Overfeeding during Lactation in Rats is Associated with Cardiovascular Insulin Resistance in the Short-Term

Childhood obesity is associated with metabolic and cardiovascular comorbidities. The development of these alterations may have its origin in early life stages such as the lactation period through metabolic programming. Insulin resistance is a common complication in obese patients and may be responsible for the cardiovascular alterations associated with this condition. This study analyzed the development of cardiovascular insulin resistance in a rat model of childhood overweight induced by overfeeding during the lactation period. On birth day, litters were divided into twelve (L12) or three pups per mother (L3). Overfed rats showed a lower increase in myocardial contractility in response to insulin perfusion and a reduced insulin-induced vasodilation, suggesting a state of cardiovascular insulin resistance. Vascular insulin resistance was due to decreased activation of phosphoinositide 3-kinase (PI3K)/Akt pathway, whereas cardiac insulin resistance was associated with mitogen-activated protein kinase (MAPK) hyperactivity. Early overfeeding was also associated with a proinflammatory and pro-oxidant state; endothelial dysfunction; decreased release of nitrites and nitrates; and decreased gene expression of insulin receptor (IR), glucose transporter-4 (GLUT-4), and endothelial nitric oxide synthase (eNOS) in response to insulin. In conclusion, overweight induced by lactational overnutrition in rat pups is associated with cardiovascular insulin resistance that could be related to the cardiovascular alterations associated with this condition.


Introduction
Obesity is a main health concern worldwide due to its association with metabolic syndrome and cardiovascular diseases [1]. Although it was previously considered that metabolic syndrome was rare in childhood, its incidence has markedly increased together with childhood obesity [2,3]. Indeed, type 2 diabetes and insulin resistance are some of the most prevalent alterations of metabolic syndrome among children [2,4].
In addition to metabolic disorders, obese children also show cardiovascular alterations [5], such as arterial hypertension, whose incidence in obese children or adolescents is around 20%-25% [6]. Furthermore, it is reported that childhood obesity is associated with morphological and functional The plasma concentrations of total lipids, triglycerides, total cholesterol, and LDL and HDL cholesterol were measured by commercial kits from Spinreact S.A.U (Girona, Spain) following the manufacturer instructions. The analysis of insulin, leptin, and adiponectin levels was performed using commercial enzyme-linked immunosorbent assay (ELISA) kits from Millipore (Dramstadt, Germany). All samples were run in duplicate and within the same assay for all analyses. The intraand inter-coefficients of variation for all analyses were < 10% and < 20%, respectively. The minimum detectable concentrations (ng/mL) were: insulin, 13.0; leptin, 4.2.

Experiments of Heart Perfusion: Langendorff
After sacrifice, hearts were immediately removed and mounted in the Langendorff perfusion system as previously described [28]. After a 30 min equilibration period with constant flow perfusion, increasing doses of insulin were added to the perfusion solution (10 −10 -10 −7 M) in the presence or absence of the blocker of the PI3K/Akt pathway wortmanin (10 −6 M), or in the presence or absence of the MAPK inhibitor SCH-772984 (3.10 −6 M). Both preincubations were performed for 30 minutes before insulin administration. Insulin (#I0516) and wortmanin (#681675) were purchased from Sigma-Aldridh (St. Louis, MO, USA) and SCH-772984 from Cayman Chemical (#19166, Ann Arbor, MI, USA). The effects of insulin administration on coronary perfusion pressure, left intraventricular pressure, dp/dt as an index of myocardial contractility, and heart rate were recorded. Control hearts were perfused during the same time without adding insulin.
Coronary perfusion pressure was measured through a lateral connection in the perfusion cannula and left ventricular pressure was measured using a latex balloon inflated to a diastolic pressure of 5-10 mmHg, both of which were connected to Statham transducers (Statham Instruments, Los Angeles, CA, USA). Left ventricular pressure was recorded and was used to calculate the first derivative of the left ventricular pressure curve (dP/dt) as an index of heart contractility and heart rate. These parameters were recorded on a computer using the PowerLab/8e data acquisition system (ADInstruments, Colorado Springs, CO, USA).
Finally, to study the effects of insulin administration in the gene or protein expression of different markers in the myocardium, hearts from both experimental groups were perfused and incubated in the presence or absence of insulin 10 −7 M for 30 min. Afterwards, hearts were collected and stored at −80 • C.

