The Effect of Insulin on the Distribution of Glucose between the Blood Plasma and the Liver

In normal fasted rats whole liver tissue contains as much glucose as the blood plasma, i.e., the ratio of the concentrations is about unity. The concentration of glucose in hepatic intracellular water is about 1.2 times higher than in plasma water. In rats injected with insulin the concentration of glucose in the liver falls to a lesser extent than in the plasma: resulting in a ratio of concentrations higher than unity. If insulin hypoglycemia is prevented by the ingestion of glucose the concentration ratio is less than in hypoglycemic rats but still significantly above unity. In normal rats the specific activities of plasma and hepatic glucose do not differ significantly at 7.5, 15, and 30 minutes after the intravenous injection of C¹⁴-labeled glucose. In rats injected with insulin the specific activity of glucose is higher in the plasma than in the liver at 7.5 and 15 minutes, but not at 30 minutes following the injection of tracer. In insulin-treated hypoglycemic rats considerably higher concentrations of labeled glucose are found in hepatic intracellular than in plasma water. The penetration of C¹⁴-glucose from plasma into hepatic intracellular water is found to be fast. Excess insulin causes an accumulation of glucose within the liver cells by retaining newly formed glucose and by the taking up of glucose from the plasma against an existing concentration gradient.     

The HISS story overview: a novel hepatic neurohumoral regulation of peripheral insulin sensitivity in health and diabetes.

Data are reviewed that are consistent with the following working hypothesis that proposes a novel mechanism regulating insulin sensitivity, which when nonfunctional, leads to severe insulin resistance. Postprandial elevation in insulin levels activates a hepatic parasympathetic reflex release of a putative hepatic insulin-sensitizing substance (HISS), which activates glucose uptake at skeletal muscle. Insulin causes HISS release in fed but not fasted animals. The reflex is mediated by acetylcholine and involves release of nitric oxide in the liver. Interruption of the release of HISS is achieved by surgical denervation of the anterior hepatic nerve plexus, muscarinic receptor blockade, or nitric oxide synthase antagonism and leads to immediate severe insulin resistance. The nitric oxide donor, SIN-1, reverses L-NAME-induced insulin resistance. Denervation-induced insulin resistance is reversed by intraportal but not intravenous administration of acetylcholine or SIN-1. Liver disease is often associated with insulin resistance; the bile duct ligation model of liver disease results in parasympathetic neuropathy and insulin resistance that is reversed by intraportal acetylcholine. Possible relevance of this HISS-dependent control of insulin action to insulin resistance in diabetes, liver disease, and obesity is discussed.     

Receptor-mediated introduction of pepstatin-asialofetuin conjugate into lysosomes of rat hepatocytes

Pepstatin was linked through a carboxyl group to asialofetuin (PS-ASF). An analysis by separation of hepatocytes from nonparenchymal cells showed that PS-ASF was taken up by hepatocytes, following intravenous injection into rats. After the injection of PS-ASF, pepstatin concentration in the liver reached a maximum at 2 h and then decreased. In an analysis by differential centrifugation of the liver homogenate from rats injected with PS-ASF, pepstatin showed a lysosomal type subcellular distribution pattern. Isolation studies of tritosomes clearly demonstrated the exclusive accumulation of pepstatin within the lysosomes of livers from rats given PS-ASF (at 2 h after administration). Pepstatin contained in tritosomes was in a free form, as determined by column chromatography of Sephadex G-15. The activity of cathepsin D in the livers was markedly inhibited in rats given PS-ASF. However, the treatment of rats with PS-ASF had no effect on the hepatic lysosomal degradation of endocytosed FITC-labeled asialofetuin (FITC-ASF). Introduction of PS-ASF into the hepatocytes was followed by the immediate and time-dependent excretion of free pepstatin into the bile. Quantification of pepstatin excreted into the bile revealed that the biliary excretion route can account for the disappearance of pepstatin from the liver.     

