Glucagon-stimulated Glucose Production Research Articles

197-OR: Glucagon Receptor Agonism Stimulates Time-Dependent Glycemic Regulation

Historically, glucagon is proposed to be a key factor in the development of diabetes. However, we demonstrated that while chronic glucagon receptor (GCGR) agonism induces hyperglycemia and glucose intolerance, it also improves insulin secretion and insulin action. These paradoxical actions suggest the mechanisms regulating GCGR-mediated glucose metabolism have yet to be fully elucidated. Glucose metabolism and glucagon-stimulated glucose production are both regulated in a circadian manner. Thus, we hypothesized that chronic GCGR signaling regulates glycemia in a time-of-day dependent manner. We treated lean mice for 4d with vehicle or the GCGR agonist IUB288 administered either during the early (ZT 3) or late (ZT9) light period. Ad libitum glycemia was assessed every 4h over a 24h period after the last injection. Independent of the time injected, IUB288-treated mice displayed improved glycemia 4h after the last injection and hyperglycemia after 12h, compared to vehicle-treated controls. This suggests that chronic GCGR agonism is not exclusively hyperglycemic and variations in GCGR-stimulated glycemia are dependent on the time after treatment, rather than the time of day. Interestingly, ad libitum insulin levels were no different between IUB288 and vehicle-treated mice, suggesting differences in insulin sensitivity. Surprisingly, 4d IUB288 treatment in diet-induced obese (DIO) mice reduced ad libitum glycemia for a greater duration than chow IUB288-treated mice. We used hyperinsulinemic-euglycemic clamps to further dissect the mechanisms of these benefits. In DIO mice, IUB288 treatment increased glucose infusion rate, suppression of endogenous glucose production, and phosphorylation of AKT (S473 and T308) . Together, these data increase our understanding of GCGR pharmacology and its therapeutic role in diabetes and obesity treatment. Disclosure S.Nason: None. T.Kim: None. J.P.Antipenko: None. B.Finan: Employee; Novo Nordisk. R.Dimarchi: None. K.M.Habegger: Consultant; Glyscend Inc., Research Support; Eli Lilly and Company, Novo Nordisk, Stock/Shareholder; Glyscend Inc. Funding T32 AI007051

Lysophosphatidic acid counteracts glucagon-induced hepatocyte glucose production via STAT3

Hepatic glucose production (HGP) is required to maintain normoglycemia during fasting. Glucagon is the primary hormone responsible for increasing HGP; however, there are many additional hormone and metabolic factors that influence glucagon sensitivity. In this study we report that the bioactive lipid lysophosphatidic acid (LPA) regulates hepatocyte glucose production by antagonizing glucagon-induced expression of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK). Treatment of primary hepatocytes with exogenous LPA blunted glucagon-induced PEPCK expression and glucose production. Similarly, knockout mice lacking the LPA-degrading enzyme phospholipid phosphate phosphatase type 1 (PLPP1) had a 2-fold increase in endogenous LPA levels, reduced PEPCK levels during fasting, and decreased hepatic gluconeogenesis in response to a pyruvate challenge. Mechanistically, LPA antagonized glucagon-mediated inhibition of STAT3, a transcriptional repressor of PEPCK. Importantly, LPA did not blunt glucagon-stimulated glucose production or PEPCK expression in hepatocytes lacking STAT3. These data identify a novel role for PLPP1 activity and hepatocyte LPA levels in glucagon sensitivity via a mechanism involving STAT3.

The Identification of Novel Protein-Protein Interactions in Liver that Affect Glucagon Receptor Activity.

