PROJECT PROPOSAL

“DIETARY INTERVENTION TO CONTROL THE
ACTIVITY OF HORMONE SENSITIVE LIPASE FOR
THE MANAGEMENT OF OBESITY AND
PREVENTION OF DIABETES”
Project Proposal
On
Yeshwanthi Singh
Ph. D. Reg. # 10BB15J08001
Department of Lipid Science
CSIR-Central Food Technological Research Institute
Mysore-570 020

Obesity is a medical condition in which excess body fat
has accumulated to the extent that it may have a negative
effect on health.
What is this obesity??

• Obesity is caused by excessive
intake of calories in relation to
energy expenditure over a long
period of time.
• The gastro intestinal tract has the
capacity to absorb large amounts of
nutrients
• Major increase in body fat can result
from even minor but chronic
differences between energy intake
and energy expenditure
Energy metabolism

• Obesity is associated with increased
number of adipocytes
• The major function of adipocytes is
storage of triglycerides
• Functions as endocrine organ and
secretes leptin, resistin, estrogens,
tumor necrosis factor alpha.
• Release fatty acids into the
circulation by lipolysis, which are
used by most organs for fuel
when glucose is limiting.
Adipose Tissue

• Alterations in adipocyte
lipolysis (TG breakdown) is
observed in obesity and
results in increased release
of fatty acids into the
circulation by,
1. Adipose triglyceride lipase
(ATGL)
2. Hormone sensitive lipase
(HSL)
3. Monoglyceride lipase
(MAGL)
Lipolysis

• HSL is a critical enzyme involved in
the hormonally regulated release of
fatty acids and glycerol from
adipocyte lipid stores
• The activation and mobilization
of HSL can be triggered by various
catecholamines and inhibited by
insulin.
• HSL has also been reported to act
mainly on diglycerides, while
triglycerides and monolglycerides
are hydrolyzed by adipose
triglyceride lipase ATGL and MGL,
respectively.
Hormone sensitive Lipase (HSL)

• The role of HSL in insulin secretion
has been studied mostly through
the use of knockout mice.
• Elevated plasma levels of free fatty
acids (FFAs) are thought to play a
major role in the pathogenesis of
insulin resistance and type 2
diabetes
• Elevated plasma FFA in type 2
diabetes has led to the proposal
that HSL may be a potential
therapeutic target for this disease,
lowering plasma FFA levels and
thereby reducing insulin resistance.
Hormone sensitive Lipase (HSL)

• Insulin reistance (IR) refers to the
situation whereby insulin
interaction with its receptor fails to
elicit downstream signalling events.
• The fat cells can produce chemicals
that lead to the release of pro-
inflammatory cytokines from
immune-related cells causing
systemic inflammation and insulin
resistance.
• As a result there is disruption in
insulin mediated control of glucose
and lipid homeostasis in primary
insulin responsive tissues : liver,
skeletal muscle, adipose tissue.
Link between obesity and insulin
resistance

• HSL thought to be the rate-limiting enzyme in adipose tissue
lipolysis
• It has been reported that there was a defect in insulin secretion
in HSL null mice (Roduit et al., 2001).
• A study performed on adipose tissue of cancer patients showed
a twofold increase in HSL mRNA that correlated with an
increase in non-esterified fatty acid levels (Holm et al., 2000).
• The accumulation of lipid droplets, likely consisting of
cholesterol esters have been observed in the adrenal cortex of
HSL knockout mice (Li et al., 2002).
• Inhibition of HSL also reduced hyperglycemia in streptozotocin-
induced diabetic rats (Wang et al., 2001)
• Treatment with a Lipolysis Inhibitor Ameliorates Adipose Tissue
Inflammation and Systemic Insulin Resistance ( Okazaki et al.,
2002)
Hormone sensitive Lipase (HSL):
study report

Hormone sensitive Lipase (HSL):
study report
• Small-molecule HSL inhibitors have been
identified (Vertesy et al., 2002; Slee et al.,
2003; De Jong et al., 2004; Ebdrup et al.,
2004; Lowe et al., 2004)
• selective HSL inhibitor BAY [227] and In
vitro SAR of (5-(2H)-isoxazolonyl) ureas,
potent inhibitors of hormone-sensitive
lipase has nonspecific toxic effects (A
Adibekian - 2013)
• These sterol mimetic inhibits PI-PLC-
dependent processes in human platelets
and neutrophils. (SA Scott - 2014)
• for carcinogenic phenotypes, limiting the
potential for on-target side effects(SA
Scott - 2014)

Our
approach
The role of elevated plasma FFA in type 2 diabetes has led to
the proposal that HSL may be a potential therapeutic target
for this disease, lowering plasma FFA levels and thereby
reducing insulin resistance

1
• Recombinant protein purification and Screening of HSL
inhibitor from food sources
2
• Invitro studies and cell-line based study for testing
efficacy of inhibitor
3
• Biochemical analysis –Administration of the molecules
and Analysis of serum parameters
4
• Toxicological evaluation of the potential extract
molecules
Objectives of the project

Why this project is required?
• The results of this research will
contribute for preventing fatty
acid induced insulin resistance
and obesity
• Side effects caused by synthetic
compounds can be addressed by
natural products.

