Lipid-Induced Peroxidation in The Intestine Is Involved in Glucose Homeostasis Imbalance in Mice
Lipid-Induced Peroxidation in The Intestine Is Involved in Glucose Homeostasis Imbalance in Mice
Lipid-Induced Peroxidation in The Intestine Is Involved in Glucose Homeostasis Imbalance in Mice
Abstract
Background: Daily variations in lipid concentrations in both gut lumen and blood are detected by specific sensors located
in the gastrointestinal tract and in specialized central areas. Deregulation of the lipid sensors could be partly involved in the
dysfunction of glucose homeostasis. The study aimed at comparing the effect of Medialipid (ML) overload on insulin
secretion and sensitivity when administered either through the intestine or the carotid artery in mice.
Methodology/Principal Findings: An indwelling intragastric or intracarotid catheter was installed in mice and ML or an
isocaloric solution was infused over 24 hours. Glucose and insulin tolerance and vagus nerve activity were assessed. Some
mice were treated daily for one week with the anti-lipid peroxidation agent aminoguanidine prior to the infusions and tests.
The intestinal but not the intracarotid infusion of ML led to glucose and insulin intolerance when compared with controls.
The intestinal ML overload induced lipid accumulation and increased lipid peroxidation as assessed by increased
malondialdehyde production within both jejunum and duodenum. These effects were associated with the concomitant
deregulation of vagus nerve. Administration of aminoguanidine protected against the effects of lipid overload and
normalized glucose homeostasis and vagus nerve activity.
Conclusions/Significance: Lipid overload within the intestine led to deregulation of gastrointestinal lipid sensing that in
turn impaired glucose homeostasis through changes in autonomic nervous system activity.
Citation: Serino M, Waget A, Marsollier N, Masseboeuf M, Payros G, et al. (2011) Lipid-Induced Peroxidation in the Intestine Is Involved in Glucose Homeostasis
Imbalance in Mice. PLoS ONE 6(6): e21184. doi:10.1371/journal.pone.0021184
Editor: Lorraine Brennan, University College Dublin, Ireland
Received November 2, 2010; Accepted May 22, 2011; Published June 16, 2011
Copyright: 2011 Serino et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: RB is the recipient of a grant from the European Association for the Study of Diabetes (EASD) and Amylin. CM is the recipient of funding from the
Agence Nationale de la Recherche (France): ANR-07-PHYSIO. The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: [email protected]
. These authors contributed equally to this work.
Introduction
It has now clearly been shown that nutrient sensing is a key
factor in the regulation of energy homeostasis, especially that of
glucose [1]. Indeed, daily variations in nutrient concentrations in
both gut lumen and blood are detected by specific sensors located
either in the gastrointestinal tract [2,3] or in specialized central
areas (mainly the hypothalamus or brainstem [4,5]). In the case of
the gastrointestinal tract, it has been known for many years that
luminal nutrients stimulate the release of regulatory peptides from
gut endocrine cells and also activate intrinsic and extrinsic neural
pathways innervating the gut, in turn conveying signals to the
brainstem through vagus afferent fibers [3,6]. It has also been
demonstrated that daily variations in nutrient concentrations in
the blood can be directly detected by nutrient sensitive neurons
(both glucose and fatty acid sensitive neurons located within the
hypothalamus [7,8]). Among nutrients, increasing evidence
suggests an important role for an intestinal lipid-induced gutbrain neuronal axis to regulate energy homeostasis [9,10] as
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Results
A 24-hour intestinal but not brain lipid infusion impaired
glucose homeostasis
The intragastric ML infusion increased the plasma TG
concentration when compared with the isocaloric infusion
(Figure 1A) though conversely no change in plasma FFA
concentrations was observed (Figure 1B). The 24 h intracarotid
ML infusion did not increase either the plasma FFA or TG
concentrations when compared with the controls (data not shown).
