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Malfunction of the liver is a central factor in metabolic disease. Glucose production by liver is complex and controlled via indirect mechanisms; insulin regulates adipose tissue lipolysis, and free fatty acids in turn regulate liver glucose output. This latter concept is confirmed by studies in L-Akt-Foxo1 knockout mice. The adipocyte is a likely locus of hepatic insulin resistance. Also, kidneys play a role in regulating glucose production; denervated kidneys abrogate the effect of fat feeding to cause insulin resistance. Glucose itself is an important regulator of liver metabolism ("glucose effectiveness"); after entering liver, glucose is phosphorylated and can be exported as lactate. Using the dynamic glucose/lactate relationship, we have been able to estimate glucose effectiveness in intact animals and human subjects. Families have been identified with a glucokinase regulatory protein defect; modeling demonstrates elevated glucokinase activity. Insulin clearance by liver is highly variable among normal individuals, and is under environmental control: high fat diet reduces clearance by 30%. Liver insulin clearance is significantly lower in African American (AA) adults and children compared to European American participants, accounting for fasting hyperinsulinemia in AA. We hypothesize that reduced hepatic insulin clearance causes peripheral insulin resistance and increased Type 2 diabetes in AA., (Copyright © 2019 Elsevier Inc. All rights reserved.)

There is wide variance among individuals in the fraction of insulin cleared by the liver (20% to 80%). Hepatic insulin clearance is 67% lower in African Americans than European Americans. Clearance is also lower in African American children 7-13 years of age. Lower hepatic insulin clearance will result in peripheral hyperinsulinemia: this exacerbates insulin resistance, which stresses the β-cells, possibly resulting in their ultimate failure and onset of type 2 diabetes. We hypothesize that lower insulin clearance can be a primary cause of type 2 diabetes in at-risk individuals., (© 2019 by the American Diabetes Association.)

Aims: Adult African American (AA) women have one third of the hepatic insulin clearance of European American (EA) women. This lower hepatic (but not extra-hepatic) insulin clearance in AA individuals is associated with higher plasma insulin concentrations. This study aims to understand whether impairment of hepatic insulin clearance is seen in AA individuals since childhood, possibly suggesting that genetic/epigenetic factors, rather than lifestyle only, contribute to this., Materials and Methods: A total of 203 children (105 male and 98 female (55 AA, 88 EA and 60 Hispanic American [HA]; ages, 7-13 years; mean BMI, 19 kg/m 2 )) underwent the frequently applied intravenous glucose tolerance test (FSIGT) at the University of Alabama at Birmingham, General Clinical Research Center and Department of Nutrition Sciences. Glucose, insulin and C-peptide levels were measured and hepatic and extra-hepatic insulin clearances were calculated using mathematical modelling., Results: Fractional hepatic insulin extraction (FE L ) was lower in AA than in EA children (mean (SD), 19% (20%) vs 33% (20%); P = 0.0007). Adjusting for age, Tanner stage and body fat, FE L was lower in AA than in EA children (P = 0.0012), and there was a slight sex-related difference (FE L, 24% (10%) vs 29% (10%) in boys vs girls; P = 0.04). Extra-hepatic insulin clearance did not differ with ethnicity (27 (12), 21 (12) and 24 (28) mL/kg/min for AA, HA and EA children, respectively; P > 0.05)., Conclusions: At a young age, FE L is lower in AAs than in EAs, which does not rule out genetic/epigenetic factors. These differences are related to hyperinsulinaemia and, over time, could possibly contribute to metabolic disorders in AA individuals., (© 2018 John Wiley & Sons Ltd.)

Hyperinsulinemia, accompanied by reduced first-pass hepatic insulin extraction (FPE) and increased secretion, is a primary response to insulin resistance. Different in vivo methods are used to estimate the clearance of insulin, which is assumed to reflect FPE. We compared two methodologically different but commonly used indirect estimates with directly measured FPE in healthy dogs ( n = 9). The indirect methods were 1) metabolic clearance rate of insulin (MCR) during the hyperinsulinemic-euglycemic clamp (EGC), a steady-state method, and 2) fractional clearance rate of insulin (FCR) during the frequently sampled intravenous glucose tolerance test (FSIGT), a dynamic method. MCR was calculated as the ratio of insulin infusion rate to steady-state plasma insulin. FCR was calculated as the exponential decay rate constant of the injected insulin. Directly measured FPE is based on the difference in insulin measurements during intraportal vs. peripheral vein insulin infusions. We found a strong correlation between indirect FCR (min -1 ) and FPE (%). In contrast, we observed a poor association between MCR (ml·min -1 ·kg -1 ) and FPE (%). Our findings in canines suggest that FCR measured during FSIGT can be used to estimate FPE. However, MCR calculated during EGC appears to be a poor surrogate for FPE.

Although the β-cells secrete insulin, the liver, with its first-pass insulin extraction (FPE), regulates the amount of insulin allowed into circulation for action on target tissues. The metabolic clearance rate of insulin, of which FPE is the dominant component, is a major determinant of insulin sensitivity (SI). We studied the intricate relationship among FPE, SI, and fasting insulin. We used a direct method of measuring FPE, the paired portal/peripheral infusion protocol, where insulin is infused stepwise through either the portal vein or a peripheral vein in healthy young dogs ( n = 12). FPE is calculated as the difference in clearance rates (slope of infusion rate vs. steady insulin plot) between the paired experiments. Significant correlations were found between FPE and clamp-assessed SI ( r s = 0.74), FPE and fasting insulin ( r s = -0.64), and SI and fasting insulin ( r s = -0.67). We also found a wide variance in FPE (22.4-77.2%; mean ± SD 50.4 ± 19.1) that is reflected in the variability of plasma insulin (48.1 ± 30.9 pmol/L) and SI (9.4 ± 5.8 × 10 4 dL · kg -1 · min -1 · [pmol/L] -1 ). FPE could be the nexus of regulation of both plasma insulin and SI., (© 2018 by the American Diabetes Association.)

African Americans (AAs) tend to have higher plasma insulin concentrations than European Americans (EAs); the increased insulin concentrations have been attributed to increased secretion and/or decreased insulin clearance by liver or other tissues. This work characterizes the contributions of hepatic versus extrahepatic insulin degradation related to ethnic differences between AAs and EAs. By using a recently developed mathematical model that uses insulin and C-peptide measurements from the insulin-modified, frequently sampled intravenous glucose tolerance test (FSIGT), we estimated hepatic versus extrahepatic insulin clearance in 29 EA and 18 AA healthy women. During the first 20 min of the FSIGT, plasma insulin was approximately twice as high in AAs as in EAs. In contrast, insulin was similar in AAs and EAs after the 20-25 min intravenous insulin infusion. Hepatic insulin first-pass extraction was two-thirds lower in AAs versus EAs in the overnight-fasted state. In contrast, extrahepatic insulin clearance was not lower in AAs than in EAs. The difference in insulin degradation between AAs and EAs can be attributed totally to liver clearance. The mechanism underlying reduced insulin degradation in AAs remains to be clarified, as does the relative importance of reduced liver clearance to increased risk for type 2 diabetes., (© 2017 by the American Diabetes Association.)