Glucagon regulates glucose homeostasis by controlling glycogenolysis and gluconeogenesis in the liver. Exaggerated and dysregulated glucagon secretion can exacerbate hyperglycemia contributing to type 2 diabetes (T2D). Thus, it is important to understand how glucagon receptor (GCGR) activity and signaling is controlled in hepatocytes. To better understand this, we sought to identify proteins that interact with the GCGR to affect ligand-dependent receptor activation. A Flag-tagged human GCGR was recombinantly expressed in Chinese hamster ovary (CHO) cells, and GCGR complexes were isolated by affinity purification (AP). Complexes were then analyzed by mass spectrometry (MS), and protein-GCGR interactions were validated by co-immunoprecipitation (Co-IP) and Western blot. This was followed by studies in primary hepatocytes to assess the effects of each interactor on glucagon-dependent glucose production and intracellular cAMP accumulation, and then in immortalized CHO and liver cell lines to further examine cell signaling. Thirty-three unique interactors were identified from the AP-MS screening of GCGR expressing CHO cells in both glucagon liganded and unliganded states. These studies revealed a particularly robust interaction between GCGR and 5 proteins, further validated by Co-IP, Western blot and qPCR. Overexpression of selected interactors in mouse hepatocytes indicated that two interactors, LDLR and TMED2, significantly enhanced glucagon-stimulated glucose production, while YWHAB inhibited glucose production. This was mirrored with glucagon-stimulated cAMP production, with LDLR and TMED2 enhancing and YWHAB inhibiting cAMP accumulation. To further link these interactors to glucose production, key gluconeogenic genes were assessed. Both LDLR and TMED2 stimulated while YWHAB inhibited PEPCK and G6Pase gene expression. In the present study, we have probed the GCGR interactome and found three novel GCGR interactors that control glucagon-stimulated glucose production by modulating cAMP accumulation and genes that control gluconeogenesis. These interactors may be useful targets to control glucose homeostasis in T2D.

Activation of cAMP Signaling Attenuates Impaired Hepatic Glucose Disposal in Aged Male p21-Activated Protein Kinase-1 Knockout Mice

p21-activated protein kinase-1 (Pak1) plays a role in insulin secretion and glucagon-like peptide-1 (GLP-1) production. Pak1(-/-) mice were found to carry a defect in ip pyruvate tolerance test (IPPTT), leading us to speculate whether Pak1 represses hepatic gluconeogenesis. We show here that the defect in IPPTT became more severe in aged Pak1(-/-) mice. In primary hepatocytes, 2,2'-dihydroxy-1,1'-dinaphthyldisulfide, a potent inhibitor of group I Paks, reduced basal glucose production (GP), attenuated forskolin- or glucagon-stimulated GP, and attenuated the stimulation of forskolin on the expression of Pck1 and G6pc. In addition, the capacity of primary hepatocytes isolated from Pak1(-/-) mice in GP at the basal level is significantly lower than that of the control littermates. These in vitro observations imply that the direct effect of Paks in hepatocytes is the stimulation of gluconeogenesis and that the impairment in IPPTT in Pak1(-/-) mice is due to the lack of Pak1 elsewhere. Consecutive ip injection of forskolin for 2 weeks increased gut proglucagon expression, associated with improved IPPTT in aged Pak1(-/-) mice and wild-type controls. In addition, administration of the DPP-IV (dipeptidyl peptidase-4) inhibitor sitagliptin for 1 week reversed the defect in IPPTT in aged Pak1(-/-) mice, associated with increased plasma GLP-1 levels. Our observations indicate a potential role of Pak1 in the gut/pancreas/liver axis in controlling glucose disposal and affirmed the therapeutic application of GLP-1 and DPP-IV inhibitors in attenuating hepatic gluconeogenesis.

Inhibitory Mechanism of an Allosteric Antibody Targeting the Glucagon Receptor

Elevated glucagon levels and increased hepatic glucagon receptor (GCGR) signaling contribute to hyperglycemia in type 2 diabetes. We have identified a monoclonal antibody that inhibits GCGR, a class B G-protein coupled receptor (GPCR), through a unique allosteric mechanism. Receptor inhibition is mediated by the binding of this antibody to two distinct sites that lie outside of the glucagon binding cleft. One site consists of a patch of residues that are surface-exposed on the face of the extracellular domain (ECD) opposite the ligand-binding cleft, whereas the second binding site consists of residues in the αA helix of the ECD. A docking model suggests that the antibody does not occlude the ligand-binding cleft. We solved the crystal structure of GCGR ECD containing a naturally occurring G40S mutation and found a shift in the register of the αA helix that prevents antibody binding. We also found that alterations in the αA helix impact the normal function of GCGR. We present a model for the allosteric inhibition of GCGR by a monoclonal antibody that may form the basis for the development of allosteric modulators for the treatment of diabetes and other class B GPCR-related diseases.

