Michael J. Pagliassotti

Sources of carbon for hepatic glycogen synthesis in the conscious dog.

To identify the source(s) of carbon for the indirect pathway of hepatic glycogen synthesis, we studied nine 42-h fasted conscious dogs given a continuous intraduodenal infusion of glucose, labeled with [1-13C]glucose and [3-3H]glucose, at 8 mg.kg-1.min-1 for 240 min. Glycogen formation by the direct pathway was measured by 13C-NMR. Net hepatic balances of glucose, gluconeogenic amino acids, lactate, and glycerol were determined using the arteriovenous difference technique. During the steady-state period (the final hour of the infusion), 81% of the glucose infused was absorbed as glucose. Net gut output of lactate and alanine accounted for 5% and 3% of the glucose infused, respectively. The cumulative net hepatic uptakes were: glucose, 15.5 +/- 3.8 g; gluconeogenic amino acids, 32.2 +/- 2.2 mmol (2.9 +/- 0.2 g of glucose equivalents); and glycerol, 6.1 +/- 0.9 mmol (0.6 +/- 0.1 g of glucose equivalents). The liver produced a net of 29.2 +/- 9.6 mmol of lactate (2.6 +/- 0.8 g of glucose equivalents). Net hepatic glycogen synthesis totaled 9.3 +/- 2.5 g (1.8 +/- 0.4 g/100 g liver), with the direct pathway being responsible for 57 +/- 10%. Thus, net hepatic glucose uptake was sufficient to account for all glycogen formed by both the direct and indirect pathways. Total net hepatic uptake of gluconeogenic precursors (gluconeogenic amino acids, glycerol, and lactate) was able to account for only 20% of net glycogen synthesis by the indirect pathway. In a net sense, our data are consistent with an intrahepatic origin for most of the three-carbon precursors used for indirect glycogen synthesis.

Chronic Hyperglycemia Enhances PEPCK Gene Expression and Hepatocellular Glucose Production Via Elevated Liver Activating Protein/Liver Inhibitory Protein Ratio

Acute hyperglycemia normally suppresses hepatic glucose production (HGP) and gluconeogenic gene expression. Conversely, chronic hyperglycemia is accompanied by progressive increases in basal HGP and is a major contributor to hyperglycemia in both type 1 and type 2 diabetes by mechanisms that are poorly understood. The aim of this study was to investigate the molecular mechanisms whereby hyperglycemia contributes to excessive gluconeogenesis in Fao hepatoma cells. Increasing glucose from 5 to 20 mmol/l resulted in loss of glucose inhibition of PEPCK gene expression after 12 h. Furthermore, 24 h of incubation with 20 mmol/l glucose increased cAMP-stimulated PEPCK mRNA by approximately 40% (P < 0.05) and similarly increased glucose production. Although total CCAAT/enhancer-binding protein beta (C/EBPbeta) protein levels were suppressed, 20 mmol/l glucose increased the liver activating protein (LAP; an active isoform of C/EBPbeta)/liver inhibitory protein (LIP; an inhibitory isoform of C/EBPbeta) ratio significantly. Chromatin immunoprecipitation studies of the endogenous PEPCK gene demonstrated an increased association of LAP with the cAMP response element of the promoter. Using transient transfection to manipulate the LAP/LIP ratio, we also demonstrate a direct relationship between this ratio and PEPCK promoter activity. An increased LAP/LIP ratio not only enhanced cAMP- and dexamethasone-induced PEPCK gene expression but also impaired the repressive effect of insulin. These results demonstrate that sustained hyperglycemia diminishes the inhibitory effect of glucose and insulin on PEPCK expression and enhances hormone-stimulated PEPCK gene expression and hepatocellular glucose production. Because prolonged hyperglycemia increases the LAP/LIP ratio and can potentiate hormone induction of PEPCK transcription, our results suggest that a hyperglycemia-driven increased LAP/LIP ratio may be a critical molecular event in the pathogenesis of increased HGP in diabetes.

