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Bioactive Constituents, Metabolites, and Functions

Morus alba leaf bioactives modulate PPAR# in the kidney of diabetic rats and impart beneficial effect Abignan Gurukar, and Nandini D Chilkunda J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01357 • Publication Date (Web): 03 Jul 2018 Downloaded from http://pubs.acs.org on July 4, 2018

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Journal of Agricultural and Food Chemistry

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Morus alba leaf bioactives modulate PPARγ in the kidney of diabetic rat and impart beneficial effect

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Mulluru Somasundara Abignan Gurukar and Nandini D. Chilkunda*

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Dept of Molecular Nutrition, CSIR-CFTRI Mysore-570 020, Karnataka, India. Academy of Scientific and Innovative Research (AcSIR), CSIR-CFTRI campus, Mysuru-570 020, Karnataka, India.

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*Corresponding Author Department of Molecular Nutrition, CSIR-Central Food Technological Research Institute, Mysuru – 570 020, Karnataka, India E-mail address: [email protected] Tel: +91-821-2514876 FAX: +91- 821- 2517233

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1 ACS Paragon Plus Environment


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ABSTRACT: Peroxisome proliferator activated receptor gamma (PPARγ) are ligand-activated nuclear receptors that can be activated or repressed by several exogenous and endogenous ligands and act by modulating genes which regulate lipid, glucose, and insulin homeostasis. In kidney, they are involved in normal kidney development and other physiological functions. In our earlier report, we showed that feeding Morus alba leaves to experimental diabetic rats ameliorated diabetic nephropathy and significantly decreased microalbuminuria. In this paper, we have attempted to look into the molecular mechanism involving PPARγ modulation by mulberry leaf bioactives by in vitro and in vivo methods and its impact on key molecules of inflammatory markers. In vitro assay by TR-FRET suggested that mulberry leaf extracts can serve as putative modulator of PPARγ . High glucose conditions in vitro and in vivo increased PPARγ levels which were ameliorated by mulberry leaves or its extracts. Interestingly, PPARγ was significantly phosphorylated at Ser112 by upstream kinases ERK42/44 in kidney of diabetic animals on feeding mulberry leaves. In vitro studies using MDCK cell line revealed that increased Ser112 phosphorylation was observed when treated with bound phenolic acid richextract but not with free phenolic acid-rich extracts. HPLC analysis and bioassay-guided activity revealed that coumaric acid was the bioactive molecule amongst bound phenolic acid-rich extract that was responsible for increased ERK 42/44-mediated phosphorylation at Ser112. Furthermore, mulberry leaf bioactives showed beneficial effect on the tested inflammatory markers.

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KEY WORDS: Morus alba/ PPARγ / diabetes /inflammation

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INTRODUCTION

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Diabetes mellitus (DM) is a chronic metabolic disorder the prevalence of which has been

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increasing steadily throughout the world. It is characterized by sustained hyperglycemia, the

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control of which is necessary to prevent various secondary complications. Diabetic nephropathy

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is one of the secondary complications which results in end-stage renal disease. As with most

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chronic diseases, the pathophysiology is multifactorial resulting in dysregulated molecular

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processes contributing to disease manifestation and progression.

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Among various metabolic regulators, PPARγ play an important role in glucose, lipid and

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insulin homeostasis 1. Furthermore, it also plays a crucial role in kidney development and has

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other important physiological functions 2. It modulates the expression of genes by binding to the

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PPRE sites. The actions of PPARγ can be accomplished by transactivation or transrepression 3.

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Compounding to the complexity, its actions can be ligand-dependent or ligand-independent

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4.

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glucose uptake into the cells through insulin-sensitizing action 1. Diabetic nephropathy was

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ameliorated by PPARγ agonists independent of the normalization of blood glucose levels

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alluding to its direct effect on the kidney 5. Though beneficial, it is associated with increased side

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effects including water accumulation in the kidney 6.

