Margaret Lautz

Insulin's direct effects on the liver dominate the control of hepatic glucose production

Insulin inhibits glucose production through both direct and indirect effects on the liver; however, considerable controversy exists regarding the relative importance of these effects. The first aim of this study was to determine which of these processes dominates the acute control of hepatic glucose production (HGP). Somatostatin and portal vein infusions of insulin and glucagon were used to clamp the pancreatic hormones at basal levels in the nondiabetic dog. After a basal sampling period, insulin infusion was switched from the portal vein to a peripheral vein. As a result, the arterial insulin level doubled and the hepatic sinusoidal insulin level was reduced by half. While the arterial plasma FFA level and net hepatic FFA uptake fell by 40-50%, net hepatic glucose output increased more than 2-fold and remained elevated compared with that in the control group. The second aim of this study was to determine the effect of a 4-fold rise in head insulin on HGP during peripheral hyperinsulinemia and hepatic insulin deficiency. Sensitivity of the liver was not enhanced by increased insulin delivery to the head. Thus, this study demonstrates that the direct effects of insulin dominate the acute regulation of HGP in the normal dog.

Brain insulin action augments hepatic glycogen synthesis without suppressing glucose production or gluconeogenesis in dogs

In rodents, acute brain insulin action reduces blood glucose levels by suppressing the expression of enzymes in the hepatic gluconeogenic pathway, thereby reducing gluconeogenesis and endogenous glucose production (EGP). Whether a similar mechanism is functional in large animals, including humans, is unknown. Here, we demonstrated that in canines, physiologic brain hyperinsulinemia brought about by infusion of insulin into the head arteries (during a pancreatic clamp to maintain basal hepatic insulin and glucagon levels) activated hypothalamic Akt, altered STAT3 signaling in the liver, and suppressed hepatic gluconeogenic gene expression without altering EGP or gluconeogenesis. Rather, brain hyperinsulinemia slowly caused a modest reduction in net hepatic glucose output (NHGO) that was attributable to increased net hepatic glucose uptake and glycogen synthesis. This was associated with decreased levels of glycogen synthase kinase 3β (GSK3β) protein and mRNA and with decreased glycogen synthase phosphorylation, changes that were blocked by hypothalamic PI3K inhibition. Therefore, we conclude that the canine brain senses physiologic elevations in plasma insulin, and that this in turn regulates genetic events in the liver. In the context of basal insulin and glucagon levels at the liver, this input augments hepatic glucose uptake and glycogen synthesis, reducing NHGO without altering EGP.

Molecular Characterization of Insulin-Mediated Suppression of Hepatic Glucose Production In Vivo

OBJECTIVE Insulin-mediated suppression of hepatic glucose production (HGP) is associated with sensitive intracellular signaling and molecular inhibition of gluconeogenic (GNG) enzyme mRNA expression. We determined, for the first time, the time course and relevance (to metabolic flux) of these molecular events during physiological hyperinsulinemia in vivo in a large animal model. RESEARCH DESIGN AND METHODS 24 h fasted dogs were infused with somatostatin, while insulin (basal or 8× basal) and glucagon (basal) were replaced intraportally. Euglycemia was maintained and glucose metabolism was assessed using tracer, 2H2O, and arterio-venous difference techniques. Studies were terminated at different time points to evaluate insulin signaling and enzyme regulation in the liver. RESULTS Hyperinsulinemia reduced HGP due to a rapid transition from net glycogen breakdown to synthesis, which was associated with an increase in glycogen synthase and a decrease in glycogen phosphorylase activity. Thirty minutes of hyperinsulinemia resulted in an increase in phospho-FOXO1, a decrease in GNG enzyme mRNA expression, an increase in F2,6P2, a decrease in fat oxidation, and a transient decrease in net GNG flux. Net GNG flux was restored to basal by 4 h, despite a substantial reduction in PEPCK protein, as gluconeogenically-derived carbon was redirected from lactate efflux to glycogen deposition. CONCLUSIONS In response to acute physiologic hyperinsulinemia, 1) HGP is suppressed primarily through modulation of glycogen metabolism; 2) a transient reduction in net GNG flux occurs and is explained by increased glycolysis resulting from increased F2,6P2 and decreased fat oxidation; and 3) net GNG flux is not ultimately inhibited by the rise in insulin, despite eventual reduction in PEPCK protein, supporting the concept that PEPCK has poor control strength over the gluconeogenic pathway in vivo.