Hepatic mechanisms of the Walnut antidiabetic effect in mice

The present study was designed to explore the mechanism of action of walnut (the seed of Juglans regia) leaf and ridge on hepatic glucose metabolism in diabetic mice. Experimental diabetes was induced by intravenous administration of streptozotocin (60 mg/kg)and confirmed with an increase of blood glucose, 90–100% of the control, 72 hours later. Isolated extracts from walnut leaf and ridges were administered in a single effective dose of 400 mg/kg orally. Firstly, blood glucose was determined every 1 hour until 5 hours post administration of extracts. In the second experiment, the liver was surgically removed, 2 hours post treatment of diabetic animals with extracts, homogenized and used for measurement of key enzymes of glycogenolysis (glycogen phosphorylase, GP) and gluconeogenesis (phosphoenolpyruvate carboxykinase, PEPCK). Treatment by both leaf and ridge extracts decreased blood glucose and liver PEPCK activity and increased blood insulin and liver GP activity. It is concluded that walnut is able to lower blood glucose through inhibition of hepatic gluconeogenesis and secretion of pancreatic insulin.     

Lack of recognition of Nepsilon-(carboxymethyl)lysine by the mouse liver reticulo-endothelial system: implications for pathophysiology

Advanced glycation end products (AGEs) are known to be associated with a number of pathological conditions, such as diabetes mellitus, Alzheimer's disease, uremia, as well as with normal aging. This study was undertaken to investigate whether Nepsilon-(carboxymethyl)lysine (CML), a major structure among numerous AGEs, engenders hepatic AGE clearance. For this purpose uptake of BSA substituted with heterogeneous AGEs or with CML only was monitored in vivo and in cultured hepatic scavenger cells. Here, we show that following intravenous administration of 125I-AGE-BSA and 125I-CML-BSA, blood radioactivity was reduced by 50% after 50s and >100 min, respectively. Recoveries from the circulation at 6 min after injection were: 5% for AGE-BSA, 95% for CML-BSA. More than 80% of the injected AGE-BSA was recovered from the liver. AGE-BSA, but not CML-BSA, was avidly endocytosed by cultured liver scavenger cells. Our results suggest that CML does not engender AGE-BSA clearance. Macromolecules substituted with CML only may escape elimination and cause pathological effects.     

Oral glucose decreases hepatic extraction of insulin.

Peripheral venous (plasma) insulin and C-peptide concentrations were measured in eight normal subjects given oral or intravenous glucose sufficient to produce similar plasma glucose concentrations. The expected increased insulin response to oral as compared with intravenous glucose was not matched by a comparable increase in C-peptide concentration. The ratio of insulin to C-peptide concentrations doubled 30 minutes after oral glucose was given; no comparable rise was seen with intravenous glucose (p = 0.01). This finding is interpreted as evidence for decreased hepatic extraction of insulin after administration of oral glucose. Such a decrease could account for at least half of the well known difference in peripheral insulin concentrations after administration of oral as compared with intravenous glucose.     

Immunocytochemical localization of platelets in baboon hepatic sinusoids using monoclonal mouse anti-human platelet glycoprotein IIIa following induction of thrombocytopenia.

A commercially available mouse monoclonal antibody to human platelet glycoprotein IIIa was used to demonstrate sequestration of platelets in hepatic biopsies obtained from baboons following intravenous infusion of echistatin, a novel fibrinogen receptor antagonist derived from the venom of the snake Echis carinatus. Biopsies of liver and spleen were taken prior to administration of echistatin. The hepatic biopsies were either snap-frozen in Freon-22/liquid nitrogen or fixed in 10% neutral buffered formalin. Biopsies of spleen were snap-frozen. During infusion of echistatin (2.3 micrograms/kg/min), circulating platelet counts decreased from 331,000/mm3 to 167,000/mm3. Selective sequestration within the liver was confirmed using whole body gamma camera imaging to demonstrate 111Indium-oxine labeled platelet accumulation within the liver during the thrombocytopenic episode. Hepatic biopsies were again taken and either snap-frozen in Freon-22/liquid nitrogen or fixed in 10% neutral buffered formalin. Biopsies of spleen and inguinal lymph node were also snap-frozen. Platelet rich plasma smears, included as positive controls, dewaxed paraffin sections, and cryosections of liver, spleen, and lymph node were stained with monoclonal mouse anti-human platelet glycoprotein IIIa using an avidin biotinylated peroxidase complex (ABC) technique. Prior to infusion of echistatin, platelet staining within the liver was minimal. After echistatin infusion, hepatic cryosections showed prominent platelet staining within hepatic sinusoids. No localization was shown in lymph node, however, the spleen showed prominent platelet staining both before and after echistatin infusion. Platelet rich plasma smears were intensely positive. No prominent platelet staining was observed in formalin-fixed, paraffin-embedded material. Thus, this immunocytochemical technique may help localize platelets in cryosections of tissues from baboons and other primate species.     

