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The contribution of hormone-independent counterregulatory signals in defense of insulin-induced hypoglycemia was determined in adrenalectomized, overnight-fasted conscious dogs receiving hepatic portal vein insulin infusions at a rate 20-fold basal. Either euglycemia was maintained () or hypoglycemia (≈45 mg/dl) was allowed to occur. There were three hypoglycemic groups: one in which hepatic autoregulation against hypoglycemia occurred in the absence of sympathetic nervous system input (), one in which autoregulation occurred in the presence of norepinephrine (NE) signaling to fat and muscle (), and one in which autoregulation occurred in the presence of NE signaling to fat, muscle, and liver (). Average net hepatic glucose balance (NHGB) during the last hour for was -0.7 ± 0.1, 0.3 ± 0.1 ( < 0.01 vs. ), 0.7 ± 0.1 ( = 0.01 vs. ), and 0.8 ± 0.1 ( = 0.7 vs. ) mg·kg·min, respectively. Hypoglycemia per se () increased NHGB by causing an inhibition of net hepatic glycogen synthesis. NE signaling to fat and muscle () increased NHGB further by mobilizing gluconeogenic precursors resulting in a rise in gluconeogenesis. Lowering glucose per se decreased nonhepatic glucose uptake by 8.9 mg·kg·min, and the addition of increased neural efferent signaling to muscle and fat blocked glucose uptake further by 3.2 mg·kg·min The addition of increased neural efferent input to liver did not affect NHGB or nonhepatic glucose uptake significantly. In conclusion, even in the absence of increases in counterregulatory hormones, the body can defend itself against hypoglycemia using glucose autoregulation and increased neural efferent signaling, both of which stimulate hepatic glucose production and limit glucose utilization.
Copyright © 2017 the American Physiological Society.
Liver glycogen is important for the counterregulation of hypoglycemia and is reduced in individuals with type 1 diabetes (T1D). Here, we examined the effect of varying hepatic glycogen content on the counterregulatory response to low blood sugar in dogs. During the first 4 hours of each study, hepatic glycogen was increased by augmenting hepatic glucose uptake using hyperglycemia and a low-dose intraportal fructose infusion. After hepatic glycogen levels were increased, animals underwent a 2-hour control period with no fructose infusion followed by a 2-hour hyperinsulinemic/hypoglycemic clamp. Compared with control treatment, fructose infusion caused a large increase in liver glycogen that markedly elevated the response of epinephrine and glucagon to a given hypoglycemia and increased net hepatic glucose output (NHGO). Moreover, prior denervation of the liver abolished the improved counterregulatory responses that resulted from increased liver glycogen content. When hepatic glycogen content was lowered, glucagon and NHGO responses to insulin-induced hypoglycemia were reduced. We conclude that there is a liver-brain counterregulatory axis that is responsive to liver glycogen content. It remains to be determined whether the risk of iatrogenic hypoglycemia in T1D humans could be lessened by targeting metabolic pathway(s) associated with hepatic glycogen repletion.
Mouse models designed to examine hepatic metabolism are critical to diabetes and obesity research. Thus, a microscale method to quantitatively assess hepatic glucose and intermediary metabolism in conscious, unrestrained mice was developed. [(13)C3]propionate, [(2)H2]water, and [6,6-(2)H2]glucose isotopes were delivered intravenously in short- (9 h) and long-term-fasted (19 h) C57BL/6J mice. GC-MS and mass isotopomer distribution (MID) analysis were performed on three 40-μl arterial plasma glucose samples obtained during the euglycemic isotopic steady state. Model-based regression of hepatic glucose and citric acid cycle (CAC)-related fluxes was performed using a comprehensive isotopomer model to track carbon and hydrogen atom transitions through the network and thereby simulate the MIDs of measured fragment ions. Glucose-6-phosphate production from glycogen diminished, and endogenous glucose production was exclusively gluconeogenic with prolonged fasting. Gluconeogenic flux from phosphoenolpyruvate (PEP) remained stable, whereas that from glycerol modestly increased from short- to long-term fasting. CAC flux [i.e., citrate synthase (VCS)] was reduced with long-term fasting. Interestingly, anaplerosis and cataplerosis increased with fast duration; accordingly, pyruvate carboxylation and the conversion of oxaloacetate to PEP were severalfold higher than VCS in long-term fasted mice. This method utilizes state-of-the-art in vivo methodology and comprehensive isotopomer modeling to quantify hepatic glucose and intermediary fluxes during physiological stress in mice. The small plasma requirements permit serial sampling without stress and the affirmation of steady-state glucose kinetics. Furthermore, the approach can accommodate a broad range of modeling assumptions, isotope tracers, and measurement inputs without the need to introduce ad hoc mathematical approximations.
Copyright © 2015 the American Physiological Society.