On the basis of studies that investigated the intraportal versus systemic insulin infusion and transendothelial transport of insulin, we proposed the "single gateway hypothesis," which supposes an indirect regulation of hepatic glucose production by insulin; the rate-limiting transport of insulin across the adipose tissue capillaries is responsible for the slow suppression of free fatty acids (FFAs), which in turn is responsible for delayed suppression of hepatic endogenous glucose production (EGP) during insulin infusion. Preventing the fall in plasma FFAs during insulin infusion either by administering intralipids or by inhibiting adipose tissue lipolysis led to failure in EGP suppression, thus supporting our hypothesis. More recently, mice lacking hepatic Foxo1 in addition to Akt1 and Akt2 (L-AktFoxo1TKO), all required for insulin signaling, surprisingly showed normal glycemia. Inhibiting the fall of plasma FFAs in these mice prevented the suppression of EGP during a clamp, reaffirming that the site of insulin action to control EGP is extrahepatic. Measuring whole-body turnover rates of glucose and FFAs in L-AktFoxo1TKO mice also confirmed that hepatic EGP was regulated by insulin-mediated control of FFAs. The knockout mouse model in combination with sophisticated molecular techniques confirmed our physiological findings and the single gateway hypothesis., (© 2017 by the American Diabetes Association.)

Insulin clearance is a highly variable and important factor that affects circulating insulin concentrations. We developed a novel model-based method to estimate both hepatic and extrahepatic insulin clearance using plasma insulin and C-peptide profiles obtained from the insulin-modified frequently sampled intravenous glucose tolerance test. Data from 100 African immigrants without diabetes (mean age 38 years, body weight 81.7 kg, fasting plasma glucose concentration 83 mg/dL, and fasting insulin concentration 37 pmol/L) were used. Endogenous insulin secretion (calculated by C-peptide deconvolution) and insulin infusion rates were used as inputs to a new two-compartment model of insulin kinetics and hepatic and extrahepatic clearance parameters were estimated. Good agreement between modeled and measured plasma insulin profiles was observed (mean normalized root mean square error 6.8%), and considerable intersubject variability in parameters of insulin clearance among individuals was identified (the mean [interquartile range] for hepatic extraction was 25.8% [32.7%], and for extrahepatic insulin clearance was 20.7 mL/kg/min [11.7 mL/kg/min]). Parameters of insulin clearance were correlated with measures of insulin sensitivity and acute insulin response to glucose. The method described appears promising for future research aimed at characterizing variability in insulin clearance and the mechanisms involved in the regulation of insulin clearance., (© 2016 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

The improvement of hepatic insulin sensitivity by the cannabinoid receptor 1 (CB1R) antagonist rimonabant (RIM) has been recently been reported to be due to upregulation of adiponectin. Several studies demonstrated that improvement in insulin clearance accompanies the enhancement of hepatic insulin sensitivity. However, the effects of RIM on hepatic insulin clearance (HIC) have not been fully explored. The aim of this study was to explore the molecular mechanism(s) by which RIM affects HIC, specifically to determine whether upregulation of liver adiponectin receptors (ADRs) and other key genes regulated by adiponectin mediate the effects. To induce insulin resistance in skeletal muscle and liver, dogs were fed a hypercaloric high-fat diet (HFD) for 6 wk. Thereafter, while still maintained on a HFD, animals received RIM (HFD+RIM; n = 11) or placebo (HFD+PL; n = 9) for an additional 16 wk. HIC, calculated as the metabolic clearance rate (MCR), was estimated from the euglycemic-hyperinsulinemic clamp. The HFD+PL group showed a decrease in MCR; in contrast, the HFD+RIM group increased MCR. Consistently, the expression of genes involved in HIC, CEACAM-1 and IDE, as well as gene expression of liver ADRs, were increased in the HFD+RIM group, but not in the HFD+PL group. We also found a positive correlation between CEACAM-1 and the insulin-degrading enzyme IDE with ADRs. Interestingly, expression of liver genes regulated by adiponectin and involved in lipid oxidation were increased in the HFD+RIM group. We conclude that in fat-fed dogs RIM enhances HIC, which appears to be linked to an upregulation of the adiponectin pathway., (Copyright © 2015 the American Physiological Society.)

The endocannabinoid system is highly implicated in the development of insulin resistance associated with obesity. It has been shown that antagonism of the CB(1) receptor improves insulin sensitivity (S(I)). However, it is unknown whether this improvement is due to the direct effect of CB(1) blockade on peripheral tissues or secondary to decreased fat mass. Here, we examine in the canine dog model the longitudinal changes in S(I) and fat deposition when obesity was induced with a high-fat diet (HFD) and animals were treated with the CB(1) antagonist rimonabant. S(I) was assessed (n = 20) in animals fed a HFD for 6 wk to establish obesity. Thereafter, while HFD was continued for 16 additional weeks, animals were divided into two groups: rimonabant (1.25 mg·kg(-1)·day(-1) RIM; n = 11) and placebo (n = 9). Euglycemic hyperinsulinemic clamps were performed to evaluate changes in insulin resistance and glucose turnover before HFD (week -6) after HFD but before treatment (week 0) and at weeks 2, 6, 12, and 16 of treatment (or placebo) + HFD. Magnetic resonance imaging was performed to determine adiposity- related changes in S(I). Animals developed significant insulin resistance and increased visceral and subcutaneous adiposity after 6 wk of HFD. Treatment with RIM resulted in a modest decrease in total trunk fat with relatively little change in peripheral glucose uptake. However, there was significant improvement in hepatic insulin resistance after only 2 wk of RIM treatment with a concomitant increase in plasma adiponectin levels; both were maintained for the duration of the RIM treatment. CB(1) receptor antagonism appears to have a direct effect on hepatic insulin sensitivity that may be mediated by adiponectin and independent of pronounced reductions in body fat. However, the relatively modest effect on peripheral insulin sensitivity suggests that significant improvements may be secondary to reduced fat mass.

Objective: Glucokinase (GCK) acts as a component of the "glucose sensor" in pancreatic β-cells and possibly in other tissues, including the brain. However, >99% of GCK in the body is located in the liver, where it serves as a "gatekeeper", determining the rate of hepatic glucose phosphorylation. Mutations in GCK are a cause of maturity-onset diabetes of the young (MODY), and GCKR, the regulator of GCK in the liver, is a diabetes susceptibility locus. In addition, several GCK activators are being studied as potential regulators of blood glucose. The ability to estimate liver GCK activity in vivo for genetic and pharmacologic studies may provide important physiologic insights into the regulation of hepatic glucose metabolism., Research Design and Methods: Here we introduce a simple, linear, two-compartment kinetic model that exploits lactate and glucose kinetics observed during the frequently sampled intravenous glucose tolerance test (FSIGT) to estimate liver GCK activity (K(GK)), glycolysis (K(12)), and whole body fractional lactate clearance (K(01)). RESULTS To test our working model of lactate, we used cross-sectional FSIGT data on 142 nondiabetic individuals chosen at random from the Finland-United States Investigation of NIDDM Genetics study cohort. Parameters K(GK), K(12), and K(01) were precisely estimated. Median model parameter estimates were consistent with previously published values., Conclusions: This novel model of lactate kinetics extends the utility of the FSIGT protocol beyond whole-body glucose homeostasis by providing estimates for indices pertaining to hepatic glucose metabolism, including hepatic GCK activity and glycolysis rate.

