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Passiflora edulis peel intake improves insulin sensitivity, increasing incretins and hypothalamic satietogenic neuropeptide in rats on a high-fat diet
Accepted Manuscript Passiflora edulis peel intake improves insulin sensitivity, increasing incretins and hypothalamic satietogenic neuropeptide in rats on a high-fat diet Glaucia Carielo Lima, Msc, Milena Morandi Vuolo, Msc, Ângela Giovana Batista, Msc, Nathália R.V. Dragano, PhD, Carina Solon, PhD, Mário Roberto Maróstica Junior, PhD PII:
S0899-9007(16)00047-2
DOI:
10.1016/j.nut.2016.01.014
Reference:
NUT 9697
To appear in:
Nutrition
Received Date: 13 August 2015 Revised Date:
11 December 2015
Accepted Date: 20 January 2016
Please cite this article as: Lima GC, Vuolo MM, Batista ÂG, Dragano NRV, Solon C, Maróstica Junior MR, Passiflora edulis peel intake improves insulin sensitivity, increasing incretins and hypothalamic satietogenic neuropeptide in rats on a high-fat diet, Nutrition (2016), doi: 10.1016/j.nut.2016.01.014. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Passiflora edulis peel intake improves insulin sensitivity, increasing incretins and hypothalamic satietogenic neuropeptide in rats on a high-fat diet Running head: P. edulis peel improves insulin sensitivity Glaucia Carielo Lima, Msca, Milena Morandi Vuolo,Msca, Ângela Giovana Batista,
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Msca, Nathália R. V. Dragano, PhDb, Carina Solon, PhDb, Mário Roberto Maróstica Junior, PhDa* a
School of Food Engineering, University of Campinas, Monteiro Lobato 80, 13083-862,
Campinas, São Paulo, Brazil
Laboratory of Cell Signaling, Faculty of Medical Sciences, University of Campinas,
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Carlos Chagas 420, 13084-970, Campinas, São Paulo, Brazil
*Correspondense: Monteiro Lobato 80, 13083-862. Campinas, São Paulo, Brazil. Phone: +55- 19-35214069; Fax: +55 19 35214060. [email protected] Authorship
GCL and MRMJ designed the study; GCL and MMV conducted the experimental work;
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GCL, AGB, NRVD and CS performed the analysis; GCL analyzed the data and wrote the article.
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Number of words: 5679 Number of figures: 5
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Number of tables: 4
Abbreviations
PEPF, Passiflora edulis peel flour; HF, high-fat; iGTT, intraperitoneal glucose tolerance test; ITT, insulin tolerance test, kITT, constant rate for glucose disappearance, EAT, epididymal adipose tissue, RAT, retroperitoneal adipose tissue
ACCEPTED MANUSCRIPT Abstract Objective: This study aimed to investigate the effect of Passiflora edulis peel flour (PEPF) intake on hypothalamic neuropeptides messenger RNA (mRNA) expression,
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insulin sensitivity and other metabolic parameters in Sprague-Dawley rats fed on highfat diet.
Methods: Sprague-Dawley rats were divided in 3 groups: Control group, fed on a normal fat diet (AIN); High-fat (HF) group, fed on a high-fat diet (35% fat (w/w)), and
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HFPF group, fed on a HF diet containing Passiflora edulis peel flour (PEPF). The rats
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from the HFPF group were kept on a high-fat diet as well as the HF group for the first 4 weeks to induce metabolic conditions related to obesity. Then the HFPF group was switched to a high-fat diet containing PEPF for additional 6 weeks. Other groups were kept on normal-fat and high-fat diet without addition of PEPF during the whole period of experiment.The glucose tolerance and insulin sensitivity were evaluated through the
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glucose tolerance test (GTT) and the insulin tolerance test (ITT). Gut hormones and adipokines were measured through an immunoassay. The hypothalamic neuropeptides
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expression was assessed by real time-PCR.
Results:The PEPF intake increased the hypothalamic cocaine- and amphetamine-
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regulated transcript (CART) expression (P < 0.05), counteracted cumulative body weight gain (P < 0.001), decreased adiposity (P < 0.05) and leptin level (P< 0.01), whereas increased adiponectin (P < 0.01), GIP (P <0.01) and GLP-1 (P<0.001), improved the insulin sensitivity in diet-induced obesity rats by increasing the kITT (P<0.01), which was calculated during the ITT. Other gut hormones (PYY, PP and amylin) and interleukins (IL-6, TNF-α, IL- 1β and MCP-1) were not changed by the PEPF intake.
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ACCEPTED MANUSCRIPT Conclusion: Our findings provide a further understanding of how the PEPF works as a dietary component to improve glucose homeostasis and demonstrating a molecular mechanism that may increase satiety by PEPF in diet-induced obesity.
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Keywords: Passiflora edulis; insulin resistance; GLP-1; pectin; obesity, neuropeptides.
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Introduction Obesity and related diseases, such as type 2 diabetes, have reached pandemic
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proportions and are among the leading causes of death worldwide [1]. Indeed, obesity
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and overweight are determinant factors for several metabolic diseases such as type 2
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diabetes, impaired glucose tolerance, fatty liver disease, cardiovascular disease, some
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types of cancers, and different mental health conditions, which results in harm for the
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health obviously, and economy, raising medical costs for their treatment and
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productivity losses [2].
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Dietary factors are importante predictors for the risk of those diseases. The
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consumption of saturated/animal fat is associated with overweight that, in turn,
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deteriorates insulin sensitivity and glucose tolerance in humans and experimental
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animals [3,4]. On the other hand, increased intake of fibre-rich foods, fruits and
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vegetables as well as limited amounts of total and saturated fats are important elements
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in the prevention of diabetes type 2 [5].
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Indeed, food components, which may play a role on appetite, energy expenditure
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or improve glucose homeostasis, have been the target of obesity-related researches [6–
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8]. Different fibers, including pectin, have been shown capacity to prevent body weight
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gain and improve insulin sensitivity [7,9], modulating gut hormones, mainly peptide
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tyrosine tyrosine (PYY) and glucagon-like peptide 1 (GLP-1) [6,10]. The gut hormones
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act on glucose homeostasis by affecting the insulin secretion and the control of food
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intake, functioning as mediators into the gut-brain-axis[11]. There are some evidences
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that GLP-1 action on satiety [12] involves the modulation of hypothalamic
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neuropeptides [6,13].
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The Passiflora edulis peel is a byproduct from juice and pulp industry. Recently,
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several studies have reported the functional properties of PEPF, particularly its dietary
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fiber content and antioxidant capacity [14–16]. These features of PEPF have boosted
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researches to evaluate its effects on health parameters, especially those related to
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antihyperglycemic action in animals [17,18] and humans [19]. Nevertheless, both
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physiological and molecular mechanisms, by which PEPF improves the glucose
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homeostasis, are still unclear. This study aimed to investigate the effect of PEPF on
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insulin sensitivity, adiposity and metabolic parameters in rats fed on a high-fat
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(saturated/animal) diet. Additionally, we investigated whether these effects are
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associated with hypothalamic neuropeptides expression, which are related to food intake
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and energetic balance.
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Material and methods
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Passiflora edulis peel flour (PEPF)
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An available commercial dried and milled Passiflora edulis peel flour produced by
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M.W.A. Com. de Produtos Alimentícios Ltda (São José do Rio Preto – SP – Brazil) was
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evaluated in this work.
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Chemical characterization of PEPF
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Proximate composition
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The PEPF was evaluated regarding its nitrogen content (Kjeldahl method) [20], total
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lipids [21], ash (met 942.05) [20] and moisture (met 934.01) [20]. The dietary fiber
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(soluble and insoluble) was determined through the enzymatic-gravimetric method,
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according to the AOAC (2005) [20]. The crude carbohydrate content was calculated
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using the percentile difference from all the other components.
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Total and soluble pectin
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The total pectin was extracted using Versene solution and 1.0 N NaOH solution until
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pH 11.5 for hydrolysis, followed by pH adjustment (5-5.5) and reaction with pectinase
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(Pectinase from Aspergillus niger, 1 U/mg; Sigma-Aldrich; St. Louis, USA). The sugar-
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free PEPF obtained using the 95% ethanol extraction was applied for soluble pectin
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determination after agitation and solubilization in water. After the carbazole reaction
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method [22], the total and soluble pectin were measured at 530 nm using a microplate
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reader Synergy HT, Biotek (Winooski, USA); with Gen5™2.0 data analysis software
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spectrophotometer. The galacturonic acid (Sigma-Aldrich; St. Louis, USA) was
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employed as standard.
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Phytochemical analysis
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The phytic acid was determined in PEPF through the colorimetric method according to
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Latta &Eskin (1980) [23]. Tannin (met 952.03) [20] and Hydrogen Cyanide (met
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915.03) [20] were analyzed in agreement with the AOAC (2005).
