Normal hepatic glucose production in the absence of GLUT2 reveals an alternative pathway for glucose release from hepatocytes.

Glucose production by liver is a major physiological function, which is required to prevent development of hypoglycemia in the postprandial and fasted states. The mechanism of glucose release from hepatocytes has not been studied in detail but was assumed instead to depend on facilitated diffusion through the glucose transporter GLUT2. Here, we demonstrate that in the absence of GLUT2 no other transporter isoforms were overexpressed in liver and only marginally significant facilitated diffusion across the hepatocyte plasma membrane was detectable. However, the rate of hepatic glucose output was normal. This was evidenced by (i) the hyperglycemic response to i.p. glucagon injection; (ii) the in vivo measurement of glucose turnover rate; and (iii) the rate of release of neosynthesized glucose from isolated hepatocytes. These observations therefore indicated the existence of an alternative pathway for hepatic glucose output. Using a [14C]-pyruvate pulse-labeling protocol to quantitate neosynthesis and release of [14C]glucose, we demonstrated that this pathway was sensitive to low temperature (12 degreesC). It was not inhibited by cytochalasin B nor by the intracellular traffic inhibitors brefeldin A and monensin but was blocked by progesterone, an inhibitor of cholesterol and caveolae traffic from the endoplasmic reticulum to the plasma membrane. Our observations thus demonstrate that hepatic glucose release does not require the presence of GLUT2 nor of any plasma membrane glucose facilitative diffusion mechanism. This implies the existence of an as yet unsuspected pathway for glucose release that may be based on a membrane traffic mechanism.

Effects of dexamethasone on hepatic glucose production and fructose metabolism in healthy humans.

This study was designed to determine whether glucocorticoids alter autoregulation of glucose production and fructose metabolism. Two protocols with either dexamethasone (DEX) or placebo (Placebo) were performed in six healthy men during hourly ingestion of[13C]fructose (1.33 mmol.kg-1.h-1) for 3 h. In both protocols, endogenous glucose production (EGP) increased by 8 (Placebo) and 7% (DEX) after fructose, whereas gluconeogenesis from fructose represented 82 (Placebo) and 72% (DEX) of EGP. Fructose oxidation measured from breath 13CO2 was similar in both protocols [9.3 +/- 0.7 (Placebo) and 9.6 +/- 0.5 mumol.kg-1.min-1 (DEX)]. Nonoxidative carbohydrate disposal, calculated as fructose administration rate minus net carbohydrate oxidation rate after fructose ingestion measured by indirect calorimetry, was also similar in both protocols [5.8 +/- 0.8 (Placebo) and 5.9 +/- 2.0 mumol.kg-1.min-1 (DEX)]. We concluded that dexamethasone 1) does not alter the autoregulatory process that prevents a fructose-induced increase in gluconeogenesis from increasing total glucose production and 2) does not affect oxidative and nonoxidative pathways of fructose. This indicates that the insulin-regulated enzymes involved in these pathways are not affected in a major way by dexamethasone.

Effects of adrenergic blockade on hepatic glucose production during ethanol administration.

Acute ethanol administration stimulates sympathetic nervous system activity. The present study was designed to determine whether this sympathetic activation affects glycogenolysis and total hepatic glucose production (HGP) during ethanol-induced inhibition of gluconeogenesis. Nineteen volunteers participated in four protocols. Two protocols aimed to study--using combined infusion of [6,6-2H2]glucose and [U-13C]glucose, VCO2 and 13CO2 measurements--the effects of ethanol infusion alone (n = 10) or with propranolol (n = 6) or phentolamine infusion (n = 4) on HGP, glucose disposal (Rd), glucose oxidation [13C]Glcox and non-oxidative glucose disposal (NOGD = Rd - [13C]Glcox). The fourth protocol assessed the effects of saline infusion alone on HGP. Using ethanol, HGP decreased by 23%, Rd by 20% and glycaemia by 9% (all P < 0.001); heart rate increased by 10%, whereas blood pressure remained unchanged. The effects were not observed with saline, except a slight (10%) decrease in HGP (P < 0.01 vs. ethanol). Ethanol did not affect [13C]Glcox but decreased NOGD by 73% (P < 0.001). Propranolol or phentolamine did not alter any of the effects of ethanol on glucose metabolism, but decreased mean arterial pressure. Propranolol prevented the ethanol-induced increase in heart rate. In conclusion, ethanol decreased blood glucose by decreasing HGP, presumably by inhibiting gluconeogenesis. Sympathetic activation prevented the decrease in blood pressure produced by ethanol but did not stimulate glycogenolysis.