Recent studies in animal and human models have revealed that free fatty acid (FFA) release from adipose tissue is oscillatory. We have shown in our laboratory that these oscillations are controlled by the sympathetic nervous system (SNS). Although FFAs have been shown to directly stimulate glucose production [endogenous glucose production (EGP)] by the liver and to reduce peripheral glucose utilization, whether the specific pattern of FFA release affects glucose metabolism is unknown. The aim of this study was to examine the effects of pulsatile vs. constant infusion of FFA on glucose homeostasis in the canine model. Euglycemic clamps with basal insulin replacement (0.1 mU.kg(-1).min(-1) insulin) were performed in dogs (n = 8) during infusion of saline (SAL) or the medium-chain fatty acid octanoate, which was given by either pulsatile infusion (PUL: 10 mmol over 2 min every 10 min) or continuous infusion (C-INF: 1 mmol/min) designed to achieve equivalent total FFA mass. Endogenous lipolytic pulses were suppressed with the beta(3)-specific adrenergic receptor antagonist bupranolol. PUL infusion elicited a pulsatile pattern of FFA in circulation with average maximum pulse height of 0.82 +/- 0.04 mM, whereas C-INF FFA levels reached 0.47 +/- 0.03 mM (fasting levels) and were maintained throughout. Glucose uptake was not affected by PUL; however, C-INF significantly reduced glucose uptake compared with both SAL and PUL. Steady-state EGP increased by >90% from basal steady state during PUL but did not change during either SAL or C-INF. Thus, pulsatile FFA infusion led to an increase in EGP while preserving glucose disposal. These data suggest that the pattern of FFA may have a role in regulation of glucose homeostasis, which may have consequences in the obese or insulin-resistant state where the SNS is known to be altered.

The full impact of the liver, through both glucose production and uptake, on systemic glucose appearance cannot be readily studied in a classical glucose clamp because hepatic glucose metabolism is regulated not only by portal insulin and glucose levels but also portal glucose delivery (the portal signal). In the present study, we modified the classical glucose clamp by giving exogenous glucose through portal vein, the "portal glucose infusion (PoG)-glucose clamp", to determine the net hepatic effect on postprandial systemic glucose supply along with the measurement of whole body glucose disposal. By comparing systemic rate of glucose appearance (R(a)) with portal glucose infusion rate (PoG(inf)), we quantified "net hepatic glucose addition (NHGA)" in the place of endogenous glucose production determined in a regular clamp. When PoG-glucose clamps (n = 6) were performed in dogs at basal insulinemia and hyperglycemia ( approximately 150 mg/dl, portal and systemic), we measured consistently higher R(a) than PoG(inf) (4.2 +/- 0.6 vs. 2.9 +/- 0.6 mg x kg(-1) x min(-1) at steady state, P < 0.001) and thus positive NHGA at 1.3 +/- 0.1 mg x kg(-1) x min(-1), identifying net hepatic addition of glucose to portal exogenous glucose. In contrast, when PoG-glucose clamps (n = 6) were performed at hyperinsulinemia ( approximately 250 pmol/l) and systemic euglycemia (portal hyperglycemia due to portal glucose infusion), we measured consistently lower R(a) than PoG(inf) (13.1 +/- 2.4 vs. 14.3 +/- 2.4 mg x kg(-1) x min(-1), P < 0.001), and therefore negative NHGA at -1.1 +/- 0.1 mg x kg(-1) x min(-1), identifying a switch of the liver from net production to net uptake of portal exogenous glucose. Steady-state whole body glucose disposal was 4.1 +/- 0.5 and 13.0 +/- 2.4 mg x kg(-1) x min(-1), respectively, determined as in a classical glucose clamp. We conclude that the PoG-glucose clamp, simulating postprandial glucose entry and metabolism, enables simultaneous assessment of the net hepatic effect on postprandial systemic glucose supply as well as whole body glucose disposal in various animal models (rodents, dogs, and pigs) with established portal vein catheterization.

Objective: Recent progress suggests that exenatide, a mimetic of glucagon-like peptide-1 (GLP-1), might lower glycemia independent of increased beta-cell response or reduced gastrointestinal motility. We aimed to investigate whether exenatide stimulates glucose turnover directly in insulin-responsive tissues dependent or independent of insulinemia., Research Design and Methods: An intraportal glucose infusion clamp was used in dogs to measure glucose turnover to encompass potent activation of the putative glucose/GLP-1 sensor in the porto-hepatic circulation with exenatide. The modified glucose clamp was performed in the presence of postprandial hyperinsulinemia and hyperglycemia with exenatide (20 microg) or saline injected at 0 min. Furthermore, the role of hyperglycemia versus hyperinsulinemia in exenatide-mediated glucose disposal was studied., Results: With hyperinsulinemia and hyperglycemia, exenatide produced a significant increase in total glucose turnover by approximately 30%, as indicated by portal glucose infusion rate (saline 15.9 +/- 1.6 vs. exenatide 20.4 +/- 2.1 mg x kg(-1) x min(-1), P < 0.001), resulting from increased whole-body glucose disposal (R(d), approximately 20%) and increased net hepatic uptake of exogenous glucose ( approximately 80%). Reducing systemic hyperglycemia to euglycemia, exenatide still increased total glucose turnover by approximately 20% (saline 13.2 +/- 1.9 vs. exenatide 15.6 +/- 2.1 mg x kg(-1) x min(-1), P < 0.05) in the presence of hyperinsulinemia, accompanied by smaller increments in R(d) (12%) and net hepatic uptake of exogenous glucose (45%). In contrast, reducing hyperinsulinemia to basal levels, exenatide-increased total glucose turnover was completely abolished despite hyperglycemia (saline 2.9 +/- 0.6 vs. exenatide 2.3 +/- 0.3 mg x kg(-1) x min(-1), P = 0.29)., Conclusions: Exenatide directly stimulates glucose turnover by enhancing insulin-mediated whole-body glucose disposal and increasing hepatic uptake of exogenous glucose, contributing to its overall action to lower postprandial glucose excursions.

