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Hindawi Journal of Nutrition and Metabolism Volume 2019, Article ID 2806519, 8 pages https://doi.org/10.1155/2019/2806519 Research Article StressduringLactationInducesInsulinResistanceAssociatedwith an Increase in Type 1 Cannabinoid Receptors in Liver and Adipose Tissue 1 1 2 3 1 Liza Fonseca, Valeska Castillo, Carolina Aguirre , Paulo Silva, Ana M. Ronco, and Miguel Llanos Laboratorio de Nutricio´n y Regulacio´n Metabo´lica, Instituto de Nutricio´n y Tecnolog´ıa de los Alimentos (INTA), Universidad de Chile, Santiago, Chile ´ ´ Departamento Ciencias de la Salud, Carrera de Nutricion y Dietetica, Facultad de Medicina, Pontificia Universidad Cato´lica de Chile, Campus San Joaqu´ın, Santiago, Chile Escuela de Nutricio´n, Universidad San Sebastia´n, Santiago, Chile Correspondence should be addressed to Miguel Llanos; mllanos@inta.uchile.cl Received 26 July 2018; Revised 19 November 2018; Accepted 28 November 2018; Published 17 January 2019 Academic Editor: Christopher L. Gentile Copyright © 2019 Liza Fonseca et al. *is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Several reports have shown that stress during lactation causes long-term metabolic and hormonal disruptions. In this study, we designed experiments to evaluate the effects of stress during lactation on the abundance of Type 1 cannabinoid/endocannabinoid receptors (CB1R) in epididymal fat and liver and development of insulin resistance in adult mice. During the whole lactation, male mice pups were daily subcutaneously injected (days 1–21) with a saline solution to produce a soft nociceptive stress (NS). Mice body weight and food intake were periodically evaluated. Adult animals were subsequently subjected to an insulin tolerance test and some days later sacrificed to evaluate the amount of epididymal fat and abundance of CB1R and adipophilin in liver and epididymal adipose tissue. Lipoprotein lipase (LPL) activity and circulating levels of leptin, adiponectin, and corticosterone were also evaluated. In this model, NS during lactation significantly increased the amount of epididymal fat and induced insulin resistance in adult mice. In addition, a significantly increased abundance of CB1R and adipophilin in epididymal fat and liver was observed, together with elevated circulating levels of leptin and corticosterone. Adult NS animals also had low plasmatic adiponectin and, although nonsignificant, had a sustained trend to a greater LPL activity associated with epididymal fat. *ese results indicate that increased abundance of CB1R in liver and epididymal fat alters tissue functionality likely associated with development of systemic metabolic alterations such as insulin resistance in adult mice. All these pathophysiological facts are long- term consequences of nociceptive stress during lactation. Stress may constitute an early stimulus leading to adi- 1. Introduction posity, overweight, and metabolic alterations in adulthood. In Evidence from epidemiological and animal studies indicates this regard, it has been observed that pups repeatedly subjected that critical windows, such as foetal and/or neonatal life, to a mild nociceptive stress during lactation show a significant represent periods in which an individual’s susceptibility for increase in body weight and epididymal fat pads [3]. long-term development of health or disease could be de- It is known that chronic stress alters physiology of different termined or “programmed” [1]. *us, foetal/neonatal pro- tissues through a diverse mechanism, including hyper- activation of the hypothalamus-pituitary-adrenal (HPA) axis gramming is defined as the “induction, suppression, or permanent change of somatic structures by an early stimulus [4]. Interestingly, the hypothalamus plays a key role in- or insult” [2]. tegrating biochemical and behavioural components involved 2 Journal of Nutrition and Metabolism in energy homeostasis [5], and one of these components is the animals had free access to purified tap water and food. A endocannabinoid system (ECS). *e ECS mainly consists of normal diet of 4 kcal/g, equivalent to 2.8 assimilated kcal/g Type 1 and 2 cannabinoid receptors (CB1R and CB2R), (Champion Co, Santiago, Chile), was used during the whole present in several tissues including the central nervous system, study [21]. adipose tissue, liver, and pancreas [6–8], with their endoge- From day 16, pregnant female mice were examined daily nous ligands, known as endocannabinoids (ECs), being at 9 : 00 and 19 : 00 h for the presence of pups. After 12–16 h arachidonoyl ethanolamide or anandamide (AEA) [9] and 2- since detection of pups, 6–8 litters of homogeneous size arachidonoyl glycerol (2-AG) [10], the most studied agonists, (12–14 pups) were put together and males were separated with endocrine, autocrine, and paracrine actions [11, 12]. from females. Afterwards, six male pups