Effects of small interfering RNA-mediated hepatic glucagon receptor inhibition on lipid metabolism in db/db mice

Hepatic glucose overproduction is a major characteristic of type 2 diabetes. Because glucagon is a key regulator for glucose homeostasis, antagonizing the glucagon receptor (GCGR) is a possible therapeutic strategy for the treatment of diabetes mellitus. To study the effect of hepatic GCGR inhibition on the regulation of lipid metabolism, we generated siRNA-mediated GCGR knockdown (si-GCGR) in the db/db mouse. The hepatic knockdown of GCGR markedly reduced plasma glucose levels; however, total plasma cholesterol was increased. The detailed lipid analysis showed an increase in the LDL fraction, and no change in VLDL HDL fractions. Further studies showed that the increase in LDL was the result of over-expression of hepatic lipogenic genes and elevated de novo lipid synthesis. Inhibition of hepatic glucagon signaling via siRNA-mediated GCGR knockdown had an effect on both glucose and lipid metabolism in db/db mice.

In vitro inhibition of α-glucosidases and glycogen phosphorylase by catechin gallates in green tea

We investigated in vitro inhibition of mammalian carbohydrate-degrading enzymes by green tea extract and the component catechins, and further evaluated their inhibitory activities in cell cultures. The extract showed good inhibition toward rat intestinal maltase and rabbit glycogen phosphorylase (GP) b, with IC 50 values of 45 and 7.4 μg/ml, respectively. The polyphenol components, catechin 3-gallate (CG), gallocatechin 3-gallate (GCG), epicatechin 3-gallate (ECG), and epigallocatechin 3-gallate (EGCG), were good inhibitors of maltase, with IC 50 values of 62, 67, 40, and 16 μM, respectively, and EGCG also showed good inhibition toward maltase expressed on Caco-2 cells, with an IC 50 value of 27 μM. The ungallated catechins, such as catechin, gallocatechin (GC), epicatechin (EC), and epigallocatechin (EGC), showed no significant inhibition toward GP b, whereas the gallated catechins CG, GCG, ECG, and EGCG inhibited the enzyme, with IC 50 values of 35, 6.3, 27, and 34 μM. From multiple inhibition studies by Dixon plots, GCG appears to bind a new allostelic site, the indole inhibitor site. These gallated catechins also inhibited glucagon-stimulated glucose production dose-dependently, with IC 50 values ranging from 33 to 55 μM. Dietary supplementation with these gallated catechins or the green tea extract containing them, which inhibits both α-glucosidases and GP in vitro and in cell culture, would contribute to the protection or improvement of type 2 diabetes.

Reduced Antidiabetic Effect of Metformin and Down-regulation of Hepatic Oct1 in Rats with Ethynylestradiol-Induced Cholestasis

To investigate the effect of 17alpha-ethynylestradiol (EE)-induced cholestasis on the expression of organic cation transporters (Octs) in the liver and kidney, as well as the pharmacokinetics and pharmacodynamics of metformin in rats. Octs mRNA and protein expression were determined. The pharmacokinetics and tissue uptake clearance of metformin were determined following iv administration (5 mg/kg). Uptake of metformin, glucagon-mediated glucose production, and AMP-activated protein kinase (AMPK) activation were measured in isolated hepatocytes. The effect of metformin (30 mg/kg) on blood glucose levels was tested using the iv glucose tolerance test (IVGTT). The mRNAs of hepatic Oct1, renal Oct1, and Oct2 were decreased by 71.1%, 37.6%, and 94.5%, respectively, by EE cholestasis. The hepatic Oct1 and renal Oct2 proteins were decreased by 30.6% and 60.2%, respectively. The systemic and renal clearance of metformin were decreased. The in vitro hepatocyte uptake of metformin was decreased by 86.4% for V (max). Suppression of glucagon-stimulated glucose production and stimulation of AMPK activation in hepatocytes by metformin were diminished. In addition, metformin did not demonstrate a glucose-lowering effect during IVGTT in EE cholestasis. The antidiabetic effect of metformin may be diminished in diabetic patients with EE cholestasis, due to impaired hepatic uptake of the drug via OCT1.