During insulin resistance, glucose homeostasis is maintained by an increase in plasma insulin via increased secretion and/or decreased first-pass hepatic insulin extraction. However, the relative importance of insulin secretion vs. clearance to compensate for insulin resistance in obesity has yet to be determined. This study utilizes the fat-fed dog model to examine longitudinal changes in insulin secretion and first-pass hepatic insulin extraction during development of obesity and insulin resistance. Six dogs were fed an isocaloric diet with an approximately 8% increase in fat calories for 12 wk and evaluated at weeks 0, 6, and 12 for changes in 1) insulin sensitivity by euglycemic-hyperinsulinemic clamp, 2) first-pass hepatic insulin extraction by direct assessment, and 3) glucose-stimulated insulin secretory response by hyperglycemic clamp. We found that 12 wk of a fat diet increased subcutaneous and visceral fat as assessed by MR imaging. Consistent with increased body fat, the dogs exhibited a approximately 30% decrease in insulin sensitivity and fasting hyperinsulinemia. Although insulin secretion was substantially increased at week 6, beta-cell sensitivity returned to prediet levels by week 12. However, peripheral hyperinsulinemia was maintained because of a significant decrease in first-pass hepatic insulin extraction, thus maintaining hyperinsulinemia, despite changes in insulin release. Our results indicate that when obesity and insulin resistance are induced by an isocaloric, increased-fat diet, an initial increase in insulin secretion by the beta-cells is followed by a decrease in first-pass hepatic insulin extraction. This may provide a secondary physiological mechanism to preserve pancreatic beta-cell function during insulin resistance.

The mechanism by which increased central adiposity causes hepatic insulin resistance is unclear. The "portal hypothesis" implicates increased lipolytic activity in the visceral fat and therefore increased delivery of free fatty acids (FFA) to the liver, ultimately leading to liver insulin resistance. To test the portal hypothesis at the transcriptional level, we studied expression of several genes involved in glucose and lipid metabolism in the fat-fed dog model with visceral adiposity vs. controls (n = 6). Tissue samples were obtained from dogs after 12 wk of either moderate fat (42% calories from fat; n = 6) or control diet (35% calories from fat). Northern blot analysis revealed an increase in the ratio of visceral to subcutaneous (v/s ratio) mRNA expression of both lipoprotein lipase (LPL) and peroxisome proliferator-activated receptor-gamma (PPARgamma). In addition, the ratio for sterol regulatory element-binding transcription factor-1 (SREBP-1) tended to be higher in fat-fed dogs, suggesting enhanced lipid accumulation in the visceral fat depot. The v/s ratio of hormone-sensitive lipase (HSL) increased significantly, implicating a higher rate of lipolysis in visceral adipose despite hyperinsulinemia in obese dogs. In fat-fed dogs, liver SREBP-1 expression was increased significantly, with a tendency for increased fatty acid-binding protein (FABP) expression. In addition, glucose-6-phosphatase (G-6-Pase) and phosphoenolpyruvate carboxykinase (PEPCK) increased significantly, consistent with enhanced gluconeogenesis. Liver triglyceride content was elevated 45% in fat-fed animals vs. controls. Moreover, insulin receptor binding was 50% lower in fat-fed dogs. Increased gene expression promoting lipid accumulation and lipolysis in visceral fat, as well as elevated rate-limiting gluconeogenic enzyme expression in the liver, is consistent with the portal theory. Further studies will need to be performed to determine whether FFA are involved directly in this pathway and whether other signals (either humoral and/or neural) may contribute to the development of hepatic insulin resistance observed with visceral obesity.

Obesity is highly correlated with insulin resistance and the development of type 2 diabetes. Insulin resistance will result in a decrease in insulin's ability to stimulate glucose uptake into peripheral tissue and will suppress glucose production by the liver. However, the development of peripheral and hepatic insulin resistance relative to one another in the context of obesity-associated insulin resistance is not well understood. To examine this phenomena, we used the moderate fat-fed dog model, which has been shown to develop both subcutaneous and visceral adiposity and severe insulin resistance. Six normal dogs were fed an isocaloric diet with a modest increase in fat content for 12 weeks, and they were assessed at weeks 0, 6, and 12 for changes in insulin sensitivity and glucose turnover. By week 12 of the diet, there was a more than twofold increase in trunk adiposity as assessed by magnetic resonance imaging because of an accumulation in both subcutaneous and visceral fat depots with very little change in body weight. Fasting plasma insulin had increased by week 6 (150% of week 0) and remained increased up to week 12 of the study (170% of week 0). Surprisingly, there appeared to be no change in the rates of insulin-stimulated glucose uptake as measured by euglycemic-hyperinsulinemic clamps throughout the course of fat feeding. However, there was an increase in steady-state plasma insulin levels at weeks 6 and 12, indicating a moderate degree of peripheral insulin resistance. In contrast to the moderate defect seen in the periphery, there was a marked impairment in insulin's ability to suppress endogenous glucose production during the clamp such that by week 12 of the study, there was a complete inability of insulin to suppress glucose production. Our results indicate that a diet enriched with a moderate amount of fat results in the development of both subcutaneous and visceral adiposity, hyperinsulinemia, and a modest degree of peripheral insulin resistance. However, there is a complete inability of insulin to suppress hepatic glucose production during the clamp, suggesting that insulin resistance of the liver may be the primary defect in the development of insulin resistance associated with obesity.

It has been proposed that the glycogenolytic and gluconeogenic pathways contributing to endogenous glucose production are interrelated. Thus a change in one source of glucose 6-phosphate might be compensated for by an inverse change in the other pathway. We therefore investigated the effects of 1,4-dideoxy-1,4-imino-D-arabinitol (DAB), a potent glycogen phosphorylase inhibitor, on glucose production in fasted conscious dogs. When dogs were treated acutely with high glucagon, glucose production rose from 1.93 +/- 0.14 to 3.07 +/- 0.37 mg x kg(-1) x min(-1) (P < 0.01). When dogs were treated acutely with DAB in addition to high glucagon infusion, the stimulation of the glycogenolytic rate was completely suppressed. Glucose production rose from 1.85 +/- 0.20 to 2.41 +/- 0.17 mg x kg(-1) x min(-1) (P < 0.05), which was due to the increase in gluconeogenesis from 0.93 +/- 0.09 to 1.54 +/- 0.08 mg x kg(-1) x min(-1) (P < 0.001). In conclusion, infusion of DAB inhibited glycogenolysis; however, the absolute contribution of gluconeogenesis to glucose production was not affected. These results suggest that inhibition of glycogenolysis could be an effective antidiabetic treatment.