PPARγ activators, namely the Thiazolidinediones, used for the treatment of diabetes, facilitates

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In recent years, emphasis on mitigating diabetes through food is gaining ground, and

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dietary factors are known to play a vital role in managing diabetes through normalization of

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blood glucose levels 7. Dietary compounds with PPARγ modulating activities are gaining

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attention to circumvent the problems associated with pharmacological activators. In our earlier

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study, we observed that feeding Morus alba leaves to experimental diabetic rats proved

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beneficial in ameliorating diabetes and diabetic nephropathy 8. Morus alba leaves are used for

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culinary purposes

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functional food on account of it being rich in various bioactives

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attempted to delineate the molecular mechanism through which mulberry can ameliorate kidney

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damage. To address it, we aimed at determining if it has putative PPARγ modulating activity

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using in vitro and in vivo approaches.

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and also for brewing tea

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. It holds a great promise in the formulation of 11

. In this paper, we have

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MATERIALS AND METHODS

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Reagents

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Antibodies against β actin, MCP-1, ERK, phospho ERK Thr202 / Tyr 204, mTOR and phospho

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mTOR Ser 2448 were purchased from Cell signaling technology, USA. Antibodies against

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NFκB, PPARγ, phospho PPARγ Ser112 were purchased from Abcam, Cambridge, UK. Glucose

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oxidase/peroxidase (GOD/POD) kit was purchased from Span Diagnostic Limited, Mumbai,

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Maharashtra, India.

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inhibitor U0126 monoethanolate and phenolic acid standards for HPLC were obtained from

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Sigma Chemical Company, St. Louis, USA. All other chemicals and reagents used were of

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analytical grade from Sisco Research Laboratories Pvt Ltd, Hyderabad, India.

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Plant materials

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Mulberry leaves were collected from Central Sericulture Research Institute, Mysuru during

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December and authenticated. The leaves were separated individually from the plant and sorted

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into young and mature leaves. The leaves from the upper part of the plant (5-6 Leaves) which

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was tender were considered as young mulberry leaves whereas the middle and bottom leaves

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portion were considered as mature. Differentiation between mature and young leaves was also

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made based on the moisture content of the leaves. Moisture content was determined by taking the

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weight difference between wet and dry weight. The collected plant material was washed in

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running tap water and dried in an oven at 60 ⁰C for 24 h after draining off the excess water. The

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dried leaves were powdered and stored in airtight containers for further use.

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Isolation of bioactive-rich extracts

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Mulberry leaves (2 g) were extracted independently with various solvents such as acetone,

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methanol, ethanol, and water on a magnetic stirrer for 2 h. The extracted mixture was centrifuged

Enhanced chemiluminescence kit, PPARγ antagonist GW 9662, ERK


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at 10,000 rpm for 20 min. The supernatant was collected and filtered. Amount of total phenolic

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acids were estimated as reported earlier 11 .

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Extraction of bound phenolic acid - rich extract

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Mulberry young leaves (2 g) were extracted separately with 75% methanol on a magnetic stirrer

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for 2 h. The residue remaining after extraction of the soluble phenolic fraction was hydrolyzed

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with 2 M NaOH at room temperature for 4 h with stirring. It was acidified to pH 2.0 with 6 M

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HCl and extracted five times with diethyl ether and ethyl acetate (1:1) and dried in a speed vac. It

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was then reconstituted in methanol and stored at -20 ºC 12 .

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HPLC analysis

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Phenolic acid-rich extracts (soluble and bound) were analyzed by HPLC. The analysis was

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carried out in an HPLC system (Shimadzu) attached with diode array detector. Filtered samples

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(20 µl) were analyzed on a C18 analytical column (250 X 4.6 mm; 5 µm) with mobile phase

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consisting of 0.1% trifluoroacetic acid as solvent A and 100 % methanol as solvent B. The total

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run was for a period of 60 min with a flow rate of 1.0 ml/min. The gradient program was as

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follows: Initial B concentration of 20% to 40 % in 40 min which was maintained for 10 min and

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then again 20 % B in next 5 min of post-run for reconditioning. Peaks were recorded

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simultaneously at 280 and 320 nm

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High-resolution mass spectrometry (HRMS)

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Putative potent bioactive-containing fraction from the HPLC was collected and the solvent was

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dried completely and reconstituted in 500 µl of MS grade methanol. About 20 µl was injected in

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the HRMS (ABSIEX Triple TOF 5600+) system, fitted with an electron spray ionization source.

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The flow rate was 20 µl/min. The data were acquired using the software Analyst TF 1.6 Build

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6211. The spectrum was recorded against intensity, cps verses m/z, the mass of sample was



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recorded in the range of 50–1000. The said spectrum was compared against the standard

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phenolic acid spectra obtained under similar conditions.

