin concentration. A 1-hour infusion of a submaximal dose of GLP-1(7-37) (5 pmol/min/kg IV) in fasted and fed Sprague-Dawley rats produced small transient increases in plasma insulin (incremental increases above basal, 72 +/- 27 and 96 +/- 28 pmol/L, respectively) and decreases in plasma glucose (to levels > or = 5.2 mmol/L). Infusion of GLP-1(7-37) (5 pmol/min/kg IV) during a hyperglycemic clamp at two sequentially increasing concentrations of glucose, 11 and 17 mmol/L, produced incremental increases in insulin of 600 and 1,200 pmol/L, respectively, relative to levels in clamped control rats. Similarly, infusion of GLP-1(7-37) (5 pmol/min/kg IV) in hyperinsulinemic, hyperglycemic Zucker diabetic fatty (ZDF) rats produced a transitory increase in plasma insulin concentration and normalized the plasma glucose concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhanced peripheral glucose utilization in transgenic mice expressing the human GLUT4 gene.

Human GLUT4 protein expression in muscle and adipose tissues of transgenic mice decreases plasma insulin and glucose levels and improves glucose tolerance compared with nontransgenic controls (Liu, M.-L., Gibbs, E. M., McCoid, S. C., Milici, A. J., Stukenbrok, H. A., McPherson, R. K., Treadway, J. L., and Pessin, J. E. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 11346-11350). We examined the basis of improved glycemic control in hGLUT4 transgenic mice by determining glucose homeostasis and metabolic profiles in vivo. Glucose turnover experiments indicated a 1.4-fold greater systemic glucose clearance in hGLUT4 mice relative to controls (p < 0.05), whereas hepatic glucose production was similar despite 26% lower (p < 0.05) glucose levels. Glucose infusion rate during an euglycemic-hyperinsulinemic clamp was 2-fold greater (p < 0.05) in hGLUT4 mice versus controls, and skeletal muscle and heart glycogen content were increased 3-5-fold (p < 0.05). The increased peripheral glucose clearance in hGLUT4 mice was associated with increased (25-32%) basal and insulin-stimulated glucose transport rate in soleus muscle (p < 0.01), and increased muscle plasma membrane-associated GLUT4 protein. Fed hGLUT4 mice displayed 20-30% lower plasma glucose and insulin levels (p < 0.05) and 43% elevated glucagon levels (p < 0.001) compared with controls. Triglycerides, free fatty acids, and beta-hydroxy-butyrate were elevated 43-63% (p < 0.05) in hGLUT4 mice due to hypoinsulinemia-induced lipolysis. Free fatty acids and beta-hydroxybutyrate levels in hGLUT4 mice increased further upon fasting, and skeletal muscle glycogen levels decreased markedly compared with controls. The data demonstrate that high level expression of hGLUT4 increases systemic glucose clearance and muscle glucose utilization in vivo and also results in marked compensatory lipolysis and muscle glycogenolysis during a fast.

Kittens choose a high leucine diet even when isoleucine and valine are the limiting amino acids.

This study was conducted to determine the effect of excess leucine on the dietary choice of kittens. The basal diets contained 16% (low nitrogen) or 24% (adequate nitrogen) amino acids and were limiting in isoleucine and valine. The addition of leucine to either of these basal diets has been shown to cause a transitory adverse effect on the growth and food intake of kittens. In separate experiments each basal diet was used to test three sets of choices: basal vs. basal + 10% leucine; basal + alanine vs. basal + leucine (isonitrogenous) and basal vs. basal + alanine. When offered basal vs. basal + excess leucine, the kittens chose significantly more of the excess leucine diet regardless of the level of nitrogen in the basal diet. When offered an isonitrogenous choice of excess alanine vs. excess leucine, the kittens selected somewhat more of the excess leucine diets over the excess alanine diets; this selection was sustained longer in kittens fed low nitrogen. In the third choice experiment (basal vs. basal + alanine), the group offered the low nitrogen diets exhibited no preferences, and the group offered adequate-nitrogen diets selected significantly more of the excess alanine diet. These results for kittens are opposite to those for rats, a species that will select a low protein basal or even a protein-free diet over a diet containing excess leucine.

Role of insulin in the blunted glucose metabolic response of septic rats to epinephrine.