Regulation of hepatic TRB3/Akt interaction induced by physical exercise and its effect on the hepatic glucose production in an insulin resistance state

To maintain euglycemia in healthy organisms, hepatic glucose production is increased during fasting and decreased during the postprandial period. This whole process is supported by insulin levels. These responses are associated with the insulin signaling pathway and the reduction in the activity of key gluconeogenic enzymes, resulting in a decrease of hepatic glucose production. On the other hand, defects in the liver insulin signaling pathway might promote inadequate suppression of gluconeogenesis, leading to hyperglycemia during fasting and after meals. The hepatocyte nuclear factor 4, the transcription cofactor PGC1-α, and the transcription factor Foxo1 have fundamental roles in regulating gluconeogenesis. The loss of insulin action is associated with the production of pro-inflammatory biomolecules in obesity conditions. Among the molecular mechanisms involved, we emphasize in this review the participation of TRB3 protein (a mammalian homolog of Drosophila tribbles), which is able to inhibit Akt activity and, thereby, maintain Foxo1 activity in the nucleus of hepatocytes, inducing hyperglycemia. In contrast, physical exercise has been shown as an important tool to reduce insulin resistance in the liver by reducing the inflammatory process, including the inhibition of TRB3 and, therefore, suppressing gluconeogenesis. The understanding of these new mechanisms by which physical exercise regulates glucose homeostasis has critical importance for the understanding and prevention of diabetes.

Normal hepatic glucose production in the absence of GLUT2 reveals an alternative pathway for glucose release from hepatocytes

Glucose production by liver is a major physiological function, which is required to prevent development of hypoglycemia in the postprandial and fasted states. The mechanism of glucose release from hepatocytes has not been studied in detail but was assumed instead to depend on facilitated diffusion through the glucose transporter GLUT2. Here, we demonstrate that in the absence of GLUT2 no other transporter isoforms were overexpressed in liver and only marginally significant facilitated diffusion across the hepatocyte plasma membrane was detectable. However, the rate of hepatic glucose output was normal. This was evidenced by (i) the hyperglycemic response to i.p. glucagon injection; (ii) the in vivo measurement of glucose turnover rate; and (iii) the rate of release of neosynthesized glucose from isolated hepatocytes. These observations therefore indicated the existence of an alternative pathway for hepatic glucose output. Using a [14C]-pyruvate pulse-labeling protocol to quantitate neosynthesis and release of [14C]glucose, we demonstrated that this pathway was sensitive to low temperature (12°C). It was not inhibited by cytochalasin B nor by the intracellular traffic inhibitors brefeldin A and monensin but was blocked by progesterone, an inhibitor of cholesterol and caveolae traffic from the endoplasmic reticulum to the plasma membrane. Our observations thus demonstrate that hepatic glucose release does not require the presence of GLUT2 nor of any plasma membrane glucose facilitative diffusion mechanism. This implies the existence of an as yet unsuspected pathway for glucose release that may be based on a membrane traffic mechanism.

Effects of infused fructose on endogenous glucose production, gluconeogenesis, and glycogen metabolism.