showing homo- Finally, enzymes synthesizing and degrading ECs are also part geneous weights were randomly selected and assigned to a of this system [13]. substitute mother so that pups received random cross lac- Stress elevates endocannabinoid levels in some areas of tation [21]. *e animals were then assigned to one of the the central nervous system which in turn activate CB1R following groups: involved in the negative feedback mechanism able to repress (1) Control mice: pups were removed daily for short the activity of the hypothalamus-pituitary-adrenal axis [14]. time from home cage using a vinyl gloved hand to In addition, overactivation of CB1R in some peripheral measure body weight. tissues has been related to overweight/obesity, insulin and (2) Stressed, nociceptive-stimulated mice (NS mice): leptin resistance, and dyslipidaemia [15, 16]. during the whole lactation (21 days), pups were Adipocytes express CB1R (a target for AEA); its acti- removed daily from home cage, and each pup was vation is involved in adipocytes growth and differentiation, gently picked up using a vinyl gloved hand, weighed, modulation of adipokines and hormones synthesis/ and subcutaneously injected in the back with sterile secretion, and stimulation of lipogenesis [15, 17, 18]. In saline solution (1 μl/g body weight) with a micro- addition, a recent study [19] reported that CB1R activation syringe (25-gauge needle). in adipose tissue alters antilipolytic activity of insulin, a fact contributing to ectopic fat deposition involved in insulin Procedures were always performed by the same exper- resistance. Furthermore, Osei-Hyiaman et al. [20] had imenter in a total time of 8–10 min/cage. Twice a week, the demonstrated that development of insulin resistance and animals were transferred to clean cages, with fresh clean hepatic steatosis due to a high-fat diet is associated with the bedding material. presence of CB1R in liver, a conclusion obtained using a At day 21 of age, the animals were separated from their liver-specific CB1R knockout mice model. Previously, we mothers, and groups of three animals were placed in new had shown that early stress produces long-term increases in cages until adulthood. Subsequently, adult 145-day-old overweight, epididymal fat content, and alterations of cir- animals were subjected to an insulin tolerance test, and culating metabolic parameters in adult mice. *is condition five days later, they were sacrificed according to the was reversed by treatment with SR141716A, the antagonist/ guidelines for rodent euthanasia provided by the American inverse agonist of CB1R, suggesting a global involvement of Veterinary Medical Association [24]. After sacrifice, the these receptors in those effects [21]. In addition, treatment whole epididymal fat pads and liver were extracted from the with AEA during lactation leads to augmented presence of abdominal area, weighed, and immediately frozen in liquid CB1R in epididymal fat, a marked increase in total body fat nitrogen for additional experiments. In addition, blood percentage and insulin resistance in adult animals [22, 23]. samples were obtained from the abdominal cava vein to With all these antecedents in mind, the aim of this study evaluate circulating levels of some metabolic markers as was to evaluate the effects of early postnatal nociceptive further described [21, 22]. stimulation on development of insulin resistance and CB1R abundance in epididymal fat pads and liver of adult mice and its association with molecules involved in lipid storage and 2.2. Body Weight and Food Intake. Body weight was recorded tissue function. every 10 days. Daily food intake per cage (containing three mice) was calculated by subtracting the lost food inside the cage due to spilling in 24 hours from day 40 to 150. 2. Materials and Methods All procedures performed in this study were approved by the 2.3. Western Blot of CB1R and Adipophilin. For western Bioethics’ Committee for Animal Experimentation of the blotting procedures, epididymal fat pads and liver from NS Institute of Nutrition and Food Technology, University of mice and control animals were homogenized as previously Chile, Santiago, Chile. described (Heidolph homogenizer DIAX 600) in 500 µl of RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% 2.1. Animals. Procedures performed in this study were sodium deoxycholate, 0.1% SDS) in the presence of protease similar to those previously described [21]. In brief, syn- inhibitor cocktail (cat. no. P2714; Sigma Chem Co, USA). chronously pregnant female CD-1 mice were kept in the *e homogenates containing 30 and 12 µg of protein from animal house under normal conditions of humidity and adipose tissue and liver, respectively, were separated in a temperature (22–24 C), on a 12 :12 h light-dark cycle. *e 10% SDS-polyacrylamide gel (Mini-PROTEAN III System; Journal of Nutrition and Metabolism 3 