Effects of morphine on glucose homeostasis in the conscious dog.

This study was designed to assess the effects of morphine sulfate on glucose kinetics and on glucoregulatory hormones in conscious overnight fasted dogs. One group of experiments established a dose-response range. We studied the mechanisms of morphine-induced hyperglycemia in a second group. We also examined the effect of low dose morphine on glucose kinetics independent of changes in the endocrine pancreas by the use of somatostatin plus intraportal replacement of basal insulin and glucagon. In the dose-response group, morphine at 2 mg/h did not change plasma glucose, while morphine at 8 and 16 mg/h caused a hyperglycemic response. In the second group of experiments, morphine (16 mg/h) caused an increase in plasma glucose from a basal 99 +/- 3 to 154 +/- 13 mg/dl (P less than 0.05). Glucose production peaked at 3.9 +/- 0.7 vs. 2.5 +/- 0.2 mg/kg per min basally, while glucose clearance declined to 1.7 +/- 0.2 from 2.5 +/- 0.1 ml/kg per min (both P less than 0.05). Morphine increased epinephrine (1400 +/- 300 vs. 62 +/- 8 pg/ml), norepinephrine (335 +/- 66 vs. 113 +/- 10 pg/ml), glucagon (242 +/- 53 vs. 74 +/- 14 pg/ml), insulin (30 +/- 9 vs. 10 +/- 2 microU/ml), cortisol (11.1 +/- 3.3 vs. 0.9 +/- 0.2 micrograms/dl), and plasma beta-endorphin (88 +/- 27 vs. 23 +/- 6 pg/ml); all values P less than 0.05 compared with basal. These results show that morphine-induced hyperglycemia results from both stimulation of glucose production as well as inhibition of glucose clearance. These changes can be explained by rises in epinephrine, glucagon, and cortisol. These in turn are part of a widespread catabolic response initiated by high dose morphine that involves activation of the sympathetic nervous system, the endocrine pancreas, and the pituitary-adrenal axis. Also, we report the effect of a 2 mg/h infusion of morphine on glucose kinetics when the endocrine pancreas is clamped at basal levels. Under these conditions, morphine exerts a hypoglycemic effect (25% fall in plasma glucose, P less than 0.05) that is due to inhibition of glucose production (by 25-43%, P less than 0.05). The hypoglycemia was independent of detectable changes in insulin, glucagon, epinephrine and cortisol, and was not reversed by concurrent infusion of a slight molar excess of naloxone. Therefore, we postulate that the hypoglycemic effect of morphine results from the interaction of the opiate with non-mu receptors either in the liver or the central nervous system.

Lack of effect of somatostatin on the stimulation of hepatic glycogenolysis by epinephrine in isolated canine hepatocytes.

The effects of somatostatin on epinephrine's ability to stimulate glucose output have been examined in hepatocytes isolated from dogs fasted overnight. Half-maximal stimulation of phosphorylase a activity and glucose output occurred at an epinephrine concentration of approx. 5 X 10(-9) M. Somatostatin at 10, 100 or 1000 ng/ml had no effect on the ability of a maximal (1 X 10(-7) M) and a submaximal (1 X 10(-8) M) dose of epinephrine to activate phosphorylase at 2 min, or to stimulate glucose output over 20 min. Since the doses of somatostatin used in the present study are up to 50-fold higher than the blood concentrations commonly found when somatostatin is used in vivo to inhibit pancreatic hormone secretion, it seems unlikely that use of somatostatin in this way would affect stimulation of hepatic glycogenolysis by epinephrine in vivo.

The role of phosphorylation in the alpha-adrenergic-mediated inhibition of rat hepatic pyruvate kinase.

Phenylephrine in the presence of 1-methyl-3-isobutylxanthine and propanolol caused a 40-50% inhibition of pyruvate kinase (type L) activity in isolated hepatocytes, which was accompanied by a 2-3-fold increase in the phosphate content of the enzyme. These changes were blocked by the alpha-adrenergic antagonist dihydroergocryptine and could not be accounted for by the slight increase in cyclic AMP-dependent protein kinase activity generated by the alpha-adrenergic agonist. It is concluded that a significant component of the inhibition of hepatic pyruvate kinase mediated by alpha-adrenergic agonists can be attributed to a cyclic AMP-independent alteration in the phosphorylation state of the enzyme.

Altered ability of the liver to produce glucose following a period of glucagon deficiency.