Epinephrine produces smaller incremental increases in plasma glucose concentration and rate of glucose appearance (Ra) in septic rats compared with nonseptic animals. In the present study, we investigated the role of insulin in the diminished response of septic rats to epinephrine-induced increases in glucose turnover. Glucose kinetics were assessed by the infusion of [6-3H]-glucose in conscious catheterized rats made septic by subcutaneous injections of live Escherichia coli. Epinephrine was infused at 1 micrograms/min/kg for 2 hours in the presence and absence of somatostatin and mannoheptulose (SRIF + MH). In comparison to nonseptic control animals, epinephrine-induced increases in plasma glucose concentration and glucose Ra were blunted by more than 50% in the septic rats. Infusion of SRIF + MH with epinephrine restored the blunted response to normal. During the infusion of epinephrine alone, the plasma insulin concentration in the septic rats was 2.8-fold higher than the nonseptic controls. SRIF + MH lowered the plasma insulin concentrations in both the nonseptic and septic rats to less than 10 microU/mL. SRIF + MH reversed the sepsis-induced hyperglucagonemia, but did not prevent a slight increase in glucagon levels during the epinephrine infusion in the nonseptic rats. In a second study, septic rats infused with SRIF + MH and replacement insulin showed a smaller increase in glucose concentration and glucose production in response to epinephrine than did septic animals administered SRIF + MH and no insulin. These results indicate that insulin plays an important role in the diminished response of septic rats to epinephrine.

Epinephrine-induced increase in glucose turnover is diminished during sepsis.

The responsiveness of septic rats to epinephrine-induced alterations in carbohydrate metabolism was studied. Nonlethal sepsis was produced by subcutaneous injections of live Escherichia coli over 18 hours in conscious catheterized rats. Glucose kinetics were assessed by IV infusion of [6-3H]-glucose. After two hours of tracer infusion, blood samples were taken for basal values. Thereafter, epinephrine was infused at 0, 0.05, 0.2, or 1.0 microgram/min/kg for an additional four hours. Compared with nonseptic rats, septic animals had increased basal values for glucose rate of appearance (Ra, 63%), glucose clearance (86%), and plasma lactate concentration (133%). Infusion of epinephrine resulted in dose-dependent increases in glucose Ra, as well as plasma glucose and lactate concentrations, and decreases in glucose clearance and muscle glycogen content. At each dose of epinephrine, the increases in response from basal of plasma glucose and glucose Ra in septic rats were 50% or less of that observed in nonseptic animals. There were no differences between septic and nonseptic rats in plasma lactate and glucose clearance responses from basal or in circulating levels of catecholamines achieved during the epinephrine infusion. The present results indicate that septic rats are less responsive than control animals to epinephrine-induced increases in glucose turnover.

Tissue glucose utilization during epinephrine-induced hyperglycemia.

The aim of this study was to investigate glucose utilization by individual tissues during epinephrine infusion. First, the applicability of the 2-deoxyglucose (2-DG) tracer technique during in vivo hyperglycemia was investigated in model systems in vitro. Epitrochlearis muscle and spleen cells were incubated with 1.25-20 mM glucose. The discrimination against 2-[14C]DG in glucose metabolic pathways, expressed by the lumped constant, remained unchanged over this wide range of glucose concentrations. It was concluded that in vivo hyperglycemia does not preclude the application of the 2-DG method. In a series of in vivo experiments, chronically catheterized conscious rats fasted for 24 h and were infused with epinephrine (0.2 microgram.kg-1.min-1), which produced a two-fold increase in plasma glucose concentration. 2-[14C]DG was injected 30 min after starting the epinephrine infusion and glucose utilization rates of individual tissues were calculated based on the concentration of phosphorylated 2-DG in samples excised at 70 min. The epinephrine infusion increased glucose utilization rates by 40-160% in hindlimb muscles, skin, ileum, liver, spleen, lung, epididymal fat, and kidney, although no change was found in the brain. Mass action of the increased plasma glucose is likely to play an important role in the enhanced rate of glucose utilization.

Adrenergic blockade prevents endotoxin-induced increases in glucose metabolism.