To determine the mechanisms that prevent an increase in gluconeogenesis from increasing hepatic glucose output, six healthy women were infused with [1-13C]fructose (22 mumol.kg-1.min-1), somatostatin, insulin, and glucagon. In control experiment, non-13C-enriched fructose was infused at the same rate without somatostatin, and [U-13C]glucose was infused to measure specifically plasma glucose oxidation. Endogenous glucose production (EGP, [6,6-2H]glucose), net carbohydrate oxidation (CHOox, indirect calorimetry), and fructose oxidation (13CO2) were measured. EGP rate did not increase after fructose infusion with (13.1 +/- 1.2 vs. 12.9 +/- 0.3 mumol.kg-1.min-1) and without (10.3 +/- 0.5 vs. 9.7 +/- 0.5 mumol.kg-1.min-1) somatostatin, despite the fact that gluconeogenesis increased. Nonoxidative fructose disposal, corresponding mainly to glycogen synthesis, was threefold net glycogen deposition, the latter calculated as fructose infusion minus CHOox (14.8 +/- 1.1 and 4.3 +/- 2.0 mumol.kg-1.min-1). It is concluded that 1) the mechanism by which EGP remains constant when gluconeogenesis from fructose increases is independent of changes in insulin and 2) simultaneous breakdown and synthesis of glycogen occurred during fructose infusion.

Effects of fructose on hepatic glucose metabolism in humans.

Hepatic and extrahepatic insulin sensitivity was assessed in six healthy humans from the insulin infusion required to maintain an 8 mmol/l glucose concentration during hyperglycemic pancreatic clamp with or without infusion of 16.7 micromol. kg(-1). min(-1) fructose. Glucose rate of disappearance (GR(d)), net endogenous glucose production (NEGP), total glucose output (TGO), and glucose cycling (GC) were measured with [6,6-(2)H(2)]- and [2-(2)H(1)]glucose. Hepatic glycogen synthesis was estimated from uridine diphosphoglucose (UDPG) kinetics as assessed with [1-(13)C]galactose and acetaminophen. Fructose infusion increased insulin requirements 2.3-fold to maintain blood glucose. Fructose infusion doubled UDPG turnover, but there was no effect on TGO, GC, NEGP, or GR(d) under hyperglycemic pancreatic clamp protocol conditions. When insulin concentrations were matched during a second hyperglycemic pancreatic clamp protocol, fructose administration was associated with an 11.1 micromol. kg(-1). min(-1) increase in TGO, a 7.8 micromol. kg(-1). min(-1) increase in NEGP, a 2.2 micromol. kg(-1). min(-1) increase in GC, and a 7.2 micromol. kg(-1). min(-1) decrease in GR(d) (P < 0. 05). These results indicate that fructose infusion induces hepatic and extrahepatic insulin resistance in humans.

Effects of fructose and glucose overfeeding on hepatic insulin sensitivity and intrahepatic lipids in healthy humans.

OBJECTIVE: To assess how intrahepatic fat and insulin resistance relate to daily fructose and energy intake during short-term overfeeding in healthy subjects. DESIGN AND METHODS: The analysis of the data collected in several studies in which fasting hepatic glucose production (HGP), hepatic insulin sensitivity index (HISI), and intrahepatocellular lipids (IHCL) had been measured after both 6-7 days on a weight-maintenance diet (control, C; n = 55) and 6-7 days of overfeeding with 1.5 (F1.5, n = 7), 3 (F3, n = 17), or 4 g fructose/kg/day (F4, n = 10), with 3 g glucose/kg/day (G3, n = 11), or with 30% excess energy as saturated fat (fat30%, n = 10). RESULTS: F3, F4, G3, and fat30% all significantly increased IHCL, respectively by 113 ± 86, 102 ± 115, 59 ± 92, and 90 ± 74% as compared to C (all P < 0.05). F4 and G3 increased HGP by 16 ± 10 and 8 ± 11% (both P < 0.05), and F3 and F4 significantly decreased HISI by 20 ± 22 and 19 ± 14% (both P < 0.01). In contrast, there was no significant effect of fat30% on HGP or HISI. CONCLUSIONS: Short-term overfeeding with fructose or glucose decreases hepatic insulin sensitivity and increases hepatic fat content. This indicates short-term regulation of hepatic glucose metabolism by simple carbohydrates.