Specific Reduction of Hepatic Glucose 6-Phosphate Transporter-1 Ameliorates Diabetes while Avoiding Complications of Glycogen Storage Disease

D-Glucose-6-phosphatase is a key regulator of endogenous glucose production, and its inhibition may improve glucose control in type 2 diabetes. Herein, 2'-O-(2-methoxy)ethyl-modified phosphorothioate antisense oligonucleotides (ASOs) specific to the glucose 6-phosphate transporter-1 (G6PT1) enabled reduction of hepatic D-Glu-6-phosphatase activity in diabetic ob/ob mice. Treatment with G6PT1 ASOs decreased G6PT1 expression, reduced G6PT1 activity, blunted glucagon-stimulated glucose production, and lowered plasma glucose concentration in a dose-dependent manner. In contrast to G6PT1 knock-out mice and patients with glycogen storage disease, excess hepatic and renal glycogen accumulation, hyperlipidemia, neutropenia, and elevations in plasma lactate and uric acid did not occur. In addition, hypoglycemia was not observed in animals during extended periods of fasting, and the ability of G6PT1 ASO-treated mice to recover from an exogenous insulin challenge was not impaired. Together, these results demonstrate that effective glucose lowering by G6PT1 inhibitors can be achieved without adversely affecting carbohydrate and lipid metabolism.

Identification and characterization of a glucagon receptor from the goldfish Carassius auratus: implications for the evolution of the ligand specificity of glucagon receptors in vertebrates.

The structural basis of ligand selectivity of G protein-coupled receptors for metabolic hormones has been an area of intense investigation, and yet it remains unresolved. One approach to delineating the mechanism of ligand-receptor interactions is to compare the ligand specificities of receptors expressed in species that emerged at different times within vertebrate evolution. In this paper we describe the isolation, functional, and phylogenetic characterization of the glucagon receptor from the goldfish Carassius auratus (Teleostei, order Cypriniformes), and compare its ligand specificity with that of the homologous rat receptor. Goldfish (gf) glucagon stimulated glucose production in a dose-dependent manner from isolated goldfish hepatocytes, resulting in 5-fold increase at 1 microm. The goldfish glucagon receptor (gfGlucR) shares 56, 51, 50, and 52% amino acid identities with frog Rana tigrina regulosa, mouse, rat, and human glucagon receptors, respectively. In competitive binding experiments, the recombinant gfGlucR displays high affinity toward goldfish, zebrafish, and human glucagons (IC(50) = 0.6, 9, and 13 nm, respectively) but not toward goldfish glucagon-like peptide-1 or human glucagon-like peptide-1 (7-36) amide. Whereas both goldfish and human glucagons stimulated dose-dependent increases in intracellular cAMP through the recombinant gfGlucR, the recombinant rat GlucR interacted only with human glucagon, analogous to the specificity of the previously characterized glucagon receptor from the frog R. tigrina regulosa. Our results demonstrate that the binding pocket of gfGlucR can accommodate a broad range of glucagon structures and that in the frogs and mammals, there is a structural switch to a more restrictive conformation of glucagon receptors.

Optimization of alkylidene hydrazide based human glucagon receptor antagonists. Discovery of the highly potent and orally available 3-cyano-4-hydroxybenzoic acid [1-(2,3,5,6-tetramethylbenzyl)-1H-indol-4-ylmethylene]hydrazide.