Combined alpha- and beta-adrenergic blockade was used to investigate the role of catecholamines in endotoxin-induced elevations in glucose kinetics. Glucose kinetics were measured before and for 4 h after the injection of endotoxin [100 micrograms/100 g body wt iv, 30% lethal dose (LD30) at 24 h]. Adrenergic blockade was achieved by the bolus injection of phentolamine and propranolol followed by their continuous infusion. Endotoxin-treated rats exhibited a transient hyperglycemia and sustained (greater than 4 h) increase in plasma lactate concentration, as well as elevated rates of glucose appearance (Ra, 83%), disappearance (Rd, 58%), recycling (160%), and metabolic clearance (23%). Adrenergic blockade prevented endotoxin-induced increases in plasma glucose concentration, Ra, Rd, and recycling but not glucose clearance. The increase in plasma lactate concentration was blunted by 35%. After 2 h, endotoxic animals infused with adrenergic antagonists developed hypoglycemia, which may have resulted from an increased plasma insulin concentration. The attenuation of elevated glucose turnover by adrenergic blockade in the endotoxin-treated animals was not due to a reduction in plasma glucagon level or differences in plasma insulin concentration. Administration of the alpha- or beta-adrenergic antagonists separately blunted but did not prevent endotoxin-induced changes in glucose kinetics, and therefore the efficacy of the adrenergic blockade could not be assigned to a single receptor class. These results indicate that catecholamines are important contributory factors to many of the early alterations in carbohydrate metabolism observed during endotoxemia.

Effects of dietary excesses of the branched-chain amino acids on growth, food intake and plasma amino acid concentrations of kittens.

The effect of excesses of the branched-chain amino acids (BCAA), particularly leucine, on growth, food intake and plasma amino acid concentrations were investigated in kittens. Effects of excess leucine were tested in kittens fed five basal diets that varied in their nitrogen and amino acid contents. Compared to rats, kittens were much less sensitive to excesses of the BCAA. Addition of 10% leucine to basal diets that provided nitrogen just at or below the minimal requirement of kittens resulted in no change or increased growth and food intake of kittens when the isoleucine and valine concentrations in the basal diet were just at or slightly in excess of the kitten's minimal requirements for those amino acids. An adverse effect of leucine added to low nitrogen basal diets was observed only when isoleucine and valine were provided below the kitten's requirement (80% of requirement). When basal diets containing adequate nitrogen (24% amino acids) were tested, the addition of leucine (10%) resulted in an adverse effect when isoleucine and valine were provided at 80% of the kitten's requirement and in mild growth depressions when isoleucine and valine were provided at 1.1 times the requirement. Leucine-induced growth depression was alleviated by the addition of isoleucine and valine at 0.5%, indicating that excess leucine caused a BCAA antagonism or an amino acid imbalance. With the addition of leucine to the basal diets, there were consistent decreases in concentrations of alanine and tyrosine in plasma but no consistent depressions in the concentrations of isoleucine and valine.

Glucose kinetics in rats infused with endotoxin-induced monokines or tumor necrosis factor.

This study was conducted to determine if macrophage elaborated monokines in general, and human recombinant tumor necrosis factor (hrTNF alpha) in particular alter glucose metabolism in a manner analogous to that observed in endotoxin-treated animals. Endotoxin-tolerant rats were infused for 3 hr with saline, E. coli endotoxin (100 micrograms/l weight) or monokines contained in conditioned media from endotoxin-stimulated RAW 264.7 cells (1 microgram/ml). Compared to saline- and endotoxin-infused rats, animals receiving the monokine mixture had no change in mean arterial blood pressure or heart rate but exhibited overt signs of morbidity including stupor and diarrhea. Monokine-infused rats remained euglycemic but had elevated lactate concentrations and a 15-30% increase in glucose rate of appearance (Ra). Nontolerant rats received a 3 hr infusion of saline, hrTNF alpha (15 micrograms/100 g), or heat-treated hrTNF alpha. HrTNF alpha infusion increased glucose Ra about 25% compared to the two control groups but did so without producing signs of morbidity seen in the monokine infused animals. Serum TNF levels were 6-fold higher in rats infused with the monokine mixture compared to animals infused with hrTNF alpha, and this reflected the different levels of TNF contained in the monokine mixture and hrTNF alpha infusates. Plasma insulin, glucagon, and catecholamine concentrations were increased in rats infused with either the monokine mixture or hrTNF alpha, but the increases were more pronounced in rats receiving the monokine mixture. The results demonstrate that monokines and hrTNF alpha increase glucose production in vivo, and that the effect may be mediated by endocrine changes known to influence glucose homeostasis.