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Animal trial and procedures 5
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Animals and treatments
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Male Sprague–Dawley rats (3 weeks old) from the Multidisciplinary Center for
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Biological Investigation on Laboratory Animal Science - University of Campinas were
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used in this investigation, which was approved by the Institutional Animal Care and Use
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Committee (Permission #3070-1). The rats were treated in accordance with the
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institutional ethical guideline, allocated in individual cages, maintained in a room with
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relative humidity (60–70%) and controlled temperature (23°C) using a 12 h light–12 h
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dark cycle. The rats were fed on American Institute of Nutrition (AIN)- 93G diet for 1
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week as an acclimatation period, and then randomized into three groups: (Control) rats
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were fed on a normolipidic diet based on AIN-93G [24] containing 12% of protein [25];
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(HF or high-fat) rats were fed on a high-fat diet containing 4% (w/w) soybean oil and
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31% (w/w) lard [25,26]; (HFPF or high-fat Passiflora flour) rats were fed on a high-fat
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diet modified (50% of the cellulose was replaced by PEPF). The rats from the HFPF
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group were kept on a high-fat diet as well as the HF group for the first 4 weeks to
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induce metabolic conditions related to obesity, according to the protocol previously
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described [25,27,28]. Then the HFPF group was switched to a high-fat diet containing
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PEPF for additional 6 weeks. Diets’composition is displayed in Table 1. Access to food
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and drink was ad libitum. Food intake was monitored three times a week and the rats
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were weighted once a week. After 10 experimental weeks, the fasting (12 h) rats were
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sacrificed by decapitation. Blood was collected and tissues were dissected immediately
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and washed with saline. Epididymal and retroperitoneal adipose tissue were weighted to
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predict the adiposity. Blood and tissues were stored at – 80 °C for further analysis.
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Intraperitoneal glucose tolerance test and insulin tolerance test
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The pheriperal sensitivity to insulin was measured through the intraperitoneal glucose
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tolerance test (iGTT) and the insulin tolerance test (ITT) methods after 9 and 10 weeks
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on treatment, respectively. The glucose was determined in blood samples collected in
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the caudal vein of the animals, using Optium Xceed (Abbott Diabetes Care).The blood
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glucose was measured at baseline (food-deprived for 12h), and then a glucose solution
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25% (1.1 mmol/kgbody weight) was injected into the peritoneal cavity. The samples
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were collected at 30, 60, 90 and 120 min after the injection of glucose solution in order
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to determine the rate of blood glucose concentrations, and then the area under curve
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ACCEPTED MANUSCRIPT (AUC) was calculated. For the ITT, the samples were collected at the following 5, 10,
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15, 20, 25 and 30 min after the intraperitoneal injection of human insulin (0.75 U/kg
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body weight; Novolin R, Novo Nordisk; Bagsvaerd, Denmark). The insulin sensitivity
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was estimated by glucose disappearance rate (kITT) from blood after the
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intravenous insulin injection during the ITT, using the following formula, 0·693/t1/2.
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The glucose t1/2 was calculated from the slope of the least-square analysis of the blood
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glucose concentrations during the linear decay phase [25].
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RNA extraction and quantitative real-time PCR
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The total RNA was extracted using the Trizol reagent (Invitrogen Corp.; Carlsbad,
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USA). RNA concentration, purity and integrity were confirmed spectrophotometrically
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by the use of a Nanodrop (ND-1000; Nanodrop Technologies, Wilmington, DE). The
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first-strand cDNA was synthesized using SuperScript III reverse transcriptase and
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random hexamer primers as described in the manufacturer’s protocol (Invitrogen Corp.;
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Carlsbad, USA) The quantitative PCR was run in order to determine the hypothalamic
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neuropeptide Y (NPY),agouti-related peptide (AgPR), pro-opiomelanocortin (POMC)
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and cocaine- and amphetamine-regulated transcript(CART) expression in rats using
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primers supplied with commercially available assays from the Applied Biosystems
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(Foster City, USA). The GAPD (Glyceraldehyde-3-phosphate dehydrogenase, Applied
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Biosystems) was used as the reference gene.The ABI Prism 7500 sequence detection
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system (Applied Biosystems) was used for Real-time PCR analysis of gene expression.
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The optimal concentration of complementary DNA and primers, as well as the
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maximum efficiency of amplification were obtained through 5-point, 2-fold dilution
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curve analysis for each gene. Amplification was performed in a 20µL final volume
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containing 40-50 ng of reverse-transcribed RNA according to the manufacturer's
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recommendations using the TaqMan PCR master mix. Real-time data were analyzed
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using the Sequence Detector System 1.7 (Applied Biosystems). Results were expressed
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as relative transcript amount as previously described [29].
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Gut hormones and adipokines
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Amylin (active), glucose-dependent insulinotropic polypeptide (GIP) (total), GLP-1
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(active), pancreatic polypeptide (PP), and PYY (total), interleukine 6 (IL-6), Monocyte
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chemoattractant protein-1(MCP-1), factores de necrose tumoral alpha (TNFα), insulin 7
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and leptin levels were measured using Rat Metabolic Hormone Magnetic Bead Panel
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(Millipore,
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Cytokine/Chemokine Magnetic Bead Panel - Immunology Multiplex Assay (Millipore,
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Darmstadt, Germany). The adiponectin concentration was measured through the
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employment of Rat Adiponectin Elisa kit (Millipore, Darmstadt, Germany).
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Statistical analysis
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The GraphPad Prism 5.0 (GraphPad Software, Inc. La Jolla, USA) was employed for
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statistical analysis. After check the fit of data to the assumptions for parametric data
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using Shapiro-Wilk normality test, the parametric data were performed using analysis of
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variance (ANOVA) followed by a Tukey’s test. ANOVA (repeated measures) and
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Tukey’s test were applied for data of body weight gain through the experimental time.
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Non-parametric data were performed using Kruskal-Wallis followed by multiple
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comparison. The correlations were analyzed by using Pearson’s correlation. The level of
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significance was set at P<0.05.
Germany).
IL-1β
level
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measured
using
Rat
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Darmstadt,
Results
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Chemical characterization of PEPF
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The PEPF indicated a large amount of dietary fibers, both insoluble and soluble
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fractions, mainly pectin (table 2). High tannin and phytic acid concentrations were
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shown in PEPF, while hydrogen cyanide was not detected in our sample.
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Food intake, body weight gain and adiposity
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The cumulative energy intake by the Control group was lower than with the animals fed
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on HF diet (P< 0.01) after 10 weeks. As expected body weight gain was significantly
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higher (P<0.001) in the HF group compared with rats fed on normal-fat diet (Control) at
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the end of experimental period.The energy intake by rats fed on HFPF was not different
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from those ones from the Control or HF groups throughout the experiment. However,
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the addition of PEPF in the HF diet counteracted the body weight gain (HF>HFPF; P<
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0.001). The femur length of the rats was measured (data not shown) to confirm that only
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body weight, and not the normal growth, has been affected by the treatments.
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In addition, the rats fed on PEPF demonstrated lower weight values for adipose tissues
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compared to the HF group. HF-fed rats showed higher weight values for epididymal
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adipose tissue (EAT), retroperitoneal adipose tissue (RAT) and the sum of both tissues
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than those rats from HFPF and Control groups (P < 0.001, HF vs Control; P < 0.05, HF
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vs HFPF).
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Hypothalamic gene expression
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The PEPF intake enhances gene expression for CART when compared with HF diet (P<
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0.05), indicating that the PEPF increases satiety in rats fed on hyperlipidic diet. The
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treatments did not alter the relative mRNA expression for POMC, NPY and AgRP (P >
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0.05).
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Glucose intolerance and insulin resistance
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The AUC values for blood glucose levels during iGTT were higher in HF group
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compared to the Control and HFPF groups (P<0.05) (figure 3). Despite a tendency to be
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lower, the values for blood glucose in the HFPF group were not different in comparison
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with the HF, indicating that the PEPF did not have effect on glucose intolerance.
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However, the PEPF significatively increased (P<0.01) the constant rate for glucose
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disappearance (kITT), suggesting that the PEPF could prevent HF diet-induced insulin
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resistance in rats.
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Gut hormones and insulin
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The PEPF increased the levels of GLP-1 when compared to HF and Control diets (both
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P<0.001) (table 4). Serum GIP levels in HFPF and Control groups were higher than HF
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(P<0.001, and P<0.01, respectively). The PP concentration was not altered by the
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PEPF. The Amylin and PYY levels were not affected by treatments (P > 0.05). The
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range for serum insulin within the groups was large, which made it harder to evidence
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any treatment-induced changes.
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Circulating concentrations of proinflammatory cytokines
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Inflammatory biomarkers measured in the serum, including IL-6 (P < 0.05), TNF-α (P <
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0.01) and MCP-1 (P < 0.01), were significantly higher in animals fed on HF diet, but
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not in HFPF, compared with those fed on Control diet (figure 4). The IL1β
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concentration was lower in Control group (P < 0.05), when compared with HF and
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HFPF group.