It is known that the effectiveness of a physiologic increment in glucagon to stimulate glucose production wanes with time even when counterregulatory insulin secretion is prevented. The aim of the present study was to establish whether the converse is true: does glucagon become a more effective stimulus of glucose production following a period of acute hypoglucagonemia? To determine this, somatostatin was infused along with basal replacement amounts of glucagon (0.65 ng/mg x min) and insulin (228 microU/kg x min) into five postabsorptive conscious dogs. After 1.5 h of basal hormone replacement, the glucagon infusion was terminated and a selective fall in the plasma glucagon level (75 +/- 6 to 30 +/- 4 pg/ml) occurred. This resulted in a drop in tracer (3H-glucose)-determined glucose production from 3.0 +/- 0.4 to 1.5 +/- 0.3 mg/kg x min. The plasma insulin level remained unchanged at 10 +/- 1 microU/ml throughout the experiment. Euglycemia (110 +/- 4 mg/dl) was maintained by exogenous glucose infusion. After 3 h of glucagon lack, restoration of the glucagon infusion returned the IRG level to control values (78 +/- 6 pg/ml) but restored glucose output only partially (42 +/- 9%), necessitating continued glucose infusion to preserve euglycemia. Repetition of the experiment in dogs whose pancreatic glucoregulatory feedback loops were broken surgically (two-stage pancreatectomy) rather than pharmacologically resulted in similar findings. It is concluded that glucagon deficiency of 3-h duration leads to a decrease, rather than an increase, in hepatic sensitivity to glucagon.

Relative importance of first- and second-phase insulin secretion in glucose homeostasis in conscious dog. II. Effects on gluconeogenesis.

The normal pancreatic response to an exogenous glucagon infusion is a biphasic release of insulin. In our study the ability of each component of insulin release to counter the effects of the glucagon on gluconeogenesis and alanine metabolism was assessed by mimicking first- and/or second-phase insulin release with infusions of somatostatin and intraportal insulin. When a fourfold increase in glucagon was brought about in the presence of fixed basal insulin release, there was a large increase in overall glucose production and gluconeogenesis. The increase in the conversion of [14C]alanine into [14C]glucose (169 +/- 42%, P less than .05) was accompanied by an increase in the fractional extraction of alanine by the liver (FEA 0.32 +/- 0.06 to 0.66 +/- 0.10, P less than .05) and net hepatic alanine uptake (NHAU 2.97 +/- 0.45 to 4.61 +/- 0.48 mumol . kg-1 . min-1, P less than .05). Simulated first-phase insulin release had no effect on the ability of glucagon to increase FEA (0.32 +/- 0.03 to 0.66 +/- 0.03, P less than .05) or NHAU (3.69 +/- 0.80 to 5.10 +/- 0.69 mumol . kg-1 . min-1, P less than .05) but did limit the increase in overall gluconeogenic conversion (114 +/- 37%). Second-phase insulin release had no effect on either the glucagon-induced increase in FEA (0.35 +/- 0.08 to 0.73 +/- 0.04) or NHAU (3.35 +/- 0.92 to 5.13 +/- 0.85 mumol . kg-1 . min-1) but completely inhibited the increase in overall gluconeogenic conversion.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of ketogenesis by epinephrine and norepinephrine in the overnight-fasted, conscious dog.

The effects on ketogenesis and lipolysis of a norepinephrine (0.04 microgram/kg-min), epinephrine (0.04 microgram/kg-min), or saline infusion were examined in the overnight-fasted, conscious dog. Plasma insulin and glucagon levels were maintained constant by means of a somatostatin infusion (0.8 microgram/kg-min) and intraportal replacement infusions of insulin and glucagon. In saline-infused dogs, plasma epinephrine (62 +/- 8 pg/ml), norepinephrine (92 +/- 29 pg/ml), blood glycerol (87 +/- 10 microM), and plasma nonesterified fatty acid (NEFA) (0.82 +/- 0.17 mM) levels did not change. Total blood ketone body levels tended to rise (62 +/- 10 to 83 +/- 11 microM) by 3 h as did total ketone body production (1.5 +/- 0.4 to 2.2 +/- 0.4 mumol/kg-min) over the same time interval. Norepinephrine infusion to produce plasma levels of 447 +/- 86 pg/ml caused a sustained 50% rise in glycerol levels (66 +/- 17 to 99 +/- 15 mumol/L, P less than 0.05) and 53% rise in nonesterified fatty acids (0.53 +/- 0.07 to 0.81 +/- 0.15 mumol/L, P less than 0.05). Total ketone body levels rose by 43% (51 +/- 8 to 73 +/- 10 mumol/L) and ketone body production rose by a similar proportion (1.5 +/- 0.2 to 2.2 +/- 0.3 mumol/kg-min), changes that did not differ significantly from control animals. A similar increment in plasma epinephrine levels (75 +/- 15 to 475 +/- 60 pg/ml) caused glycerol levels to rise by 82% (105 +/- 23 to 191 +/- 26 mumol/L) in 30 min, but this rise was not sustained and the level fell to 146 +/- 14 mumol/L by 120 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Similar dose responsiveness of hepatic glycogenolysis and gluconeogenesis to glucagon in vivo.