Blood glucose and plasma insulin concentration remained unchanged after both types of infusions. The GLP-1 concentration in
the portal vein was markedly increased by the intragastric ML
perfusion (Figure 1C; t0). To determine the impact of the intestinal
or intracarotid lipid overload on the control of glucose homeostasis
we first investigated the time-course of glycemia in response to an
oral glucose tolerance test (OGTT). At the end of the intragastric
ML infusion, the blood glucose profile during OGTT was
significantly higher in ML when compared with the isocal. In ML
mice treated with the antagonist GLP1 receptor exendin 9 (Ex9)
hyperglycemia during OGTT was much significantly higher than in
Figure 1. Effects of lipid infusion on glucose homeostasis in mice infused intragastrically for 24 hours with Medialipid (ML) or
isocaloric solution (A, B, C, D, E, F) ; and in the brain (intracarotid) with ML or isocal (G, H, I). A: Plasma TG (g/l). B: FFA (mM). C: Plasma
portal GLP1 concentration (pM) at the end of the intragastric perfusion (t0) and 15 min after glucose gavage. D: Time course of glycemia (mM) during
the OGTT. E: Plasma insulin (pg/ml) 20 min before and 15 min after glucose challenge. F: Time course of glycemia during ITT. Results in 24 h
intracarotid infused mice: G: Time course of glycemia (mM) during OGTT. H: Plasma insulin (pg/ml) 20 min before and 15 min after glucose. I: Time
course of glycemia during ITT. (n = 8) *p,0.05, **p,0.01, ***p,0.001 compared to controls.
doi:10.1371/journal.pone.0021184.g001
Figure 2. Intestinal lipid content and markers of inflammation in isocaloric or ML intragastrically infused mice. A: TG content (mg/g of
tissue) in jejunum. B: Electronic microscopy of jejunum (64000). C,D,E, F : mRNA expression of TNFa (C), IL1b (D), PAI-1 (E) and F4/80 (F). G: Number
of macrophages (F4/80), H: F4/80 immunohistochemistry. (n = 8) *p,0.05, **p,0.01, ***p,0.001 compared to controls.
doi:10.1371/journal.pone.0021184.g002
Figure 3. Markers of oxidative stress in intragastric-infused mice. A,B,C: mRNA expression of NADPH oxidase (A), GST (B), catalase (C).
D: Activity of glutathione reductase in duodenum and jejunum. E: Lipid peroxidation by the expression of MDA (mM/mg protein). F, G, H, I: Infusion of
isocaloric and Medialipid solution over 6 h. F: MDA production. G: Glutathione reductase activity in duodenum and jejunum. H: Plasma insulin
(pg/ml) 20 min before and 15 min after glucose challenge. I: Time course of glycemia (mM) during OGTT. (n = 8) *p,0.05, **p,0.01, ***p,0.001
compared to controls.
doi:10.1371/journal.pone.0021184.g003
Interestingly, brain ML infusion did not increase MDA production suggesting that the duration of the infusion was not sufficient
(data not shown).
Discussion
The present study showed that in mice a 24 h intragastric but
not an intracarotid lipid overload, that mimicked the daily fat load
during high-fat feeding, impaired both glucose and insulin
intolerance and hence the overall glucose homeostasis. This was
linked to an increased lipid content in the jejunum. Furthermore
Figure 4. Effects of aminoguanidine on glucose homeostasis. Effect of aminoguanidine treatment on ML intragastrically infused mice. A:
Malondialdehyde (MDA) production in duodenum and jejunum at the end of the infusion. B: FFA (mM). C: TG (g/l) concentration before (t0) and after
(24 h) the perfusion. D: Time course of glycemia (mM) during OGTT and E: Plasma insulin (pg/ml) 20 min before and 15 min after glucose challenge.
F: Time course of glycemia after insulin load. (n = 8) *p,0.05, when compared to ML.
doi:10.1371/journal.pone.0021184.g004
Figure 5. Activity of the vagal nerve. A: Recordings during 5 min before (upper panel) and 5 min after (lower panel) the glucose load in
isocaloric (isocal), Medialipid (ML) or, aminoguanidine treated-ML (ML+ amino) conditions. B: Frequency of the vagal activity recorded before and
during OGTT. (n = 8). **p,0.01 vs isocaloric.
doi:10.1371/journal.pone.0021184.g005
Methods
Animals and research design
Ethical statement. The following animal experimental
procedures were approved by the local ethical committee of the
Rangueil hospital and by the local ethical committee of the
University of Paris Diderot (permit number: A75-13-17).