Highly potent human glucagon receptor (hGluR) antagonists have been prepared employing both medicinal chemistry and targeted libraries based on modification of the core (proximal) dimethoxyphenyl group, the benzyl ether linkage, as well as the (distal) benzylic aryl group of the lead 2, 3-cyano-4-hydroxybenzoic acid (3,5-dimethoxy-4-isopropylbenzyloxybenzylidene)hydrazide. Electron-rich proximal aryl moieties such as mono- and dimethoxy benzenes, naphthalenes, and indoles were found to be active. The SAR was found to be quite insensitive regarding the linkage to the distal aryl group, since long and short as well as polar and apolar linkers gave highly potent compounds. The presence of a distal aryl group was not crucial for obtaining high binding affinity to the hGluR. In many cases, however, the affinity could be further optimized with substituted distal aryl groups. Representative compounds have been tested for in vitro metabolism, and structure-metabolism relationships are described. These efforts lead to the discovery of 74, NNC 25-2504, 3-cyano-4-hydroxybenzoic acid [1-(2,3,5,6-tetramethylbenzyl)-1H-indol-4-ylmethylene]hydrazide, with low in vitro metabolic turnover. 74 was a highly potent noncompetitive antagonist of the human glucagon receptor (IC(50) = 2.3 nM, K(B) = 760 pM) and of the isolated rat receptor (IC(50) = 430 pM, K(B) = 380 pM). Glucagon-stimulated glucose production from isolated primary rat hepatocytes was inhibited competitively by 74 (K(i) = 14 nM). This compound was orally available in dogs (F(po) = 15%) and was active in a glucagon-challenged rat model of hyperglucagonemia and hyperglycemia.

Small-molecule glucagon receptor antagonists

The development of small-moiecule giucagon antagonists reported in the scientific literature is summarized. Peptidic and nonpeptidic giucagon antagonists have been shown to attenuate giucagon-induced glucose production in animal models. To date, various classes of compounds having affinity for the glucagon receptor have been identified. Recently, a competitive human small-molecule glucagon antagonist was reported to block glucagon-stimulated glucose production in humans. Thus, small molecules that antagonize the glucagon receptor are potential therapeutic agents for the control of diabetes.

Acute effects of leptin on glucose metabolism of in situ rat perfused livers and isolated hepatocytes.

To investigate whether leptin interferes directly with glycogenolysis and gluconeogenesis in isolated rat hepatocytes and also in in situ rat perfused livers. Male albino rats (200-250 g) were used in all experiments. D-glucose, L-lactate and pyruvate production. In the present study, no differences were found for the rates of glycolysis, as expressed by the areas under the curves, among control (24.2+5.0 mmol¿g), leptin (32.0+4.5 mmol¿g), glucagon (24.7+3.0 mmol¿g), and the leptin + glucagon (23.8+3.4 mmol¿g) groups. No difference was found for the rates of glycogenolysis between the control and the leptin perfused livers (15.2+3.9 and 15.0+3.2 mmol¿g, respectively). In the presence of glucagon, the areas under the curves for the rate of glycogenolysis rose to 108.6+3.8 mmol¿g. When leptin was combined with glucagon, the area under the curve for glycogenolysis was 43. 7+4.3 mmol¿g. In fact, leptin caused a reduction of almost 60% (P<0. 001) in the rate of glucagon-stimulated glycogenolysis. Under basal conditions, the addition of leptin (100 ng¿ml) to the incubation medium did not elicit any alteration in glucose production by isolated hepatocytes. However, in the presence of leptin, the production of glucose from glycerol (2 mM), L-lactate (2 mM). L-alanine (5 mM) and L-glutamine (5 mM) by the isolated hepatocytes was significantly reduced (30%, 30%, 23% and 25%, respectively). The rate of glucose production (glycogenolysis) by isolated hepatocytes was not different between the control and the leptin incubated groups (445.0+/-91.0 and 428.0+/-72.0 nmol¿106 cells¿h, respectively). We conclude that leptin per se does not directly affect either liver glycolysis or its glucose production, but a physiological leptin concentration is capable of acutely inducing a direct marked reduction on the rate of glucagon-stimulated glucose production in in situ rat perfused liver. Leptin is also capable of reducing glucose production from different gluconeogenic precursors in isolated hepatocytes.

Acute troglitazone action in isolated perfused rat liver.