Time sequence of the intensification of the liver glucose production induced by high-fat diet in mice

It is well established that the development of insulin resistance shows a temporal sequence in different organs and tissues. Moreover, considering that the main aspect of insulin resistance in liver is a process of glucose overproduction from gluconeogenesis, we investigated if this metabolic change also shows temporal sequence. For this purpose, a well-established experimental model of insulin resistance induced by high-fat diet (HFD) was used. The mice received HFD (HFD group) or standard diet (COG group) for 1, 7, 14 or 56?days. The HFD group showed increased (P?<?0.05 versus COG) epididymal, retroperitoneal and inguinal fat weight from days 1 to 56. In agreement with these results, the HFD group also showed higher body weight (P?<?0.05 versus COG) from days 7 to 56. Moreover, the changes induced by HFD on liver gluconeogenesis were progressive because the increment (P?<?0.05 versus COG) in glucose production from l-lactate, glycerol, l-alanine and l-glutamine occurred 7, 14, 56 and 56 days after the introduction of the HFD schedule, respectively. Furthermore, glycaemia and cholesterolemia increased (P?<?0.05 versus COG) 14?days after starting the HFD schedule. Taken together, the results suggest that the intensification of liver gluconeogenesis induced by an HFD is not a synchronous all-or-nothing process but is specific for each gluconeogenic substrate and is integrated in a temporal manner with the progressive augmentation of fasting glycaemia. Copyright (c) 2012 John Wiley & Sons, Ltd.

Maturation of endogenous glucose production in preterm and term calves

Glucose disposability is often impaired in neonatal calves and even more in preterm calves. The objective of this study was to investigate ontogenic maturation of endogenous glucose production (eGP) in calves and its effects on postnatal glucose homeostasis. Calves (n = 7 per group) were born preterm (PT; delivered by section 9 d before term) or at term (T; spontaneous vaginal delivery), or spontaneously born and fed colostrum for 4 d (TC). Blood samples were taken immediately after birth and before and 2h after feeding at 24h after birth (PT; T) or on d 4 of life (TC) to determine metabolic and endocrine changes. After birth (PT and T) or on d 3 of life (TC), fasted calves were gavaged with deuterium-labeled water to determine gluconeogenesis (GNG) and intravenously infused with [U(13)C]-glucose to measure eGP and glucose oxidation (GOx) in blood plasma. After slaughter at 26h after birth (PT, T) or on d 4 of life (TC), glycogen concentrations in liver and hepatic mRNA concentrations and enzyme activities of pyruvate carboxylase, phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase were measured. Preterm calves had the lowest plasma concentrations of cortisol and 3,5,3'-triiodothyronine at birth. Plasma glucose concentrations from d 1 to 2 decreased more, but plasma concentrations of lactate and urea and glucagon:insulin ratio were higher in PT than in T and TC calves. The eGP, GNG, GOx, as well as hepatic glycogen concentrations and PEPCK activities, were lowest in PT calves. Results indicate impaired glucose homeostasis due to decreased eGP in PT calves and maturation of eGP with ontogenic development.

Intestinal glucose absorption but not endogenous glucose production differs between colostrum- and formula-fed neonatal calves