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ACCEPTED MANUSCRIPT Leptin and adiponectin
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The HF rats demonstred elevated serum leptin concentrations compared to Control
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(P<0.001) and HFPF (P<0.05) (figure 5), whereas the adiponectin levels were lower in
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the HF group in comparison with other groups (both P <0.01). The adipokines levels
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were correlated to the adipose tissue content in the rats.The leptin level has strong
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positive relation with the adipose tissue content in animals (r = 0.8498, P< 0.0001, r2 =
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0.72) whereas the adiponectin is negatively related to adiposity (r= - 0.8018, P< 0.0001,
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r2 = 0.64).
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Discussion
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The saturated/animal fat was used in this study to induce the obesity in animals.
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Indeed, the HF diet provoked changes in metabolic parameters related to obesity, such
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as increased mass gain, impairment of the insulin sensitivity, hyperleptinemia, reduction
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of adiponectin levels and higher concentration of proinflammatory interleukins in the
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HF group, when compared with those animals from Control group. On the other hand,
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the addition of PEPF in high-fat diet protected the animals against the excessive body
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weight gain and fatness. The average of body weight gain and sum of RAT+EAT in HF
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group was 12.01% and 21.72%, respectively, higher than those ones from HFPF-fed
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rats. Furthermore, we noticed high gene expression for CART in HFPF-fed rats,
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suggesting that the PEPF may interfere in satiety and food intake, implying in protective
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factor against obesity. Although this finding was not followed by significant decrease in
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total energy intake compared to HF group, it is consistent with the observation that food
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intake was similar between the control group and HFPF-fed rats in our trial, indicating
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an effect on food intake control. Similarly, resistant starch (RS), which has also a
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similar role as the soluble fiber, has also shown increased hypothalamic POMC, another
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satietogenic neuropeptide, reflecting on food intake in rats [6]. Additionally, the RS was
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able to decrease body fatness and increased plasma GLP-1 [6] as we have observed with
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PEPF. Apart from its incretin effects, evidences that intestinal GLP-1 afects satiety have
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also been reported [12]. It is well-known that GLP-1 analogs induce weight loss and
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improve glucose homeostasis in rodents and humans and recently, it was reported that
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Liraglutide (comercially available GLP-1 analogue for type 2 diabetes treatment)
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treatment increased CART mRNA levels in the hypothalamic arcuate nucleus in diet-
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induced obese rats [13]. Together, these evidences and similiarities between our
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findings and those with RS could suggest that GLP-1 increment elicited by fermentable
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fibersmay be related with food intake control via action on anorexigenics neuropeptides.
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However, further researches are needed to elucidate whether physiological GLP-1, and
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not only its mimetics, is able to act directly in central food intake control. As the adiposity and the satiety signals, the glucose metabolism has been
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affected by the PEPF intake as well. The iGTT confirmed the impairment of glucose
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tolerance, induced by HF diet, which was indicated by the higher AUC values presented
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by HF diet-fed rats. Although the HFPF-fed rats have not presented decreased values
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for AUC in comparison with the HF group during iGTT, those animals demonstrated no
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difference compared to normal-fat diets. Besides, we noticed that the addition of PEPF
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in high-fat diet has improved insulin sensitivity, which was shown by increased kITT
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values during ITT. The kITT rate is considered a reflection of insulin sensitivity, once it
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indicates the rate of glucose cleareance by increasing glucose uptake in insulin sensitive
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tissues and suppression of hepatic glucose production in response to exogenous insulin
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[25]. We could not find statistical differences among groups regarding fasting insulin
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leveldue to the large variation of values presented in the groups. Nevertheless, animals
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fed on HF diet showed the highest average comparing to other groups, but this
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difference did not reach statistical significance. These findings, associated with those
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found in iGTT and iITT, suggest that the PEPF could be effective to control glucose
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homeostasis in diet-induced obesity.
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The improvement of glucose metabolism by the PEPF may be associated with
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the ability of PEPF to modulate the incretins GLP-1 and GIP, which were increased in
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animals fed on HFPF. Both incretins stimulate insulin secretion by β-cells in the islet of
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Langerhans in a glucose-dependent way [30]. GLP-1plays a role on satiety and likely
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helps long-term body weight regulation [12].We found high amount of pectin in PEPF,
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representing almost the total soluble fraction of dietary fiber. In agreement with our
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findings, increasing amounts of the pectin in the diet proportionately decrease food
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intake, body weight gain and body fat content, while increase circulating PYY and
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GLP-1 levels in rats. The strong relationship of those findings and the gut hypertrophy
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found in already mentioned study supports indicate that the increase of these hormones
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is probably a result of the L-cell proliferation [10]. Indeed, it is described in the
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literature the capacity of the pectin to produce short-chain fatty acids as products of its
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fermentation in the intestine [31]. Besides, studies have shown that PEPF intake results
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fermentation of pectin from the PEPF in intestine could be involved in the modulation
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of gut hormones, especially GLP-1, once the SCFA have been linked to enhanced
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release of this incretin [33,34]. In vitro assay has shown that butyrate stimulated the
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release of GLP-1 from intestinal L-cell [33] as well as acetate and propionate pointed
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out the same using of a mix colonic cell [34]. In addition, mice lacking SCFA receptors
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exhibited SCFA-triggered GLP-1 secretion, acompanied by impairment of glucose
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tolerance [34]. Furthermore, butyrate and propionate suplementation has shown
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protection against diet-induced obesity and insulin resistance, by inducing gut hormones
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and food intake inhibition in mice [35]. These evidences support that theincrementof
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GLP-1 may be induced by SCFA elicited through fiber fermentation, which could
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indicate a likely mechanism of action of the pectin from PEPF in order to increase the
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incretin and, therefore, other metabolic parameters related.
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Besides incretins, PEPF also modulated adipokines in diet-induced obesity rats.
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This modulation was associated with body fat changes, demonstrated by Pearson’s
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correlation analysis. The increase of the adiponectin levels in animals fed with HFPF
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could be related with the enhancement of insulin sensitivity, since serum adiponectin
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levels are inversely correlated with obesity and type 2 diabetes and positively associated
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with insulin sensitivity [36,37]. Moreover, rats fed on HFPF diet showed lower leptin
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levels than those fed on HF diet. This adipokine plays a role on energy balance, by
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signalling pathways in the hypothalamus in order to decrease the food intake and
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increase the energy expenditure. Nevertheless, this role is disrupted in prolonged
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obesity, which is well-known as leptin resistance [38]. The hyperleptinemia is needed
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for
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syndrome [40]. Thus, the decreased leptinemia in PEPF-fed rats could be considered a
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protective factor against those conditions.
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the development of leptin resistance [39] and the increased risk of metabolic
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It has been well established that obesity and insulin resistance are associated
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with a chronic low-grade inflammation. Pro-inflammatory interleukins, such as IL-6,
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IL-1, MCP-1 and TNF- α are elevated in these conditions [41]. The rats fed on HF diet
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showed higher serum concentration of IL-6, MCP-1 and TNF- α compared to those fed
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on normal-fat diet (Control group). Together, these results indicates that the saturated
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fat used in our study contributes to increase the inflammation in rats, which is
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associated with the obesity and impairment of insulin sensitivity [42]. Although there 12
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was not significant different between HF and HFPF groups, the interleukins
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concentration was similar between control and HFPF-fed rats in our experiment, except
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for IL 1 β, indicating that PEPF could attenuate obesity- related inflammation in rats. Apart of pectin, other phytochemicals in PEPF have also shown effects on
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glucose metabolism and fatness: the tannic acid has indicated antihyperglicemic effect
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in vitro assay by stimulating phosphorylation of protein factors in the insulin-mediated
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glucose transport pathway and inducing GLUT 4 translocation, using 3T3-L1 cells as
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model [43]. In vivo, tannic acid has reverted the glucose and the insulin level into the
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normal range in diabetic rats [44]. The addition of phytic acid or rice bran in HF diet
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showed positive effects on glucose metabolism and body weight gain in mice [45]. The
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antioxidant potential of these compounds could be linked to those effects. In fact,
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oxidative stress plays a role in thedevelopment and progression of obesity and diabetes,
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and antioxidants activity of dietary components could prevent hyperglycemia under
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obesogenic diet [45]. The antioxidant capacity of the PEPF was also reported in
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previous studies [14–16]. Thereby, it is possible that this component of the PEPF plays
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some role in the findings described here, by synergic action of polyphenols and fibers.
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Nevertheless, driving this approach in future researches is necessary to elucidate this
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hypothesis.
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Together, our findings demonstrate the physiological mechanisms by which the
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PEPF could assist in the prevention of deleterious effects demonstrated by the intake of
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a high-fat (saturated/animal) diet.
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Conclusion
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Our findings provide a further understanding of how the PEPF, source of pectin, works
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as a dietary ingredient to improve glucose homeostasis. Besides, the increased CART
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expression in the hypothalamus demonstrates a molecular mechanism in order to
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enhance satiety by PEPF in diet-induced obesity. These results represent physiological
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effects of PEPF not described before, and support the evidence that the PEPF may be a
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useful dietary component to prevent body fatness and insulin resistance.