Our laboratory has previously demonstrated that hypoglycemic detection occurs in the portal vein, not the liver. To ascertain whether hypoglycemic detection may also occur in the hepatic artery, normoglycemia was established across the liver via a localized hepatic artery glucose infusion. Male mongrel dogs (n = 7) were infused with insulin (5.0 mU x kg(-1) x min(-1)) via the jugular vein to induce systemic hypoglycemia. Animals participated in two hyperinsulinemic-hypoglycemic clamp experiments distinguished by the site of glucose infusion. During the liver irrigation protocol, glucose was infused via the hepatic artery (HA protocol) to maintain liver normoglycemia as systemic glucose concentrations were systematically lowered over 260 min (nadir = 2.2 +/- 0.01 mmol/l). During control experiments, glucose was infused peripherally (PER protocol) to control reductions in blood glucose. Arterial glucose concentrations were not significantly different at any time between the two protocols (P = 0.73). Hepatic artery and liver glucose concentrations were significantly elevated in the HA versus PER protocol throughout the duration of the progressive hyperinsulinemic-hypoglycemic clamp. During the PER protocol, epinephrine and norepinephrine concentrations increased significantly above basal values (0.53 +/- 0.06 and 0.85 +/- 0.2 nmol/l, respectively) to plateaus of 4.4 +/- 0.86 (P = 0.0001) and 3.6 +/- 0.69 nmol/l (P = 0.001), respectively. There were no significant differences between the two protocols in the epinephrine (P = 0.81) and the norepinephrine (P = 0.68) response to hypoglycemia. The current findings indicate that glucosensors important to hypoglycemic detection do not reside in the hepatic artery. Furthermore, these data confirm our previous findings that glucosensors important to hypoglycemic detection are not present in the liver, but are in fact localized to the portal vein.

Troglitazone is an antidiabetic agent that improves the ability of adipocytes to store triglycerides by enhancing their insulin sensitivity. Although potent in insulin-resistant states, the effect of troglitazone on lipid and glucose turnover in normal animals has not been assessed. Euglycemic clamps were performed as an insulin dose response in normal mongrel dogs (n = 6). Somatostatin was infused without hormone replacement (zero insulin) for 90 min. Insulin was then either portally replaced (1.8 pmol x min(-1) x kg(-1), overreplaced (5.4 pmol x min(-1) x kg(-1)), or overreplaced peripherally to match the systemic levels of the portal overreplacement dose (2.3 pmol x min(-1) x kg(-1)) for 180 min. A total of 600 mg troglitazone was then given orally each day for 3 weeks and continued throughout a second experimental phase, at which point the euglycemic clamps were repeated. In concordance with previous studies, endogenous glucose production (EGP) was similar whether insulin was delivered portally or peripherally, both before and during troglitazone treatment. Although free fatty acids (FFAs) at zero insulin were not affected, there was a leftward shift of the insulin-FFA dose response curve secondary to a suppression of FFA release into plasma. EGP was paradoxically elevated by troglitazone treatment because of an elevation of both gluconeogenesis and glycogenolysis. In conclusion, troglitazone reduced hepatic sensitivity to FFAs. Because EGP is a primary determinant of fasting blood glucose, we hypothesize that a protective mechanism exists in normal animals, preventing hypoglycemia during insulin sensitization with troglitazone.

Aims/hypothesis: Insulin control of glucose output is a major mechanism by which appropriate amounts of glucose are produced to supply energy to the central nervous system, without causing long-term increases of the plasma glucose concentration. It is hypothesised that the primary route by which insulin maintains control over glucose production is indirect and is mediated by regulation of non-esterified fatty acid release from the adipocyte. The question arises as to why evolution has chosen insulin to be secreted into the portal vein, if control of the liver is partially or primarily indirect. It is suggested that alterations in hepatic insulin clearance which attend increases in central adiposity are an important part of the compensation for insulin resistance and limit the necessity for up-regulation of insulin secretion in insulin resistance secondary to central adiposity., Methods: Review of research from author's group and other laboratories., Results: Data over the previous decade indicate that suppression of glucose output by increased insulin is a relatively slow process, much slower than the rate of binding of insulin to hepatocytes. One explanation is that insulin acts on an extrahepatic tissue, which in turn alters a signal to the liver, reducing glucose output. Additional evidence for an extrahepatic primary effect of insulin emerges from experiments in which insulin was given portally or peripherally at half the portal dose. Endogenous glucose production was related to systemic, not portal insulin, supporting the concept that the primary step in insulin's action on liver is on some other tissue, altering signalling to the liver itself. Strong correlation between plasma non-esterified fatty acids (NEFA) and liver glucose output suggests that the primary effect is on the adipocyte. The primacy of the adipocyte locus for the insulin effect included data that insulin's action on liver is prevented when plasma NEFA are maintained, as well as data showing proportional decline in glucose production and fatty acids when antilipolysis is induced by an adenosine agonist. Why then, from an evolutionary point of view is insulin secreted into the portal vein? Institution of central adiposity in dogs with fat feeding causes hepatic insulin resistance, at least partially due to the provision of NEFA in portal blood. The initial response to resistance is enhanced beta-cell sensitivity to glucose; a secondary compensation is, however, a substantial reduction in liver clearance, allowing for a greater proportion of secreted insulin to reach muscle, where it can more efficiently stimulate glucose utilisation., Conclusion/interpretation: Non-esterified fatty acids act as a signal as well as a metabolic substrate. They can regulate glucose utilisation in muscle and apparently are important signals to the liver and the beta cells as well. The importance of portal vein NEFA concentrations to the function of the liver could explain insulin resistance of the liver with central pattern obesity.

The combined model approach uses kinetic analysis of both plasma insulin and C-peptide dynamics to estimate prehepatic insulin secretion rates and parameters of insulin and C-peptide kinetics. The original model used single-compartment kinetics to describe both insulin and C-peptide despite knowledge that C-peptide follows two-compartment kinetics. The performance of the model under rapidly changing secretory conditions has come into question. Thus a more complex combined model is introduced, incorporating two-compartmental C-peptide disappearance. The addition of two-compartment C-peptide kinetics required a novel numerical approach to allow estimation of model parameters. This simulation study was undertaken to 1) compare the performance of the original combined model and 2) examine the numerical method used to identify parameters for the extended combined model with two-compartment C-peptide kinetics under simulated conditions of rapidly changing insulin and C-peptide. Monte Carlo simulation revealed that the original combined model does not provide accurate estimates of prehepatic insulin secretion under rapid kinetics. However, the extended combined model provides accurate reconstruction of prehepatic insulin secretory profile without separate quantification of C-peptide kinetics.