Bio-Rad, USA) and subsequently transferred overnight to a Whitney U test was utilized to establish statistical differences PVDF membrane at 4 C [22, 23]. Rabbit polyclonal antibody between experimental groups. Statistical significance was set at P≤ 0.05. for CB1R (Cayman Chemical, CA, USA) and adipophilin (*ermo Fisher Scientific, USA) was used as the primary antibody, and a peroxidase-conjugated anti-rabbit antibody 3. Results was used as the secondary antibody (Rockland Immuno- 3.1. Plasma Corticosterone, Leptin, and Adiponectin. First, to chemicals, USA); CB1R and adipophilin were visualized by validate the model, plasma levels of immunoreactive cor- chemiluminescence (Western Lightning Plus-ECL, en- ticosterone (ir-CTT) and leptin, in both NS and control hanced chemiluminescence substrate; Perkin Elmer, USA). mice, were measured. As expected, adult animals subjected *e obtained protein bands were normalized against β-actin to nociceptive stress during lactation had significantly higher expression and quantified using the Gel-Pro Analyzer 3.1 levels of ir-CTT and leptin than control mice. Here, we also programme [22]. Appropriate positive and negative controls incorporated measurements of plasma adiponectin which is with brain tissue and the corresponding blocking peptide to significantly lowered in NS mice (Table 1). the CB1R antibody, respectively, were also performed (Cayman Chemical, CA, USA). 3.2. Food Intake, Body Weight, and Epididymal Fat. Daily food intake from day 40 to 150, measured in five cages (three 2.4. Plasma Hormonal Levels. Corticosterone levels were animals per cage), show significant differences between both determined in duplicate using a commercial ELISA kit groups only at 60 days old (Figure 1). Body weight gain according to the manufacturer’s instruction (cat. no. 500651; during lactation and adulthood in both groups of mice is Cayman Chemical, USA). Plasma leptin and adiponectin shown in Figure 2. In this set of experiments, there were no levels were assessed in duplicate using a commercial col- significant differences in body weight between control and orimetric sandwich ELISA kit (cat. no. MOB00 and NS mice. However, in spite of no differences in body weight, MRP300, respectively; R&D Systems, USA). 150-day-old NS mice had a marked increase in epididymal fat content (Figure 3) when compared to control mice. 2.5. Lipoprotein Lipase Activity of Epididymal Adipose Tissue. Epididymal fat pads, from either nonfasting animals or after 3.3. Western Blot Analysis of CB1R and Adipophilin in Epi- 5 h fasting, were homogenized in 0.05 M sodium phosphate buffer pH 8.0, and the samples were then treated with didymal Fat Pads and Liver. Figures 4(a) and 4(b) show CB1R protein levels in epididymal fat and liver of 150-day- heparin (0.5 U). After treatment, lipoprotein lipase (LPL) old control and NS mice, respectively. It may be observed activity was assessed using the CONFLUOLIP Continuous that NS mice almost had as much as twice the levels of CB1R Fluorometric Lipase Test (cat.no. PR2003; Progen Bio- (60 kDa) in epididymal fat than control animals (P< 0.05). technik GmbH, Germany) according to the manufacturer’s In liver, there was also a 55% increase in abundance of CB1R protocol. Fluorescence of quenched pyrene, after adding protein, which is in the limit of significance (P � 0.06). In lipase-containing samples, was measured in a kinetic assay addition, adipophilin (a protein marker for lipid accumu- for 2 hours, using 342 nm excitation and 400 nm emission lation in somatic cells; 48 kDa) was found to be about 60% wavelengths in a 96-well plate at 37 C. Fluorescence intensity higher in epididymal fat and liver of NS mice than in that of was measured with a SpectraMax Gemini EM Microplate control animals (Figures 5(a) and 5(b); P< 0.05). In both Reader (Molecular Devices, USA). *e activity of LPL in the cases, β-actin (42 kDa) was utilized as a loading control. assay was calculated as the ratio delta fluorescence/time and was expressed as pmol/min/mg protein. Serial dilutions of the fluorescent standard were used for calibration of 3.4. Lipoprotein Lipase Activity of Epididymal Fat. No sig- measurement. nificant differences in epididymal fat LPL activity were found between both groups of fasted animals; however, there 2.6. Insulin Tolerance Test. *e insulin tolerance test (ITT) was a consistent, although nonsignificant, higher LPL ac- tivity in NS mice, showing a sustained 25.9% increase than was performed in 6 h fasted mice [22]. *e animals were intraperitoneally injected at time 0 with recombinant human those control mice (Figure 6(a)). *is trend was also ob- served in nonfasted mice although in a minor extent of 17% insulin (0.75 U/kg; Eli Lilly), and tail blood glucose levels were subsequently measured (basal and 15, 30, 45, and increase (Figure 6(b)). 