Effects of Insulin on the Metabolic Control of Hepatic Gluconeogenesis In Vivo

OBJECTIVE Insulin represses the expression of gluconeogenic genes at the mRNA level, but the hormone appears to have only weak inhibitory effects in vivo. The aims of this study were 1) to determine the maximal physiologic effect of insulin, 2) to determine the relative importance of its effects on gluconeogenic regulatory sites, and 3) to correlate those changes with alterations at the cellular level. RESEARCH DESIGN AND METHODS Conscious 60-h fasted canines were studied at three insulin levels (near basal, 4×, or 16×) during a 5-h euglycemic clamp. Pancreatic hormones were controlled using somatostatin with portal insulin and glucagon infusions. Glucose metabolism was assessed using the arteriovenous difference technique, and molecular signals were assessed. RESULTS Insulin reduced gluconeogenic flux to glucose-6-phosphate (G6P) but only at the near-maximal physiological level (16× basal). The effect was modest compared with its inhibitory effect on net hepatic glycogenolysis, occurred within 30 min, and was associated with a marked decrease in hepatic fat oxidation, increased liver fructose 2,6-bisphosphate level, and reductions in lactate, glycerol, and amino acid extraction. No further diminution in gluconeogenic flux to G6P occurred over the remaining 4.5 h of the study, despite a marked decrease in PEPCK content, suggesting poor control strength for this enzyme in gluconeogenic regulation in canines. CONCLUSIONS Gluconeogenic flux can be rapidly inhibited by high insulin levels in canines. Initially decreased hepatic lactate extraction is important, and later reduced gluconeogenic precursor availability plays a role. Changes in PEPCK appear to have little or no acute effect on gluconeogenic flux.

Interaction Between the Central and Peripheral Effects of Insulin in Controlling Hepatic Glucose Metabolism in the Conscious Dog

The importance of hypothalamic insulin action to the regulation of hepatic glucose metabolism in the presence of a normal liver/brain insulin ratio (3:1) is unknown. Thus, we assessed the role of central insulin action in the response of the liver to normal physiologic hyperinsulinemia over 4 h. Using a pancreatic clamp, hepatic portal vein insulin delivery was increased three- or eightfold in the conscious dog. Insulin action was studied in the presence or absence of intracerebroventricularly mediated blockade of hypothalamic insulin action. Euglycemia was maintained, and glucagon was clamped at basal. Both the molecular and metabolic aspects of insulin action were assessed. Blockade of hypothalamic insulin signaling did not alter the insulin-mediated suppression of hepatic gluconeogenic gene transcription but blunted the induction of glucokinase gene transcription and completely blocked the inhibition of glycogen synthase kinase-3β gene transcription. Thus, central and peripheral insulin action combined to control some, but not other, hepatic enzyme programs. Nevertheless, inhibition of hypothalamic insulin action did not alter the effects of the hormone on hepatic glucose flux (production or uptake). These data indicate that brain insulin action is not a determinant of the rapid (<4 h) inhibition of hepatic glucose metabolism caused by normal physiologic hyperinsulinemia in this large animal model.

