Subscriber access provided by Kaohsiung Medical University
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
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 40
Journal of Agricultural and Food Chemistry
2
Morus alba leaf bioactives modulate PPARγ in the kidney of diabetic rat and impart beneficial effect
3
Mulluru Somasundara Abignan Gurukar and Nandini D. Chilkunda*
4 5 6
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.
1
7
*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
8
1 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 2 of 40
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
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.
27
KEY WORDS: Morus alba/ PPARγ / diabetes /inflammation
28
2 ACS Paragon Plus Environment
Page 3 of 40
29
Journal of Agricultural and Food Chemistry
INTRODUCTION
30
Diabetes mellitus (DM) is a chronic metabolic disorder the prevalence of which has been
31
increasing steadily throughout the world. It is characterized by sustained hyperglycemia, the
32
control of which is necessary to prevent various secondary complications. Diabetic nephropathy
33
is one of the secondary complications which results in end-stage renal disease. As with most
34
chronic diseases, the pathophysiology is multifactorial resulting in dysregulated molecular
35
processes contributing to disease manifestation and progression.
36
Among various metabolic regulators, PPARγ play an important role in glucose, lipid and
37
insulin homeostasis 1. Furthermore, it also plays a crucial role in kidney development and has
38
other important physiological functions 2. It modulates the expression of genes by binding to the
39
PPRE sites. The actions of PPARγ can be accomplished by transactivation or transrepression 3.
40
Compounding to the complexity, its actions can be ligand-dependent or ligand-independent
41
4.
42
glucose uptake into the cells through insulin-sensitizing action 1. Diabetic nephropathy was
43
ameliorated by PPARγ agonists independent of the normalization of blood glucose levels
44
alluding to its direct effect on the kidney 5. Though beneficial, it is associated with increased side
45
effects including water accumulation in the kidney 6.
PPARγ activators, namely the Thiazolidinediones, used for the treatment of diabetes, facilitates
46
In recent years, emphasis on mitigating diabetes through food is gaining ground, and
47
dietary factors are known to play a vital role in managing diabetes through normalization of
48
blood glucose levels 7. Dietary compounds with PPARγ modulating activities are gaining
49
attention to circumvent the problems associated with pharmacological activators. In our earlier
50
study, we observed that feeding Morus alba leaves to experimental diabetic rats proved
51
beneficial in ameliorating diabetes and diabetic nephropathy 8. Morus alba leaves are used for
52
culinary purposes
53
functional food on account of it being rich in various bioactives
54
attempted to delineate the molecular mechanism through which mulberry can ameliorate kidney
55
damage. To address it, we aimed at determining if it has putative PPARγ modulating activity
56
using in vitro and in vivo approaches.
9
and also for brewing tea
10
. It holds a great promise in the formulation of 11
. In this paper, we have
57
3 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 4 of 40
58
MATERIALS AND METHODS
59
Reagents
60
Antibodies against β actin, MCP-1, ERK, phospho ERK Thr202 / Tyr 204, mTOR and phospho
61
mTOR Ser 2448 were purchased from Cell signaling technology, USA. Antibodies against
62
NFκB, PPARγ, phospho PPARγ Ser112 were purchased from Abcam, Cambridge, UK. Glucose
63
oxidase/peroxidase (GOD/POD) kit was purchased from Span Diagnostic Limited, Mumbai,
64
Maharashtra, India.
65
inhibitor U0126 monoethanolate and phenolic acid standards for HPLC were obtained from
66
Sigma Chemical Company, St. Louis, USA. All other chemicals and reagents used were of
67
analytical grade from Sisco Research Laboratories Pvt Ltd, Hyderabad, India.
68
Plant materials
69
Mulberry leaves were collected from Central Sericulture Research Institute, Mysuru during
70
December and authenticated. The leaves were separated individually from the plant and sorted
71
into young and mature leaves. The leaves from the upper part of the plant (5-6 Leaves) which
72
was tender were considered as young mulberry leaves whereas the middle and bottom leaves
73
portion were considered as mature. Differentiation between mature and young leaves was also
74
made based on the moisture content of the leaves. Moisture content was determined by taking the
75
weight difference between wet and dry weight. The collected plant material was washed in
76
running tap water and dried in an oven at 60 ⁰C for 24 h after draining off the excess water. The
77
dried leaves were powdered and stored in airtight containers for further use.
78
Isolation of bioactive-rich extracts
79
Mulberry leaves (2 g) were extracted independently with various solvents such as acetone,
80
methanol, ethanol, and water on a magnetic stirrer for 2 h. The extracted mixture was centrifuged
Enhanced chemiluminescence kit, PPARγ antagonist GW 9662, ERK
4 ACS Paragon Plus Environment
Page 5 of 40
Journal of Agricultural and Food Chemistry
81
at 10,000 rpm for 20 min. The supernatant was collected and filtered. Amount of total phenolic
82
acids were estimated as reported earlier 11 .
83
Extraction of bound phenolic acid - rich extract
84
Mulberry young leaves (2 g) were extracted separately with 75% methanol on a magnetic stirrer
85
for 2 h. The residue remaining after extraction of the soluble phenolic fraction was hydrolyzed
86
with 2 M NaOH at room temperature for 4 h with stirring. It was acidified to pH 2.0 with 6 M
87
HCl and extracted five times with diethyl ether and ethyl acetate (1:1) and dried in a speed vac. It
88
was then reconstituted in methanol and stored at -20 ºC 12 .