Experiments of Vascular Reactivity
For the vascular reactivity experiments, the aorta was carefully dissected, cut into 2 mm segments, and kept in cold isotonic saline solution. The assembly of the segments was performed as previously described [23]. The changes in isometric force were recorded using a PowerLab data acquisition system (ADInstruments, Colorado Springs, CO, USA). After applying an optimal passive tension of 1 g, vascular segments were allowed to equilibrate for 60-90 min. Afterwards, segments were stimulated with potassium chloride (KCl 100 mM, #1.04936, Sigma-Aldrich, St. Louis, MO, USA) to determine the contractility of smooth muscle. Segments that failed to contract at least 0.5 g to KCl were discarded. After equilibration, the segments were precontracted with 10 −7.5 . M phenylephrine (#P1250000, Sigma-Aldrich, St. Louis, MO, USA) to subsequently perform a cumulative dose-response curve in response to insulin (10 −11 -10 −6 M), acetylcholine (10 −9 -10 −4 M), and sodium nitroprusside (10 −9 -10 −4 M) (#A6625 and # 71778, Sigma-Aldrich, St. Louis, MO, USA) . The relaxation in response to insulin was determined based on the percentage of the active tone achieved by the nitric oxide (NO) donor sodium nitroprusside (10 −5 M).

Nitrite and Nitrate Determination in the Culture Medium
Nitrite and nitrate concentrations, as indicators of nitric oxide release, were measured in the culture medium after incubation of aorta segments from both L12 and L3 rats in the presence or absence of insulin by a modified Griess assay method, described by Miranda et al. 2001 [29]. Briefly, 100 µL of vanadium chloride (#208272, Sigma-Aldrich, St. Louis, MO, USA) was added to 100 µl of culture medium on a 96-well plate. Immediately after, the Griess reagent (1:1 mixture of 1 % sulfanilamide (Merck Millipore, Darmstadt, Germany), and 0.1 % naphthylethylenediamine dihydrochloride (#106237, Merck Millipore, Darmstadt, Germany)) was added to each well and incubated at 37 • C for 30 min. The absorbance was measured at 540 nm. Nitrite and nitrate concentrations were calculated using a NaNO 2 (#237213, Sigma-Aldrich, St. Louis, MO, USA) standard curve and was expressed in µM.

Protein Quantification by Western Blot
First, 100 mg of myocardial (ventricular) or arterial tissue was homogenized using radioimmunoprecipitation assay buffer (RIPA buffer). After centrifugation (12000 rpm, 4 • C, 20 min), supernatant was collected and total protein content was measured by the Bradford method (Sigma-Aldrich, St. Louis, MO, USA). In each assay, the same amount of protein was loaded in each well (100 µg). After electrophoresis using resolving acrylamide sodium dodecyl sulfate (SDS) gels Afterwards, membranes were also incubated with GAPDH (1:500; Ambion life technologies, Waltham, Massachusetts, USA) in order to normalize each sample for gel-loading variability. For each sample, relative protein expression levels were calculated in relation to protein expression levels in samples from L12 rats.