This study was undertaken to determine whether the dose-dependent effect of glucagon on gluconeogenesis parallels its effect on hepatic glycogenolysis in conscious overnight-fasted dogs. Endogenous insulin and glucagon secretion were inhibited by somatostatin (0.8 micrograms X kg-1 X min-1), and intraportal replacement infusions of insulin (213 +/- 28 microU X kg-1 X min-1) and glucagon (0.65 ng X kg-1 X min-1) were given to maintain basal hormone concentrations for 2 h (12 +/- 2 microU/ml and 108 +/- 23 pg/ml, respectively). The glucagon infusion was then increased 2-, 4-, 8-, or 12-fold for 3 h, whereas the rate of insulin infusion was left unchanged. Glucose production (GP) was determined with 3-[3H]glucose, and gluconeogenesis (GNG) was assessed with tracer (U-[14C]alanine conversion to [14C]glucose) and arteriovenous difference (hepatic fractional extraction of alanine, FEA) techniques. Increases in plasma glucagon of 53 +/- 8, 199 +/- 48, 402 +/- 28, and 697 +/- 149 pg/ml resulted in initial (15-30 min) increases in GP of 1.1 +/- 0.4 (N = 4), 4.9 +/- 0.5 (N = 4), 6.5 +/- 0.6 (N = 6), and 7.7 +/- 1.4 (N = 4) mg X kg-1 X min-1, respectively; increases in GNG (approximately 3 h) of 48 +/- 19, 151 +/- 50, 161 +/- 25, and 157 +/- 7%, respectively; and increases in FEA (3 h) of 0.14 +/- 0.07, 0.37 +/- 0.05, 0.42 +/- 0.04, and 0.40 +/- 0.17, respectively. In conclusion, GNG and glycogenolysis were similarly sensitive to stimulation by glucagon in vivo, and the dose-response curves were markedly parallel.

Expression of the human beta(3)-adrenergic receptor gene in SK-N-MC cells is under the control of a distal enhancer.

Mechanisms of transcriptional regulation of the human beta(3)-adrenergic receptor were studied using SK-N-MC cells, a human neuroblastoma cell line that expresses beta(3)- and beta(1)-adrenergic receptors endogenously. Deletions spanning different portions of a 7-kb 5'-flanking region of the human beta(3)-adrenergic receptor gene were linked to a luciferase reporter and transfected in SK-N-MC, CV-1, and HeLa cells. Maximal luciferase activity was observed when a 200-bp region located between -6.5 and -6.3 kb from the translation start site was present. This region functioned only in SK-N-MC cells. Electrophoretic mobility shift assays of nuclear extracts from SK-N-MC, CV-1, and HeLa cells using double stranded oligonucleotides spanning different portions of the 200-bp region as probes and transient transfection studies revealed the existence of three cis-acting regulatory elements: A) -6.468 kb-AGGTGGACT--6.458 kb, B) -6.448 kb-GCCTCTCTGGGGAGCAGCTTCTCC-6.428 kb, and C) -6.405 kb-20 repeats of CCTT-6.385 kb. These elements act together to achieve full transcriptional activity. Mutational analysis, antibody supershift, and electrophoretic mobility shift assay competition experiments indicated that element A binds the transcription factor Sp1, element B binds protein(s) present only in nuclear extracts from SK-N-MC cells and brown adipose tissue, and element C binds protein(s) present in both SK-N-MC and HeLa cells. In addition, element C exhibits characteristics of an S1 nuclease-hypersensitive site. These data indicate that cell-specific positive cis-regulatory elements located 6.5 kb upstream from the translation start site may play an important role in transcriptional regulation of the human beta(3)-adrenergic receptor. These data also suggest that brown adipose tissue-specific transcription factor(s) may be involved in the tissue-specific expression of the beta(3)-adrenergic receptor gene.