Suppression of hepatic glucose output (HGO) has been shown to be primarily mediated by peripheral rather than portal insulin concentrations; however, the mechanism by which peripheral insulin suppresses HGO has not yet been determined. Previous findings by our group indicated a strong correlation between free fatty acids (FFA) and HGO, suggesting that insulin suppression of HGO is mediated via suppression of lipolysis. To directly test the hypothesis that insulin suppression of HGO is causally linked to the suppression of adipose tissue lipolysis, we performed euglycemic-hyperinsulinemic glucose clamps in conscious dogs (n = 8) in which FFA were either allowed to fall or were prevented from falling with Liposyn plus heparin infusion (LI; 0.5 ml/min 20% Liposyn plus 25 U/min heparin with a 250 U prime). Endogenous insulin and glucagon were suppressed with somatostatin (1 microgram/min/kg), and insulin was infused at a rate of either 0.125 or 0.5 mU/min/kg. Two additional experiments were performed at the 0.5 mU/min/kg insulin dose: a double Liposyn infusion (2 x LI; 1.0 ml/min 20% Liposyn, heparin as above), and a glycerol infusion (19 mg/min). With the 0.125 mU/min/kg insulin infusion, FFA fell 40% and HGO fell 33%; preventing the fall in FFA with LI entirely prevented this decline in HGO. With 0.5 mU/min/kg insulin infusion, FFA levels fell 64% while HGO declined 62%. Preventing the fall in FFA at this higher insulin dose largely prevented the fall in HGO; however, steady state HGO still declined by 18%. Doubling the LI infusion did not further affect HGO, suggesting that the effect of FFA on HGO is saturable. Elevating plasma glycerol levels did not alter insulin's ability to suppress HGO. These data directly support the concept that insulin suppression of HGO is not direct, but rather is mediated via insulin suppression of adipose tissue lipolysis. Thus, resistance to insulin control of hepatic glucose production in obesity and/or non-insulin-dependent diabetes mellitus may reflect resistance of the adipocyte to insulin suppression of lipolysis.

Overproduction of glucose by the liver in the face of insulin resistance is a primary cause of hyperglycemia in non-insulin-dependent diabetes mellitus (NIDDM). However, mechanisms involved in control of hepatic glucose output (HGO) remain less than clear, even in normal individuals. Recent results have supported an indirect extrahepatic effect of insulin as the primary locus of insulin action to restrain HGO. One suggested extrahepatic site is the pancreatic alpha-cell. To examine whether insulin's extrahepatic site is independent of the alpha-cells, HGO suppression was examined independent of changes in glucagon secretion or insulin antagonism of glucagon action. Euglycemic glucose clamps (n = 40) with somatostatin infusion were performed in conscious dogs (n = 5). Paired experiments were conducted in which insulin was infused either portally (1.2, 3.0, 6.0 pmol.min-1.kg-1) or peripherally at half the portal infusion rate (0.6, 1.5, 3.0 pmol.min-1.kg-1). Additional zero and saturating portal-dose experiments (100 pmol.min-1.kg-1) were also performed. For the paired experiments, portal insulin infusion resulted in portal insulin concentrations approximately two to three times higher than in the corresponding peripheral insulin infusion experiments, while at the same time peripheral insulin concentrations were approximately matched. Equal peripheral insulin concentration resulted in equivalent HGO suppression irrespective of the portal concentrations. Thus, insulin affects a signal at a peripheral site, other than alpha-cell, that in turn suppresses hepatic glucose production. To investigate the nature of this signal, we measured alanine, lactate, and free fatty acids (FFAs).(ABSTRACT TRUNCATED AT 250 WORDS)

The significance of the portohepatic glucosensors for counterregulation in deep hypoglycemia (i.e., glycemia < 2.8 mM) was studied in chronically cannulated male mongrel dogs in the conscious state. A total of 16 experiments were carried out on 6 dogs using the liver clamp technique under hyperinsulinemic conditions (insulin infusion, 39 pmol.min-1.kg-1, 0-150 min). The level of glycemia presented to the liver was made to differ from the systemic arterial glucose level via portal glucose infusion. Tracer-determined rates of glucose clearance and hepatic glucose output (HGO) were assessed using D-[3-3H]glucose (0.26 microCi.min-1). Three protocols were used. In protocol I, liver clamp, systemic hypoglycemia at 2.60 +/- 0.09 mM, and liver glycemia at 3.86 +/- 0.05 mM were achieved with portal glucose infusion (28.2 +/- 3.0 mumol.min-1.kg-1). For protocol II, glucose was infused peripherally (18.2 +/- 4.3 mumol.min-1.kg-1), while systemic and liver glycemia were sustained at deep hypoglycemia, 2.50 +/- 0.08 mM. In protocol III, via peripheral glucose infusion (62.9 +/- 5.8 mumol.min-1.kg-1), systemic and liver glycemia were maintained at a level matched to the liver glycemia during protocol I (3.98 +/- 0.05 mM, P > 0.10). When compared with protocols I and III, the catecholamine response above basal was significantly greater during protocol II with liver and systemic deep hypoglycemia (7.30 +/- 1.51 and 2.89 +/- 0.5 nM for epinephrine and norepinephrine, respectively, P < 0.005). These values reflect net increases in the catecholamine responses of 100% and 85% for epinephrine and norepinephrine when compared with protocol I.(ABSTRACT TRUNCATED AT 250 WORDS)

The impact of hepatic glucose concentration on the sympathetic response to progressive hypoglycemia was examined in chronically cannulated conscious male dogs (n = 6). Graded hypoglycemia was induced via peripheral insulin infusion (30 pmol.kg-1.min-1) with either peripheral (PER) or portal (POR) glucose infusion. Over the 260-min experimental period, arterial glycemia was adjusted from 5.2 +/- 0.1 to 2.5 +/- 0.1 mM in decrements of approximately 0.5 mM every 40 min. Arterial glycemias were not significantly different between PER and POR at any measured level. However, hepatic glycemia was significantly elevated at all times during POR (8.4 +/- 0.8 to 3.4 +/- 0.2 mM) when compared to PER (5.2 +/- 0.2 to 2.5 +/- 0.1 mM). Plasma epinephrine values were significantly greater during PER vs. POR at all arterial glycemias below 4.0 mM. At the lowest level of arterial glycemia studied (2.5 +/- 0.2 mM) the epinephrine response above basal was 3-fold greater for PER (8.7 +/- 1.7 nM) when compared to POR (2.6 +/- 0.6 nM) (P < 0.01). Plasma norepinephrine results were similar for the two protocols, with PER demonstrating a 3-fold greater response above basal when compared to POR at 2.5 mM arterial glycemia (P < 0.05). While the sympathetic response was markedly different between protocols when expressed as a function of arterial glycemia, when expressed as a function of hepatic glycemia this discrepancy was largely eliminated. This latter observation supports the liver as the primary locus for glycemic detection relevant to the sympathoadrenal response when hypoglycemia develops slowly--i.e., over a period of 2-3 h. A comparison of the current findings with our previous observations suggests that the hepatic glucosensors may play a greater role in hypoglycemic counterregulation as the rate of fall in glycemia is less.