89
HPLC analysis
90
Phenolic acid-rich extracts (soluble and bound) were analyzed by HPLC. The analysis was
91
carried out in an HPLC system (Shimadzu) attached with diode array detector. Filtered samples
92
(20 µl) were analyzed on a C18 analytical column (250 X 4.6 mm; 5 µm) with mobile phase
93
consisting of 0.1% trifluoroacetic acid as solvent A and 100 % methanol as solvent B. The total
94
run was for a period of 60 min with a flow rate of 1.0 ml/min. The gradient program was as
95
follows: Initial B concentration of 20% to 40 % in 40 min which was maintained for 10 min and
96
then again 20 % B in next 5 min of post-run for reconditioning. Peaks were recorded
97
simultaneously at 280 and 320 nm
98
High-resolution mass spectrometry (HRMS)
99
Putative potent bioactive-containing fraction from the HPLC was collected and the solvent was
100
dried completely and reconstituted in 500 µl of MS grade methanol. About 20 µl was injected in
101
the HRMS (ABSIEX Triple TOF 5600+) system, fitted with an electron spray ionization source.
102
The flow rate was 20 µl/min. The data were acquired using the software Analyst TF 1.6 Build
103
6211. The spectrum was recorded against intensity, cps verses m/z, the mass of sample was
5 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 6 of 40
104
recorded in the range of 50–1000. The said spectrum was compared against the standard
105
phenolic acid spectra obtained under similar conditions.
106
Animal cell culture
107
A kidney tubular epithelial cell line, Madin-Darby Canine Kidney Epithelial Cells (MDCK) was
108
procured from NCCS, Pune. It was cultured in Minimum Essential Medium (MEM) with 5 mM
109
glucose maintained at 37 ºC in a humidified atmosphere containing 5 % CO2. The cells were
110
initially seeded in a 6-well plate with normal glucose (5 mM). After 24 h, the wells were
111
replenished with media containing either normal glucose or high glucose (30 mM). After further
112
24 h, fresh media was added and treated individually with Pioglitazone (25 µM), GW 9662
113
(PPARγ antagonist, 10 µM), mulberry extracts (20 µM as Gallic acid equivalent) or ERK
114
inhibitor (U0126 monoethanolate, 10 µM). Cells treated with mannitol (MN, 25 mM) was used
115
as an osmotic control for high glucose conditions. The cells were washed with PBS and extracted
116
with cell lysis buffer at the end of experimental period.
117
Animals
118
Male Wistar rats of 60 days of age weighing around 115 ± 10.0 g were used for the experiment.
119
The study had prior animal ethical clearance from the Institute Animal Ethical Committee
120
(IAEC). The animals were acclimatized for 2 weeks using standard diet and made sure that they
121
had free access to food and water.
122
Induction of diabetes mellitus and grouping of rats
123
Experimental diabetes was induced in a group of rats by injecting Streptozotocin (STZ)
124
intraperitoneally at 37 mg/kg body weight dissolved in freshly prepared citrate buffer (0.1 M, pH
125
4.5)
126
water for 48 h soon after STZ injection to prevent drug-induced hypoglycemic shock. After one
13
. Control rats were sham injected with citrate buffer. The rats were fed with 5% glucose
6 ACS Paragon Plus Environment
Page 7 of 40
Journal of Agricultural and Food Chemistry
127
week of STZ injection, diabetic status was confirmed by measuring fasting blood glucose levels.
128
The animals were again sub-grouped into three groups, diabetic control, pioglitazone and
129
mulberry leaf-supplemented diabetic groups based on fasting blood glucose level as shown
130
schematically (Figure. 1). The experiment was carried out for 2 months from the day of
131
treatment initiation after the induction of diabetes.
132
Diet
133
Mulberry leaves (5%) were incorporated into the diet by replacing equal quantities of starch.
134
Pioglitazone was gavaged daily at 10 mg/Kg body weight in 0.5% carboxymethyl cellulose
135
according to established method 14. Animals were maintained for two months after the induction
136
of diabetes with their respective diets.
137
Collection of kidney and analysis of various parameters in blood and urine
138
Blood was drawn from retro-orbital plexus into tubes containing heparin (20 U/ml blood). Blood
139
glucose was determined by Glucose oxidase-peroxidase method
140
Albumin in urine was measured by using Albumin Blue 580 method 16. Creatinine was estimated
141
in urine and serum by Jaffe’s method 17 using a commercially available kit. Glomerular filtration
142
rate was determined by estimating creatinine levels in urine and serum using the formula as
143
reported earlier. The kidney was harvested after euthanizing the rats.
144
Western blot analysis
145
About 50 – 100 mg of kidney stored in liquid nitrogen was taken in 1 ml of RIPA buffer. The
146
tissue was homogenized and centrifuged at 15,000 rpm for 30 min. The supernatant was
147
aliquoted and stored at -80 °C for future use. An aliquot of protein (50 µg) was loaded on to a 10
15
, after prior fasting for 12 h.
7 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 8 of 40
148
% SDS PAGE gel and transferred to PVDF membrane and blocked with 5% BSA in TBST
149
overnight. Primary antibodies were diluted with 5 % BSA in TBST at room temperature and
150
treated for 2 h. The secondary antibody was diluted with 2.5 % BSA in TBST and treated for 2 h
151
at room temperature. The blots were then washed with TBST for 5 min thrice and developed by
152
enhanced chemiluminescence kit and documented using a Bio-Rad gel-doc system.
153
Statistical analysis
154
Statistical analysis of data was performed by one-way analysis of variance with a Tukey’s
155
multiple comparison post-test. Comparison between control (C) and diabetic (D) has been
156
denoted by †, whereas the comparison between diabetic (D) and treated D groups has been
157
denoted by ‡. The significance levels are indicated as follows; *P