New potent antihyperglycemic agents in db/db mice: synthesis and structure-activity relationship studies of (4-substituted benzyl) (trifluoromethyl)pyrazoles and -pyrazolones.

The synthesis, structure-activity relationship (SAR) studies, and antidiabetic characterization of 1,2-dihydro-4-[[4-(methylthio)phenyl]methyl]-5-(trifluoromethyl)-3H- pyrazol-3-one (as the hydroxy tautomer; WAY-123783, 4) are described. Substitution of 4-methylthio, methylsulfinyl, or ethyl to a benzyl group at C4, in combination with trifluoromethyl at C5 of pyrazol-3-one, generated potent antihyperglycemic agents in obese, diabetic db/db mice (16-30% reduction in plasma glucose at 2 mg/kg). The antihyperglycemic effect was associated with a robust glucosuria (> 8 g/dL) observed in nondiabetic mice. Chemical trapping of four of the seven possible tautomeric forms of the heterocycle by mono- and dialkylation at the acidic hydrogens provided several additional potent analogs (39-43% reduction at 5 mg/kg) of the lead 4 as well as a dialkylated pair of regioisomers that showed separation of the associated glucosuric effect produced by all of the active analogs in normal mice. Further pharmacological characterization of the lead WAY-123783 (ED50 = 9.85 mg/kg, po in db/db mice), in oral and subcutaneous glucose tolerance tests, indicated that unlike the renal and intestinal glucose absorption inhibitor phlorizin, pyrazolone 4 does not effectively block intestinal glucose absorption. SAR and additional pharmacological data reported herein suggest that WAY-123783 represents a new class of potent antihyperglycemic agents which correct hyperglycemia by selective inhibition of renal tubular glucose reabsorption.

Perfluorocarbon-based antidiabetic agents.

In a preliminary communication (J. Med. Chem. 1989, 32, 11-13) a series of perfluoro-N-[4-(1H-tetrazol-5ylmethyl)phenyl]alkana mides (perfluoro anilides I), designed as novel analogues of ciglitazone, were reported to possess oral antidiabetic activity in two genetic animal models of non-insulin-dependent diabetes mellitus (NIDDM): obese (ob/ob) and diabetic (db/db) mice. In this report, the results from a structure-activity relationship (SAR) study of the series I are described. Comprehensive statistical analysis among the 86 analogues screened for blood glucose lowering in ob/ob mice was achieved by a new application of a general statistical procedure which made it possible to make meaningful comparisons between more than 140 separate experiments (N = 2966). Perfluoro anilides I lowered plasma glucose in the hyperglycemic ob/ob and db/db mice but not in euglycemic normal rats. In the hyperinsulinemic ob/ob mouse, decreases in plasma insulin levels paralleled the decline in plasma glucose. Potency and efficacy in the series was shown to be dependent on the length of the perfluorocarbon chain (RF) of I. Optimal activity occurred with the C7 and C8 RF chains. The more extensive SAR studies reported here, indicated that the lipophilic RF chain is the most important structural element of I since neither the phenyl nor tetrazole rings present in anilides I were necessary for antihyperglycemic activity while medium length (C7-C8) RF chains, especially the C7F15 chain, were shown to confer antihyperglycemic activity in ob/ob mice to a wide variety of structures.

Oral hypoglycemic agents. Pyrimido[1,2-a]indoles and related compounds.

A series of pyrimido[1,2-a]indoles were synthesized and studied for their hypoglycemic activity following oral administration at a standard dose of 100 mg/kg to fed rats. The effect of 10-alkoxyalkyl, 10-alkyl, 10-aryl, and 3,3-dialkyl substitution on the activity of 10-hydroxypyrimido[1,2-a]indoles was investigated. Relative potencies of a number of the most active compounds were defined by three-point dose-response studies. The most potent compounds were those with either 3,3-dimethyl substituents, compounds 21, 22, and 38, or 3,3-spirocyclohexane substituents, compounds 39 and 49. 10-Aminopyrimido[1,2-a]indoles were in general less active than the 10-hydroxy analogues, and potency was further decreased by derivatizing the 10-amino group. The most potent 10-amino derivatives were 57 and 58.