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Acknowledgments
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We thank Professor Lício Velloso for his collaboration and the Obesity and Comorbities
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Research Center for providing methodological advice. 13
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This work was supported by FAPESP (grant number: 2012/12322-0) and CNPq.
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FAPESP and CNPq had no role in the design, analysis or writing of this article.
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The authors declare that they have no conflicts of interest.
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ACCEPTED MANUSCRIPT doi:10.2337/db06-1491.P.D.C. [42] Cani PD, Amar J, Iglesias MA, Poggi M, Knauf C, Bastelica D, et al. Original Article. Diabetes 2007;56:1761–72. doi:10.2337/db06-1491.P.D.C. [43] Liu X, Kim J, Li Y, Li J, Liu F, Chen X. Tannic acid stimulates glucose transport and inhibits adipocyte differentiation in 3T3-L1 cells. J Nutr 2005;135:165–71. S, Chandirasegaran G. TANNIC ACID IN RATS. Int J Curr Res
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ACCEPTED MANUSCRIPT Figures Subtitles Figure 1- Effect of Passiflora edulis peel flour intake on body weight throughout the experimental weeks, total body weight gain after 10 experimental weeks and total energy (kJ) intake after 10 experimental weeks.
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C, Control group fed on AIN 93G diet; HF, High-fat group fed on AIN 93G diet modified with 35% fat (w/w); HFPF, High-fat Passiflora Flour group fed on high-fat diet added 2.5% (w/w) Passiflora edulis peel flour.
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Data are presented as average (SD), n=8. * HF group > Control group (P < 0.05); ** HF group > Control and HFPF groups (both P < 0.01) by analysis of variance (ANOVA)
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(repetead measures) followed by Tukey’s test. Different letters represent statistical difference by one-way ANOVA, following by Tukey's test; P < 0.05. Figure 2 - Effect of Passiflora edulis peel flour intake on hypothalamic expression of neuropeptide Y (NPY), agouti-related peptide (AgRP), pro-opiomelanocortin (POMC)
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and cocaine- and amphetamine-regulated transcript (CART) expression. C, Control group fed on AIN 93G diet; HF, High-fat group fed on AIN 93G diet modified with 35% fat (w/w); HFPF, High-fat Passiflora Flour group fed on high-fat
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diet added 2.5% (w/w) Passiflora edulis peel flour. Data are presented as an average (SD), n=5. Different letters represent statiscal difference by Kruskal-Wallis and
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multiple comparision tests.
Figure 3- Effect of Passiflora edulis peel flour intake on glucose tolerance and insulin sensitivity evaluated by iGTT and kITT iGTT, intraperitoneal glucose tolerance test; AUC, area under curve; ITT, insulin tolerance test; kITT, glucose disappearance rate; Control, Control group fed on AIN 93G diet; HF, High-fat group fed on AIN 93G diet modified with 35% fat (w/w); HFPF, High-fat Passiflora Flour group fed on high-fat diet added 2.5% (w/w) Passiflora edulis peel flour. Data are presented as average (SD), n=5. Different letters represent statiscal difference by analysis of variance (ANOVA) followed by Tukey’s test (P < 0.05). 19
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Figure 4 - Effect of Passiflora edulis peel flour intake on serum proinflammatory interleukins levels.
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Control, Control group fed on AIN 93G diet; HF, High-fat group; HFPF, modified highfat diet containing Passiflora edulis peel flour; IL-6, interleukin 6; IL-1β, interleukin 1β; TNF α, tumoral necrose factor α; MCP 1, Monocyte Chemoattractant Protein-1.
* Data are presented as average (SD), n=6. Different letters represent statistical
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difference by analysis of variance (ANOVA) followed by Tukey’s test; p<0.05).
Figure 5 - Effect of Passiflora edulis peel flour intake on serum adiponectin and leptin
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levels.
Control, Control group fed on AIN 93G diet; HF, High-fat group fed on AIN 93G diet modified with 35% fat (w/w); HFPF, High-fat Passiflora Flour group fed on high-fat diet added 2.5% (w/w) Passiflora edulis peel flour.
Data are presented as average (SD), n=6. Different letters represent statistical difference
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by analysis of variance (ANOVA) followed by Tukey’s test (P < 0.05).
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ACCEPTED MANUSCRIPT Table 1 Composition of experimental diets (g/Kg of diet)* Control
HF
HFPF
Cornstarch
435.12
258.3
258.3
Casein (85.5%)
140.35
140.35
140.35
Dextrinized cornstarch
144.51
85.79
85.79
Sucrose
109.57
65.05
65.05
Soybean oil
70.00
40.00
40.00
310.00
310.00
50.00
25.00
-
25.00
35.00
35.00
10.00
10.00
10.00
3.00
3.00
3.00
2.50
2.50
2.50
0.014
0.014
0.014
15.4
21.9
21.9
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Cellulose
50.00
PEPF
-
Vitamin mix (AIN-93G-VX) L-Cystine Choline bitartrate Tert-butylhydroquinone
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Energy density (kJ/g diet)
35.00
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Mineral mix (AIN-93G-MX)
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Lard
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Ingredient
Control, Control group fed on AIN 93G diet; HF, High-fat group fed on AIN 93G diet modified with 35% fat (w/w); HFPF, High-fat Passiflora Flour group fed on AIN 93G modified with 35% fat (w/w) added 2.5% (w/w) Passiflora edulis peel
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flour.
*Diets were prepared according to American Institute of Nutrition for AIN 93-G [24]
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with modified protein content to 12%, according Dragano et al. (2013) [25].
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ACCEPTED MANUSCRIPT Table 2 Proximate and phytochemical composition of Passiflora edulis peel flour Content (g/100g) 7.42 ± 0.25
Ash
6.00 ± 0.16
Lipids
3.39 ± 0.18
Protein
8.87 ± 0.10
Carbohydrate†
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Moisture
14.24 ± 0.34
Total dietary fiber
60.08 ± 0.02
Insoluble dietary fiber
20.13 ± 0.64
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Soluble dietary fiber
39.96 ± 0.66
Soluble pectin
Phytochemicals Phytic acid (mg/100g) Hydrogen cyanid (mg/ Kg) Tannin (mg/100g)
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Total pectin
4.85 ± 0.53 17.27 ± 0.11
37.0 ± 0.02 LOD < 0.30 308.21 ± 1.12
LOD,limit of detection. † Calculated by difference, excludes the dietary fiber fraction.
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Data were expressed with an average (SD), n=3.
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ACCEPTED MANUSCRIPT Table 3 Liver, epididymal adipose tissue (EAT) and retroperitoneal adipose tissue (RAT) % ratio weights (g/100gtotal body weight)
Liver
Control 2.80 ± 0.32
HF 2.94 ± 0.52
HFPF 2.88 ± 0.32
EAT
1.94 ± 0.46 b
3.00 ± 0.76 a
2.38 ± 0.64 b
RAT
2.12 ± 0.62 b
3.04 ± 0.50 a
2.51 ± 0.53 b
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EAT+RAT 3.79 ± 1.32 b 6.04 ± 1.22 a 4.73 ± 1.13 b Control group fed on AIN 93G diet; HF, High-fat group fed on AIN 93G diet modified with 35% fat (w/w); HFPF, High-fat Passiflora Flour group fed on AIN 93G modified with 35% fat (w/w) added 2.5% (w/w) Passiflora edulis peel flour. Values are an average
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ACCEPTED MANUSCRIPT Table 4 - Effect of Passiflora edulis peel flour intake on serum gut hormones and insulin levels (ng/ml) in rats HF
HFPF
Amylin
38.93 ± 3.32
48.53 ± 4.63
50.86 ± 5.04
GIP
75.12 ± 5.12 a
44.09 ± 2.66 b
63.99 ± 11.26 a
GLP-1
66.10 ± 8.62 c
82.10 ± 10.89 b
127.25 ± 5.32 a
PP
29.14 ± 4.64 b
47.39 ± 7.44 a
PYY
51.71 ± 10.78
60.04 ± 13.08
395.75 ± 319.84
1277.00 ± 749.76
Insulin
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Control
48.86 ± 10.60 a 66.00 ± 7.57
628.40 ± 257.26
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Control group fed on AIN 93G diet; HF, High-fat group fed on AIN 93G diet modified with 35% fat (w/w); HFPF, High-fat Passiflora Flour group fed on AIN 93G modified
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with 35% fat (w/w) added 2.5% (w/w) Passiflora edulis peel flour. Values are an average and SD, n=6. Data were analyzed using ANOVA followed by Tukey’s test (P
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< 0.05). Difference letters in the same row represent statistical difference.