Platelet-activating factor-induced increases in glucose kinetics.

Platelet-activating factor (PAF) is a postulated mediator of many of the early hemodynamic effects of endotoxin. The aim of the present study was to determine whether in vivo administration of PAF could produce alterations in whole-body glucose metabolism that would mimic those seen during endotoxemia. Glucose kinetics were assessed in chronically catheterized conscious rats by the constant infusion of [6-3H]- and [U-14C]glucose before and for 4 h after either a bolus injection (300 ng/kg) or a constant infusion (20 or 220 ng.min-1.kg-1) of PAF. The bolus injection of PAF produced a 30% decrease in blood pressure by 5 min that returned to control levels by 30 min. Increased plasma glucose (40%) and lactate (150%) levels after injection of PAF were also transient. In contrast, the bolus injection of PAF elevated the rate of glucose appearance (Ra; 44%) for 1.5 h. The lower PAF infusion rate decreased blood pressure 11% to 104 mmHg, whereas the higher infusion rate decreased pressure 34% to 77 mmHg. Both PAF infusion rates produced elevations in plasma glucose (28, 150%) and glucose Ra (20, 60%) throughout the 4-h infusion period in a dose-related manner. The PAF infusions also induced dose-related increases in plasma glucagon and catecholamine levels throughout the infusion period. In a separate group of experiments a complete adrenergic blockade, produced by the constant infusion of propranolol and phentolamine, completely prevented PAF-induced increases in glucose kinetics and the hyperglucagonemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic blockade does not abolish elevated glucose turnover during bacterial infection.

Infusions of adrenergic antagonists were used to investigate the role of catecholamines in infection-induced elevations of glucose kinetics. Infection was produced in conscious catheterized rats by repeated subcutaneous injections of live Escherichia coli over 24 h. Glucose kinetics were measured by the constant intravenous infusion of [6-3H]- and [U-14C]glucose. Compared with noninfected rats, infected animals were hyperthermic (+1.4 degrees C) and showed increased rates of glucose appearance (45%), clearance (43%), and recycling (140%) as well as mild hyperlacticacidemia. Plasma catecholamine concentrations were increased by 50-70% in the infected rats, but there were no differences in plasma glucagon, corticosterone, and insulin levels. Adrenergic blockade was produced by primed constant infusion of both propranolol (beta-blocker) and phentolamine (alpha-blocker). A 2-h administration of adrenergic antagonists did not attenuate the elevated glucose kinetics or plasma lactate concentration in the infected rats, although it abolished the hyperthermia. In a second experiment, animals were infused with propranolol and phentolamine beginning 1 h before the first injection of E. coli and throughout the course of infection. Continuous adrenergic blockade failed to attenuate infection-induced elevations in glucose kinetics and plasma lactate. These results indicate that the adrenergic system does not mediate the elevated glucose metabolism observed in this mild model of infection.

Alterations in glucose kinetics induced by pentobarbital anesthesia.

Because pentobarbital is often used in investigations related to carbohydrate metabolism, the in vivo effect of this drug on glucose homeostasis was studied. Glucose kinetics, assessed by the constant intravenous infusion of [6-3H]- and [U-14C]glucose, were determined in three groups of catheterized fasted rats: conscious, anesthetized and body temperature maintained, and anesthetized but body temperature not maintained. After induction of anesthesia, marked hypothermia (5 degrees C decrease in core temperature) developed in rats not provided with external heat. Anesthetized rats that developed hypothermia showed a decrease in mean arterial blood pressure (25%) and heart rate (40%), whereas no differences were seen in blood pressure and heart rate of conscious and euthermic anesthetized rats. Likewise, the plasma lactate concentration and the rates of glucose appearance, recycling, and metabolic clearance were reduced by 30-50% in the hypothermic anesthetized rats. Changes in whole-body carbohydrate metabolism were prevented when body temperature was maintained. Because plasma pentobarbital levels were similar between the euthermic and hypothermic rats during the first 2 h of the experiment, the rapid reduction in glucose metabolism in this latter group appears related to the decrease in body temperature. The continuous infusion of epinephrine produced alterations in glucose kinetics that were not different between conscious animals and anesthetized rats with body temperature maintained. However, marked differences were seen in hypothermic rats. Thus pentobarbital-anesthetized rats became hypothermic when kept at room temperature and exhibited marked decreases in glucose metabolism. Such changes were absent when body temperature was maintained during anesthesia.