1. The thiazolidinedione compound, troglitazone, enhances insulin action and reduces plasma glucose concentrations when administered chronically to type 2 diabetic patients. 2. To analyse to what extent thiazolidinediones interfere with liver function, we examined the acute actions of troglitazone (0.61 and 3.15 microM) on hepatic glucose and lactate fluxes, bile secretion, and portal pressure under basal, insulin- and/or glucagon-stimulated conditions in isolated perfused rat livers. 3. During BSA-free perfusion, high dose troglitazone increased basal (P < 0.01), but inhibited glucagon-stimulated incremental glucose production by approximately 75% (10.0 +/- 2.5 vs control: 40.0 +/- 7.2 micromol g liver(-1), P < 0.01). In parallel, incremental lactate release rose approximately 6 fold (13.1 +/- 5.9 vs control: 2.2 +/- 0.8 mmol g liver(-1), P < 0.05), while bile secretion declined by approximately 67% [0.23 +/- 0.02 vs control: 0.70 +/- 0.05 mg g liver(-1) min(-1)), P < 0.001]. Low dose troglitazone infusion did not enhance the inhibitory effect of insulin on glucagon-stimulated glucose production, but rapidly increased lactate release (P < 0.0005) and portal venous pressure (+0.17 +/- 0.07 vs +0.54 +/- 0.07 cm buffer height, P < 0.0001). 4. These results indicate that troglitazone exerts both insulin-like and non-insulin-like hepatic effects, which are blunted by addition of albumin, possibly due to troglitazone binding.

Glucose metabolism during exercise in man: the role of insulin and glucagon in the regulation of hepatic glucose production and gluconeogenesis.

This study was designed to further characterize the role of insulin and glucagon in the regulation of glucose production and gluconeogenesis during a 2-h mild intensity exercise (40% VO2max) in 14 h fasted healthy male subjects. Endogenous insulin and glucagon secretions were suppressed by the infusion of somatostatin. The pancreatic hormones were replaced singly or in combination to match the hormonal concentrations observed during exercise in control subjects. Glucose turnover was determined by a tracer method using the stable isotope D-[2,3,4,6,6-2H]glucose. Gluconeogenesis was estimated by the simultaneous infusion of L-[1,2,3-13C]alanine to follow the conversion of alanine to glucose. Hepatic glucose production significantly increased from a resting rate of 12.1 +/- 0.2 to 27.6 +/- 1.4 mumol.kg-1.min-1 during exercise (p < 0.05). In the absence of glucagon, this increase in hepatic glucose production during exercise was totally abolished (p < 0.05). When insulin was made deficient, in the presence of glucagon, there was an overshoot in the increase in hepatic glucose production during exercise to 36.4 +/- 1.6 mumol.kg-1.min-1 (p < 0.05). The normal increase in hepatic glucose output during exercise was reproduced when both insulin and glucagon were replaced. Exercise increased gluconeogenesis by 47% above the resting level (p < 0.05). When glucagon was made deficient, in the absence or presence of insulin, this increase in gluconeogenesis was totally suppressed (p < 0.05). Furthermore, glucagon replacement during exercise in the absence of insulin resulted in a further increase in gluconeogenesis to 93% above resting value (p < 0.05). From these observations, it is concluded that during prolonged mild intensity exercise in healthy subjects, the rise in glucagon is essential for the increase in hepatic glucose production and the increase in gluconeogenesis. It is also suggested that the lower level of insulin during exercise still exerts a restraining effect on glucagon-stimulated glucose production and gluconeogenesis, thus preventing hyperglycemia.

Lack of effect of islet amyloid polypeptide on hepatic glucose output in the in situ-perfused rat liver

Islet amyloid polypeptide (IAPP), a novel peptide isolated from islet amyloid deposits in patients with insulinoma and non-insulin-dependent diabetes mellitus (NIDDM), has been reported to be cosecreted with insulin from pancreatic β cells and to inhibit glucose uptake and glycogen synthesis in muscle tissue in vitro. We investigated the effects of the synthesized, rat-amidated form of IAPP on hepatic glucose output, and IAPP extraction, using an in situ flow-through perfusion system in rats to elucidate the actions of IAPP on the liver. The IAPP (10 −8 mol/L) alone had no effects on the hepatic glucose release. Infusion of 6 × 10 −11 mol/L glucagon alone resulted in an expected elevation in glucose production ( 30.0 ± 1.7 μmol 35 min g liver ). Insulin (3 × 10 −10 mol/L) submaximally decreased the glucagon-stimulated glucose production to 73% ( from 30.0 ± 1.7 to 22.0 ± 1.4 μmol 35 min g liver ; n = 7, P < .01). A simultaneous infusion of 10 −8 mol/L IAPP did not influence the glucagon-stimulated glucose production ( 27.6 ± 1.2 μmol 35 min g liver ) or the insulin-dependent inhibition of glucagon-stimulated glucose production ( 22.6 ± 1.3 μmol 35 min g liver ). IAPP extraction by the liver in a single passage was minimal, in contrast to approximately 50% hepatic insulin extraction. These results indicate that IAPP does not play any important role in modulating glycogen metabolism in the liver.