This study compares the dynamics and sensitivity of hepatic and peripheral insulin action in conscious dogs. Glucose turnover was measured simultaneously by HOT GINF tracer methodology and by hepatic AV differences. SRIF was infused during euglycemic clamps to suppress endogenous insulin and glucagon secretion. Basal plasma glucagon levels were recreated by intraportal replacement (2 ng.min-1 x kg-1), and insulin was infused intraportally at 0, 3, 6, 10, or 20 pmol.min-1 x kg-1 for 3 h. Steady-state HGO and NHGO were suppressed by insulin with EC50s of 164 and 95 pM, respectively. As expected, these were lower than the EC50 for Rd stimulation (516 pM), demonstrating greater hepatic than peripheral insulin sensitivity. In contrast to sensitivity, dynamics for suppression of HGO and NHGO and for stimulation of Rd by insulin were indistinguishable: half-times averaged 43 +/- 5, 42 +/- 9, and 45 +/- 5 min, respectively (P > 0.77). For all three variables, the half-time of insulin effect was independent of insulin dose (P > 0.26). The striking similarity of the time courses for suppression of glucose production and stimulation of glucose uptake suggests that both effects are secondary manifestations of a single, rate-limiting phenomenon. We hypothesize this single gateway to insulin action is transendothelial insulin transport, which we previously have shown to be rate limiting for insulin's effect on glucose uptake in vivo.

We assessed hepatic glucagon sensitivity in overnight-fasted, conscious dogs. Six pancreatic replacement protocols were performed in each of five animals. Somatostatin was infused to inhibit endogenous insulin and glucagon, insulin was replaced intraportally at 200 microU.min-1.kg-1, and glucagon was infused intraportally at 0, 0.6, 1, 2, 5, or 20 ng.min-1.kg-1. One intravenous glucose tolerance test was also performed in each animal for measurement of insulin sensitivity (SI). During hormone replacement at a given glucagon dose, plasma glucose differed substantially among animals (P = 0.003). Therefore the dose required for restoration of euglycemia ("glucagon requirement") varied nearly sevenfold among animals, suggesting appreciable differences in glucagon sensitivity (GS). The latter was quantitated in individual animals as the initial slope of integrated glucose output vs. glucagon concentration. GS varied from 0.22 to 3.9 mg.kg-1.pg-1.ml among various animals and was inversely and significantly related to glucagon requirement. SI varied less (approximately 4-fold) and was not associated with glucagon requirement. These observations suggested that interanimal differences in glucose during hormone replacement were the result of substantial differences in GS. In addition, we found the GS of a given animal to be highly associated (P = 0.01) with its fasting glucose level. We conclude that GS varies substantially, and as such may be an important determinant of the fasting glucose level in normal animals.

To ascertain whether hepatic glucoreceptors are important to hypoglycemic counterregulation, a localized euglycemic clamp was employed across the liver during general hypoglycemia. Dogs were infused peripherally with insulin (18-21 pmol.kg-1.min-1) for 150 min to induce systemic hypoglycemia. During the liver-clamp (LC) protocol, glucose was infused via the portal vein to maintain euglycemia at the liver. In control experiments, i.e., matched infusion (MI), glucose was infused peripherally at a rate determined to yield similar arterial glycemia levels in the two protocols. Arterial glucose concentrations were not different between protocols during the final hour of insulin infusion (3.26 +/- 0.21 and 3.25 +/- 0.21 mM during LC and MI, respectively; P = 0.91). Calculated hepatic glucose concentrations during the same period were significantly higher for LC (5.22 +/- 0.23 mM) than for MI (3.25 +/- 0.21 mM). During MI, both epinephrine and norepinephrine rose significantly from basal values of 562 +/- 87 pM and 1.21 +/- 0.19 nM to plateaus of 3691 +/- 1097 pM (P = 0.0001) and 2.38 +/- 0.35 nM (P = 0.0002), respectively. However, during LC, the elevation in epinephrine was suppressed by 42 +/- 8% (P = 0.015) relative to MI. Six of seven animals demonstrated a suppression in the norepinephrine response, averaging 32 +/- 13% (NS, P = 0.068). The glucagon response to hypoglycemia was unaffected by the level of hepatic glycemia. Hepatic hypoglycemia is essential to produce the full sympathoadrenal response to insulin-induced hypoglycemia.

After a meal or glucose load, most carbons of hepatic glycogen are derived from gluconeogenesis. In vitro, hepatic glycogen accumulation is sluggish with glucose alone but markedly enhanced in the presence of gluconeogenic substrates. These findings conflict with the classical view that glucose is the major precursor of hepatic glycogen and have been termed the "glucose paradox." In this review, we attempt to elucidate the central mechanism underlying the glucose paradox by critically examining the in vitro data of hepatic glycogen accumulation. Our analysis is inconsistent with the current hypothesis that glucose phosphorylation is rate limiting for hepatic glycogen accumulation from glucose and that gluconeogenesis enhances glycogen accumulation primarily by increasing substrate flux to the hepatic glucose 6-phosphate pool. Instead, our analysis leads us to the conclusion that the rate-limiting step is the net incorporation of glucose 6-phosphate into glycogen, which is synergistically facilitated with glucose and gluconeogenic substrates. Thus gluconeogenic substrates are involved in the regulation of key enzyme(s) of glycogen metabolism. In addition, in the livers from fasted rats there is substantial cycling through glycogen, and that suppression of glycogen degradation may be a major mechanism in the enhancement of glycogen accumulation by gluconeogenic substrates. Thus we propose a specific hypothesis of the role of gluconeogenic substrates in glycogen metabolism (i.e., inhibition of phosphorylase), which can be tested by future studies.

Insulin may suppress hepatic glucose production directly, or indirectly via suppression of release of gluconeogenic substrates from extrasplanchnic tissues. To compare these mechanisms, we performed insulin dose-response experiments in conscious dogs at euglycemia, during somatostatin infusion, and intraportal glucagon replacement. Insulin was sequentially infused either intraportally (0.05, 0.20, 0.40, 1.0, 1.4, and/or 3.0; protocol I) or systemically at half the intraportal rate (0.025, 0.10, 0.20, 0.50, 0.70, and/or 1.5 mU.min-1.kg-1; protocol II). Exogenous glucose infused during clamps was labeled with 3-[3H]glucose (2 microCi/g) to prevent a fall in plasma specific activity (P greater than 0.2) that may have contributed to previous underestimations of hepatic glucose output (HGO). Portal insulins were up to threefold higher during intraportal infusion, but peripheral insulin levels were not different between the intraportal and systemic protocols [7 +/- 5 vs. 9 +/- 1, 12 +/- 4 vs. 13 +/- 6, 16 +/- 3 vs. 27 +/- 5, 70 +/- 23 vs. 48 +/- 8, 83 +/- 3 vs. 86 +/- 21, and 128 vs. 120 +/- 14 microU/ml for paired insulin doses; P greater than 0.06 by analysis of variance (ANOVA)]. Despite higher portal insulin levels in protocol I, HGO suppression was equivalent in the two protocols when systemic insulin was matched, from 3.3 +/- 0.1 to near-total suppression at 0.3 mg.min-1.kg-1 at the highest insulin infusion rate (3.0 mU.min-1.kg-1; P less than 0.0001) with intraportal insulin, from 2.9 +/- 0.8 to -0.8 +/- 0.2 mg.min-1.kg-1 in protocol II (P less than 0.001). Suppression of HGO was similar at matched systemic insulin, regardless of portal insulin, suggesting the primacy of insulin's action on the periphery in its restraint of hepatic glucose production.