Detection of Glucose Transporter 4 (GLUT-4) in the Heart by Immunofluorescence
Hearts were fixed in 4% paraformaldehyde diluted in PBS solution for 24 hours and embedded in paraffin using the automatic equipment (Leica TP 1020, Leica, Switzerland). Longitudinal sections (3 µm-thick, HM 325, Microm) were stained with hematoxylin-eosin.
For GLUT-4 detection by immunofluorescence, ventricular sections were incubated with a specific antibody for human glucose transporter 4 (GLUT 4, rabbit anti-human GLUT4, ab654, Abcam), diluted 1/500 in PBS for 1 h, rinsed extensively with PBS, and incubated with the corresponding secondary antibody (goat anti-rabbit, Alexa Fluor 488; Molecular Probes) diluted to 1:1000. Then, to determine the translocation of GLUT-4 to the plasmatic membrane of cardiomyocytes, wheat germ agglutinin (WGA, Alexa Fluor™ 594) diluted to 1:200 in PBS was added and incubated for 45 min at room temperature in the dark. Finally, samples were rinsed successively with PBS, distilled water, and ethanol, and mounted with a drop of VECTASHIELD Antifade Mounting Medium containing 4',6-diamidino-2-phenylindole (DAPI) (H-12000 Vector Laboratories), which fluoresces when bound to DNA. Images were obtained and scanned using a Leica SP8 Confocal Microscopy System coupled to a DMi6000 inverted microscope. Images were acquired with a 63x magnification, 1.4 NA PlanApo Oil objective and an additional confocal zoom of 2. As a source of excitation for the green and red channels, a white light laser was used, adjusting the wavelengths to the absorption optics of the fluorescent molecules. For visualization of DAPI, a 405 nm laser was used. Ten cells in each slide were randomly selected per sample for analysis of the GLUT4 in the heart. Quantification was made using a macro program and ImageJ software.

Statistical Analysis
All data are represented as mean ± SEM. Differences between L3 and L12 rats variables were examined by Student's t-test or by two-way ANOVA followed by Bonferroni's test for data obtained from experiments performed in the presence or absence of insulin. Differences were considered significant when p < 0.05.

Body and Organ Weight
At birth, body weight did not differ between rats raised in control and reduced litters (Table 1). However, L3 rats showed increased body weight at weaning (p < 0.001), as well as increased visceral (p < 0.001), subcutaneous (p < 0.001), brown (p < 0.01), and periaortic (p < 0.05) fat weights compared to L12 rats. Regarding muscle mass, both gastrocnemius and heart weights were also significantly increased in L3 rats compared to L12 (p < 0.01 and p < 0.05 respectively). Table 1. Body and organ weights from L12 (lean) and L3 (overfed) rats.

L12 L3
Body weight at birth (g) 6.8 ± 1.1 6.9 ± 1.1 Body weight at weaning (g) 50 Table 2 shows a significant increase of glucose and insulin plasma levels in L3 rats compared to L12 (p < 0.05 for both). Likewise, plasma concentrations of leptin (p < 0.01), adiponectin (p < 0.01), total lipids (p < 0.01), and total cholesterol (p < 0.05) were significantly higher in overfed rats compared to controls. On the contrary, postnatal overfeeding induced a significant reduction in the plasma levels of HDL cholesterol (p < 0.05). No changes were found in the plasma levels of triglycerides and LDL cholesterol between experimental groups.

mRNA Levels of Insulin Receptor and Glucose Transporter 4 in the Myocardium and GLUT-4 Localization
The mRNA levels of insulin receptor and glucose transporter 4 are shown in Figure 1. Overfed rats showed an upregulation in the gene expression of both IR (p < 0.05; Figure 1A) and GLUT-4 (p < 0.05; Figure 1B) in the myocardium compared to control rats. However, quantification of GLUT-4 by immunofluorescence showed a reduced localization of GLUT-4 in the cell membrane of cardiomyocytes in hearts from overfed pups compared to controls (p < 0.001; Figure 1C,D)

Changes in Heart Rate, Coronary Perfusion Pressure and Heart Contractility (dp/dt) in Response to Insulin Administration
Basal heart rate was 280 ± 16 and 297 ± 12 beats/min in L12 and L3, respectively, and it was not modified by insulin treatment (data not shown).
The changes in coronary perfusion pressure and heart contractility (dp/dt) in response to insulin administration are represented in Figure 2A,B, respectively. Insulin administration to perfused hearts from L12 rats induced vasodilatation of coronary arteries at 10 −8 and 10 −9 M concentrations, and vasoconstriction at the highest concentration used (10 −7 M), whereas in hearts from L3 rats only the vasoconstriction with 10 −7 M concentration was observed.
Before insulin treatment, the hearts from rats raised in reduced litters showed decreased contractility compared to hearts from rats raised in control litters (1699 ± 7 vs. 2331 ± 6 mmHg/s; p < 0.05). Insulin administration to perfused hearts induced a significant increase in heart contractility, both in L12 and in L3 rats, with this increase being significantly lower in hearts from overweight rats at insulin concentrations of 10 −9 and 10 −8 M (p < 0.05 for both). Preincubation with the blocker of the PI3K/Akt pathway wortmanin blunted the increase in dp/dt in both control and in overfed hearts at all dosages studied. Finally, preincubation with the blocker of the MAPK pathway SCH-772984 before the insulin dose-response curve significantly attenuated the early overfeeding-induced decrease in heart contractility in response to insulin at 10 −10 , 10 −9 , and 10 −7 M (p < 0.05 for all).