A High-Fat, High-Fructose Diet Accelerates Nutrient Absorption and Impairs Net Hepatic Glucose Uptake in Response to a Mixed Meal in Partially Pancreatectomized Dogs

The aim of this study was to elucidate the impact of a high-fat, high-fructose diet (HFFD; fat, 52%; fructose, 17%), in the presence of a partial (~65%) pancreatectomy (PPx), on the response of the liver and extrahepatic tissues to an orally administered, liquid mixed meal. Adult male dogs were fed either a nonpurified, canine control diet (CTR; fat, 26%; no fructose; n = 5) or a HFFD (n = 5) for 8 wk. Diets were provided in a quantity to maintain neutral or positive energy balance in CTR or HFFD, respectively. Dogs underwent a sham operation or PPx at wk 0, portal and hepatic vein catheterization at wk 6, and a mixed meal test at wk 8. Postprandial glucose concentrations were significantly greater in the HFFD group (14.5 ± 2.0 mmol/L) than in the CTR group (9.2 ± 0.5 mmol/L). Impaired glucose tolerance in HFFD was due in part to accelerated gastric emptying and glucose absorption, as indicated by a more rapid rise in arterial plasma acetaminophen and the rate of glucose output by the gut, respectively, in HFFD than in CTR. It was also attributable to lower net hepatic glucose uptake (NHGU) in the HFFD group (5.5 ± 3.9 μmol · kg(-1) · min(-1)) compared to the CTR group (26.6 ± 7.0 μmol · kg(-1) · min(-1)), resulting in lower hepatic glycogen synthesis (GSYN) in the HFFD group (10.8 ± 5.4 μmol · kg(-1) · min(-1)) than in the CTR group (30.4 ± 7.0 μmol · kg(-1) · min(-1)). HFFD also displayed aberrant suppression of lipolysis by insulin. In conclusion, HFFD feeding accelerates gastric emptying and diminishes NHGU and GSYN, thereby impairing glucose tolerance following a mixed meal challenge. These data reveal a constellation of deleterious metabolic consequences associated with consumption of a HFFD for 8 wk.

Insulin-induced hypoglycemia increases hepatic sensitivity to glucagon in dogs

In individuals with type 1 diabetes, hypoglycemia is a common consequence of overinsulinization. Under conditions of insulin-induced hypoglycemia, glucagon is the most important stimulus for hepatic glucose production. In contrast, during euglycemia, insulin potently inhibits glucagons effect on the liver. The first aim of the present study was to determine whether low blood sugar augments glucagons ability to increase glucose production. Using a conscious catheterized dog model, we found that hypoglycemia increased glucagons ability to overcome the inhibitory effect of insulin on hepatic glucose production by almost 3-fold, an effect exclusively attributable to marked enhancement of the effect of glucagon on net glycogen breakdown. To investigate the molecular mechanism by which this effect comes about, we analyzed hepatic biopsies from the same animals, and found that hypoglycemia resulted in a decrease in insulin signaling. Furthermore, hypoglycemia and glucagon had an additive effect on the activation of AMPK, which was associated with altered activity of the enzymes of glycogen metabolism.

Liver Glycogen Loading Dampens Glycogen Synthesis Seen in Response to Either Hyperinsulinemia or Intraportal Glucose Infusion

The purpose of this study was to determine the effect of liver glycogen loading on net hepatic glycogen synthesis during hyperinsulinemia or hepatic portal vein glucose infusion in vivo. Liver glycogen levels were supercompensated (SCGly) in two groups (using intraportal fructose infusion) but not in two others (Gly) during hyperglycemic-normoinsulinemia. Following a 2-h control period during which fructose infusion was stopped, there was a 2-h experimental period in which the response to hyperglycemia plus either 4× basal insulin (INS) or portal vein glucose infusion (PoG) was measured. Increased hepatic glycogen reduced the percent of glucose taken up by the liver that was deposited in glycogen (74 ± 3 vs. 53 ± 5% in Gly+INS and SCGly+INS, respectively, and 72 ± 3 vs. 50 ± 6% in Gly+PoG and SCGly+PoG, respectively). The reduction in liver glycogen synthesis in SCGly+INS was accompanied by a decrease in both insulin signaling and an increase in AMPK activation, whereas only the latter was observed in SCGly+PoG. These data indicate that liver glycogen loading impairs glycogen synthesis regardless of the signal used to stimulate it.