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ACCEPTED MANUSCRIPT Highlights: Passiflora edulis peel intake improves insulin sensitivity in high-fat fed rats Passiflora edulis peel intake increases incretins GIP and GLP-1 in high-fat fed rats Passiflora edulis peel intake modulates adiponectin and leptin in high-fat fed rats
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Passiflora edulis peel intake increases satietogenic neuropeptide in high-fat fed rats
S0899-9007(16)00047-2
DOI:
10.1016/j.nut.2016.01.014
Reference:
NUT 9697
To appear in:
Nutrition
Received Date: 13 August 2015 Revised Date:
11 December 2015
Accepted Date: 20 January 2016
Please cite this article as: Lima GC, Vuolo MM, Batista ÂG, Dragano NRV, Solon C, Maróstica Junior MR, Passiflora edulis peel intake improves insulin sensitivity, increasing incretins and hypothalamic satietogenic neuropeptide in rats on a high-fat diet, Nutrition (2016), doi: 10.1016/j.nut.2016.01.014. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Passiflora edulis peel intake improves insulin sensitivity, increasing incretins and hypothalamic satietogenic neuropeptide in rats on a high-fat diet Running head: P. edulis peel improves insulin sensitivity Glaucia Carielo Lima, Msca, Milena Morandi Vuolo,Msca, Ângela Giovana Batista,
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Msca, Nathália R. V. Dragano, PhDb, Carina Solon, PhDb, Mário Roberto Maróstica Junior, PhDa* a
School of Food Engineering, University of Campinas, Monteiro Lobato 80, 13083-862,
Campinas, São Paulo, Brazil
Laboratory of Cell Signaling, Faculty of Medical Sciences, University of Campinas,
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b
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Carlos Chagas 420, 13084-970, Campinas, São Paulo, Brazil
*Correspondense: Monteiro Lobato 80, 13083-862. Campinas, São Paulo, Brazil. Phone: +55- 19-35214069; Fax: +55 19 35214060. [email protected] Authorship
GCL and MRMJ designed the study; GCL and MMV conducted the experimental work;
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GCL, AGB, NRVD and CS performed the analysis; GCL analyzed the data and wrote the article.
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Number of words: 5679 Number of figures: 5
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Number of tables: 4
Abbreviations
PEPF, Passiflora edulis peel flour; HF, high-fat; iGTT, intraperitoneal glucose tolerance test; ITT, insulin tolerance test, kITT, constant rate for glucose disappearance, EAT, epididymal adipose tissue, RAT, retroperitoneal adipose tissue
ACCEPTED MANUSCRIPT Abstract Objective: This study aimed to investigate the effect of Passiflora edulis peel flour (PEPF) intake on hypothalamic neuropeptides messenger RNA (mRNA) expression,
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insulin sensitivity and other metabolic parameters in Sprague-Dawley rats fed on highfat diet.
Methods: Sprague-Dawley rats were divided in 3 groups: Control group, fed on a normal fat diet (AIN); High-fat (HF) group, fed on a high-fat diet (35% fat (w/w)), and
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HFPF group, fed on a HF diet containing Passiflora edulis peel flour (PEPF). The rats
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from the HFPF group were kept on a high-fat diet as well as the HF group for the first 4 weeks to induce metabolic conditions related to obesity. Then the HFPF group was switched to a high-fat diet containing PEPF for additional 6 weeks. Other groups were kept on normal-fat and high-fat diet without addition of PEPF during the whole period of experiment.The glucose tolerance and insulin sensitivity were evaluated through the
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glucose tolerance test (GTT) and the insulin tolerance test (ITT). Gut hormones and adipokines were measured through an immunoassay. The hypothalamic neuropeptides
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expression was assessed by real time-PCR.
Results:The PEPF intake increased the hypothalamic cocaine- and amphetamine-
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regulated transcript (CART) expression (P < 0.05), counteracted cumulative body weight gain (P < 0.001), decreased adiposity (P < 0.05) and leptin level (P< 0.01), whereas increased adiponectin (P < 0.01), GIP (P <0.01) and GLP-1 (P<0.001), improved the insulin sensitivity in diet-induced obesity rats by increasing the kITT (P<0.01), which was calculated during the ITT. Other gut hormones (PYY, PP and amylin) and interleukins (IL-6, TNF-α, IL- 1β and MCP-1) were not changed by the PEPF intake.
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ACCEPTED MANUSCRIPT Conclusion: Our findings provide a further understanding of how the PEPF works as a dietary component to improve glucose homeostasis and demonstrating a molecular mechanism that may increase satiety by PEPF in diet-induced obesity.
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Keywords: Passiflora edulis; insulin resistance; GLP-1; pectin; obesity, neuropeptides.
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Introduction Obesity and related diseases, such as type 2 diabetes, have reached pandemic
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proportions and are among the leading causes of death worldwide [1]. Indeed, obesity
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and overweight are determinant factors for several metabolic diseases such as type 2
5
diabetes, impaired glucose tolerance, fatty liver disease, cardiovascular disease, some
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types of cancers, and different mental health conditions, which results in harm for the
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health obviously, and economy, raising medical costs for their treatment and
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productivity losses [2].
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Dietary factors are importante predictors for the risk of those diseases. The
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consumption of saturated/animal fat is associated with overweight that, in turn,
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deteriorates insulin sensitivity and glucose tolerance in humans and experimental
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animals [3,4]. On the other hand, increased intake of fibre-rich foods, fruits and
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vegetables as well as limited amounts of total and saturated fats are important elements
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in the prevention of diabetes type 2 [5].
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Indeed, food components, which may play a role on appetite, energy expenditure
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or improve glucose homeostasis, have been the target of obesity-related researches [6–
17
8]. Different fibers, including pectin, have been shown capacity to prevent body weight
18
gain and improve insulin sensitivity [7,9], modulating gut hormones, mainly peptide
19
tyrosine tyrosine (PYY) and glucagon-like peptide 1 (GLP-1) [6,10]. The gut hormones
20
act on glucose homeostasis by affecting the insulin secretion and the control of food
21
intake, functioning as mediators into the gut-brain-axis[11]. There are some evidences
22
that GLP-1 action on satiety [12] involves the modulation of hypothalamic
23
neuropeptides [6,13].
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The Passiflora edulis peel is a byproduct from juice and pulp industry. Recently,
25
several studies have reported the functional properties of PEPF, particularly its dietary
26
fiber content and antioxidant capacity [14–16]. These features of PEPF have boosted
27
researches to evaluate its effects on health parameters, especially those related to
28
antihyperglycemic action in animals [17,18] and humans [19]. Nevertheless, both
29
physiological and molecular mechanisms, by which PEPF improves the glucose
30
homeostasis, are still unclear. This study aimed to investigate the effect of PEPF on
31
insulin sensitivity, adiposity and metabolic parameters in rats fed on a high-fat
32
(saturated/animal) diet. Additionally, we investigated whether these effects are
33
associated with hypothalamic neuropeptides expression, which are related to food intake
34
and energetic balance.
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Material and methods
36
Passiflora edulis peel flour (PEPF)
38
An available commercial dried and milled Passiflora edulis peel flour produced by
39
M.W.A. Com. de Produtos Alimentícios Ltda (São José do Rio Preto – SP – Brazil) was
40
evaluated in this work.
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41 42
Chemical characterization of PEPF
43
Proximate composition
45
The PEPF was evaluated regarding its nitrogen content (Kjeldahl method) [20], total
46
lipids [21], ash (met 942.05) [20] and moisture (met 934.01) [20]. The dietary fiber
47
(soluble and insoluble) was determined through the enzymatic-gravimetric method,
48
according to the AOAC (2005) [20]. The crude carbohydrate content was calculated
49
using the percentile difference from all the other components.
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Total and soluble pectin
52
The total pectin was extracted using Versene solution and 1.0 N NaOH solution until
53
pH 11.5 for hydrolysis, followed by pH adjustment (5-5.5) and reaction with pectinase
54
(Pectinase from Aspergillus niger, 1 U/mg; Sigma-Aldrich; St. Louis, USA). The sugar-
55
free PEPF obtained using the 95% ethanol extraction was applied for soluble pectin
56
determination after agitation and solubilization in water. After the carbazole reaction
57
method [22], the total and soluble pectin were measured at 530 nm using a microplate
58
reader Synergy HT, Biotek (Winooski, USA); with Gen5™2.0 data analysis software
59
spectrophotometer. The galacturonic acid (Sigma-Aldrich; St. Louis, USA) was
60
employed as standard.
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Phytochemical analysis
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The phytic acid was determined in PEPF through the colorimetric method according to
64
Latta &Eskin (1980) [23]. Tannin (met 952.03) [20] and Hydrogen Cyanide (met
65
915.03) [20] were analyzed in agreement with the AOAC (2005).