Magnitude of Negative Arterial-Portal Glucose Gradient Alters Net Hepatic Glucose Balance in Conscious Dogs

To examine the relationship between the magnitude of the negative arterial-portal glucose gradient and net hepatic glucose uptake, two groups of 42-h fasted, conscious dogs were infused with somatostatin, to suppress endogenous insulin and glucagon secretion, and the hormones were replaced intraportally to create hyperinsulinemia (3- to 4-fold basal) and basal glucagon levels. The hepatic glucose load to the liver was doubled and different negative arterial-portal glucose gradients were established by altering the ratio between portal and peripheral vein glucose infusions. In protocol 1 (n = 6) net hepatic glucose uptake was 42.2 ± 6.7, 35.0 ± 3.9, and 33.3 ± 4.4 μmol · kg−1 · min−1 at arterial-portal plasma glucose gradients of −4.1 ± 0.9, −1.8 ± 0.4, and −0.8 ± 0.1 mM, respectively. In protocol 2 (n = 6) net hepatic glucose uptake was 26.1 ± 2.8 and 12.2 ± 1.7 μmol · kg−1 · min−1 at arterial-portal plasma glucose gradients of −0.9 ± 0.2 and −0.4 ± 0.1 mM, respectively. No changes in the hepatic insulin or glucose loads were evident within a given protocol. Although net hepatic glucose uptake was lower in protocol 2 when compared with protocol 1 (26.1 ± 2.8 vs. 33.3 ± 4.4 μmol · kg−1 · min−1) in the presence of a similar arterial-portal plasma glucose gradient (−0.9 vs. −0.8 mM) the difference could be attributed to the hepatic glucose load being lower in protocol 2 (i.e., hepatic fractional glucose extraction was not significantly different) primarily as a result of lower hepatic blood flow. In conclusion, in the presence of fixed hepatic glucose and insulin loads, the magnitude of the negative arterial-portal glucose gradient can modify net hepatic glucose uptake in vivo.

Effect of Spontaneous Gestational Diabetes on Fetal and Postnatal Hepatic Insulin Resistance in Leprdb/+ Mice

Infant macrosomia is a classic feature of a gestational diabetes mellitus (GDM) pregnancy and is associated with increased risk of adult obesity and type II diabetes mellitus, however mechanisms linking GDM and later disease remain poorly understood. The heterozygous leptin receptor-deficient (Leprdb/+) mouse develops spontaneous GDM and the fetuses display characteristics similar to infants of GDM mothers. We examined the effects of GDM on maternal insulin resistance, fetal growth, and postnatal development of hepatic insulin resistance. Fetal body weight on d 18 of gestation was 6.5% greater (p < 0.05) in pups from ad libitum-fed db/+ mothers compared with wild-type (WT) controls. Pair-feeding db/+ mothers to the intake of WT mothers normalized fetal weight despite less than normal maternal insulin sensitivity. More stringent caloric restriction reduced insulin and glucose levels below WT controls and resulted in fetal intrauterine growth restriction. The level of hepatic insulin receptor protein was decreased by 28% to 31% in both intrauterine growth restriction and fetuses from ad libitum-fed GDM mothers compared with offspring from WT mothers. In 24-wk-old adult offspring from GDM mothers, body weight was similar to WT offspring, however, the females from GDM mothers were fatter and hyperinsulinemic compared with offspring from WT mothers. Insulin-stimulated phosphorylation of Akt, a key intermediate in insulin signaling, was severely decreased in the livers of adult GDM offspring. Hepatic glucose-6-phosphatase activity was also inappropriately increased in the adult offspring from GDM mothers. These results suggest that spontaneous GDM in the pregnant Leprdb/+ mouse is triggered by overfeeding, and this effect results in obesity and insulin resistance in the livers of the adult offspring. The specific decrease in Akt phosphorylation in livers of adult offspring suggests that this may be a mechanism for reduced insulin-dependent physiologic events, such as suppression of hepatic glucose production, a defect associated with susceptibility to type II diabetes mellitus.

Regulation of hepatic lactate balance during exercise

The rate of exchange of lactate across the liver gives important insights into intracellular processes during muscular work. At the onset of exercise hepatic glycogenolysis increases rapidly, resulting in high rates of glycolytic flux and a transient rise in lactate output. With increasing exercise duration, gluconeogenesis is accelerated and the liver gradually shifts from a lactate-producing to a lactate-consuming state. Exercise-induced changes in hormone levels are critical in the regulation of hepatic glycogenolysis and gluconeogenesis and, therefore, net hepatic lactate balance. The fall in insulin stimulates hepatic glycogenolysis, glycolytic flux, and, as a result, hepatic lactate output. On the other hand, the stimulatory effects of glucagon on gluconeogenesis elicit an increase in hepatic lactate uptake. The rise in epinephrine may regulate gluconeogenesis during prolonged exercise by stimulating peripheral lactate mobilization, thereby providing gluconeogenic substrate to the liver. Chronic hepatic-denervation leads to an increase in gluconeogenesis and net hepatic lactate uptake at rest without altering total glucose production. However, the response to exercise is unaffected by the absence of hepatic nerves. Hence, the direction and magnitude of the hepatic lactate balance during exercise yields important information regarding flux through the gluconeogenic and glycolytic pathways, such that high rates of gluconeogenesis correspond to accelerated rates of hepatic lactate uptake and high rates of hepatic glycolytic flux lead to increased rates of hepatic lactate output.