Effects of islet amyloid polypeptide on hepatic insulin resistance and glucose production in the isolated perfused rat liver.

The impact of (pancreatic) islet amyloid polypeptide on glucose metabolism and insulin sensitivity was examined in isolated rat livers perfused in a non-recirculating system. Continuous infusion of 10(-7) mol/l islet amyloid polypeptide affected neither basal nor glucagon (10(-9) mol/l)-stimulated glucose output by livers from fed rats, but it did increase the hepatic cyclic AMP release within 44 min (7.91 +/- 12.07 vs control: 0.07 +/- 0.03 pmol x 100 g body weight-1). The effect of the peptide on the ability of insulin to inhibit glucagon-induced hepatic glycogenolysis was measured in three experimental groups (n = 6). As expected glucagon (7 x 10(-11) mol/l) increased integral hepatic glucose release within 84 min (763.4 +/- 161.7 vs -25.7 +/- 73.2 mumol x 100 g body weight-1 in the control group, p less than 0.001), while insulin (100 mU/l) decreased the glucagon-stimulated glucose production (395.2 +/- 180.0 mumol x 100 g body weight-1, p less than 0.01). Simultaneous infusion of 10(-7) mol/l islet amyloid polypeptide however, was not able to reverse insulin-dependent inhibition of glucagon-stimulated hepatic glucose output (370.0 +/- 102.5 mumol x 100 g body weight-1, NS) or to enhance lactate-induced gluconeogenesis of livers from 24 h fasted rats (n = 8). The glucose production stimulated by 10(-9) mol/l glucagon was slightly greater in islet amyloid polypeptide-pre-treated livers than in a control group without addition of islet amyloid polypeptide (5 min: 3.60 +/- 3.36 vs 1.67 +/- 1.28 mumol.min-1 x 100 g body weight-1).(ABSTRACT TRUNCATED AT 250 WORDS)

Hormone-stimulated glucose production from glycogen in hepatocytes from streptozotocin diabetic rats

The contribution of hormone-stimulated glycogenolysis to hepatic glucose production was studied in hepatocytes from streptozotocin diabetic rats. To this end, the activation of glycogen phosphorylase by glucagon, vasopressin, and the α 1-adrenergic agonist phenylephrine was compared in hepatocytes from normal and diabetic rats and related to glycogen content, glucose production, and microsomal glucose-6-phosphatase activity. Streptozotocin-induced diabetes reduced the glycogen content and the amount of total ( a + b) phosphorylase in hepatocytes proportionally to the severity of the disease. In cells from severely diabetic rats (group 1), the responsiveness of activation of phosphorylase to the hormones was reduced by about half, consistent with a 45% reduction in total phosphorylase. In addition, the sensitivity of phosphorylase activation to all hormones investigated was decreased by about 1 order of magnitude or more in cells of this group. In hepatocytes from rats with milder diabetes (group 2), maximal phosphorylase activation reached an intermediate value between that of the control group and of group 1. In response to all hormones investigated, group 2 diabetic rat hepatocytes produced less glucose than control rat liver cells, while in group 1 there was no increase in glucose production at all, presumably because glycogen concentration was too low. However, in group 2 diabetic rat hepatocytes, glucagon-stimulated glucose production, unlike phosphorylase activation, did not show decrease sensitivity, presumably because glucose-6-phosphatase activity is increased by diabetes. Our results thus indicate that hormone-stimulated liver glycogenolysis is unlikely to contribute to enhanced glucose production in insulin-deficient diabetes, despite increased glucose-6-phosphatase activity.