Studies on new acidic azoles as glucose-lowering agents in obese, diabetic db/db mice.

Bioisosteric substitution was used as a tool to generate several new structural alternatives to the thiazolidine-2,4-dione and tetrazole heterocycles as potential antidiabetic agents. Among the initial leads that emerged from this strategy, a family of acidic azoles, isoxazol-3- and -5-ones and a pyrazol-3-one, showed significant plasma glucose-lowering activity (17-42% reduction) in genetically obese, diabetic db/db mice at a dose of 100 mg/kg/day x4. Structure-activity relationship studies determined that 5-alkyl-4-(arylmethyl)pyrazol-3-ones, which exist in solution as aromatic enol/iminol tautomers, were the most promising new class of potential antidiabetic agent (32-45% reduction at 20 mg/kg/d x4). Included in this work are convenient syntheses for several types of acidic azoles that may find use as new acidic bioisosteres in medicinal chemistry such as the antidiabetic lead 5-(trifluoromethyl)pyrazol-3-one (hydroxy tautomer) and aza homologs of the pyrazolones, 1,2,3-triazol-5-ones (hydroxy tautomer) and 1,2,3,4-tetrazol-5-one heterocycles. log P and pKa data for 15 potential acidic bioisosteres, all appended to a 2-naphthalenylmethyl residue so as to maintain a similar distance between the acidic hydrogen and arene nucleus, are presented. This new data set allows comparison of a wide variety of potential acid mimetics (pKa 3.78-10.66; log P -0.21 to 2.76) for future drug design.

The effects of epinephrine on ketogenesis in the dog after a prolonged fast.

The effects of a selective increase in epinephrine on ketogenesis and lipolysis were determined in the conscious dog following a prolonged fast (7 days). Plasma insulin and glucagon were fixed at basal levels by infusion of somatostatin (0.8 micrograms/kg/min) and basal intraportal replacement amounts of insulin (210 +/- 20 microU/kg/min) and glucagon (0.65 ng/kg/min). Following a 40-minute control period, saline or epinephrine (0.04 microgram/kg/min) was infused for 3 hours. Plasma insulin, glucagon, and norepinephrine levels did not change during saline (6 +/- 1 microU/mL, 83 +/- 17 pg/mL, and 137 +/- 38 pg/mL, respectively) or epinephrine (10 +/- 1 microU/mL, 73 +/- 18 pg/ml, 98 +/- 13 pg/mL, respectively) infusion. Plasma epinephrine levels increased from 80 +/- 26 to 440 +/- 47 pg/mL in response to infusion of the catecholamine, but remained unchanged during saline infusion. Glycerol levels (93 +/- 10 mumol/L) remained unchanged during saline infusion, but increased in response to epinephrine (108 +/- 9 to 170 +/- 18 mumol/L by 30 minutes). The glycerol level had returned to baseline and to the value apparent in saline controls by 60 minutes. The nonesterified fatty acid (NEFA) level declined slowly during the 3-hour saline infusion, but was elevated in response to epinephrine infusion (1.27 +/- 0.16 to 1.97 +/- 0.25 mmol/L at 30 minutes). After the initial epinephrine-induced increase, the NEFA level declined so that by 3 hours it was not significantly different from the basal or saline values.(ABSTRACT TRUNCATED AT 250 WORDS)

Lack of effect of somatostatin on epinephrine-stimulated glucose production in the dog.

The effects of somatostatin on epinephrine-stimulated hepatic glucose production were assessed in the conscious overnight fasted dog. Glucose production was measured using a primed constant infusion of 3-3H-glucose. Two experiments were performed on each of four animals, each experiment consisting of a tracer equilibration period (80 minutes), a control period (40 minutes), and a test period (180 minutes). In the first experiment, epinephrine was infused at 0.08 microgram/kg/min during the test period so that the plasma concentration rose from 138 +/- 4 to 727 +/- 109 pg/mL. In the second experiment two weeks later, epinephrine was again infused (112 +/- 17 to 727 +/- 148 pg/mL) but with somatostatin (0.8 microgram/kg/min) and intraportal replacement amounts of insulin and glucagon. The pancreatic hormones were administered in such a way as to mimic the insulin and glucagon levels observed during epinephrine infusion in the first experiment. In the first experiment, epinephrine caused changes in insulin and glucagon levels at five minutes of plus 9 +/- 1 microU/mL and minus 1 +/- 3 pg/mL, respectively, and averaged plus 6 +/- 2 microU/mL and minus 9 +/- 5 pg/mL over the first hour. Glucose production peaked at 15 minutes (increment of 0.83 +/- 0.24 mg/kg/min) and increased by an average of 0.38 +/- 0.12 mg/kg/min in the first hour. In the second experiment, intraportal replacement of insulin and glucagon during epinephrine infusion resulted in changes in insulin and glucagon levels at five minutes of plus 8 +/- 3 microU/mL and plus 2 +/- 2 pg/mL, respectively, and averaged plus 4 +/- 2 microU/mL and minus 7 +/- 6 pg/mL over the first hour.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of epinephrine's glycogenolytic effect in isolated canine hepatocytes.