Effects of vagal blockade on the counterregulatory response to insulin-induced hypoglycemia in the dog.

Our aim was to determine whether vagal transmission is required for the hormonal response to insulin-induced hypoglycemia in 18-h-fasted conscious dogs. Hollow coils were placed around the vagus nerves, with animals under general anesthesia, 2 wk before an experiment. On the day of the study they were perfused with -15 degrees C ethanol for the purpose of blocking vagal transmission, either coincident with the onset of insulin-induced hypoglycemia or after 2 h of established hypoglycemia. In a separate study the coils were perfused with 37 degrees C ethanol in a sham cooling experiment. The following parameters were measured: heart rate, arterial plasma glucose, insulin, pancreatic polypeptide, glucagon, cortisol, epinephrine, norepinephrine, glycerol, free fatty acids, and endogenous glucose production. In response to insulin-induced hypoglycemia (42 mg/dl), plasma glucagon peaked at a level that was double the basal level, and plasma cortisol levels quadrupled. Plasma epinephrine and norepinephrine levels both rose considerably to 2,135 +/- 314 and 537 +/- 122 pg/ml, respectively, as did plasma glycerol (330 +/- 60%) and endogenous glucose production (150 +/- 20%). Plasma free fatty acids peaked at 150 +/- 20% and then returned to basal levels by the end of the study. The hypoglycemia-induced changes were not different when vagal cooling was initiated after the prior establishment of hypoglycemia. Similarly, when vagal cooling occurred concurrently with the initiation of insulin-induced hypoglycemia (46 mg/dl), there were no significant differences in any of the parameters measured compared with the control. Thus vagal blockade did not prevent the effect on either the hormonal or metabolic responses to low blood sugar. Functioning vagal afferent nerves are not required for a normal response to insulin-induced hypoglycemia.Our aim was to determine whether vagal transmission is required for the hormonal response to insulin-induced hypoglycemia in 18-h-fasted conscious dogs. Hollow coils were placed around the vagus nerves, with animals under general anesthesia, 2 wk before an experiment. On the day of the study they were perfused with -15°C ethanol for the purpose of blocking vagal transmission, either coincident with the onset of insulin-induced hypoglycemia or after 2 h of established hypoglycemia. In a separate study the coils were perfused with 37°C ethanol in a sham cooling experiment. The following parameters were measured: heart rate, arterial plasma glucose, insulin, pancreatic polypeptide, glucagon, cortisol, epinephrine, norepinephrine, glycerol, free fatty acids, and endogenous glucose production. In response to insulin-induced hypoglycemia (42 mg/dl), plasma glucagon peaked at a level that was double the basal level, and plasma cortisol levels quadrupled. Plasma epinephrine and norepinephrine levels both rose considerably to 2,135 ± 314 and 537 ± 122 pg/ml, respectively, as did plasma glycerol (330 ± 60%) and endogenous glucose production (150 ± 20%). Plasma free fatty acids peaked at 150 ± 20% and then returned to basal levels by the end of the study. The hypoglycemia-induced changes were not different when vagal cooling was initiated after the prior establishment of hypoglycemia. Similarly, when vagal cooling occurred concurrently with the initiation of insulin-induced hypoglycemia (46 mg/dl), there were no significant differences in any of the parameters measured compared with the control. Thus vagal blockade did not prevent the effect on either the hormonal or metabolic responses to low blood sugar. Functioning vagal afferent nerves are not required for a normal response to insulin-induced hypoglycemia.

Insulin is required for the liver to respond to intraportal glucose delivery in the conscious dog