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. Concomitant fourfold increases in the insulin and glucagon infusion rates caused glucose production to fall from 3.03 ± 0.23 to 0.76 ± 0.39 mg/kg/min by 1 hour and to remain similarly suppressed for 3 hours. Glucose utilization rose from 2.95 ± 0.20 to 8.44 ± 0.87 mg/kg/min and euglycemia was again maintained by glucose infusion. Gluconeogenic conversion increased (67 ± 12%) in the studies in which insulin and glucagon were kept at basal values, but the level, fractional extraction, and hepatic uptake of alanine did not change significantly. The selective increase in glucagon caused gluconeogenic conversion (169% ± 42%), hepatic fractional alanine extraction (0.32 ± 0.05 to 0.66 ± 0.10), and hepatic alanine uptake (2.96 ± 0.45 to 4.54 ± 0.43 μmol/kg/min) to increase, while the alanine level decreased (387 ± 40 to 272 ± 48 μmol/L). The selective increase in insulin was associated with an increase in gluconeogenic conversion (40% ± 25%) similar to that apparent in the control group, no change in the fractional extraction or uptake of alanine by the liver, and a small fall in alanine level (337 ± 33 to 249 ± 55 μmol/L). Concomitant increases in both insulin and glucagon left gluconeogenic conversion unchanged (26% ± 21%), but increased the fractional extraction of alanine by the liver (0.39 ± 0.05 to 0.64 ± 0.03). Hepatic alanine uptake did not change, but a decrease in the alanine level (353 ± 40 to 174 ± 16 μmol/L) was observed. These studies indicate that in the overnight fasted conscious dog, insulin is a potent inhibitor of the stimulatory effects of glucagon on hepatic glycogenolysis and gluconeogenesis. However, insulin is unable to limit alanine uptake by suppression of hepatic fractional extraction, instead it prevents increased alanine uptake by limiting the net release of alanine from extrahepatic tissues.

Reversal of in vitro hepatic insulin resistance in chronic pancreatitis by pancreatic polypeptide in the rat.

In vitro isolated liver perfusion in a rat model of chronic pancreatitis (CP) has been shown to demonstrate hepatic resistance to insulin. The ability of pancreatic polypeptide (PP) to reverse the resistance to insulin on glucagon-stimulated hepatic glucose production was therefore investigated in this model. CP was induced in 250 to 300 gm Sprague-Dawley rats by infusion of 50 microliters of 99% oleic acid into the pancreas via the common bile duct. After 6 to 8 weeks, isolated liver perfusion was performed on livers from both CP rats and sham-operated control animals (n = 12, 14), both with and without PP administration. Glucagon infusion (100 pg/ml for 30 minutes) produced a five- to sixfold increase in hepatic glucose production. The integrated hepatic glucose output (IHGO) response to glucagon alone was comparable in pancreatic and sham-operated animals; during period 1 (0 to 10 minutes) IHGO was 7.1 +/- 0.5 mg/gm-min for sham-operated controls (n = 8) and 7.1 +/- 0.4 mg/gm-min for pancreatitic animals (n = 6) without PP treatment. Animals that received PP (100 ng intraperitoneally 5 hours before liver harvest and perfusion with 4.2 ng/ml from 10 to 30 minutes) demonstrated an IHGO for period 1 for the sham (n = 6) and pancreatitic animals (n = 6) of 5.6 +/- 0.6 and 4.8 +/- 0.8 mg/gm-min, respectively. Insulin infusion (100 microU/ml added to perfusate from 10 to 30 minutes) in CP livers without PP revealed impaired responsiveness to insulin; the ratio of period 3 (20 to 30 minutes)/period 1 IHGO was 110% +/- 5% in CP livers compared with 77% +/- 5% in sham controls (p less than 0.01). In contrast, PP treatment restored hepatic responsiveness to insulin to control levels; the period 3/period 1 IHGO was 75% +/- 13% in CP livers treated with PP, which was indistinguishable from the 67% +/- 9% response seen in sham-operated control animals. These data provide the first in vitro evidence of a primary hepatic glucoregulatory role of PP. Therefore PP deficiency may contribute to altered glucose metabolism through the induction of a reversible hepatic resistance to insulin.