These studies were designed to test whether a putative gastrointestinal factor (separate from that stimulating insulin release) is involved in the enhancement of liver glycogen storage during oral glucose ingestion. To do this, we compared net hepatic glucose uptake in conscious dogs, following oral glucose administration, with hepatic uptake during intraportal glucose infusion. The rate of intraportal glucose infusion was calculated to match the time course of gut glucose absorption in the oral administration experiments. In control studies, intragastric instillation of tap water [90 +/- 2.4 (SE) in four dogs had no effect on basal rates of gastrointestinal (GI) glucose uptake (16 +/- 1 mg/min) oe hepatic glucose production (97 +/- 3 mq/min). Net basal GI lactate production was equal to GI glucose utilization (P greater than 0.1); glycolytic conversion of glucose to lactate accounted for all basal GI glucose utilization. In oral experiments, gastric instillation of 1.2 g/kg glucose (N = 8) caused GI glucose absorption to increase within 5 min (P less than 0.01). Net glucose absorption from the gut was maximal (355 1.55 mg/min) at 60 min, and was complete at 165-240 min (mean = 186 min). During absorption, liver switched from production to net uptake by 30 min (P less than 0.01); production was resumed by 3 h. Total glucose taken up by liver was 7.19 +/- 1.8 g (23% oral load). No net metabolism of the instilled glucose to lactate occurred during absorption; GI lactate production was the same during absorption (13.0 +/- 5.0 mg/min) as before glucose instillation (11.2 +/- 2.0 mg/min; P greater than 0.45). In intraportal experiments, intraportal glucose infusion (total = 1.09 g/kg) induced liver to take up glucose by 15 min (P less than 0.01); total hepatic uptake (4.6 +/- 1.5 g) was not significantly different from the oral experiments (P greater than 0.15). Also, nonsplanchnic glucose uptake was the same in the oral (25.1 +/- 2.2) and intraportal (25.2 +/- 1.4) studies. The lack of difference between hepatic and extrahepatic fates of administered glucose with oral and intraportal administration indicates that no putative gut factor need be invoked to explain hepatic glycogen deposition during oral glucose, and it seems probable that no such factor exists in the dog.

Metabolic fluxes (Ra and Rd) are calculated in the nonsteady state using Steele's equations. These calculations require estimates of the values and rates of change of glucose and specific activity at discrete sampling times. Data smoothing minimizes the effect of measurement error that confounds the Ra and Rd calculations. We compared three smoothing methods: a) moving average, b) polynomial fitting, and c) optimal segments, a new technique that utilizes optimization methods. Experimental designs were simulated: 1) constant infusion of glucagon in depancreatized dogs, and 2) glucose oscillations generated by constant high-level glucose infusion. Measurement error was added to raw data. After smoothing, fluxes were calculated and compared to the "actual" Ra and Rd. Ra calculated from unsmoothed noisy data were in error by an average 39%. Error was reduced by smoothing to: 23%, moving average; 18%, polynomial fitting; 15%, optimal segments. Optimal segments was best for calculating Ra (P less than 0.01) and was better than or equal to other methods for Rd. Distortion in flux patterns was greatest for polynomial fitting (P less than 0.01) and least for optimal segments (P less than 0.001). Optimal segments is the method of choice for smoothing tracer data; it improves calculations of Ra and Rd with minimal distortion.

A previously introduced method by which prehepatic beta-cell secretion is calculated in vivo from plasma measurements of insulin and C-peptide was applied to data derived from iv glucose tolerance tests performed in normal women. Prehepatic secretory rates calculated using the combined model appeared biphasic in nature after glucose injection. Basal insulin secretion was 63.9 +/- 9.8 pmol/min. The duration of first phase was approximately 5 min, with secretion reaching a peak of 2033 +/- 342 pmol/min. The first phase was followed by a significant refractory period in which the secretory rate fell below basal values. The magnitude of second phase secretion was small relative to first phase secretion and appeared pulsatile in nature. Total integrated insulin secretion was 22.2 +/- 2.7 nmol, of which first phase accounted for 32%, and second phase accounted for the remaining 68%. Total incremental integrated secretion was 10.6 +/- 1.4 nmol, accounting for approximately half of the total insulin secretion. Proportions of first and second phase secretion changed to 66.5% and 33.5%, respectively, with incremental data. This study shows that the combined model of insulin and C-peptide is capable of estimating prehepatic insulin secretion from the iv glucose tolerance test and may provide a useful tool to measure secretion in vivo under various pathological conditions.

Incorporation of Glc and Fru into glycogen was measured in perfused livers from 24-h fasted rats using [6-3H]Glc and [U-14C]Fru. For the initial 20 min, livers were perfused with low Glc (2 mM) to deplete hepatic glycogen and were perfused for the following 30 min with various combinations of Glc and Fru. With constant Fru (2 mM), increasing perfusate Glc increased the relative contribution of Glc carbons to glycogen (7.2 +/- 0.4, 34.9 +/- 2.8, and 59.1 +/- 2.7% at 2, 10, and 20 mM Glc, respectively; n = 5 for each). During perfusion with substrate levels seen during refeeding (10 mM Glc, 1.8 mumol/g/min gluconeogenic flux from 2 mM Fru), Fru provided 54.7 +/- 2.7% of the carbons for glycogen, while Glc provided only 34.9 +/- 2.8%, consistent with in vivo estimations. However, the estimated rate of Glc phosphorylation was at least 1.10 +/- 0.11 mumol/g/min, which exceeded by at least 4-fold the glycogen accumulation rate (0.28 +/- 0.04 mumol of glucose/g/min). The total rate of glucose 6-phosphate supply via Glc phosphorylation and gluconeogenesis (2.9 mumol/g/min) exceeded reported in vivo rates of glycogen accumulation during refeeding. Thus, in perfused livers of 24-h fasted rats there is an apparent redundancy in glucose 6-phosphate supply. These results suggest that the rate-limiting step for hepatic glycogen accumulation during refeeding is located between glucose 6-phosphate and glycogen, rather than at the step of Glc phosphorylation or in the gluconeogenic pathway.

The rates of storage and release of carbohydrate by the liver are determined by the plasma concentrations of several blood-borne signals; most important are the concentrations of glucose, and of the hormones insulin and glucagon. To understand the complex control relationships of these three signals as they affect the liver, their individual dynamic influences have been determined experimentally, and the findings have been integrated by means of a computer simulation of the pathways of hepatic glycogen metabolism. The simulation studies have led to specific hypotheses about the biochemical effects of glucose and insulin on the liver. The simulation studies have also led to the conclusion that glucose exerts a rapid moment-to-moment influence on the rate of uptake of glucose by the liver. Insulin, however, by exerting a slower influence on the sensitivity of the liver to glucose, is very effective in "optimizing" the amount of glycogen which the liver stores during food intake. Thus, integrated experimental and simulation studies can lead to a view of a physiological regulating system which does not emerge from either approach used alone.-

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.