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Prolonged Fasting Induces Peripheral Insulin Resistance, Which Is Not Ameliorated by High-Dose Salicylate

Departments of Endocrinology and Metabolism (S.N.v.d.C., G.A., H.P.S.) and Clinical Chemistry (M.T.A., E.E.), Laboratory of Endocrinology, Academic Medical Center, University of Amsterdam, 1100 DD Amsterdam; and Department of Endocrinology (J.A.R.), Leiden University Medical Center, 2300 RC Leiden, The Netherlands

Address all correspondence and requests for reprints to: S. N. van der Crabben, University Medical Center Utrecht (Wilhelmina Children’s Hospital), DBG Department of Clinical Genetics, KC04.084.2, Lundlaan 6, P.O. Box 85090, 3508 AB Utrecht, The Netherlands. E-mail: saskiananette{at}hotmail.com.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Context: Elevated plasma free fatty acids, excess reactive oxygen species, inflammation, and gluco-counterregulatory hormones induce insulin resistance (IR) through activation of Jun NH₂-terminal kinase and nuclear factor-B inhibitor B kinase, which leads to hyperphosphorylation of the insulin receptor substrate type 1. Aspirin blocks nuclear factor-B inhibitor B kinase and improves IR in type 2 diabetes mellitus.

Objective: We hypothesized that high-dose aspirin would also attenuate fasting-induced IR in healthy lean subjects.

Design: Glucose and glutathione (GHS) metabolism was studied after 12 and 60 h of fasting on two occasions: with and without aspirin (6 g/d).

Setting: The study took place at the Academic Medical Center, Metabolic Research Unit.

Participants: Six healthy lean men participated.

Intervention: Intervention included 60 h of fasting with or without aspirin (6 g/d).

Main Outcome Measure: Main outcome measures included glucose and GSH metabolism.

Results: Fasting decreased insulin-mediated peripheral glucose uptake by 46% after 60 h (P = 0.03). Aspirin did not alter this effect of 60 h of fasting on insulin sensitivity (P = 0.03). GSH concentration decreased during fasting, but the fractional synthetic rate of GSH was unaffected either with or without aspirin. Fasting did not affect inflammatory parameters, although aspirin increased soluble TNF receptors I and II.

Conclusion: Prolonged fasting induces profound peripheral IR. In contrast to type 2 diabetes mellitus, high-dose salicylate does not affect fasting-induced peripheral IR.

	Introduction

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In the insulin-resistant state of obesity, elevated plasma free fatty acid (FFA) levels, increased inflammatory cytokines, elevated levels of gluco-counterregulatory hormones, and excess reactive oxygen species (ROS) are considered to be involved in the inhibition of the insulin receptor substrate type 1 (IRS-1) pathway (1, 2).

Insulin resistance (IR) is induced by the inhibitory phosphorylation of serine residues of IRS-1. IRS-1 phosphorylation is activated by several serine/threonine kinases, which include protein kinase C, Jun NH₂-terminal kinase, and nuclear factor-B inhibitor-B kinase (IKK) (3). Finally, IRS-1 serine phosphorylation interferes with glucose transporter 4 translocation to the cell membrane (4).

The mechanisms of fasting-induced IR have not been completely elucidated. Considering the increased plasma FFA levels, the mechanisms might be comparable to the insulin-resistant state of obesity and type 2 diabetes mellitus (T2DM).

FFA induces activation of nuclear factor-B (5). Conversely, peripheral IR in T2DM can be reduced by high-dose aspirin treatment mainly via inhibition of IKK (β) and/or cyclooxygenase activity (6, 7). We hypothesized that if fasting-induced IR is caused by the same mechanism as in T2DM, high-dose aspirin would be able to attenuate IR induced by prolonged fasting in healthy lean subjects. Therefore, peripheral glucose metabolism was studied during hyperinsulinemic euglycemic clamp conditions in healthy lean subjects after 12 and 60 h of fasting with and without administration of aspirin (6 g/d). The influence of other factors potentially involved in the inhibition of the IRS-1 pathway, glutathione (GSH; human’s main antioxidant) metabolism and the plasma concentrations of highly sensitive C-reactive protein (hs-CRP), IL-6, and soluble TNF receptor (sTNF-R) I and II as parameters of inflammation, were measured during the basal state only.

	Subjects and Methods

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Subjects

After informed consent, six healthy, nonsmoking, male volunteers [age, 20 (19–24) yr; weight, 73.2 (65.7–77.7) kg; body mass index, 21.7 (19–22.8) kg/m²] were included. The study was approved by the Medical Ethical Committee of the Academic Medical Center.