60 min after injection). Glycemia was determined with a highly sensitive, wide-range Accu-Chek Performa gluc- 3.5. Insulin Tolerance Test. In Figure 7(a), it is observed that ometer (Roche Diagnostics, Mannheim, Germany). *e area adult NS mice show an insulin resistance condition which is under the curve (AUC) between 0 and 60 min was de- not observed in nonstressed control mice, where glycemia termined using the trapezoidal method [25]. reached about 50% of the initial value after 30–60 min of insulin injection, where NS mice only reached a 70%. In 2.7. Statistical Analysis. Data were expressed as mean- addition, area under the curve obtained with glycemia values ± SEM. Shapiro–Wilk’s test was first performed to evaluate from NS mice is higher and significantly different from the normal distribution of data. *e nonparametric Mann– area obtained in control animals (Figure 7(b)). 4 Journal of Nutrition and Metabolism Table 1: Plasma levels of hormones in control and nociceptive- already shown [21], the amount of epididymal fat becomes stimulated adult male mice (NS mice). normal when these animals are orally treated with SR141716A, a well-known antagonist/inverse agonist of Plasma levels Control NS mice CB1R, indicating a role for these receptors in adipose tissue Corticosterone (ng/ml) 23.9± 4.8 42.4± 7.3 accumulation. Fatty acids availability for lipid synthesis Leptin (ng/ml) 4.6± 1.6 19.2± 4.0 and storage in adiposomes may be explained in part by Adiponectin (μg/ml) 5.5± 0.5 4.1± 0.1 increased LPL activity observed in response to CB1R Data represent mean± SEM of 5 mice from each group. P< 0.05. stimulation [15]. In this regard, it has been shown that primary cultured adipocytes treated with a CB1R agonist (WIN-55, 212) increased LPL activity in a dose-dependent manner [15], which in turn increases availability of fatty acids to be incorporated in adipocytes. Although our present results indicate a nonsignificant 17–25% increase in LPL activity of adult NS mice, this increase is chronically sustained during nonfasting and fasting. *is condition may result in a long-term higher accumulation of epi- didymal fat in adult NS mice, as previously observed [3, 21]. In this study, we have observed a clear state of insulin resistance in those adult mice previously stressed during lactation. In this regard, it has recently been shown that activation of CB1R affects antilipolytic activity of insulin in 40 50 60 70 80 90 100 110 120 130 140 150 adipose tissue [19]. *is effect may result in ectopic de- Days position of lipids in tissues involved in glucose homeo- Control NS mice stasis, leading to insulin resistance [28]. Interestingly, in this study, we have also observed an increase in hepatic Figure 1: Twenty-four hours of food intake measured in gram (g), CB1R, and previously, we showed a decreased activity of every 10 days from 40 to 150 days. P< 0.05 (Mann–Whitney U FAAH in the liver of NS mice; both conditions may test; mean± SEM; n � 5 cages with 3 mice each/group). converge in hyperactivation of CB1R. In this context, Osei- Hyiaman et al. [20] working in a liver-specific CB1R 4. Discussion knockout mice model concluded that endocannabinoid *is study shows that stress during lactation leads to activation of hepatic CB1R contributes to high-fat-diet- systemic insulin resistance and a significant increased induced insulin resistance. *us, NS mice insulin re- abundance of CB1R in epididymal fat pads, concomitant sistance shown in the present study may be likely asso- ciated with a chronic overactivity of CB1R in adipose tissue with an increased amount of this tissue [21]. *is finding, in addition to a reduced expression and activity of FAAH, and liver. In its role as an endocrine organ, adipose tissue produces previously shown in our laboratory [21, 26], indicates a concerted condition where increased levels of both, the the hormone adiponectin, whose plasma levels are negatively associated with obesity [29]. In this study, plasma concen- agonist AEA (due to low FAAH) and its receptor, may likely result in overactivity of CB1R, with consequences in trations of adiponectin in stressed animals were significantly adipose tissue accumulation and altered physiology. In this lower than those in controls. Adiponectin has a role in re- sense, a significantly higher expression of adipophilin in ducing the expression of enzymes involved in lipogenesis and epididymal fat pads and liver of stressed animals was clearly increases fatty acid oxidation in the muscle, reducing body observed. *is 50 kDa protein, a marker of lipid accu- weight in mice [30]. Interestingly, data regarding CB1R ac- mulation [27], is associated with the formation of adipo- tivation and decreased plasma adiponectin levels have already been reported. *us, CB1R blocking with SR141716A in rats somes in several types of cells, having a role in the regulation of lipid storage and reserves. Recently, it has also and adipocyte