Role of hepatic STAT3 in brain-insulin action on hepatic glucose production

STAT3 regulates glucose homeostasis by suppressing the expression of gluconeogenic genes in the liver. The mechanism by which hepatic STAT3 is regulated by nutritional or hormonal status has remained unknown, however. Here, we show that an increase in the plasma insulin concentration, achieved either by glucose administration or by intravenous insulin infusion, stimulates tyrosine phosphorylation of STAT3 in the liver. This effect of insulin was mediated by the hormone's effects in the brain, and the increase in hepatic IL-6 induced by the brain-insulin action is essential for the activation of STAT3. The inhibition of hepatic glucose production and of expression of gluconeogenic genes induced by intracerebral ventricular insulin infusion was impaired in mice with liver-specific STAT3 deficiency or in mice with IL-6 deficiency. These results thus indicate that IL-6-STAT3 signaling in the liver contributes to insulin action in the brain, leading to the suppression of hepatic glucose production.     

Time dependent effects of dexamethasone on serum insulin level and insulin receptors in rat liver and erythrocytes

The effects of glucocorticoid excess on regulation of insulin receptors were investigated in dexamethasone-treated rats. Glucocorticoid excess was produced by administration of dexamethasone (0.5 mg/100 g b.w.) 30 min, 4, 12, 18, 24, 42 or 70 h before experiments. This treatment caused time-dependent changes of glucose and insulin concentration in blood, as well as in amounts of specific insulin binding and insulin receptors of liver cells and erythrocytes. The time intervals in which dexamethasone produced the increase in insulin concentration were accompanied with decrease in insulin binding to receptors in membranes of liver cells, while significant changes in insulin binding to receptors of erythrocytes were not observed under the same experimental conditions. The effect is maximal 18 and 42 h after dexamethasone treatment that increase insulin blood level by about 85% and 60%, respectively. Receptor analysis revealed that changes in specific binding of insulin could be due to significant changes in amount of binding sites on cell surface rather than to mild alteration in receptor affinity. These findings suggest that besides the changes in insulin level, the alterations in insulin receptor number and affinity may play a major role in the states of altered insulin sensitivity which accompany glucocorticoid excess.     

Factors influencing the inhibition of biliary glutathione efflux induced by biliary obstruction

The aim of this study was to investigate mechanisms responsible for the inhibition of biliary glutathione efflux in rats with secondary biliary cirrhosis. Rats were studied after bile duct obstruction for 28 days. The biliary secretion of reduced glutathione (GSH), oxidised glutathione (GSSG) and cysteine were completely inhibited in biliary obstructed rats. Hepatic gamma glutamyltranspeptidase (gamma-GT) activity increased significantly, but following its inhibition by acivicin administration GSH, GSSG and cysteine were still absent in bile. Biliary obstruction resulted in a significant increase of the permeability of the paracellular pathway, as shown by the higher bile/plasma ratio and hepatic clearance of [14C]sucrose. GSH and GSSG were, however, significantly lower in the carotid artery and hepatic vein of obstructed animals and the arteriovenous difference across the liver was reduced. The concentration of GSH was significantly reduced and that of GSSG increased in the liver of obstructed rats. Biliary obstruction induced an increase in the hepatic concentration of cysteine and an inhibition of both gamma glutamylcysteine synthetase and methionine adenosyl transferase activities. Dichlorofluorescein (DCF) and the GSSG/GSH ratio and thiobarbituric acid reactive substances (TBARS) concentration, markers of reactive oxygen species production and lipid peroxidation, respectively, were significantly increased. Our data indicate that increased degradation or blood reflux of glutathione do not participate in the disruption of its secretion into bile and support the view that impairment of glutathione synthesis and oxidative stress could contribute to the decline in biliary glutathione output.     