Protocol

Subjects fasted twice: once during a control setting [weight, 73.5 kg (range, 64.7–78.0 kg)] and once during intake of high-dose aspirin [6 g/d; aspirin setting: weight, 71.8 kg (range, 65.7–79 kg); P = 0.753]. The studies were performed in random order and were separated by at least 4 wk. The intake of enteric-coated aspirin (Bayer, Pittsburgh, PA) was started after finishing the 12-h clamp study. The drug (1200 mg at each time point) was ingested at 0700, 1100, 1500, 1900, and 2300 h to achieve plasma salicylate levels between 250 and 350 mg/liter and was continued until the morning of the 60-h study. During measurement of glucose metabolism after 60 h of fasting, aspirin was ingested every hour to maintain constant levels of plasma salicylate (7). The aspirin concentrations achieved in the present study are estimated to inhibit IKKβ activity 90% (6).

Measurements of glucose kinetics ([6,6-²H₂]glucose at 8.0 µmol/kg prime and 0.11 µmol/kg·min continuous infusion), GSH metabolism ([3,3-²H₂]cysteine at 5.0 µmol/kg prime and 0.08 µmol/kg·min continuous infusion) (both isotopes were >98% pure and >99% enriched; ARC Laboratories BV, Apeldoorn, The Netherlands), counterregulatory hormones, and inflammatory markers were measured after 12 and 60 h of fasting in the control and the aspirin study in the basal state. Glucose kinetics were also measured during a 2.5-h hyperinsulinemic-euglycemic clamp (insulin infusion, 20 mU/BSA·min).

Analytical procedures

Isotope enrichment (tracer/tracee ratio) of [6,6-²H₂]glucose, [3,3-²H₂]cysteine, and d₂-GSH were determined by gas chromatography-mass spectrometry (8, 9, 10). For [3,3-²H₂]cysteine, enrichment erythrocytes were washed three times with 0.9% saline directly after withdrawal. GSH synthesis takes place in the erythrocytes. The tracer/tracee ratio of [3,3-²H₂]cysteine in erythrocytes is much lower than in plasma (data not shown), which makes washing of the erythrocytes necessary.

Insulin, cortisol, glucagon, adiponectin, FFA, sTNF-RI, and sTNF-RII were determined as described earlier (11). Hs-CRP was determined with a particle-enhanced immunoturbidimetric assay (P800 analyzer; Roche, Mannheim, Germany). Salicylate was determined on a TDxFLx system (Abbott Diagnostics, Abbott Park, IL) with a fluorescence polarization immunoassay.

Calculation and statistics

Peripheral glucose uptake [rate of disposal (Rd)] was calculated using the modified form of the Steele equations as described previously (12). Glutathione kinetics [concentration and fractional synthetic rate (FSR)] are calculated as described by Humbert et al. (10) and Darmaun et al. (13).

To analyze the effect of aspirin, results [median (range)]) were compared using Wilcoxon’s signed rank test (version 11.5.2; SPSS Inc., Chicago, IL). To quantify the effect of aspirin on glucose and GSH parameters, the difference between 12 and 60 h of fasting as percentage of the 12-h value ( percentage) are presented. P values of <0.05 were considered statistically significant.

	Results

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All results are shown in Tables 1 and 2.

Control study Weight loss was 1.8 kg (1.0–2.4 kg, P = 0.028).

Basal plasma glucose concentration decreased by 32%, and GSH concentrations decreased by 9% (P < 0.05). FSR of GSH was not different.

During hyperinsulinemia, insulin concentrations [179 (158–224) vs. 163 (138–209) pmol/liter, P = 0.028] were about 11% lower, Rd was about 48% lower, and plasma FFA levels [0.03 (0.01–0.07) vs. 0.32 (0.31–0.45) pmol/liter, P = 0.027] were increased.

Aspirin study Aspirin ingestion resulted in plasma salicylate levels of 282 (207–356) mg/liter. Weight loss was 2.5 kg (2.0–3.5 kg, P = 0.028).

Basal plasma glucose concentration decreased by 29%, and GSH concentrations decreased by 12% (P < 0.05). FSR of GSH was not altered.

During hyperinsulinemia, plasma insulin concentrations [202 (174–224) vs. 225 (184–235) pmol/liter, P = 0.046] increased by about 6%, Rd decreased by about 38%, and plasma FFA levels were increased [0.05 (0.02–0.07) vs. 0.36 (0.30–0.49) pmol/liter, P = 0.027].

Comparison of control vs. aspirin

Weight loss with aspirin was higher (P = 0.046). Aspirin did not affect the decrease in Rd during fasting. There were no differences in GSH concentrations or kinetics between both conditions. Plasma levels of insulin, FFA, adiponectin, glucagon, cortisol, and (nor)epinephrine were not different between both study conditions.

Despite identical insulin infusion rates, plasma insulin levels during the hyperinsulinemic-euglycemic clamp studies were 30% higher in the aspirin studies compared with the control studies (P = 0.028).