culture results in increased expression and plasma concentration of adiponectin [7]. been attributed to adiposomes, the ability to incorporate AEA, suggesting that AEA is not only released in response Another hormone secreted primarily by adipose tissue is leptin, which crosses the blood-brain barrier and acts on the to demand but also complemented by its intracellular dynamic equilibrium according to transport and avail- hypothalamus indicating body energy reserves and con- ability in adiposomes [11]. *ereby, the significantly higher trolling EC levels [31, 32]. Plasma leptin levels in NS mice protein levels of adipophilin found in epididymal fat and were significantly higher compared to those in controls, liver of stressed animals not only indicate an increased which are concordant with higher amounts of epididymal fat abundance of adiposomes and lipid accumulation but also found, confirming the positive correlation between these indirectly suggest a higher AEA storage. In this sense, it is two parameters. When analyzing immunoreactive corticosterone results, important to mention that a high-fat-diet-induced accu- mulation of lipids in adipose tissue and liver involves plasma levels of this hormone remained high until adult- hood only in NS mice. *is result is in agreement with hyperactivation of CB1R associated with elevated levels of AEA and CB1R in both tissues [8, 15]. In addition, as previous studies showing that postnatal daily stress induces Daily food intake (g/cage) Journal of Nutrition and Metabolism 5 20 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 30 40 50 60 70 80 90 100 110 120 130 140 150 Age (days) Age (days) Control Control NS mice NS mice (a) (b) Figure 2: Time course of body weight of control and NS mice (a) during lactation and (b) at dierent ages from day 30 to 150 (mean ± SEM; n 14 mice/group). 2.5 2.0 1.5 1.0 0.5 0.0 Control NS mice Treatment Figure 3: Eect of nociceptive stress during lactation on total amount of epididymal fat of adult control and NS mice. P < 0.05 (Mann–Whitney U test; mean ± SEM; n 14 mice/group). CB1R β-actin 250 250 200 200 150 150 100 100 50 50 0 0 Control NS mice Control NS mice Treatment Treatment (a) (b) Figure 4: Western blot analysis of relative levels of CB1R in epididymal fat (a) and liver (b) of control and NS mice at 150 days. For densitometry quantiˆcation purposes, β-actin was used as a loading control. Results are expressed as percentage relative to CB1R expression in control mice. P < 0.05; P 0.06 (Mann–Whitney U test; mean ± SEM; n 5 mice/group). Four representative blots are shown. Body weight (g) CB1R protein (% of control) Epididymal fat (g) CB1R protein (% of control) Body weight (g) 6 Journal of Nutrition and Metabolism Adipophilin β-actin 200 200 150 150 100 100 50 50 0 0 Control NS mice Control NS mice Treatment Treatment (a) (b) Figure 5: Western blot analysis of relative levels of adipophilin in epididymal fat (a) and liver (b) of control and NS mice at 150 days. For densitometry quantiˆcation purposes, β-actin was used as a loading control. Results are expressed as percentage relative to adipophilin expression in control mice. P < 0.05 (Mann–Whitney U test; mean ± SEM; n 5 mice/group). Four representative blots are shown. 4 4 3 3 2 2 1 1 0 0 Control NS mice Control NS mice Treatment Treatment (a) (b) Figure 6: Lipoprotein lipase activity in adipocytes of epididymal fat pads from fasting (a) and nonfasting (b) adult control and NS mice. No signiˆcantly dierent values (Mann–Whitney U test; mean ± SEM; n 5 mice/group). 40 3000 Control NS mice 0 15 30 45 60 Treatment Time (min) Control NS mice (a) (b) Figure 7: (a) Insulin tolerance test (ITT) in adult, 145-day-old control and NS mice. (b) Area under the curve of ITT obtained from both animal groups. Results are expressed as mean ± SEM (n 6 mice/group; P < 0.05; Mann–Whitney U test). Glycemia (% of initial value) LPL activity (pmol/min/mg protein) Adipophilin protein (% of control) AUC (mg/dl·min) LPL activity (pmol/min/mg protein) Adipophilin protein (% of control) Journal of Nutrition and Metabolism 7 hormonal changes and metabolic alterations that may persist Acknowledgments during adulthood, including elevated circulating levels of *is study received financial support from Fondecyt-Chile corticosterone and ACTH [3]. In this sense, it has also been (Grant No. 1130106) and Direccion ´ de Investigacion, ´ INTA, reported that mice chronically exposed to corticosterone Universidad de Chile (Grant No. CC1004-2018) to ML. develop metabolic syndrome [33]. Interestingly, it was also demonstrated that corticosterone-stimulated metabolic syn- drome depends on CB1R activity. Indeed, CB1R “knockout” References animals do not show such a metabolic alteration in this mouse model [33]. Moreover, we had previously shown that NS mice [1] R. A. Waterland and R. L. 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