Animals housing. Eleven-week-old C57BL6/J (Charles
River, LArbresle, France) male mice were housed in a controlled
environment (inverted 12-h daylight cycle, lights off at 10:00 a.m.)
with free access to food and water. All mice were fed with a normal
carbohydrate diet (NC : proteins 22%, glucides 67%, lipids 11% of
total kcal).
Research design. An indwelling catheter was installed in the
stomach or in the carotid artery towards the brain. Following
insertion of the catheters, mice were allowed to recover for one
week post-surgery and to reach their pre-surgical body weight. On
the day of experiment the catheters were connected to infusion
systems that enabled the animal to remain in its cage. Mice were
infused over 24 h with a triglyceride emulsion (Medialipid 20%;
18 Kcal/24 h, KabeVitrum, Stockholm, Sweden) or an isocaloric
solution (Nutriflex lipid, B Braun, France). Medialipid contains
200 g/L of lipids (mainly soy oil) and Nutriflex is composed of
57 g/L of amino acids, 144 g/L of glucose and 40 g/L of lipids. It
is noteworthy that this lipid infusion rate corresponds to the
amount of lipid absorbed over 24 hours by mouse fed a high-fat
diet [41]. A subset of mice was infused for six hours only, to
evaluate the effect of a very short-term infusion on glucose
homeostasis. At the completion of the infusions, different
metabolic analyses were performed as described below. The
duodenum, jejunum and the hypothalamus were collected and
stored at 280uC. In another set of experiments, a group of mice
had free access to drinking water complemented with a solution of
7
Tolerance tests
Oral glucose tolerance test (OGTT). After the 24 hinfusion, mice were disconnected from the infusion system,
allowing free moving in the cage. After twenty minutes,
intragastric infused mice were gavaged with a glucose solution
(2 g/kg). The glycemia was determined by a glucometer (Accu
Chek, France) from 2 ml collected from the tip of the tail vein at
times 0, 15, 30, 60, 90 and 120 min. In addition 20 ml of blood
were sampled 20 min before, and at 15 and 60 min after the
glucose gavage, in order to measure insulinemia. Blood was
immediately centrifuged and plasma was frozen until insulin assay.
Insulin tolerance test (ITT). A single dose of insulin was
injected (0.05 U/ml, 10 ml/g, ip). The glycemia was measured in
tail blood at times 0, 5, 10, 15, 20, 30, 40, 50 and 60 min.
GLP-1 sample collection. In mice allocated for assessment
of portal vein GLP-1 concentrations, blood was collected in the
presence of Diprotin A (Ile-pro-ile, 0.1 nM, Sigma-Aldrich, Saint
Louis MO) and heparin at the end of the 24-hour infusion, and
fifteen minutes following the glucose challenge. A rapid anesthesia
was induced by an intraperitoneal injection of a mix of Ketamine
1000 (Vibrac, France) and Xylazine (Rompum 2%, Bayer health
care, France, 100 and 10 mg/kg i.p., respectively) to obtain the
portal vein samples.
Plasma parameters. Plasma insulin concentrations were
determined in 10 ml using the mouse ultrasensitive insulin ELISA
kit (Mercodia, Upsala, Sweden) and plasma GLP-1 concentrations
were determined in 100 ml using the Glucagon-Like Peptide-1
(Active) ELISA kit (Linco Research). Plasma FFA concentration
was assessed by the NEFA C kit (WAKO) using 8 ml. Plasma
triglyceride concentration was determined in 3 ml using the
triglycerides enzymatic PAP150 kit (Biomerieux).
Biochemical assays
Protein extraction. Whole intestine and hypothalamus were
solubilized by a RIPA solution, (Tris 1 M pH 7,5 (Sigma), Triton
106 (Sigma), NaCl 5 M, NaF 1 M (Sigma)) with antiprotease solution (aprotinin at 1.5 mg/ml (Euromedex), leupeptin at
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Author Contributions
Conceived and designed the experiments: J-CT AN-S SL RB CC-G CM.
Performed the experiments: MS AW NM MM GP CK JD AL RB CC-G
CM. Analyzed the data: MS AW NM MM RB CC-G CM. Wrote the
paper: RB CM.
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