Myocardial Activation of PI3K/Akt and MAPK Pathways in Response to Insulin Administration
The two-way ANOVA analysis revealed no interaction between factors for p-Akt ( Figure 3A), total Akt ( Figure 3B), p-Akt/Akt ratio ( Figure 3C), p-MAPK ( Figure 3D), and total MAPK ( Figure 3D); and a significant effect of insulin in both types of litters for p-Akt, p-Akt/Akt ratio, and p-MAPK (p < 0.01 for all). However, there was significant interaction between the two factors for p-MAPK/MAPK ratio ( Figure 3F; F = 11.60; p < 0.01) and a significant effect of insulin was found only in hearts from rats raised in reduced litters (p < 0.001). Protein levels of (A) ratio of phosphorylated to total Akt (p-Akt/Akt), (B) total Akt (Akt), (C) ratio of phosphorylated to total mitogen-activated protein kinase (MAPK) (p-MAPK/MAPK), and (D) total MAPK (MAPK) in perfused hearts from rats raised in L12 or L3 litters, incubated either with vehicle (control) or with insulin (10 −7 M) for 30 min. Note: * p < 0.05 difference between control hearts from L3 and L12; ## p < 0.01; ### p < 0.001 difference between hearts incubated with insulin and control. Values are represented as mean ± SEM (n = 4-5 rats/experimental group) and expressed as % vs. L12. Data were analyzed by two-way ANOVA followed by Bonferroni post-hoc test.
No changes between experimental groups were found in the myocardial levels of p-eNOS, eNOS, and the ratio p-eNOS/eNOS (data not shown).

Vascular Reactivity of Aortic Rings in Response to Acetylcholine (Ach) and Sodium Nitroprusside (NTP)
The vascular response of aortic rings from L12 and L3 rats to Ach and NTP are shown in Figure 4A,B, respectively. Aortic rings from overfed rats showed decreased vasorelaxation in response to high concentrations of Ach (10 −6 and 10 −5 M) (p < 0.05 for both). Likewise, vasorelaxation in response to NTP was significantly reduced in L3 rats compared to controls at dosages between 10 −8 and 10 −6 M (p < 0.05 for all).
Results of nitrite and nitrate release and eNOS activation in aorta segments from both lean and overfed rats in response to insulin are shown in Figure 5B,C, respectively.
In both cases, we found no interaction between both factors. In response to insulin 10 −7 M, nitrite and nitrate concentrations (µM) were significantly up-regulated in the culture medium of aortic rings from L12 rats (p < 0.01), but not in aortic rings from L3 rats ( Figure 5B). Likewise, insulin significantly increased the levels of p-eNOS in the aorta from lean (p < 0.05) but not from overfed rats ( Figure 5C) whereas the content of total eNOS was unchanged ( Figure 5D) , ratio of phosphorylated to total eNOS (p-eNOS/eNOS) (C) and total eNOS (eNOS) (D) in aorta segments from rats raised in L12 or L3 litters incubated over 30 days with either vehicle (control) or insulin (10 −7 M). Note: * p < 0.05; ** p < 0.01 difference between aortic rings from L3 and L12; # p < 0.05; $ p < 0.05 difference between hearts incubated with insulin from L3 and L12. Values are represented as mean ± SEM (n = 4-5 rats/experimental group) and expressed as % vs. L12. (n = 6-9 rats/experimental group). Data were analyzed by two-way ANOVA followed by Bonferroni post-hoc test.