Mechanism of rapid elimination of lysophosphatidic acid and related lipids from the circulation of mice

Lysophosphatidic acid (LPA) is a bioactive lipid mediator. Concentrations of the major LPA species in mouse plasma decreased uniformly following administration of a potent selective inhibitor of the LPA-generating lysophospholipase D autotaxin, identifying an active mechanism for removal of LPA from the circulation. LPA, akylglycerol phosphate (AGP), sphingosine 1-phosphate (S1P), and a variety of structural mimetics of these lipids, including phosphatase-resistant phosphonate analogs of LPA, were rapidly eliminated (t1/2 < 30 s) from the circulation of mice following intravenous administration of a single bolus dose without significant metabolism in situ in the blood. These lipids accumulated in the liver. Elimination of intravenously administered LPA was blunted by ligation of the hepatic circulation, and ∼90% of LPA administered through the portal vein was accumulated by the isolated perfused mouse liver at first pass. At early times following intravenous administration, more LPA was associated with a nonparenchymal liver cell fraction than with hepatocytes. Primary cultures of nonparenchymal liver cells rapidly assimilated exogenously provided LPA. Our results identify hepatic uptake as an important determinant of the bioavailability of LPA and bioactive lysophospholipid mimetics and suggest a mechanism to explain changes in circulating LPA levels that have been associated with liver dysfunction in humans.     

Fasting and postprandial concentrations of GLP-1 in intestinal lymph and portal plasma: evidence for selective release of GLP-1 in the lymph system

Glucagon like peptide 1 (GLP-1) is an intestinal hormone that plays an important role in glucose metabolism. GLP-1 is released from mucosal L cells following nutrient ingestion and contributes to the incretin effect, with the enhancement of insulin secretion occurring with enteral compared with intravenous glucose administration. The mechanisms linking nutrient absorption and GLP-1 secretion are unknown, and studies addressing this topic, particularly in small animal models, have been hampered by the relatively low concentrations of GLP-1 in the circulation. We hypothesized that GLP-1 levels would be higher in samples of intestinal lymph compared with plasma and could provide a novel system in which to study meal-induced hormone secretion. We addressed this hypothesis in conscious rats with indwelling catheters in the portal vein and distal intestinal lymph duct. These animals had plasma and lymph sampled before and for 240 min after instillation of a liquid meal in the gastrointestinal tract. Lymph contained detectable concentrations of glucose, insulin, and GLP-1 that were reliably measured using our assays. Before and after the Ensure feeding, plasma insulin levels were approximately two times as high in portal plasma as intestinal lymph. In marked contrast, GLP-1 levels were five to six times higher in lymph relative to portal plasma following nutrient administration. This relative difference in GLP-1 levels was even greater when lymph was compared with peripheral plasma and dramatically exceeded the ratio of lymph to plasma peptide tyrosine-tyrosine concentrations. This is the first observation of a gastrointestinal hormone being disproportionately transported in lymph. The remarkable levels of GLP-1 in intestinal lymph demonstrate the potential for lymphatic sampling as a more sensitive means of studying the secretory physiology of this hormone in vivo. In addition, these data raise the possibility that intestinal lymph may serve as a specialized signaling conduit for regulatory peptides secreted by gastrointestinal endocrine cells.     

Reduction of hepatic insulin clearance after oral glucose ingestion is not mediated by glucagon-like peptide 1 or gastric inhibitory polypeptide in humans

Changes in hepatic insulin clearance can occur after oral glucose or meal ingestion. This has been attributed to the secretion and action of gastric inhibitory polypeptide (GIP) and glucagon-like peptide (GLP)-1. Given the recent availability of drugs based on incretin hormones, such clearance effects may be important for the future treatment of type 2 diabetes. Therefore, we determined insulin clearance in response to endogenously secreted and exogenously administered GIP and GLP-1. Insulin clearance was estimated from the molar C-peptide-to-insulin ratio calculated at basal conditions and from the respective areas under the curve after glucose, GIP, or GLP-1 administration. Oral glucose administration led to an approximately 60% reduction in the C-peptide-to-insulin ratio (P < 0.0001), whereas intravenous glucose administration had no effect (P = 0.09). The endogenous secretion of GIP or GLP-1 was unrelated to the changes in insulin clearance. The C-peptide-to-insulin ratio was unchanged after the intravenous administration of GIP or GLP-1 in the fasting state (P = 0.27 and P = 0.35, respectively). Likewise, infusing GLP-1 during a meal course did not alter insulin clearance (P = 0.87). An inverse nonlinear relationship was found between the C-peptide-to-insulin ratio and the integrated insulin levels after oral and during intravenous glucose administration. Insulin clearance is reduced by oral but not by intravenous glucose administration. Neither GIP nor GLP-1 has significant effects on insulin extraction. An inverse relationship between insulin concentrations and insulin clearance suggests that the secretion of insulin itself determines the rate of hepatic insulin clearance.     