A Soluble Guanylate Cyclase–Dependent Mechanism Is Involved in the Regulation of Net Hepatic Glucose Uptake by Nitric Oxide in Vivo

OBJECTIVE We previously showed that elevating hepatic nitric oxide (NO) levels reduced net hepatic glucose uptake (NHGU) in the presence of portal glucose delivery, hyperglycemia, and hyperinsulinemia. The aim of the present study was to determine the role of a downstream signal, soluble guanylate cyclase (sGC), in the regulation of NHGU by NO. RESEARCH DESIGN AND METHODS Studies were performed on 42-h–fasted conscious dogs fitted with vascular catheters. At 0 min, somatostatin was given peripherally along with 4× basal insulin and basal glucagon intraportally. Glucose was delivered at a variable rate via a leg vein to double the blood glucose level and hepatic glucose load throughout the study. From 90 to 270 min, an intraportal infusion of the sGC inhibitor 1H-[1,2,4] oxadiazolo[4,3-a] quinoxalin-1-one (ODQ) was given in −sGC (n = 10) and −sGC/+NO (n = 6), whereas saline was given in saline infusion (SAL) (n = 10). The −sGC/+NO group also received intraportal SIN-1 (NO donor) to elevate hepatic NO from 180 to 270 min. RESULTS In the presence of 4× basal insulin, basal glucagon, and hyperglycemia (2× basal ), inhibition of sGC in the liver enhanced NHGU (mg/kg/min; 210–270 min) by ∼55% (2.9 ± 0.2 in SAL vs. 4.6 ± 0.5 in −sGC). Further elevating hepatic NO failed to reduce NHGU (4.5 ± 0.7 in −sGC/+NO). Net hepatic carbon retention (i.e., glycogen synthesis; mg glucose equivalents/kg/min) increased to 3.8 ± 0.2 in −sGC and 3.8 ± 0.4 in −sGC/+NO vs. 2.4 ± 0.2 in SAL (P < 0.05). CONCLUSIONS NO regulates liver glucose uptake through a sGC-dependent pathway. The latter could be a target for pharmacologic intervention to increase meal-associated hepatic glucose uptake in individuals with type 2 diabetes.

Inhaled Insulin Is Associated with Prolonged Enhancement of Glucose Disposal in Muscle and Liver in the Canine

Diabetic patients treated with inhaled insulin exhibit reduced fasting plasma glucose levels. In dogs, insulin action in muscle is enhanced for as long as 3 h after insulin inhalation. This study was designed to determine whether this effect lasts for a prolonged duration such that it could explain the effect observed in diabetic patients. Human insulin was administered via inhalation (Exubera; n = 9) or infusion (Humulin R; n = 9) in dogs using an infusion algorithm that yielded matched plasma insulin kinetics between the two groups. Somatostatin was infused to prevent insulin secretion, and glucagon was infused to replace basal plasma levels of the hormone. Glucose was infused into the portal vein at 4 mg/kg/min and into a peripheral vein to maintain the arterial plasma glucose level at 160 mg/dl. Arterial and hepatic sinusoidal insulin and glucose levels were virtually identical in the two groups. Notwithstanding, glucose utilization was greater when insulin was administered by inhalation. At its peak, the peripheral glucose infusion rate was 4 mg/kg/min greater in the inhalation group, and a 50% difference between groups persisted over 8 h. Inhalation of insulin caused a greater increase in nonhepatic glucose uptake in the first 3 h after inhalation; thereafter, net hepatic glucose uptake was greater. Inhalation of insulin was associated with greater than expected (based on insulin levels) glucose disposal. This may explain the reduced fasting glucose concentrations observed in humans after administration of certain inhaled insulin formulations compared with subcutaneous insulin.