The liver responds to an increase in blood glucose levels in the postprandial state by uptake of glucose and conversion to glycogen. Liver glycogen synthase (GYS2), a key enzyme in glycogen synthesis, is controlled by a complex interplay between the allosteric activator glucose-6-phosphate (G6P) and reversible phosphorylation through glycogen synthase kinase-3 and the glycogen-associated form of protein phosphatase 1. Here, we initially performed mutagenesis analysis and identified a key residue (Arg(582)) required for activation of GYS2 by G6P. We then used GYS2 Arg(582)Ala knockin (+/R582A) mice in which G6P-mediated GYS2 activation had been profoundly impaired (60-70%), while sparing regulation through reversible phosphorylation. R582A mutant-expressing hepatocytes showed significantly reduced glycogen synthesis with glucose and insulin or glucokinase activator, which resulted in channeling glucose/G6P toward glycolysis and lipid synthesis. GYS2(+/R582A) mice were modestly glucose intolerant and displayed significantly reduced glycogen accumulation with feeding or glucose load in vivo. These data show that G6P-mediated activation of GYS2 plays a key role in controlling glycogen synthesis and hepatic glucose-G6P flux control and thus whole-body glucose homeostasis.
Net hepatic glucose uptake (NHGU) is an important contributor to postprandial glycemic control. We hypothesized that NHGU is reduced during normal pregnancy and in a pregnant diet-induced model of impaired glucose intolerance/gestational diabetes mellitus (IGT/GDM). Dogs (n = 7 per group) that were nonpregnant (N), normal pregnant (P), or pregnant with IGT/GDM (pregnant dogs fed a high-fat and -fructose diet [P-HFF]) underwent a hyperinsulinemic-hyperglycemic clamp with intraportal glucose infusion. Clamp period insulin, glucagon, and glucose concentrations and hepatic glucose loads did not differ among groups. The N dogs reached near-maximal NHGU rates within 30 min; mean ± SEM NHGU was 105 ± 9 µmol·100 g liver⁻¹·min⁻¹. The P and P-HFF dogs reached maximal NHGU in 90-120 min; their NHGU was blunted (68 ± 9 and 16 ± 17 µmol·100 g liver⁻¹·min⁻¹, respectively). Hepatic glycogen synthesis was reduced 20% in P versus N and 40% in P-HFF versus P dogs. This was associated with a reduction (>70%) in glycogen synthase activity in P-HFF versus P and increased glycogen phosphorylase (GP) activity in both P (1.7-fold greater than N) and P-HFF (1.8-fold greater than P) dogs. Thus, NHGU under conditions mimicking the postprandial state is delayed and suppressed in normal pregnancy, with concomitant reduction in glycogen storage. NHGU is further blunted in IGT/GDM. This likely contributes to postprandial hyperglycemia during pregnancy, with potential adverse outcomes for the fetus and mother.
The cellular events mediating the pleiotropic actions of portal vein glucose (PoG) delivery on hepatic glucose disposition have not been clearly defined. Likewise, the molecular defects associated with postprandial hyperglycemia and impaired hepatic glucose uptake (HGU) following consumption of a high-fat, high-fructose diet (HFFD) are unknown. Our goal was to identify hepatocellular changes elicited by hyperinsulinemia, hyperglycemia, and PoG signaling in normal chow-fed (CTR) and HFFD-fed dogs. In CTR dogs, we demonstrated that PoG infusion in the presence of hyperinsulinemia and hyperglycemia triggered an increase in the activity of hepatic glucokinase (GK) and glycogen synthase (GS), which occurred in association with further augmentation in HGU and glycogen synthesis (GSYN) in vivo. In contrast, 4 weeks of HFFD feeding markedly reduced GK protein content and impaired the activation of GS in association with diminished HGU and GSYN in vivo. Furthermore, the enzymatic changes associated with PoG sensing in chow-fed animals were abolished in HFFD-fed animals, consistent with loss of the stimulatory effects of PoG delivery. These data reveal new insight into the molecular physiology of the portal glucose signaling mechanism under normal conditions and to the pathophysiology of aberrant postprandial hepatic glucose disposition evident under a diet-induced glucose-intolerant condition.
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.