Glucose supply markedly changes during the transition to extrauterine life. In this study, we investigated diet effects on glucose metabolism in neonatal calves. Calves were fed colostrum (C; n = 7) or milk-based formula (F; n = 7) with similar nutrient content up to d 4 of life. Blood plasma samples were taken daily before feeding and 2 h after feeding on d 4 to measure glucose, lactate, nonesterified fatty acids, protein, urea, insulin, glucagon, and cortisol concentrations. On d 2, additional blood samples were taken to measure glucose first-pass uptake (FPU) and turnover by oral [U-(13)C]-glucose and i.v. [6,6-(2)H(2)]-glucose infusion. On d 3, endogenous glucose production and gluconeogenesis were determined by i.v. [U-(13)C]-glucose and oral deuterated water administration after overnight feed deprivation. Liver tissue was obtained 2 h after feeding on d 4 and glycogen concentration and activities and mRNA abundance of gluconeogenic enzymes were measured. Plasma glucose and protein concentrations and hepatic glycogen concentration were higher (P < 0.05), whereas plasma urea, glucagon, and cortisol (d 2) concentrations as well as hepatic pyruvate carboxylase mRNA level and activity were lower (P < 0.05) in group C than in group F. Orally administered [U-(13)C]-glucose in blood was higher (P < 0.05) but FPU tended to be lower (P < 0.1) in group C than in group F. The improved glucose status in group C resulted from enhanced oral glucose absorption. Metabolic and endocrine changes pointed to elevated amino acid degradation in group F, presumably to provide substrates to meet energy requirements and to compensate for impaired oral glucose uptake.

Supplementation of conjugated linoleic acid in dairy cows reduces endogenous glucose production during early lactation.

Trans-10,cis-12 conjugated linoleic acid (CLA) supplementation causes milk fat depression in dairy cows, but CLA effects on glucose metabolism are not clear. The objective of the study was to investigate glucose metabolism, especially endogenous glucose production (eGP) and glucose oxidation (GOx), as well as hepatic genes involved in endogenous glucose production in Holstein cows supplemented either with 50 g of rumen-protected CLA (9% trans-10,cis-12 and 10% cis-9,trans-11; CLA; n=10) or 50 g of control fat (24% C18:2; Ctrl; n=10) from wk 2 before parturition to wk 9 of lactation. Animal performance data were recorded and blood metabolites and hormones were taken weekly from 2 wk before to 12 wk after parturition. During wk 3 and 9 after parturition, glucose tolerance tests were performed and eGP and GOx were measured by [U-(13)C] glucose infusion. Liver biopsies were taken at the same time to measure total fat and glycogen concentrations and gene expression of pyruvate carboxylase, cytosolic phosphoenolpyruvate carboxykinase, glucose-6-phosphatase, and carnitine palmitoyl-transferase 1. Conjugated linoleic acid feeding reduced milk fat, but increased milk lactose output; milk yield was higher starting 5 wk after parturition in CLA-fed cows than in Ctrl-fed cows. Energy balance was more negative during CLA supplementation, and plasma concentrations of glucose were higher immediately after calving in CLA-fed cows. Conjugated linoleic acid supplementation did not affect insulin release during glucose tolerance tests, but reduced eGP in wk 3, and eGP and GOx increased with time after parturition. Hepatic gene expression of cytosolic phosphoenolpyruvate carboxykinase tended to be lower in CLA-fed cows than in Ctrl-fed cows. In spite of lower eGP in CLA-fed cows, lactose output and plasma glucose concentrations were greater in CLA-fed cows than in Ctrl-fed cows. This suggests a CLA-related glucose sparing effect most likely due to lower glucose utilization for milk fat synthesis and probably because of a more efficient whole-body energy utilization in CLA-fed cows.

Effects of infused amino acids on glucose production and utilization in healthy human subjects.

Amino acids have been reported to increase endogenous glucose production in normal human subjects during hyperinsulinemia: however, controversy exists as to whether insulin-mediated glucose disposal is inhibited under these conditions. The effect of an amino acid infusion on glucose oxidation rate has so far not been determined. Substrate oxidation rates, endogenous glucose production, and [13C]glucose synthesis from [13C]bicarbonate were measured in six normal human subjects during sequential infusions of exogenous glucose and exogenous glucose with (n = 5) or without (n = 5) exogenous amino acids. Amino acids increased endogenous glucose production by 84% and [13C]glucose synthesis by 235%. Glucose oxidation estimated from indirect calorimetry decreased slightly after amino acids, but glucose oxidation estimated from [13C]glucose-13CO2 data was increased by 14%. It is concluded that gluconeogenesis is the major pathway of amino acid degradation. During amino acid administration, indirect calorimetry underestimates the true rate of glucose oxidation, whereas glucose oxidation calculated from the 13C enrichment of expired CO2 during [U-13C]glucose infusion does not. A slight stimulation of glucose oxidation during amino acid infusion, concomitant with an increased plasma insulin concentration, indicates that amino acids do not inhibit glucose oxidation.