1. Bacterial expression and purification of Lipases: Gene construct will
be transformed into E.coli BL21 (AI3) cells and expression of the protein
will be induced with 0.5 mM isopropylthio-ßgalactoside (IPTG) for 4 h at
37 ºc.
Gene expression will be confirmed by immunoblot blot analysis using
anti-His6 monoclonal antibody (1:5000, v/v). The expressed protein will
be purified with Ni2+-NTA agarose beads.
The amount of purified recombinant protein will be estimated using
Lowry's method.
2. Preparation of extracts: Shortlisting food sources and various extracts
preaparation.
3. High-throughput screening of lipase inhibitor from food source by
activity based proteome profiling (ABPP).
ABPP is a powerful proteomic tool allowing inhibitor activity profiling in
complex proteomes
The recombinant protein will be incubated with small molecule inhibitors
and then treated with fluorophosphonate (FP), a active site probes which
are specific for active serine hydrolases detected and quantified of by
immunoblot, fluorescent gel imaging or mass spectrometry.
Objective 1

1. In vitro HSL assay: Lipase activity will be measured by
monitoring the release of fatty acid from TAG, MAG and /or other
substrates
The assays will be performed in the presence or absence of
inhibitor using recombinant proteins or cell free lysate as enzyme
source.
The lipids were extracted and then resolved on a silica-TLC plate
using petroleum ether:diethyl ether: acetic acid (70:30:1, v/v) as
the solvent system.
2.Cell line study: Culture of 3T3-L1 cell lines. Differentiated 3T3-L1
cells will be treated with identified extracts for various
concentration and different time intervals. The cells will be
maintained at 37℃ with 5% CO2 throughout the experiments.
3. Diet induced mouse model for obesity and insulin resistance.
The experiments will be initiated with Male 4-week-old C57BL/6J
mice. Mice will be randomly divided into two groups normal or
control diet and high fat diet (HFD)
Objective 2

1. Biochemical analysis: The biochemical analysis will be
performed for different time interval in serum samples. Levels
of triglyceride, total cholesterol, HDL, LDL, alanine
aminotransferase (ALT), aspartate aminotransferase (AST),
blood urea nitrogen (BUN), and creatinine were analyzed with
an automatic analyser .
The concentrations of serum leptin and adiponectin will be
measured with mouse enzyme-linked immunosorbent assay kits.
The absorbance was measured using a microplate
spectrophotometer
1. Adipose tissue weight and morphology analysis. To assess the
sizes of the adipocytes, the area of 20 adipocyte cells was
measured in representative sections by light microscopy and an
image analysis program.
After the treatment period the white adipose tissues will be
removed from the mice and weighed immediately.
Objective 3

1. Protein expressional profile. The impact of identified small
molecules and Hormone sensitive lipase inhibition on protein
expression will be monitored by immunoblot analysis using
gene specific antibodies.
2. Toxicological studies: Repeated dose toxicity testing using oral
administration of a test substance in rodents for 28 and 90 days
is used to evaluate chronic toxic effects, primarily effects on
various organ systems, and to establish a no observed effect
level.
The endpoints for repeat dose testing consist of an evaluation
of clinical observations, blood analysis, whole body gross necropsy,
and microscopic examination of all organs and tissues
(histopathology)
Objective 4

. The deliverable will be the food-based potent Hormone
sensitive lipase inhibitor molecules for human welfare by
management as well as to prevent major life-style diseases,
obesity and diabetes. Food as a medicine to treat insulin
resistance mediated pathogenesis in obesity and diabetes.
Outcome from this project

Institute Manpo
wer
Budget
Consum
ables
Travel Equipme
nt
Conting
encies
Overhea
d
Costs
Total
Central
Food
Technolo
gical
Research
Institute,
MYSORE
DISTRICT
4,32,000 30,00,00
0
1,00,000 14,66,25
6
70,000 7,60,238 58,28,494
Total 4,32,000 30,00,00
0
1,00,000 14,66,25
6
70,000 7,60,238 58,28,494
BUDGET ESTIMATES

PROJECT PROPOSAL

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