Jatropha curcas Protein Concentrate Stimulates Insulin Signaling, Lipogenesis, Protein Synthesis and the PKCα Pathway in Rat Liver Liliana León-López, Claudia C. Márquez-Mota, Laura A. VelázquezVillegas, Amanda Gálvez-Mariscal, Daniel Arrieta-Báez, et al. Plant Foods for Human Nutrition ISSN 0921-9668 Plant Foods Hum Nutr DOI 10.1007/s11130-015-0502-9
1 23
Your article is protected by copyright and all rights are held exclusively by Springer Science +Business Media New York. This e-offprint is for personal use only and shall not be selfarchived in electronic repositories. If you wish to self-archive your article, please use the accepted manuscript version for posting on your own website. You may further deposit the accepted manuscript version in any repository, provided it is only made publicly available 12 months after official publication or later and provided acknowledgement is given to the original source of publication and a link is inserted to the published article on Springer's website. The link must be accompanied by the following text: "The final publication is available at link.springer.com”.
1 23
Author's personal copy Plant Foods Hum Nutr DOI 10.1007/s11130-015-0502-9
ORIGINAL PAPER
Jatropha curcas Protein Concentrate Stimulates Insulin Signaling, Lipogenesis, Protein Synthesis and the PKCα Pathway in Rat Liver Liliana León-López 1 & Claudia C. Márquez-Mota 2 & Laura A. Velázquez-Villegas 2 & Amanda Gálvez-Mariscal 3 & Daniel Arrieta-Báez 4 & Gloria Dávila-Ortiz 1 & Armando R. Tovar 2 & Nimbe Torres 2
# Springer Science+Business Media New York 2015
Abstract Jatropha curcas is an oil seed plant that belongs to the Euphorbiaceae family. Nontoxic genotypes have been reported in Mexico. The purpose of the present work was to evaluate the effect of a Mexican variety of J. curcas protein concentrate (JCP) on weight gain, biochemical parameters, and the expression of genes and proteins involved in insulin signaling, lipogenesis, cholesterol and protein synthesis in rats. The results demonstrated that short-term consumption of JCP increased serum glucose, insulin, triglycerides and cholesterol levels as well as the expression of transcription factors involved in lipogenesis and cholesterol synthesis (SREBP-1 and LXRα). Moreover, there was an increase in insulin signaling mediated by Akt phosphorylation and mTOR. JCP also increased PKCα protein abundance and the activation of downstream signaling pathway targets such as the AP1 and NF-κB transcription factors typically activated Electronic supplementary material The online version of this article (doi:10.1007/s11130-015-0502-9) contains supplementary material, which is available to authorized users. * Nimbe Torres
[email protected]
by phorbol esters. These results suggested that phorbol esters are present in JCP, and that they could be involved in the activation of PKC which may be responsible for the high insulin secretion and consequently the activation of insulindependent pathways. Our data suggest that this Mexican Jatropha variety contains toxic compounds that produce negative metabolic effects which require caution when using in the applications of Jatropha-based products in medicine and nutrition. Keywords Jatropha . Lipogenesis . PKCα . Insulin signaling
Abbreviations Akt (v-akt murine thymoma viral oncogene homolog protein kinase B) AP1 Activator protein 1 c-FOS Cellular Finkel-Biskis-Jinkins murine osteosarcoma viral oncogene c-JUN Cellular jun proto-oncogene FAS Fatty acid synthase LXRα Liver X receptor mTOR Mechanistic target of rapamycin NFκ-B Nuclear factor κ-light-chain enhancer of activated B cells PKCα Protein kinase C SREBP1 Sterol regulatory element binding protein-1 SREBP2 Sterol regulatory element binding protein-2
1
Departamento de Ingenieria Bioquímica, Escuela Nacional de Ciencias Biológicas-IPN, Prolongación de Carpio y Plan de Ayala S/N, CP 11340 México, D.F., Mexico
2
Departamento de Fisiología de la Nutrición, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Vasco de Quiroga No 15, CP14000 México, D.F., Mexico
3
Facultad de Química, Universidad Nacional Autónoma de México, Circuito de la Investigación Científica S/N Conjunto E, UNAM, CP04510 México, D.F., Mexico
Introduction
Instituto Politécnico Nacional-CNMN, Calle Luis Enrique Erro s/n, Unidad Profesional Adolfo López Mateos, Col. Zacatenco, México, D.F. CP 07738, Mexico
Jatropha curcas (J. curcas) is a tree of the Euphorbiaceae family, which is used due to the high oil content (40–60 %)
4
Author's personal copy Plant Foods Hum Nutr
of its seeds [1] as an alternative source of biodiesel. The protein concentrate obtained from the seed cake is a good source of protein [2]. However, the presence of some toxic components, such as phorbol esters (PEs) [3], remains the main factor that prevents its utilization as a food ingredient. Phorbol esters are naturally occurring products that act as potent tumor promoters. They activate isoforms of protein kinase C (PKCs) and the mechanistic target of rapamycin complex 1 (mTORC1), and they regulate the control of mRNA translation, cell growth, and metabolism in response to diverse stimuli. Inappropriate activation of mTORC1 can lead to cancer [4]. Edible or nontoxic sources of J. curcas that contain negligible amounts of phorbol esters have been reported to exist in Mexico [5]. Thus, protein concentrates obtained from seed cake from these sources, which are thought to be nontoxic genotypes, could be used in the food industry for the development of diets for human and animal consumption. The purpose of this work was to assess whether J. curcas protein concentrate can affect nutritional and biochemical parameters, and to determine its role in the regulation of the protein kinase C (PKC) pathway that is typically induced by phorbol esters in rat liver.
