Lakhasly

OBJECTIVE--Insulin represses the expression of gluconeo- genic genes at the mRNA level, but the hormone appears to have only weak inhibitory effects in vivo.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. 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 alter- ations at the cellular level.No further diminu- tion 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 gluconeo- genic regulation in canines.In the healthy state, small increases (twofold) in the plasma insulin level can result in near-complete inhibition of the net contribution of glycogen to hepatic glucose production (HGP) (1).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.RESULTS--Insulin reduced gluconeogenic flux to glucose-6- phosphate (G6P) but only at the near-maximal physiological level (16
basal).Initially decreased hepatic lactate extraction is important, and later reduced gluconeogenic precursor availability plays a role.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.CONCLUSIONS--Gluconeogenic flux can be rapidly inhibited by high insulin levels in canines.Diabetes 58: 2766-2775, 2009 Hepatic glycogen metabolism in vivo is ex- tremely sensitive to the effects of insulin.On the other hand, the effects of insulin on hepatic gluconeogenesis are less potent, are more complex, and occur through multiple mechanisms.Corresponding author: Dale S. Edgerton, [email protected] ahead of print at http://diabetes.diabetesjournals.org on 15 September 2009.See http://creativecommons.org/licenses/by -nc-nd/3.0/ for details.The direct inhibitory effect of insulin on the From the Vanderbilt University Medical Center, Nashville, Tennessee.Changes in PEPCK appear to have little or no acute effect on gluconeogenic flux.This effect has been as- cribed to activation of glycogen synthesis (2).Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.DOI: 10.2337/db09-0328.(C) 2009 by the American Diabetes Association.The costs of publication of this article were defrayed in part by the payment of page charges.Received 17 March 2009 and accepted 8 August 2009.

OBJECTIVE—Insulin represses the expression of gluconeo- genic 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 alter- ations 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 diminu- tion 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 gluconeo- genic 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. Diabetes 58: 2766–2775, 2009
Hepatic glycogen metabolism in vivo is ex- tremely sensitive to the effects of insulin. In the healthy state, small increases (twofold) in the plasma insulin level can result in near-complete inhibition of the net contribution of glycogen to hepatic glucose production (HGP) (1). This effect has been as- cribed to activation of glycogen synthesis (2). On the other hand, the effects of insulin on hepatic gluconeogenesis are less potent, are more complex, and occur through multiple mechanisms. The direct inhibitory effect of insulin on the
From the Vanderbilt University Medical Center, Nashville, Tennessee. Corresponding author: Dale S. Edgerton, [email protected]. Received 17 March 2009 and accepted 8 August 2009. Published ahead of print
at http://diabetes.diabetesjournals.org on 15 September 2009. DOI:
10.2337/db09-0328.
© 2009 by the American Diabetes Association. Readers may use this article as
long as the work is properly cited, the use is educational and not for profit, and the work is not altered. See http://creativecommons.org/licenses/by -nc-nd/3.0/ for details.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
transcription and activity of key hepatic gluconeogenic enzymes through forkhead box class O-1 (FOXO1) phos- phorylation, including phosphoenolpyruvate carboxyki- nase (PEPCK) (3,4), is well established. Further, insulin inhibits the secretion of glucagon, a known activator of gluconeogenesis (5), thereby bringing about an indirect inhibitory effect on the process in the liver. In addition, insulin inhibits lipolysis (6), which reduces circulating glycerol and nonesterified free fatty acid (NEFA) levels. Glycerol is an important gluconeogenic (GNG) precursor, and NEFAs provide energy for gluconeogenesis (7). Net hepatic lactate uptake is reduced by insulin through several mechanisms. First, the hormone influences sub- strate cycling through the glycolytic/GNG pathways via allosteric and phosphorylation-mediated changes in the activity of key enzymes (e.g., bifunctional enzyme and pyruvate kinase) (4,8). Second, hepatic lactate flux is regulated by insulin secondary to its inhibitory effect on lipolysis (e.g., via reduced citrate, which results in in- creased phosphofructokinase-1 [PFK-1] activity) (4,8,9). Insulin decreases net amino acid release from muscle via inhibition of proteolysis and potentially by increased pro- tein synthesis (10), although the effect of reduced GNG amino acid precursor availability is offset to some degree by an increase in hepatic amino acid transport (11). Finally, evidence in rodents suggests that hepatic glucone- ogenesis may also be inhibited as the result of insulin action in the hypothalamus. It is postulated that brain insulin action increases vagal transmission, thereby in- creasing the phosphorylation of signal transducer and activator of transcription 3 (STAT3) in the liver and in turn reducing PEPCK and glucose-6-phosphatase (G6Pase) transcription (12–14).