Epinephrine (10(-7) mol/L) addition to isolated canine hepatocytes activates glycogen phosphorylase from 12.3 +/- 0.4 to 28.6 +/- 2.6 U/g and glucose output from 42 +/- 3 to 170 +/- 24 nmol/mg/h. Preincubation of hepatocytes with propranolol (2 X 10(-5) mol/L) caused a 73% inhibition of phosphorylase activation and a 77% inhibition of the stimulation of glucose output by epinephrine. Phentolamine (2 X 10(-5) mol/L) on the other hand, caused a 16% inhibition of phosphorylase activation and a 27% inhibition of the stimulation of glucose output by epinephrine. These results were unaffected by the sex of the animal. In the dog the glycogenolytic effects of epinephrine appear to be mediated primarily by a beta-adrenergic mechanism.

Effects of insulin on glucagon-stimulated glucose production in the conscious dog.

The relative importance of insulin and glucagon as primary regulators of glucose metabolism in vivo was assessed in 18-hour fasted conscious dogs. Glucose turnover was determined using [3-3H]glucose and gluconeogenesis was assessed using tracer ([14C]alanine) and A-V difference techniques during a 40-minute control period and a 3-hour period during which various hormonal perturbations were brought about. During the infusion of somatostatin and basal intraportal replacement amounts of insulin and glucagon for the entire study, the plasma glucose concentration (109 +/- 5 mg/dL), glucose production (3.24 +/- 0.30 mg/kg/min), and glucose utilization (3.17 +/- 0.32 mg/kg/min) remained unchanged. When the glucagon infusion rate was increased fourfold at the end of the control period, the plasma glucose level increased from 107 +/- 4 to 225 +/- 23 mg/dL by 1 hour and remained elevated. Glucose production increased from 3.14 +/- 0.29 to 7.66 +/- 0.51 mg/kg/min by 15 minutes and decreased to 4.23 +/- 0.35 mg/kg/min by 3 hours. Glucose utilization rose from a basal value of 3.20 +/- 0.26 to 5.46 +/- 0.27 mg/kg/min by 3 hours. When a fourfold increase in the insulin infusion rate was brought about at the end of the control period, glucose production decreased from 2.83 +/- 0.20 to 1.16 +/- 0.57 mg/kg/min by 1 hour, after which it increased slightly (1.62 +/- 0.81 mg/kg/min). Glucose utilization increased from 2.92 +/- 0.30 to 8.12 +/- 1.12 mg/kg/min by 3 hours. Euglycemia was maintained by glucose infusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of alanine concentration independent of changes in insulin and glucagon on alanine and glucose homeostasis in the conscious dog.

The effect of an alanine load per se on hepatic alanine balance and hepatic glucose production is unclear. To examine this question, alanine was infused into six postabsorptive dogs at a rate of 6 mumol/kg-min, while maintaining insulin and glucagon levels using the pancreatic clamp technique. The arterial alanine concentration rose from a basal level of 227 +/- 16 mumol/L to 497 +/- 40 mumol/L during alanine infusion (P less than .01). The net hepatic fractional extraction of alanine remained unchanged, while hepatic alanine uptake increased from 3.0 +/- 0.3 to 6.0 +/- 0.4 mumol/kg-min (P less than .01). Conversion of alanine into glucose increased 87% to 2.7 +/- 0.3 mumol/kg-min during alanine infusion (P less than .01) while gluconeogenic efficiency remained essentially unchanged. Despite the increased gluconeogenic rate, the total rate of glucose production was unchanged. These data suggest that an increase in the alanine load to the liver causes a proportional increase in net hepatic alanine uptake and the gluconeogenic rate, but that in an overnight fasted animal this increase is insufficient to significantly increase glucose production.