66 67
Animal trial and procedures 5
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Animals and treatments
70
Male Sprague–Dawley rats (3 weeks old) from the Multidisciplinary Center for
71
Biological Investigation on Laboratory Animal Science - University of Campinas were
72
used in this investigation, which was approved by the Institutional Animal Care and Use
73
Committee (Permission #3070-1). The rats were treated in accordance with the
74
institutional ethical guideline, allocated in individual cages, maintained in a room with
75
relative humidity (60–70%) and controlled temperature (23°C) using a 12 h light–12 h
76
dark cycle. The rats were fed on American Institute of Nutrition (AIN)- 93G diet for 1
77
week as an acclimatation period, and then randomized into three groups: (Control) rats
78
were fed on a normolipidic diet based on AIN-93G [24] containing 12% of protein [25];
79
(HF or high-fat) rats were fed on a high-fat diet containing 4% (w/w) soybean oil and
80
31% (w/w) lard [25,26]; (HFPF or high-fat Passiflora flour) rats were fed on a high-fat
81
diet modified (50% of the cellulose was replaced by PEPF). The rats from the HFPF
82
group were kept on a high-fat diet as well as the HF group for the first 4 weeks to
83
induce metabolic conditions related to obesity, according to the protocol previously
84
described [25,27,28]. Then the HFPF group was switched to a high-fat diet containing
85
PEPF for additional 6 weeks. Diets’composition is displayed in Table 1. Access to food
86
and drink was ad libitum. Food intake was monitored three times a week and the rats
87
were weighted once a week. After 10 experimental weeks, the fasting (12 h) rats were
88
sacrificed by decapitation. Blood was collected and tissues were dissected immediately
89
and washed with saline. Epididymal and retroperitoneal adipose tissue were weighted to
90
predict the adiposity. Blood and tissues were stored at – 80 °C for further analysis.
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Intraperitoneal glucose tolerance test and insulin tolerance test
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The pheriperal sensitivity to insulin was measured through the intraperitoneal glucose
94
tolerance test (iGTT) and the insulin tolerance test (ITT) methods after 9 and 10 weeks
95
on treatment, respectively. The glucose was determined in blood samples collected in
96
the caudal vein of the animals, using Optium Xceed (Abbott Diabetes Care).The blood
97
glucose was measured at baseline (food-deprived for 12h), and then a glucose solution
98
25% (1.1 mmol/kgbody weight) was injected into the peritoneal cavity. The samples
99
were collected at 30, 60, 90 and 120 min after the injection of glucose solution in order
100
to determine the rate of blood glucose concentrations, and then the area under curve
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ACCEPTED MANUSCRIPT (AUC) was calculated. For the ITT, the samples were collected at the following 5, 10,
102
15, 20, 25 and 30 min after the intraperitoneal injection of human insulin (0.75 U/kg
103
body weight; Novolin R, Novo Nordisk; Bagsvaerd, Denmark). The insulin sensitivity
104
was estimated by glucose disappearance rate (kITT) from blood after the
105
intravenous insulin injection during the ITT, using the following formula, 0·693/t1/2.
106
The glucose t1/2 was calculated from the slope of the least-square analysis of the blood
107
glucose concentrations during the linear decay phase [25].
108
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RNA extraction and quantitative real-time PCR
110
The total RNA was extracted using the Trizol reagent (Invitrogen Corp.; Carlsbad,
111
USA). RNA concentration, purity and integrity were confirmed spectrophotometrically
112
by the use of a Nanodrop (ND-1000; Nanodrop Technologies, Wilmington, DE). The
113
first-strand cDNA was synthesized using SuperScript III reverse transcriptase and
114
random hexamer primers as described in the manufacturer’s protocol (Invitrogen Corp.;
115
Carlsbad, USA) The quantitative PCR was run in order to determine the hypothalamic
116
neuropeptide Y (NPY),agouti-related peptide (AgPR), pro-opiomelanocortin (POMC)
117
and cocaine- and amphetamine-regulated transcript(CART) expression in rats using
118
primers supplied with commercially available assays from the Applied Biosystems
119
(Foster City, USA). The GAPD (Glyceraldehyde-3-phosphate dehydrogenase, Applied
120
Biosystems) was used as the reference gene.The ABI Prism 7500 sequence detection
121
system (Applied Biosystems) was used for Real-time PCR analysis of gene expression.
122
The optimal concentration of complementary DNA and primers, as well as the
123
maximum efficiency of amplification were obtained through 5-point, 2-fold dilution
124
curve analysis for each gene. Amplification was performed in a 20µL final volume
125
containing 40-50 ng of reverse-transcribed RNA according to the manufacturer's
126
recommendations using the TaqMan PCR master mix. Real-time data were analyzed
127
using the Sequence Detector System 1.7 (Applied Biosystems). Results were expressed
128
as relative transcript amount as previously described [29].
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129 130
Gut hormones and adipokines
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Amylin (active), glucose-dependent insulinotropic polypeptide (GIP) (total), GLP-1
132
(active), pancreatic polypeptide (PP), and PYY (total), interleukine 6 (IL-6), Monocyte
133
chemoattractant protein-1(MCP-1), factores de necrose tumoral alpha (TNFα), insulin 7
ACCEPTED MANUSCRIPT 134
and leptin levels were measured using Rat Metabolic Hormone Magnetic Bead Panel
135
(Millipore,
136
Cytokine/Chemokine Magnetic Bead Panel - Immunology Multiplex Assay (Millipore,
137
Darmstadt, Germany). The adiponectin concentration was measured through the
138
employment of Rat Adiponectin Elisa kit (Millipore, Darmstadt, Germany).
139
Statistical analysis
140
The GraphPad Prism 5.0 (GraphPad Software, Inc. La Jolla, USA) was employed for
141
statistical analysis. After check the fit of data to the assumptions for parametric data
142
using Shapiro-Wilk normality test, the parametric data were performed using analysis of
143
variance (ANOVA) followed by a Tukey’s test. ANOVA (repeated measures) and
144
Tukey’s test were applied for data of body weight gain through the experimental time.
145
Non-parametric data were performed using Kruskal-Wallis followed by multiple
146
comparison. The correlations were analyzed by using Pearson’s correlation. The level of
147
significance was set at P<0.05.
Germany).
IL-1β
level
was
measured
using
Rat
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Darmstadt,
Results
150
Chemical characterization of PEPF
151
The PEPF indicated a large amount of dietary fibers, both insoluble and soluble
152
fractions, mainly pectin (table 2). High tannin and phytic acid concentrations were
153
shown in PEPF, while hydrogen cyanide was not detected in our sample.
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Food intake, body weight gain and adiposity
156
The cumulative energy intake by the Control group was lower than with the animals fed
157
on HF diet (P< 0.01) after 10 weeks. As expected body weight gain was significantly
158
higher (P<0.001) in the HF group compared with rats fed on normal-fat diet (Control) at
159
the end of experimental period.The energy intake by rats fed on HFPF was not different
160
from those ones from the Control or HF groups throughout the experiment. However,
161
the addition of PEPF in the HF diet counteracted the body weight gain (HF>HFPF; P<
162
0.001). The femur length of the rats was measured (data not shown) to confirm that only
163
body weight, and not the normal growth, has been affected by the treatments.
164
In addition, the rats fed on PEPF demonstrated lower weight values for adipose tissues
165
compared to the HF group. HF-fed rats showed higher weight values for epididymal
166
adipose tissue (EAT), retroperitoneal adipose tissue (RAT) and the sum of both tissues
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ACCEPTED MANUSCRIPT 167
than those rats from HFPF and Control groups (P < 0.001, HF vs Control; P < 0.05, HF
168
vs HFPF).
169
Hypothalamic gene expression
171
The PEPF intake enhances gene expression for CART when compared with HF diet (P<
172
0.05), indicating that the PEPF increases satiety in rats fed on hyperlipidic diet. The
173
treatments did not alter the relative mRNA expression for POMC, NPY and AgRP (P >
174
0.05).
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Glucose intolerance and insulin resistance
177
The AUC values for blood glucose levels during iGTT were higher in HF group
178
compared to the Control and HFPF groups (P<0.05) (figure 3). Despite a tendency to be
179
lower, the values for blood glucose in the HFPF group were not different in comparison
180
with the HF, indicating that the PEPF did not have effect on glucose intolerance.
181
However, the PEPF significatively increased (P<0.01) the constant rate for glucose
182
disappearance (kITT), suggesting that the PEPF could prevent HF diet-induced insulin
183
resistance in rats.
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Gut hormones and insulin
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The PEPF increased the levels of GLP-1 when compared to HF and Control diets (both
187
P<0.001) (table 4). Serum GIP levels in HFPF and Control groups were higher than HF
188
(P<0.001, and P<0.01, respectively). The PP concentration was not altered by the
189
PEPF. The Amylin and PYY levels were not affected by treatments (P > 0.05). The
190
range for serum insulin within the groups was large, which made it harder to evidence
191
any treatment-induced changes.
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Circulating concentrations of proinflammatory cytokines
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Inflammatory biomarkers measured in the serum, including IL-6 (P < 0.05), TNF-α (P <
195
0.01) and MCP-1 (P < 0.01), were significantly higher in animals fed on HF diet, but
196
not in HFPF, compared with those fed on Control diet (figure 4). The IL1β
197
concentration was lower in Control group (P < 0.05), when compared with HF and
198
HFPF group.
199
9
ACCEPTED MANUSCRIPT Leptin and adiponectin
201
The HF rats demonstred elevated serum leptin concentrations compared to Control
202
(P<0.001) and HFPF (P<0.05) (figure 5), whereas the adiponectin levels were lower in
203
the HF group in comparison with other groups (both P <0.01). The adipokines levels
204
were correlated to the adipose tissue content in the rats.The leptin level has strong
205
positive relation with the adipose tissue content in animals (r = 0.8498, P< 0.0001, r2 =
206
0.72) whereas the adiponectin is negatively related to adiposity (r= - 0.8018, P< 0.0001,
207
r2 = 0.64).