Studies were carried out on conscious female non-pregnant (NP) and pregnant (P; third-trimester) dogs (n 16; eight animals per group) to define the role of the liver in mixed meal disposition with arteriovenous difference and tracer techniques. Hepatic and hindlimb substrate disposal was assessed for 390 min during and after an intragastric mixed meal infusion labelled with [¹⁴C]glucose. The P dogs exhibited postprandial hyperglycaemia compared with NP dogs (area under the curve (AUC; change from basal over 390 min) of arterial plasma glucose: 86 680 (sem 12 140) and 187 990 (sem 33 990) mg/l in NP and P dogs, respectively; P < 0·05). Plasma insulin concentrations did not differ significantly between the groups (AUC: 88 230 (sem 16 314) and 69 750 (sem 19 512) pmol/l in NP and P dogs, respectively). Net hepatic glucose uptake totalled 3691 (sem 508) v. 5081 (sem 1145) mg/100 g liver in NP and P dogs, respectively (P = 0·38). The AUC of glucose oxidation by the gut and hindlimb were not different in NP and P dogs, but hepatic glucose oxidation (84 (sem 13) v. 206 (sem 30) mg/100 g liver) and glycogen synthesis (0·4 (sem 0·5) v. 26 (sem 0·7) g/100 g liver) were greater in P dogs (P < 0·05). The proportion of hepatic glycogen deposited via the direct pathway did not differ between the groups. Hindlimb glucose uptake and skeletal muscle glycogen synthesis was similar between the groups, although final glycogen concentrations were higher in NP dogs (9·6 (sem 0·6) v. 70 (sem 0·6) mg/g muscle; P < 0·05). Thus, hepatic glucose oxidation and glycogen storage were augmented in late pregnancy. Enhanced hepatic glycogen storage following a meal probably facilitates the maintenance of an adequate glucose supply to maternal and fetal tissues during the post-absorptive period.
The effects of a glycogen phosphorylase inhibitor (GPI) and metformin (MT) on hepatic glucose fluxes (μmol · kg(-1) · min(-1)) in the presence of basal and 4-fold basal levels of plasma glucagon were investigated in 18-h fasted conscious dogs. Compared with the vehicle treatment, GPI infusion suppressed net hepatic glucose output (NHGO) completely (-3.8 ± 1.3 versus 9.9 ± 2.8) despite increased glucose 6-phosphate (G-6-P) neogenesis from gluconeogenic precursors (8.1 ± 1.1 versus 5.5 ± 1.1). MT infusion did not alter those parameters. In response to a 4-fold rise in plasma glucagon levels, in the vehicle group, plasma glucose levels were increased 2-fold, and NHGO was increased (43.9 ± 5.7 at 10 min and 22.7 ± 3.4 at steady state) without altering G-6-P neogenesis (3.7 ± 1.5 and 5.5 ± 0.5, respectively). In the GPI group, there was no increase in NHGO due to decreased glucose-6-phosphatase flux associated with reduced G-6-P concentration. A lower G-6-P concentration was the result of increased net glycogenesis without altering G-6-P neogenesis. In the MT group, the increment in NHGO (22.2 ± 4.4 at 10 min and 12.1 ± 3.6 at steady state) was approximately half of that of the vehicle group. The lesser NHGO was associated with reduced glucose-6-phosphatase flux but a rise in G-6-P concentration and only a small incorporation of plasma glucose into glycogen. In conclusion, the inhibition of glycogen phosphorylase a activity decreases basal and glucagon-induced NHGO via redirecting glucose 6-phosphate flux from glucose toward glycogen, and MT decreases glucagon-induced NHGO by inhibiting glucose-6-phosphatase flux and thereby reducing glycogen breakdown.
OBJECTIVE - The objective of this study was to determine how increasing the hepatic glycogen content would affect the liver's ability to take up and metabolize glucose.
RESEARCH DESIGN AND METHODS - During the first 4 h of the study, liver glycogen deposition was stimulated by intraportal fructose infusion in the presence of hyperglycemic-normoinsulinemia. This was followed by a 2-h hyperglycemic-normoinsulinemic control period, during which the fructose infusion was stopped, and a 2-h experimental period in which net hepatic glucose uptake (NHGU) and disposition (glycogen, lactate, and CO(2)) were measured in the absence of fructose but in the presence of a hyperglycemic-hyperinsulinemic challenge including portal vein glucose infusion.
RESULTS - Fructose infusion increased net hepatic glycogen synthesis (0.7 ± 0.5 vs. 6.4 ± 0.4 mg/kg/min; P < 0.001), causing a large difference in hepatic glycogen content (62 ± 9 vs. 100 ± 3 mg/g; P < 0.001). Hepatic glycogen supercompensation (fructose infusion group) did not alter NHGU, but it reduced the percent of NHGU directed to glycogen (79 ± 4 vs. 55 ± 6; P < 0.01) and increased the percent directed to lactate (12 ± 3 vs. 29 ± 5; P = 0.01) and oxidation (9 ± 3 vs. 16 ± 3; P = NS). This change was associated with increased AMP-activated protein kinase phosphorylation, diminished insulin signaling, and a shift in glycogenic enzyme activity toward a state discouraging glycogen accumulation.
CONCLUSIONS - These data indicate that increases in hepatic glycogen can generate a state of hepatic insulin resistance, which is characterized by impaired glycogen synthesis despite preserved NHGU.