To determine whether insulin is essential for the augmented hepatic glucose uptake observed in the presence of intraportal glucose delivery, SRIF was used to induce acute insulin deficiency in 5 conscious dogs, and glucose was infused into the portal vein or a peripheral vein in two sequential, randomized periods. Insulin and C-peptide levels were below the limits of detection after SRIF infusion, and the load of glucose presented to the liver was approximately doubled and equivalent during the portal and peripheral periods. Net hepatic glucose output was 2.9 ± 0.9 and 2.1 ± 1.1 μmol · kg−1 · min−1 during portal and peripheral glucose delivery, respectively. In an additional set of protocols, pancreatectomized dogs were used to investigate the effects of prolonged insulin deficiency (n = 5) and acute insulin replacement (n = 4) on the hepatic response to intraportal glucose delivery. In the prolonged insulin deficiency protocol, SRIF was used to lower glucagon and thereby reduce circulating glucose levels, and glucose was infused into the portal or peripheral circulations in two sequential, randomized periods. As with acute insulin deficiency, net hepatic glucose output was still evident and similar (3.6 ± 1.1 and 4.2 ± 1.3 μmol · kg−1 · min−1) during portal and peripheral glucose delivery, respectively. When the pancreatectomized dogs were restudied using a similar protocol, but one in which insulin was replaced (4X-basal), and the glucose load to the liver was matched to that which occurred in the prolonged insulin deficiency protocol, net hepatic glucose uptake was 23.6 ± 6.1 μmol · kg−1 · min−1 during portal glucose delivery but only 10.3 ± 3.5 μmol · kg−1 · min−1 during peripheral glucose delivery. These results suggest that the induction of net hepatic glucose uptake and the augmented hepatic response to intraportal glucose delivery require the presence of insulin.

The head arterial glucose level is not the reference site for generation of the portal signal in conscious dogs

Experiments were performed on twelve 42-h-fasted, conscious dogs to determine whether the head arterial glucose level is used as a reference standard for comparison with the portal glucose level in bringing about the stimulatory effect of portal glucose delivery on net hepatic glucose uptake (NHGU). Each experiment consisted of an 80-min equilibration, a 40-min control, and two 90-min test periods. After the control period, somatostatin was given along with insulin (7.2 pmol ⋅ kg-1 ⋅ min-1; 3.5-fold increase) and glucagon (0.6 ng ⋅ kg-1 ⋅ min-1; basal) intraportally. Glucose was infused intraportally (22.2 μmol ⋅ kg-1 ⋅ min-1) and peripherally as needed to double the hepatic glucose load. In one test period, glucose was infused into both vertebral and carotid arteries (HEADG; 22.2 ± 0.8 μmol ⋅ kg-1 ⋅ min-1); in the other test period, saline was infused into the head arteries (HEADS). One-half of the dogs received HEADG first. When all dogs are considered, the blood arterial-portal glucose gradients (-0.52 ± 0.07 vs. -0.49 ± 0.03 mM) and the hepatic glucose loads (339 ± 14 vs. 334 ± 20 μmol ⋅ kg-1 ⋅ min-1) were similar in HEADG and HEADS. NHGU was 24.1 ± 3.8 and 25.1 ± 4.6 μmol ⋅ kg-1 ⋅ min-1, and nonhepatic glucose uptake was 46.1 ± 4.2 and 48.8 ± 7.0 μmol ⋅ kg-1 ⋅ min-1in HEADG and HEADS, respectively. The head arterial glucose level is not the reference standard used for comparison with the portal glucose level in the generation of the portal signal.Experiments were performed on twelve 42-h-fasted, conscious dogs to determine whether the head arterial glucose level is used as a reference standard for comparison with the portal glucose level in bringing about the stimulatory effect of portal glucose delivery on net hepatic glucose uptake (NHGU). Each experiment consisted of an 80-min equilibration, a 40-min control, and two 90-min test periods. After the control period, somatostatin was given along with insulin (7.2 pmol. kg(-1). min(-1); 3.5-fold increase) and glucagon (0.6 ng. kg(-1). min(-1); basal) intraportally. Glucose was infused intraportally (22.2 micromol. kg(-1). min(-1)) and peripherally as needed to double the hepatic glucose load. In one test period, glucose was infused into both vertebral and carotid arteries (HEAD(G); 22.2 +/- 0.8 micromol. kg(-1). min(-1)); in the other test period, saline was infused into the head arteries (HEAD(S)). One-half of the dogs received HEAD(G) first. When all dogs are considered, the blood arterial-portal glucose gradients (-0.52 +/- 0.07 vs. -0.49 +/- 0.03 mM) and the hepatic glucose loads (339 +/- 14 vs. 334 +/- 20 micromol. kg(-1). min(-1)) were similar in HEAD(G) and HEAD(S). NHGU was 24.1 +/- 3.8 and 25.1 +/- 4.6 micromol. kg(-1). min(-1), and nonhepatic glucose uptake was 46.1 +/- 4.2 and 48.8 +/- 7.0 micromol. kg(-1). min(-1) in HEAD(G) and HEAD(S), respectively. The head arterial glucose level is not the reference standard used for comparison with the portal glucose level in the generation of the portal signal.