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Discussion
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The saturated/animal fat was used in this study to induce the obesity in animals.
211
Indeed, the HF diet provoked changes in metabolic parameters related to obesity, such
212
as increased mass gain, impairment of the insulin sensitivity, hyperleptinemia, reduction
213
of adiponectin levels and higher concentration of proinflammatory interleukins in the
214
HF group, when compared with those animals from Control group. On the other hand,
215
the addition of PEPF in high-fat diet protected the animals against the excessive body
216
weight gain and fatness. The average of body weight gain and sum of RAT+EAT in HF
217
group was 12.01% and 21.72%, respectively, higher than those ones from HFPF-fed
218
rats. Furthermore, we noticed high gene expression for CART in HFPF-fed rats,
219
suggesting that the PEPF may interfere in satiety and food intake, implying in protective
220
factor against obesity. Although this finding was not followed by significant decrease in
221
total energy intake compared to HF group, it is consistent with the observation that food
222
intake was similar between the control group and HFPF-fed rats in our trial, indicating
223
an effect on food intake control. Similarly, resistant starch (RS), which has also a
224
similar role as the soluble fiber, has also shown increased hypothalamic POMC, another
225
satietogenic neuropeptide, reflecting on food intake in rats [6]. Additionally, the RS was
226
able to decrease body fatness and increased plasma GLP-1 [6] as we have observed with
227
PEPF. Apart from its incretin effects, evidences that intestinal GLP-1 afects satiety have
228
also been reported [12]. It is well-known that GLP-1 analogs induce weight loss and
229
improve glucose homeostasis in rodents and humans and recently, it was reported that
230
Liraglutide (comercially available GLP-1 analogue for type 2 diabetes treatment)
231
treatment increased CART mRNA levels in the hypothalamic arcuate nucleus in diet-
232
induced obese rats [13]. Together, these evidences and similiarities between our
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findings and those with RS could suggest that GLP-1 increment elicited by fermentable
234
fibersmay be related with food intake control via action on anorexigenics neuropeptides.
235
However, further researches are needed to elucidate whether physiological GLP-1, and
236
not only its mimetics, is able to act directly in central food intake control. As the adiposity and the satiety signals, the glucose metabolism has been
238
affected by the PEPF intake as well. The iGTT confirmed the impairment of glucose
239
tolerance, induced by HF diet, which was indicated by the higher AUC values presented
240
by HF diet-fed rats. Although the HFPF-fed rats have not presented decreased values
241
for AUC in comparison with the HF group during iGTT, those animals demonstrated no
242
difference compared to normal-fat diets. Besides, we noticed that the addition of PEPF
243
in high-fat diet has improved insulin sensitivity, which was shown by increased kITT
244
values during ITT. The kITT rate is considered a reflection of insulin sensitivity, once it
245
indicates the rate of glucose cleareance by increasing glucose uptake in insulin sensitive
246
tissues and suppression of hepatic glucose production in response to exogenous insulin
247
[25]. We could not find statistical differences among groups regarding fasting insulin
248
leveldue to the large variation of values presented in the groups. Nevertheless, animals
249
fed on HF diet showed the highest average comparing to other groups, but this
250
difference did not reach statistical significance. These findings, associated with those
251
found in iGTT and iITT, suggest that the PEPF could be effective to control glucose
252
homeostasis in diet-induced obesity.
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The improvement of glucose metabolism by the PEPF may be associated with
254
the ability of PEPF to modulate the incretins GLP-1 and GIP, which were increased in
255
animals fed on HFPF. Both incretins stimulate insulin secretion by β-cells in the islet of
256
Langerhans in a glucose-dependent way [30]. GLP-1plays a role on satiety and likely
257
helps long-term body weight regulation [12].We found high amount of pectin in PEPF,
258
representing almost the total soluble fraction of dietary fiber. In agreement with our
259
findings, increasing amounts of the pectin in the diet proportionately decrease food
260
intake, body weight gain and body fat content, while increase circulating PYY and
261
GLP-1 levels in rats. The strong relationship of those findings and the gut hypertrophy
262
found in already mentioned study supports indicate that the increase of these hormones
263
is probably a result of the L-cell proliferation [10]. Indeed, it is described in the
264
literature the capacity of the pectin to produce short-chain fatty acids as products of its
265
fermentation in the intestine [31]. Besides, studies have shown that PEPF intake results
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267
fermentation of pectin from the PEPF in intestine could be involved in the modulation
268
of gut hormones, especially GLP-1, once the SCFA have been linked to enhanced
269
release of this incretin [33,34]. In vitro assay has shown that butyrate stimulated the
270
release of GLP-1 from intestinal L-cell [33] as well as acetate and propionate pointed
271
out the same using of a mix colonic cell [34]. In addition, mice lacking SCFA receptors
272
exhibited SCFA-triggered GLP-1 secretion, acompanied by impairment of glucose
273
tolerance [34]. Furthermore, butyrate and propionate suplementation has shown
274
protection against diet-induced obesity and insulin resistance, by inducing gut hormones
275
and food intake inhibition in mice [35]. These evidences support that theincrementof
276
GLP-1 may be induced by SCFA elicited through fiber fermentation, which could
277
indicate a likely mechanism of action of the pectin from PEPF in order to increase the
278
incretin and, therefore, other metabolic parameters related.
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Besides incretins, PEPF also modulated adipokines in diet-induced obesity rats.
280
This modulation was associated with body fat changes, demonstrated by Pearson’s
281
correlation analysis. The increase of the adiponectin levels in animals fed with HFPF
282
could be related with the enhancement of insulin sensitivity, since serum adiponectin
283
levels are inversely correlated with obesity and type 2 diabetes and positively associated
284
with insulin sensitivity [36,37]. Moreover, rats fed on HFPF diet showed lower leptin
285
levels than those fed on HF diet. This adipokine plays a role on energy balance, by
286
signalling pathways in the hypothalamus in order to decrease the food intake and
287
increase the energy expenditure. Nevertheless, this role is disrupted in prolonged
288
obesity, which is well-known as leptin resistance [38]. The hyperleptinemia is needed
289
for
290
syndrome [40]. Thus, the decreased leptinemia in PEPF-fed rats could be considered a
291
protective factor against those conditions.
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It has been well established that obesity and insulin resistance are associated
293
with a chronic low-grade inflammation. Pro-inflammatory interleukins, such as IL-6,
294
IL-1, MCP-1 and TNF- α are elevated in these conditions [41]. The rats fed on HF diet
295
showed higher serum concentration of IL-6, MCP-1 and TNF- α compared to those fed
296
on normal-fat diet (Control group). Together, these results indicates that the saturated
297
fat used in our study contributes to increase the inflammation in rats, which is
298
associated with the obesity and impairment of insulin sensitivity [42]. Although there 12
ACCEPTED MANUSCRIPT 299
was not significant different between HF and HFPF groups, the interleukins
300
concentration was similar between control and HFPF-fed rats in our experiment, except
301
for IL 1 β, indicating that PEPF could attenuate obesity- related inflammation in rats. Apart of pectin, other phytochemicals in PEPF have also shown effects on
303
glucose metabolism and fatness: the tannic acid has indicated antihyperglicemic effect
304
in vitro assay by stimulating phosphorylation of protein factors in the insulin-mediated
305
glucose transport pathway and inducing GLUT 4 translocation, using 3T3-L1 cells as
306
model [43]. In vivo, tannic acid has reverted the glucose and the insulin level into the
307
normal range in diabetic rats [44]. The addition of phytic acid or rice bran in HF diet
308
showed positive effects on glucose metabolism and body weight gain in mice [45]. The
309
antioxidant potential of these compounds could be linked to those effects. In fact,
310
oxidative stress plays a role in thedevelopment and progression of obesity and diabetes,
311
and antioxidants activity of dietary components could prevent hyperglycemia under
312
obesogenic diet [45]. The antioxidant capacity of the PEPF was also reported in
313
previous studies [14–16]. Thereby, it is possible that this component of the PEPF plays
314
some role in the findings described here, by synergic action of polyphenols and fibers.
315
Nevertheless, driving this approach in future researches is necessary to elucidate this
316
hypothesis.
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Together, our findings demonstrate the physiological mechanisms by which the
318
PEPF could assist in the prevention of deleterious effects demonstrated by the intake of
319
a high-fat (saturated/animal) diet.
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Conclusion
322
Our findings provide a further understanding of how the PEPF, source of pectin, works
323
as a dietary ingredient to improve glucose homeostasis. Besides, the increased CART
324
expression in the hypothalamus demonstrates a molecular mechanism in order to
325
enhance satiety by PEPF in diet-induced obesity. These results represent physiological
326
effects of PEPF not described before, and support the evidence that the PEPF may be a
327
useful dietary component to prevent body fatness and insulin resistance.