Assessment of gluconeogenesis in vivo is complicated by the sensitive effects of insulin on hepatic glycogen metabolism. Because higher insulin concentrations are required to suppress gluconeogenesis than to inhibit gly- cogenolysis or increase glycogen synthesis (15,16), glu- coneogenically derived glucose-6-phosphate (G6P) can be diverted into hepatic glycogen even during mild hyperin- sulinemia. As a result, redirection of the product of GNG flux into hepatic glycogen can decrease gluconeogenesis, per se, in the absence of a fall in GNG flux to G6P (1,17).
Despite the numerous mechanisms by which insulin can inhibit the GNG process, acute physiologic increases in insulin have minimal impact on GNG flux in humans and large animals (1,15,17–20). Much of what is known about the regulation of gluconeogenesis is derived from in vitro studies on tissues or cells that lack important inputs for GNG control (e.g., GNG precursor load, NEFA availability, neuronal transmission, glycogen, hormonal milieu). Like- wise, many studies of GNG regulation have been per- formed in rodents, and it is not clear how control of the process may differ in those species compared with larger
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D.S. EDGERTON AND ASSOCIATES
animals (e.g., basal rates of gluconeogenesis are 5–10 times higher in rats and mice than in humans or canines; glycogen stores are completely depleted during fasting in rodents but not in humans or canines).
Given the difficulty in detecting an inhibitory effect of insulin on GNG flux in vivo, conditions were optimized in the present study to allow such an effect to be seen. First, canines were fasted for 60 h to increase the percentage contribution of gluconeogenesis to HGP. In addition, be- cause we previously failed to observe any suppression of GNG flux in overnight fasted canines when the level of insulin was increased 4-fold (20), we examined both 4- and 16-fold increases in insulin. Finally, insulin was elevated for 5 h to provide sufficient time for changes in insulin signaling to affect gene transcription and for the latter to translate into substantial effects on GNG enzyme levels and activities. To focus on the effects of insulin, glucagon and glucose were clamped at basal levels. In addition, liver biopsies were taken at the end of each study to allow the effects of insulin on molecular signaling and gene tran- scription to be correlated with alterations in whole-body metabolic flux rates.
RESEARCH DESIGN AND METHODS
Animal care and surgical procedures. Sixteen conscious mongrel canines of either sex were studied after a 60-h fast. Housing and diet have been described previously (1). The surgical facility met the standards published by the American Association for the Accreditation of Laboratory Animal Care, and the protocols were approved by the Vanderbilt University Medical Center Animal Care Committee. All canines underwent a laparotomy 2 weeks before the experiment to implant portal vein infusion catheters into the jejunal and splenic veins, sampling catheters into the femoral artery and portal and hepatic veins, and ultrasonic flow probes (Transonic Systems, Ithaca, NY) around the hepatic artery and portal vein, as described elsewhere (1). All canines studied were healthy, as indicated by 1) leukocyte count 18,000/ mm3, 2) hematocrit 35%, and 3) good appetite and normal stools. Experimental design. Animals were allowed to rest quietly in a Pavlov harness for 60 min before the experiments started. Each study consisted of a basal period (40 to 0 min) and an experimental period (0–300 min). Somatostatin (0.8 g
kg1
min1; Bachem, Torrance, CA) was infused (0–300 min) to inhibit endogenous pancreatic hormone secretion. During the same period, intraportal infusions of glucagon (0.5 ng
kg1
min1; Lilly, Indianapolis, IN) and insulin (300, 1,200, or 5,000 U
kg1
min1; Lilly) were given in the control (n  5), 4
(n  5), or 16
(n  6) groups, respectively. Previously, when the plasma glucose level was maintained at basal levels by titration of intraportal insulin in 60-h fasted canines, the insulin requirement was 215  24 U
kg1
min1 (21). Therefore, in the present study, insulin was infused in the control group at a rate slightly above basal. Glucose was infused intravenously to maintain euglycemia.
Immediately after the final sampling time, each animal was anesthetized
and three sections of liver lobes were freeze clamped in situ and stored at
70°C as previously described (1). All animals were then killed, and the
correct positions of the catheter tips were confirmed.
Analytical procedures. Hematocrit; plasma glucose, glucagon, insulin, cor-
tisol, and NEFA; and blood alanine, glycine, serine, threonine, lactate,
glutamine, glutamate, glycerol, and -hydroxybutyrate concentrations
were determined as previously described (1). RNA extraction, cDNA
synthesis, real-time PCR, SDS-PAGE, and Western blotting procedures
were performed by standard methods, details of which are provided in the
online appendix at http://diabetes.diabetesjournals.org/content/suppl/2009/
08/ 25/db09-0328.DC1/DB09-0328_online_appendix.pdf. Fructose 2,6-
bisphosphate (F2,6P2) levels and pyruvate kinase activity were determined
as described in the online appendix.