Blocking VLDL secretion causes hepatic steatosis but does not affect peripheral lipid stores or insulin sensitivity in mice

The liver secretes triglyceride-rich VLDLs, and the triglycerides in these particles are taken up by peripheral tissues, mainly heart, skeletal muscle, and adipose tissue. Blocking hepatic VLDL secretion interferes with the delivery of liver-derived triglycerides to peripheral tissues and results in an accumulation of triglycerides in the liver. However, it is unclear how interfering with hepatic triglyceride secretion affects adiposity, muscle triglyceride stores, and insulin sensitivity. To explore these issues, we examined mice that cannot secrete VLDL [due to the absence of microsomal triglyceride transfer protein (Mttp) in the liver]. These mice exhibit markedly reduced levels of apolipoprotein B-100 in the plasma, along with reduced levels of triglycerides in the plasma. Despite the low plasma triglyceride levels, triglyceride levels in skeletal muscle were unaffected. Adiposity and adipose tissue triglyceride synthesis rates were also normal, and body weight curves were unaffected. Even though the blockade of VLDL secretion caused hepatic steatosis accompanied by increased ceramides and diacylglycerols in the liver, the mice exhibited normal glucose tolerance and were sensitive to insulin at the whole-body level, as judged by hyperinsulinemic euglycemic clamp studies. Normal hepatic glucose production and insulin signaling were also maintained in the fatty liver induced by Mttp deletion. Thus, blocking VLDL secretion causes hepatic steatosis without insulin resistance, and there is little effect on muscle triglyceride stores or adiposity

Liver hyperplasia and paradoxical regulation of glycogen metabolism and glucose-sensitive gene expression in GLUT2-null hepatocytes. Further evidence for the existence of a membrane-based glucose release pathway.

We investigated the impact of GLUT2 gene inactivation on the regulation of hepatic glucose metabolism during the fed to fast transition. In control and GLUT2-null mice, fasting was accompanied by a approximately 10-fold increase in plasma glucagon to insulin ratio, a similar activation of liver glycogen phosphorylase and inhibition of glycogen synthase and the same elevation in phosphoenolpyruvate carboxykinase and glucose-6-phosphatase mRNAs. In GLUT2-null mice, mobilization of glycogen stores was, however, strongly impaired. This was correlated with glucose-6-phosphate (G6P) levels, which remained at the fed values, indicating an important allosteric stimulation of glycogen synthase by G6P. These G6P levels were also accompanied by a paradoxical elevation of the mRNAs for L-pyruvate kinase. Re-expression of GLUT2 in liver corrected the abnormal regulation of glycogen and L-pyruvate kinase gene expression. Interestingly, GLUT2-null livers were hyperplasic, as revealed by a 40% increase in liver mass and 30% increase in liver DNA content. Together, these data indicate that in the absence of GLUT2, the G6P levels cannot decrease during a fasting period. This may be due to neosynthesized glucose entering the cytosol, being unable to diffuse into the extracellular space, and being phosphorylated back to G6P. Because hepatic glucose production is nevertheless quantitatively normal, glucose produced in the endoplasmic reticulum may also be exported out of the cell through an alternative, membrane traffic-based pathway, as previously reported (Guillam, M.-T., Burcelin, R., and Thorens, B. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 12317-12321). Therefore, in fasting, GLUT2 is not required for quantitative normal glucose output but is necessary to equilibrate cytosolic glucose with the extracellular space. In the absence of this equilibration, the control of hepatic glucose metabolism by G6P is dominant over that by plasma hormone concentrations.