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Acknowledgments
330
We thank Professor Lício Velloso for his collaboration and the Obesity and Comorbities
331
Research Center for providing methodological advice. 13
ACCEPTED MANUSCRIPT 332
This work was supported by FAPESP (grant number: 2012/12322-0) and CNPq.
333
FAPESP and CNPq had no role in the design, analysis or writing of this article.
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The authors declare that they have no conflicts of interest.
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ACCEPTED MANUSCRIPT doi:10.2337/db06-1491.P.D.C. [42] Cani PD, Amar J, Iglesias MA, Poggi M, Knauf C, Bastelica D, et al. Original Article. Diabetes 2007;56:1761–72. doi:10.2337/db06-1491.P.D.C. [43] Liu X, Kim J, Li Y, Li J, Liu F, Chen X. Tannic acid stimulates glucose transport and inhibits adipocyte differentiation in 3T3-L1 cells. J Nutr 2005;135:165–71. S, Chandirasegaran G. TANNIC ACID IN RATS. Int J Curr Res
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ACCEPTED MANUSCRIPT Figures Subtitles Figure 1- Effect of Passiflora edulis peel flour intake on body weight throughout the experimental weeks, total body weight gain after 10 experimental weeks and total energy (kJ) intake after 10 experimental weeks.
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C, Control group fed on AIN 93G diet; HF, High-fat group fed on AIN 93G diet modified with 35% fat (w/w); HFPF, High-fat Passiflora Flour group fed on high-fat diet added 2.5% (w/w) Passiflora edulis peel flour.
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Data are presented as average (SD), n=8. * HF group > Control group (P < 0.05); ** HF group > Control and HFPF groups (both P < 0.01) by analysis of variance (ANOVA)
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(repetead measures) followed by Tukey’s test. Different letters represent statistical difference by one-way ANOVA, following by Tukey's test; P < 0.05. Figure 2 - Effect of Passiflora edulis peel flour intake on hypothalamic expression of neuropeptide Y (NPY), agouti-related peptide (AgRP), pro-opiomelanocortin (POMC)
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and cocaine- and amphetamine-regulated transcript (CART) expression. C, Control group fed on AIN 93G diet; HF, High-fat group fed on AIN 93G diet modified with 35% fat (w/w); HFPF, High-fat Passiflora Flour group fed on high-fat
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diet added 2.5% (w/w) Passiflora edulis peel flour. Data are presented as an average (SD), n=5. Different letters represent statiscal difference by Kruskal-Wallis and
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multiple comparision tests.
Figure 3- Effect of Passiflora edulis peel flour intake on glucose tolerance and insulin sensitivity evaluated by iGTT and kITT iGTT, intraperitoneal glucose tolerance test; AUC, area under curve; ITT, insulin tolerance test; kITT, glucose disappearance rate; Control, Control group fed on AIN 93G diet; HF, High-fat group fed on AIN 93G diet modified with 35% fat (w/w); HFPF, High-fat Passiflora Flour group fed on high-fat diet added 2.5% (w/w) Passiflora edulis peel flour. Data are presented as average (SD), n=5. Different letters represent statiscal difference by analysis of variance (ANOVA) followed by Tukey’s test (P < 0.05). 19
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Figure 4 - Effect of Passiflora edulis peel flour intake on serum proinflammatory interleukins levels.
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Control, Control group fed on AIN 93G diet; HF, High-fat group; HFPF, modified highfat diet containing Passiflora edulis peel flour; IL-6, interleukin 6; IL-1β, interleukin 1β; TNF α, tumoral necrose factor α; MCP 1, Monocyte Chemoattractant Protein-1.
* Data are presented as average (SD), n=6. Different letters represent statistical
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difference by analysis of variance (ANOVA) followed by Tukey’s test; p<0.05).
Figure 5 - Effect of Passiflora edulis peel flour intake on serum adiponectin and leptin
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levels.
Control, Control group fed on AIN 93G diet; HF, High-fat group fed on AIN 93G diet modified with 35% fat (w/w); HFPF, High-fat Passiflora Flour group fed on high-fat diet added 2.5% (w/w) Passiflora edulis peel flour.
Data are presented as average (SD), n=6. Different letters represent statistical difference
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by analysis of variance (ANOVA) followed by Tukey’s test (P < 0.05).
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ACCEPTED MANUSCRIPT Table 1 Composition of experimental diets (g/Kg of diet)* Control
HF
HFPF
Cornstarch
435.12
258.3
258.3
Casein (85.5%)
140.35
140.35
140.35
Dextrinized cornstarch
144.51
85.79
85.79
Sucrose
109.57
65.05
65.05
Soybean oil
70.00
40.00
40.00
310.00
310.00
50.00
25.00
-
25.00
35.00
35.00
10.00
10.00
10.00
3.00
3.00
3.00
2.50
2.50
2.50
0.014
0.014
0.014
15.4
21.9
21.9
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Cellulose
50.00
PEPF
-
Vitamin mix (AIN-93G-VX) L-Cystine Choline bitartrate Tert-butylhydroquinone
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Energy density (kJ/g diet)
35.00
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Mineral mix (AIN-93G-MX)
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Lard
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Ingredient
Control, Control group fed on AIN 93G diet; HF, High-fat group fed on AIN 93G diet modified with 35% fat (w/w); HFPF, High-fat Passiflora Flour group fed on AIN 93G modified with 35% fat (w/w) added 2.5% (w/w) Passiflora edulis peel
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flour.
*Diets were prepared according to American Institute of Nutrition for AIN 93-G [24]
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with modified protein content to 12%, according Dragano et al. (2013) [25].
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ACCEPTED MANUSCRIPT Table 2 Proximate and phytochemical composition of Passiflora edulis peel flour Content (g/100g) 7.42 ± 0.25
Ash
6.00 ± 0.16
Lipids
3.39 ± 0.18
Protein
8.87 ± 0.10
Carbohydrate†
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Moisture
14.24 ± 0.34
Total dietary fiber
60.08 ± 0.02
Insoluble dietary fiber
20.13 ± 0.64
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Soluble dietary fiber
39.96 ± 0.66
Soluble pectin
Phytochemicals Phytic acid (mg/100g) Hydrogen cyanid (mg/ Kg) Tannin (mg/100g)
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Total pectin
4.85 ± 0.53 17.27 ± 0.11
37.0 ± 0.02 LOD < 0.30 308.21 ± 1.12
LOD,limit of detection. † Calculated by difference, excludes the dietary fiber fraction.
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Data were expressed with an average (SD), n=3.
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ACCEPTED MANUSCRIPT Table 3 Liver, epididymal adipose tissue (EAT) and retroperitoneal adipose tissue (RAT) % ratio weights (g/100gtotal body weight)
Liver
Control 2.80 ± 0.32
HF 2.94 ± 0.52
HFPF 2.88 ± 0.32
EAT
1.94 ± 0.46 b
3.00 ± 0.76 a
2.38 ± 0.64 b
RAT
2.12 ± 0.62 b
3.04 ± 0.50 a
2.51 ± 0.53 b
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EAT+RAT 3.79 ± 1.32 b 6.04 ± 1.22 a 4.73 ± 1.13 b Control group fed on AIN 93G diet; HF, High-fat group fed on AIN 93G diet modified with 35% fat (w/w); HFPF, High-fat Passiflora Flour group fed on AIN 93G modified with 35% fat (w/w) added 2.5% (w/w) Passiflora edulis peel flour. Values are an average
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and SD, n=8. Data were analyzed using analysis of variance (ANOVA) followed by
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ACCEPTED MANUSCRIPT Table 4 - Effect of Passiflora edulis peel flour intake on serum gut hormones and insulin levels (ng/ml) in rats HF
HFPF
Amylin
38.93 ± 3.32
48.53 ± 4.63
50.86 ± 5.04
GIP
75.12 ± 5.12 a
44.09 ± 2.66 b
63.99 ± 11.26 a
GLP-1
66.10 ± 8.62 c
82.10 ± 10.89 b
127.25 ± 5.32 a
PP
29.14 ± 4.64 b
47.39 ± 7.44 a
PYY
51.71 ± 10.78
60.04 ± 13.08
395.75 ± 319.84
1277.00 ± 749.76
Insulin
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Control
48.86 ± 10.60 a 66.00 ± 7.57
628.40 ± 257.26
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Control group fed on AIN 93G diet; HF, High-fat group fed on AIN 93G diet modified with 35% fat (w/w); HFPF, High-fat Passiflora Flour group fed on AIN 93G modified
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with 35% fat (w/w) added 2.5% (w/w) Passiflora edulis peel flour. Values are an average and SD, n=6. Data were analyzed using ANOVA followed by Tukey’s test (P
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ACCEPTED MANUSCRIPT Highlights: Passiflora edulis peel intake improves insulin sensitivity in high-fat fed rats Passiflora edulis peel intake increases incretins GIP and GLP-1 in high-fat fed rats Passiflora edulis peel intake modulates adiponectin and leptin in high-fat fed rats
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Passiflora edulis peel intake increases satietogenic neuropeptide in high-fat fed rats