Vascular
Reactivity of Aortic Rings in Response to Insulin in Presence/Absence of Meclofenamate, Apamine/Charibdotoxine, L-NAME, or Wortmanin Figure 6 shows the vascular response of aortic segments from both control and overfed rats to insulin in presence or absence of meclofenamate (A), apamine or charibdotoxine (B), L-NAME (C), or wortmanin (D).
In all cases, no significant interaction was found between factors (litter size and treatment with blockers). As indicated by the Area Under the Curve (AUC) values, insulin-induced vasodilation of aortic rings was partially reduced in L12 rats in the presence of L-NAME (p < 0.01) and wortmanin (p < 0.01). However, insulin-induced vasodilation of aortic rings from L3 rats was only attenuated in the presence of L-NAME (p < 0.01). Note: * p < 0.05 difference between aortic rings from L3 and L12; # p < 0.05; ## p < 0.01 difference between aortic rings incubated with the blockers and control. Values are expressed as percentage of initial tone and represented as mean ± SEM; n = 6-13 rats/experimental group. Data were analyzed by two-way ANOVA followed by Bonferroni post-hoc test.

Activation of PI3K/Akt and MAPK Pathways in Response to Insulin Administration in Arterial Tissue
The activation of PI3K/Akt and MAPK pathways in arterial tissue in the presence or absence of insulin is shown if Figure 7.
In response to insulin 10 −7 M, no interaction and no significant changes were found in the activation of MAPK pathway in either aorta segments from L12 or in aorta segments form L3 rats. On the contrary, insulin induced a significant activation of the PI3K/Akt pathway in arterial tissue from lean rats, as is shown by the significant interaction (F = 5.44, p < 0.05) and the increase in the arterial content of p-Akt (p < 0.05, Figure 7A) and the arterial p-Akt/Akt ratio (p < 0.01, Figure 7C) only in arterial segments from L12 rats. Note: # p < 0.05; difference between hearts incubated with insulin and control. Values are represented as mean ± SEM (n = 6-9 rats/experimental group) and expressed as % vs. L12. Data were analyzed by two-way ANOVA followed by Bonferroni post-hoc test. Figure 8 shows the mRNA levels of IR (A), GLUT-4 (B), and eNOS (C) in aorta segments from L12 and L3 rats in the presence or absence of insulin.