First-phase insulin secretion: does it exist in real life? Considerations on shape and function

To fulfill its preeminent function of regulating glucose metabolism, insulin secretion must not only be quantitatively appropriate but also have qualitative, dynamic properties that optimize insulin action on target tissues. This review focuses on the importance of the first-phase insulin secretion to glucose metabolism and attempts to illustrate the relationships between the first-phase insulin response to an intravenous glucose challenge and the early insulin response following glucose ingestion. A clear-cut first phase occurs only when the beta-cell is exposed to a rapidly changing glucose stimulus, like the one induced by a brisk intravenous glucose administration. In contrast, peripheral insulin concentration following glucose ingestion does not bear any clear sign of biphasic shape. Coupling data from the literature with the results of a beta-cell model simulation, a close relationship between the first-phase insulin response to intravenous glucose and the early insulin response to glucose ingestion emerges. It appears that the same ability of the beta-cell to produce a pronounced first phase in response to an intravenous glucose challenge can generate a rapidly increasing early phase in response to the blood glucose profile following glucose ingestion. This early insulin response to glucose is enhanced by the concomitant action of incretins and neural responses to nutrient ingestion. Thus, under physiological circumstances, the key feature of the early insulin response seems to be the ability to generate a rapidly increasing insulin profile. This notion is corroborated by recent experimental evidence that the early insulin response, when assessed at the portal level with a frequent sampling, displays a pulsatile nature. Thus, even though the classical first phase does not exist under physiological conditions, the oscillatory behavior identified at the portal level does serve the purpose of rapidly exposing the liver to elevated insulin levels that, also in virtue of their up-and-down pattern, are particularly effective in restraining hepatic glucose production.     

Inhibitors of sterol synthesis. A major role of chylomicrons in the metabolism of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one in the rat

The metabolism of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one (I), a potent regulator of cholesterol (Chol) metabolism which has significant hypocholesterolemic activity upon oral administration to animals, has been investigated in male rats. After intragastric administration of [2,4-3H] I and [4-14C]Chol in triolein to intestinal lymph duct-cannulated rats, most of the 3H of the lymph was associated with chylomicrons. Most of the 3H in the chylomicrons was associated with fatty acid esters of I and the oleate ester represented the major species of the esters of I. After intravenous injection of the isolated doubly-labeled chylomicrons to intact rats, rapid clearance of 3H and 14C from blood was observed which was associated with a rapid and selective uptake of 3H and 14C by liver. The rate of disappearance of 3H from blood and the rate of uptake of 3H by liver were similar, if not identical, to those for 14C. In contrast, the disappearance of 3H from the liver was much more rapid than that of 14C. Studies of the distribution of 3H in liver demonstrated rapid formation of free I and the formation of [3H]Chol. In addition, significant amounts of the 3H in liver were associated with polar materials, a finding which was not observed in the case of 14C. After intravenous administration of the doubly-labeled chylomicrons to bile duct-cannulated rats, very rapid and substantial metabolism of the administered 3H to polar biliary metabolites was observed. The bulk of the 3H not recovered in bile at 49 h after the injection of the labeled chylomicrons was recovered in blood and tissues and almost all (integral of 94%) of this material was associated with Chol and Chol esters. The combined results indicate an important role for chylomicrons in the overall metabolism of I. The selective delivery of I to liver as its oleate ester in chylomicrons (or, more probably, as chylomicron remnants) and the subsequent metabolism of the oleate ester of I in liver has important consequences with respect to the actions of I which are discussed herein.     