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
High-dose aspirin treatment ameliorates peripheral IR in obese patients with T2DM (7). In these patients, IR is associated with increased plasma levels of FFA and inflammatory cytokines and excess ROS (1, 2).

This study shows that prolonged fasting in lean healthy men induces profound IR on Rd concomitantly with higher plasma FFA levels. High-dose aspirin treatment did not attenuate this. The study also indicates that fasting-induced IR is not associated with inflammation, based on the absence of changes in plasma levels of the inflammatory mediators. The results of this study show, for the first time, that fasting decreases erythrocyte GSH concentrations without any change in FSR. The explanation for this dissociation is unclear. The unchanged FSR of GSH indicates that there is no fasting-related lack of substrates for GSH synthesis. The previously shown reduction in ROS production in leukocytes during a 48-h fast makes an increased use of GSH through this pathway also unlikely (14).

A potential confounding factor in the present study is the difference in plasma insulin levels between the control and aspirin studies during the hyperinsulinemic-euglycemic clamp studies after 60 h of fasting. Because insulin infusion rates were identical in both settings, the increased plasma insulin levels during aspirin intake are most likely due to a decrease of plasma insulin clearance caused by aspirin (7, 15). Because insulin levels after 60 h of fasting were 30% higher in the aspirin than in the control study, these differences in insulin concentrations are not affecting the conclusions of the present study.

Another distorting factor might have been the increased weight loss in the aspirin study. However, the difference was small (0.7 kg, or 1% of body weight).

The results of aspirin treatment in the present study differ from a recent study that examined the effects of aspirin pretreatment on lipid-infusion-induced IR in healthy subjects (16). In that study, aspirin pretreatment ameliorated lipid-induced IR by 17%. Two-hour hyperinsulinemic-euglycemic clamps were performed after an overnight fast with and without 4 h of lipid-heparin infusion, both with and without 4-g aspirin pretreatment. The first dose was given 24 h before the test, and the next three doses were given every 8 h for a total aspirin amount of 4 g. Stable isotopes were not used to examine glucose metabolism, and insulin sensitivity was expressed using M-values. If M-values would have been used in our study, high-dose aspirin treatment would have tended to increase the M-value by 17% (P = 0.058) after 60 h of fasting, erroneously suggesting that high-dose aspirin treatment decreased fasting-induced IR. However, in our study, insulin levels did not fully suppress endogenous glucose production, resulting in an M-value underestimating real Rd. Other differences with our study relate to a lower aspirin dosage and the nonphysiological induction of IR. Although prolonged administration of aspirin enhances insulin sensitivity in chronic states of IR, short-term administration of aspirin is ineffective in improving IR during the induction of IR by short-term starvation.

Prolonged fasting with high-dose aspirin caused ambiguous changes in levels of inflammatory parameters; IL-6 did not change, hs-CRP tended to increase, and sTNF-RI and -II increased. The tendency for increased hs-CRP levels is similar to the results found in a recent study but remains unexplained (16). Increased levels of sTNF-RI and -II suggest that high-dose aspirin increased TNF. Although TNF is certainly one important mediator of sTNF-R release, other factors are also involved (1). In addition, sTNF-RI and -II are proteins, and protein metabolism and clearance is influenced by fasting (17).

In conclusion, prolonged fasting induces profound peripheral IR in healthy lean men. These data are consistent with the fact that counterregulatory hormones and FFA are responsible for IR. This is not affected by high-dose aspirin. There is no evidence in our data that aspirin exerted an antiinflammatory effect. Therefore, the effect of aspirin on peripheral IR in patients with T2DM is different from fasting-induced IR in healthy subjects. This observation points to differences in underlying mechanisms inducing IR in both conditions. This indicates that the model used for the study of the pathophysiology of IR is of utmost importance but also indicates that data obtained in a certain model cannot be considered to be applicable to the pathophysiology of IR in general.

	Acknowledgments

 
We thank An Ruiter and Barbara Voermans for their excellent analytical support and the laboratory of pharmacology for their kind assistance.

	Footnotes

 
Disclosure Statement: The authors have nothing to disclose.

First Published Online December 4, 2007

¹ S.N.v.d.C. and G.A. made an equal contribution to this work.

Abbreviations: FFA, Free fatty acids; FSR, fractional synthetic rate; GSH, glutathione; hs-CRP, highly sensitive C-reactive protein; IKK, inhibitor-B kinase; IR, insulin resistance; IRS-1, insulin receptor substrate type 1; Rd, rate of disposal; ROS, reactive oxygen species; sTNF-R, soluble TNF receptor; T2DM, type 2 diabetes mellitus.

Received November 13, 2006.

Accepted November 26, 2007.

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