Attenuation of endotoxin-induced increase in glucose metabolism by platelet-activating factor antagonist.

Platelet-activating factor (PAF) has recently been proposed as a putative mediator of various pathophysiologic events during endotoxemia. The aim of the present study was to determine the relative importance of PAF in producing the alterations in carbohydrate metabolism following endotoxin. Chronically catheterized conscious rats were treated with SRI 63-441, a specific PAF receptor antagonist, or saline prior to Escherichia coli endotoxin (100 micrograms/100 g body weight, LD10) administration. Hemodynamic and whole-body glucose kinetic changes, the latter assessed by a constant intravenous infusion of [6-3H] glucose, were determined throughout the 4-hr experimental protocol. Endotoxin induced a transient 30-35% reduction in mean arterial blood pressure (MABP) in animals treated with saline. The PAF-antagonist attenuated this hypotensive effect, and MABP was only reduced by 14-18%. Endotoxin increased plasma glucose and lactate levels, as well as the rate of glucose appearance (Ra) in saline-treated rats. The PAF antagonist reduced the hyperglycemia by 60-75% and tended to prevent the hyperlactacidemia. The endotoxin-induced elevation in glucose Ra was also attenuated by 55%. A similar degree of hyperglucagonemia was observed following endotoxin in both groups, and plasma insulin concentrations were not different. However, plasma catecholamine levels were significantly lower (30-70%) in endotoxemic rats treated with the PAF antagonist. These results suggest that the enhanced production of PAF following endotoxin may be responsible, at least in part, for the early hemodynamic changes. The role of PAF as a mediator of endotoxin-induced glucose dyshomeostasis, however, may be secondary to its hemodynamic effects.

Leucine and isoleucine requirements of the kitten.

In separate experiments the isoleucine and leucine requirements of the kitten were determined on the basis of growth and nitrogen retention. The dietary concentrations of isoleucine tested were (g/kg diet) 1.4, 2.2, 3.0, 3.8, 4.6 and 9.0 with adequate (12.0 g/kg diet) leucine. The levels of leucine tested were (g/kg diet) 5.0, 7.5, 9.0, 10.5, 12.0 and 20.0 in diets containing adequate (9.0 g/kg diet) isoleucine. In both experiments six male and six female kittens received each dietary level of isoleucine or leucine for periods of 10 d in a balanced 6 X 6 Latin-square experimental design. Asymptotic curves were fitted to the response relationships and the minimal dietary requirements for maximal response were estimated from the values at 0.95 of the asymptote. On this basis, the requirements for maximal growth were 6.2 g isoleucine/kg and 7.8 g leucine/kg diet. The requirements for maximal N retention were higher; 8.4 g isoleucine and 10.6 g leucine/kg diet. The isoleucine requirements suggested by this method are probably overestimations and might be slightly above 4.6 g/kg diet. Plasma isoleucine and leucine concentrations were not useful in estimating the requirements. Plasma leucine increased rectilinearly with increasing dietary leucine while the response of plasma isoleucine to increasing dietary isoleucine was non-rectilinear. Neither response relationship exhibited a breakpoint at the level of requirement. Below the suggested minimal requirement for leucine there were significant increases in the concentrations of isoleucine and valine in the plasma. Dietary isoleucine below the level of requirement had no effect on plasma valine and leucine. Dietary leucine had no effect on the plasma concentrations of methionine, phenylalanine and threonine, suggesting that the effect of decreasing dietary leucine on plasma isoleucine and valine is a result of decreased oxidation rather than decreased protein anabolism. In a separate experiment six kittens, presented a diet containing 2.2 g isoleucine/kg, developed crusty exudates around their eyes within 27 d and six kittens, presented diets containing 3.8 g isoleucine/kg, showed this clinical sign but with less severity within 47 d. Cultures of conjunctival swabs taken from the most severely affected kittens showed the presence of staphylococcal species, suggesting that in isoleucine-deficient kittens there was impaired resistance to these dermal microbes.