Calculations. Net hepatic substrate balances were calculated with the
arteriovenous (A-V) difference method using the following formula: net
hepatic balance  load – load , where load  [H]
HF and load  [A] outinout in

AF [P]
PF and where [H], [A], and [P] are the substrate concentrations in hepatic vein, femoral artery, and portal vein blood or plasma, respectively, and HF, AF, and PF are the blood or plasma flow in the hepatic vein, hepatic artery, and portal vein, respectively, as determined by the ultrasonic flow probes. With this calculation, a positive value represents net output by the liver, whereas a negative value represents net hepatic uptake. Plasma glucose
values were multiplied by 0.73 to convert them to blood glucose values as validated elsewhere (20). Net hepatic fractional extraction was calculated by dividing net hepatic substrate balance by hepatic substrate load. Nonhepatic glucose uptake was calculated as the glucose infusion rate plus net hepatic glucose balance, with changes in the glucose mass accounted for when deviations from steady state were present. The approximate insulin and glucagon levels in plasma entering the liver sinusoids were calculated using the formula [A]
%AF [P]
%PF, where [A] and [P] are arterial and portal vein hormone concentrations, respectively, and %AF and %PF are the respec- tive fractional contributions of arterial and portal flow to total hepatic blood flow.
Gluconeogenesis is the synthesis and subsequent hepatic release of glu- cose from noncarbohydrate precursors. Because carbon produced from flux through the GNG pathway can also be stored in glycogen, we make a distinction between gluconeogenesis and GNG flux to G6P. Hepatic GNG flux to G6P was determined by summing net hepatic uptake rates of GNG precursors (alanine, glycine, serine, threonine, glutamine, glutamate, glycerol, lactate, and pyruvate); these rates were divided by two to account for the incorporation of three carbon precursors into the six-carbon glucose mole- cule. Glycolytic flux was estimated by summing the net hepatic output rates (when such occurred) of the substrates noted above (in glucose equivalents) and hepatic glucose oxidation. In earlier studies, glucose oxidation was 0.2  0.1 mg
kg1
min1 even when the concentrations of circulating insulin, glucose, and NEFAs varied widely (22,23). Because glucose oxidation did not change appreciably under conditions similar to the present study, glucose oxidation was assumed to be constant (0.2 mg
kg1
min1). Net hepatic glycogenolytic flux was estimated by subtracting GNG flux from the sum of net hepatic glucose balance and glycolytic flux. A positive number therefore represents net glycogen breakdown, whereas a negative number indicates net glycogen synthesis.
The assumptions related to using the A-V difference technique for assessing GNG flux to G6P should be considered. Ideally, GNG flux would be calculated using unidirectional hepatic uptake and output rates for each substrate, but this would be difficult because it would require the simultaneous use of multiple stable isotopes that could themselves induce a mild perturbation of the metabolic state; therefore, net hepatic balance was used instead. There is little or no production of GNG amino acids or glycerol by the liver, so in this case the compromise is of little consequence. This may not be the case for lactate, however. The estimate of GNG flux to G6P will be quantitatively accurate only if lactate flux is unidirectional at a given moment (i.e., either in or out of the liver). In a given cell, this is a reasonable assumption in light of the reciprocal control of gluconeogenesis and glycolysis (4). Spatial separa- tion of metabolic pathways may exist, however, so that predominantly GNG periportal and glycolytic perivenous hepatocytes (24) could simultaneously take up and release lactate, respectively. Total GNG flux will be underesti- mated to the extent that this occurs and to the extent that intrahepatic precursors (GNG amino acids) contribute to the process. The method also assumes that there is 100% conversion of GNG precursors taken up by the liver into G6P (they are not oxidized or used in the synthesis of proteins or fatty acids). The errors due to these assumptions are difficult to assess but appear to be small and in fact are offsetting. Results obtained using the A-V difference technique described here were previously compared with results obtained with independent GNG measurements that are not subject to these assumptions, and they were similar (1,25), suggesting that these assumptions appear reasonable.
It should be noted that our estimates of gluconeogenesis and glycogenol- ysis relate solely to the liver. In a previous study, net renal glucose balance was close to zero in 60-h fasted canines, indicating that in a net sense the liver is the only source of glucose in such animals (21). To the extent that the kidney makes an absolute contribution to whole-body glucose production, we would slightly underestimate whole-body GNG flux.
Statistical analysis. Statistical comparisons were carried out using two-way repeated measure ANOVA (group
time) (SigmaStat). One-way ANOVA comparison tests were used post hoc when significant F ratios were obtained. Significance was determined as P  0.05.
RESULTS
Hormone levels. Plasma insulin levels did not increase appreciably in the control group, whereas they increased an average of 4- and 16-fold in the 4
and 16
groups, respectively (Fig. 1). The plasma arterial and hepatic sinusoidal glucagon and arterial cortisol levels remained basal and were similar over time and between groups (not shown).