Materials and Methods Preparation of Raw Material and Protein Concentrate The J. curcas seeds used in this study were obtained from ripe fruits harvested in Yautepec, Morelos, Mexico. The whole seeds (kernels plus shells) were partially defatted by mechanical pressing. The seed cake was obtained as follows. Seeds were milled using a blender, and the flour obtained was passed through a sieve (20mesh screen) [2]. The seed cake flour was suspended in distilled water (1:10,w/v). This suspension was adjusted to pH 11 using concentrated NaOH and stirred at constant temperature for 1 h. The suspension was then centrifuged at 18,000×g, 30 min at 15 °C. The supernatant was filtered and collected. The pH of this solution was adjusted to pH 5 with 1 N HCl for protein precipitation. The precipitated proteins were collected by centrifugation (18,000 × g, 30 min, 4 °C) and freeze-dried. In order to remove residual oil, the protein concentrate was defatted by mixing the freeze-dried concentrate with ice-cold hexane (1:10, w/v) for 12 h in an ice bath on a stir plate. Hexane was removed by decanting and the protein concentrate was air-dried overnight in a laboratory hood to remove residual hexane. After hexane removal, the protein concentrate was autoclaved (121 °C, 15 min) to inactivate heat-labile anti-nutritional compounds.
Amino Acid Profiles The amino acid profiles in all protein concentrates used in the study were determined by Silliker Iberica (Barcelona, Spain) by HPLC, using derivatization with O-phtalaldehyde (OPA). Animals and Experimental Protocol Sprague–Dawley rats (n=75) were purchased from HarlanTeklad (México, DF) and weighed between 180 and 200 g. Rats were placed in individual cages, maintained on a constant 12 h light/dark cycle at 22 °C and randomly assigned to one of the three experimental diets (25 rats per group) as follows. (1) Control group: 20 % casein (CAS) as recommended by the American Institute of Nutrition [6], (2) Soy group: 20 % soy protein (SOY) as protein source; and (3) JCP group, a diet containing 20 %J. curcas protein (JCP) concentrate. To synchronize food intake, rats were trained to consume the experimental diets during a restricted period from 09:00 to 14:30 h for 21 days with free access to water to evaluate the short-term effect of JPC. The diets were designed according to the AIN93G [6]. Animals were weighed every day and food consumption was also measured every day. On day 21, five rats from each group were anesthetized with CO2 and then killed by decapitation at 0, 30, 60, 90 and 120 min after they started to eat their meal. The rat livers were rapidly excised, frozen in liquid nitrogen and stored at −70 °C. Institutional guidelines for animal care and use were followed. The animal protocol was approved by the Institutional Animal Care and Research Advisory Committee of the National Institute of Medical Sciences and Nutrition Salvador Zubiran in Mexico City. Serum Measurements Blood was collected in tubes with gel and clot activator (BD Vacutainer, Franklin Lakes, NJ) and centrifuged at 1000×g for 15 min to obtain serum. Please refer to Electronic Supplementary Materials for detailed method description. Western Blot Liver proteins were extracted and protein detection was performed by Western blot analysis as described in the Electronic Supplementary Materials. Preparation of Nuclear Extracts and Electrophoretic Mobility Shift Assay (EMSA) Nuclear extracts from rat livers were prepared and used for EMSA as described in the Electronic Supplementary Materials.
Author's personal copy Plant Foods Hum Nutr
ESI-MS of J. curcas Protein Concentrate (JPC) Electrospray ionization (ESI) analysis was done on a Bruker micrOTOF-Q II (Bruker Daltonics, Bremen, Germany). Please refer to Electronic Supplementary Materials for detailed method description. Statistical Analysis The results are reported as the means±SEM. Data were tested using 2-way ANOVA with the type of protein and the time after feeding as independent variables. One-way ANOVA was used to analyze EMSA densitometries. Significant differences between groups were analyzed by t-test. Differences between means were compared at a level of significance of p