Effects of Y. pestis mouse toxin on carbohydrate metabolism in rats

Effects of intravenous Y. pestis mouse toxin (LD50) injection on glucose, lactate glucagon, insulin blood levels and cAMP liver content in dynamics of intoxication development were studied. Hypoglycemia, observed 2 hours after toxin administration seems not to be due to the enhanced glucose utilization in peripheral tissues because insulin blood level during this period was decreased and lactate concentration has not been changed. Glucagon content by 2-5 hour of shock was strong elevated. Proposal is made that Y. pestis mouse toxin might induce carbohydrate metabolism alterations via direct liver glucose synthesising enzymes inhibition rather than cAMP-dependent glycogenolysis and gluconeogenesis regulation disturbances in this organ.     

Acute fructose intake suppresses fasting-induced hepatic gluconeogenesis through the AKT-FoxO1 pathway

Excessive intake of fructose increases lipogenesis in the liver, leading to hepatic lipid accumulation and development of fatty liver disease. Metabolic alterations in the liver due to fructose intake have been reported in many studies, but the effect of fructose administration on hepatic gluconeogenesis is not fully understood. The aim of this study was to evaluate the acute effects of fructose administration on fasting-induced hepatic gluconeogenesis. C57BL/6J mice were administered fructose solution after 14 h of fasting and plasma insulin, glucose, free fatty acids, and ketone bodies were analysed. We also measured phosphorylated AKT and forkhead box O (FoxO) 1 protein levels and gene expression related to gluconeogenesis in the liver. Furthermore, we measured glucose production from pyruvate after fructose administration. Glucose-administered mice were used as controls. Fructose administration enhanced phosphorylation of AKT in the liver, without increase of blood insulin levels. Blood free fatty acids and ketone bodies concentrations were as high as those in the fasting group after fructose administration, suggesting that insulin-induced inhibition of lipolysis did not occur in mice administered with fructose. Fructose also enhanced phosphorylation of FoxO1 and suppressed gluconeogenic gene expression, glucose-6-phosphatase activity, and glucose production from pyruvate. The present study suggests that acute fructose administration suppresses fasting-induced hepatic gluconeogenesis in an insulin-independent manner.     

Polymethacrylic acid: induction of lymphocytosis and tissue distribution.

Polmethacrylic acid (PMAA) induces up to a three-fold increase in the lymphocyte population of peripheral blood in rats, goats and calves after intravenous administration. Other routes of administration are less effective. A maximum lymphocytosis is achieved after 3 hr with all doses in excess of 30 mg PMAA/kg body weight; over the next few hours the lymphocyte level declines to normal. Granulocytes increase steadily for the first 7 hr before declining. Multiple doses of PMAA 2 hr apart failed to maintain or significantly alter the lymphocytosis. PMAA was labelled with 125I and 14C, and was traced to various sites in the rat. The greatest accumulation of radioactivity was in the spleen, lungs, liver, kidney, adrenals and mesenteric lymph nodes (with 14C-PMAA). The accumulation appeared more specific for spleen and lymph nodes since there was only a small loss of activity following removal of blood by whole body perfusion. This supports previous findings indicating that these two tissues play a major role in the development of lymphocytosis. Accumulation in the bone marrow may be indicative of stem cell mobilization. The results are discussed in terms of the lymphocytosis-inducing mechanism and the site of action of PMAA and the possible clinical application to ECIB therapy is considered.     

Extrahpetic distribution of sulfobromophthalein.

Plasma clearance of sulfobromophthalein (BSP) is widely used as a measure of hepatic function. Its validity depends upon its exclusive elimination from the body via bile. For example, in the present study, when BSP was administered intravenously (i.v.) to rats at four different doses (18.75, 37.5, 75, and 150 mg/kg), less than 0.5% of each dose was excreted into the urine and between 70 and 85% was excreted into the bile within 6 h after administration. It has been assumed that the distribution of BSP is limited to the blood and liver witith very little appearing in other tissues. When we measured the amount of BSP in the plasma, liver, and the bile 10 min after the i.v. administration of either a high (150 mg/kg) or a low (18.75 mg/kg) dose of BSP, only 60% of the dose was accounted for. The concentration of BSP and 12-I-labelled albumin (RISA) was measured in various tissue samples 10 min after administration of 17.5 or 150 mg of BSP or RISA per kilogram. More BSP was found in all tissues than was contained in the plasma entrapped therein. Thus, the distribution of BSP is not limited to the liver and plasma. During excretion BSP leaves other tissue (kidney, spleen, lung, etc.) and is ultimately excreted into the bile.