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Animal cell culture

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A kidney tubular epithelial cell line, Madin-Darby Canine Kidney Epithelial Cells (MDCK) was

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procured from NCCS, Pune. It was cultured in Minimum Essential Medium (MEM) with 5 mM

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glucose maintained at 37 ºC in a humidified atmosphere containing 5 % CO2. The cells were

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initially seeded in a 6-well plate with normal glucose (5 mM). After 24 h, the wells were

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replenished with media containing either normal glucose or high glucose (30 mM). After further

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24 h, fresh media was added and treated individually with Pioglitazone (25 µM), GW 9662

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(PPARγ antagonist, 10 µM), mulberry extracts (20 µM as Gallic acid equivalent) or ERK

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inhibitor (U0126 monoethanolate, 10 µM). Cells treated with mannitol (MN, 25 mM) was used

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as an osmotic control for high glucose conditions. The cells were washed with PBS and extracted

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with cell lysis buffer at the end of experimental period.

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Animals

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Male Wistar rats of 60 days of age weighing around 115 ± 10.0 g were used for the experiment.

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The study had prior animal ethical clearance from the Institute Animal Ethical Committee

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(IAEC). The animals were acclimatized for 2 weeks using standard diet and made sure that they

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had free access to food and water.

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Induction of diabetes mellitus and grouping of rats

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Experimental diabetes was induced in a group of rats by injecting Streptozotocin (STZ)

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intraperitoneally at 37 mg/kg body weight dissolved in freshly prepared citrate buffer (0.1 M, pH

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4.5)

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water for 48 h soon after STZ injection to prevent drug-induced hypoglycemic shock. After one

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. Control rats were sham injected with citrate buffer. The rats were fed with 5% glucose


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week of STZ injection, diabetic status was confirmed by measuring fasting blood glucose levels.

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The animals were again sub-grouped into three groups, diabetic control, pioglitazone and

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mulberry leaf-supplemented diabetic groups based on fasting blood glucose level as shown

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schematically (Figure. 1). The experiment was carried out for 2 months from the day of

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treatment initiation after the induction of diabetes.

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Diet

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Mulberry leaves (5%) were incorporated into the diet by replacing equal quantities of starch.

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Pioglitazone was gavaged daily at 10 mg/Kg body weight in 0.5% carboxymethyl cellulose

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according to established method 14. Animals were maintained for two months after the induction

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of diabetes with their respective diets.

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Collection of kidney and analysis of various parameters in blood and urine

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Blood was drawn from retro-orbital plexus into tubes containing heparin (20 U/ml blood). Blood

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glucose was determined by Glucose oxidase-peroxidase method

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Albumin in urine was measured by using Albumin Blue 580 method 16. Creatinine was estimated

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in urine and serum by Jaffe’s method 17 using a commercially available kit. Glomerular filtration

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rate was determined by estimating creatinine levels in urine and serum using the formula as

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reported earlier. The kidney was harvested after euthanizing the rats.

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Western blot analysis

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About 50 – 100 mg of kidney stored in liquid nitrogen was taken in 1 ml of RIPA buffer. The

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tissue was homogenized and centrifuged at 15,000 rpm for 30 min. The supernatant was

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aliquoted and stored at -80 °C for future use. An aliquot of protein (50 µg) was loaded on to a 10

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, after prior fasting for 12 h.



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% SDS PAGE gel and transferred to PVDF membrane and blocked with 5% BSA in TBST

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overnight. Primary antibodies were diluted with 5 % BSA in TBST at room temperature and

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treated for 2 h. The secondary antibody was diluted with 2.5 % BSA in TBST and treated for 2 h

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at room temperature. The blots were then washed with TBST for 5 min thrice and developed by

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enhanced chemiluminescence kit and documented using a Bio-Rad gel-doc system.

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Statistical analysis

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Statistical analysis of data was performed by one-way analysis of variance with a Tukey’s

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multiple comparison post-test. Comparison between control (C) and diabetic (D) has been

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denoted by †, whereas the comparison between diabetic (D) and treated D groups has been

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denoted by ‡. The significance levels are indicated as follows; *P

Morus alba - American Chemical Society

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