Discussion
In this study we show that early overnutrition in rats is associated with cardiovascular insulin resistance, both in the heart and in the aorta, and that this state may be responsible, at least in part, for the development of the cardiovascular alterations associated to this condition.
Our results show that insulin induces vasodilation in aorta segments of rats from both experimental groups. Arterial vasodilatation in response to insulin has been described before and is mediated, at least in part, by the release of endothelial nitric oxide [32]. Nitric oxide also seems to mediate insulin-induced vasodilation of aorta segments in our rats, as the relaxation in response to insulin was significantly reduced by the nitric oxide inhibitor L-NAME and incubation with insulin significantly stimulated nitrite release in aorta segments from L12 rats.
It is reported that both the metabolic and vascular effects of insulin are mainly mediated by activation of the PIK3/Akt pathway [13,14]. Likewise, in this study insulin-induced vasodilation and incubation of aorta segments with insulin increased the p-AKT/Akt ratio in aorta segments from lean rats, and insulin-induced vasodilatation was reduced in the presence of the inhibitor of the PIK3/Akt pathway wortmanin.
Obesity is associated with a reduced effect of insulin on glucose cell uptake, and it has been described that it also impairs its vasodilating arterial effect, which may contribute to the development of metabolic syndrome [17,33]. The reduced vasodilator effect in response to insulin in overfed rats was also associated with a decreased gene expression of insulin receptor, Glut-4 transporter, and eNOS gene expression, which resulted in decreased NO production. These results clearly indicate an impairment of the intracellular mechanisms that insulin triggers, resulting in a state of vascular insulin resistance, as occurred in previous studies that reported a decreased activation of this pathway in states of insulin resistance, both in vivo [34,35] and in vitro [36]. The reduction of insulin-induced vasodilatation in overfed rats may be related to a general impairment of arterial vasodilatation during the first stages of weight gain, as both the relaxation in response to acetylcholine, which is mediated by nitric oxide release from the endothelium, and to sodium nitroprusside, which acts directly on the vascular smooth muscle, were reduced in early overfed rats. Likewise, it is reported that vasodilatation to reactive hyperemia correlates negatively with body fat in adolescents [37]. As previously reported [38,39], the vascular insulin resistance may be due to both increased vascular inflammation and oxidative stress, since both the mRNA levels of pro-inflammatory and pro-oxidant markers were upregulated in overfed rats. However, adiponectin levels were also increased in over-nourished rats, which may be related to the unchanged activation of the MAPK pathway in arterial tissue.
The insulin effect on the heart has been less studied than that in the arteries, but there are several studies reporting a positive inotropic effect [12]. As expected, our results also show a positive inotropic effect of insulin in the heart, as indicated by the increased intraventricular pressure and dP/dt in response to insulin in both experimental groups, which agrees with previous studies [40,41]. As previously described [42], this effect is also mediated by the activation of the PI3K/Akt pathway, as cardiac insulin administration significantly increases the p-Akt/Akt ratio in the myocardium, and this effect is blocked by wortmanin. The increased myocardial contractility in response to insulin was reduced in hearts of overweight rats, indicating that insulin resistance is also present in the cardiac function. This decreased cardiac insulin sensitivity in overfed rats could be related, at least in part, with the decreased heart contractility previously described in this experimental model of early overnutrition [21]. However, the decreased contractility seemed not to be related with morphological or functional myocardial alterations, as we did not find any changes in either the cardiomyocyte area or in the expression of contractile proteins between L12 and L3 rats.
Our results also show that the mechanisms of insulin resistance may be different in the heart and in the arteries. In the myocardium, the increase in the p-Akt/Akt ratio induced by insulin was not different in either experimental groups. Moreover, the expression of insulin receptor and Glut-4 transporter was not reduced but increased in overweight rats, although its translocation to the cardiomyocyte cell membrane was significantly lower. Since the activation of the PI3K/Akt pathway in response to insulin was similar in hearts from control and overfed rats, these results suggest that this pathway may not be the one responsible for cardiac insulin resistance in this experimental model of early overnutrition. Thus, we explored the activation of the MAPK pathway in response to insulin and we observed that the ratio p-MAPK/MAPK was enhanced by insulin to a greater extent in overweight rats than in controls. It has been described that the activation of the MAPK pathway inhibits myocardial contraction [43,44] by enhancing dephosphorylation of alpha-tropomyosin [45], and is involved in the development of insulin resistance in the heart [46]. Our results support this idea, as preincubation of hearts with a MAPK blocker attenuates the early overfeeding-induced decrease in heart contractility in response to insulin. Therefore, the activation of this pathway in overfed rats may counteract the activation of the PI3K/Akt pathway and result in a reduced cardiac contractility in response to insulin. The hyperactivity of the MAPK pathway may be related to the pro-inflammatory state in the hearts of overweight rats, where we found an increased expression of proinflammatory cytokines. Likewise, MAPK activation drives inflammatory processes in cardiomyocytes after LPS stimulation [47], doxorubicin toxicity [48], or diabetes [49]. Inflammation in obesity induces cardiomyocyte cell death [50] and is a predictor of cardiac disease in states of insulin resistance [51]. For this reason, MAPK inhibitors are being studied as possible cardioprotective agents [52].
Altogether, our results show that early postnatal overfeeding is associated with a state of cardiovascular insulin resistance that affects both the aorta and the myocardium. However, the intracellular mechanisms that result in this state seem to be different in the heart and in the vessels, with the decreased activation of the PI3K/Akt pathway being responsible for vascular resistance in the aorta and the overactivation of the MAPK pathway being responsible for cardiac insulin resistance in the myocardium.
In conclusion, the results of the present study indicate that early overnutrition in rats is associated with decreased insulin sensitivity in the short-term, both in the heart and in the aorta. This suggests that in a context of increased nutrients and energy supply, alterations of insulin signaling in the vasculature and heart arise early, possibly explaining the finding of heart dysfunction in obese children [53][54][55][56] and highlighting the importance of an early intervention.