diabetes insulin dependent diabetes mellitus non-insulin dependent diabetes mellitus gestational...
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DIABETES
Insulin Dependent Diabetes Mellitus
Non-Insulin Dependent Diabetes Mellitus
Gestational Diabetes
•Glucose Intolerance
•Trunal Obesity
•Hypertension
•Dyslipidemia
•Increased risk of coronary artery disease, etc.
Insulin Dependent Diabetes Mellitus
Non-Insulin Dependent Diabetes Mellitus
Gestational Diabetes
The cells of the Pancreas fail to produce insulin
So insulin is administered sub cutaneous to the patient
This develops during pregnancy and may disappear later. The patients develop a high risk of Type II Diabetes
This is due to Insulin Resistance i.e., low secretion of Insulin or insulin insensitivity of the cells.
Type II Diabetes mellitus
Current Therapeutic Agents
Glucosidase Inhibitors
Sulfonyl Ureas
Metformin
Thiazolidinediones
Insulin Injections
Side Effects
•Weight gain
•Hypoglycemia
•Gastrointestinal disturbances
•Lactic acidosis
•Edema
•Anemia
cellPreproinsulin
Endoplasmic Reticulum
Secretary granules
cytoplasm
Proinsulin
Insulin
C-peptide
Golgi
Insulin Secretion
IRS 1/2
P110
P85
Raf
MEK
MAPK
PIP3
PKC
PDK 1
Akt/PKB
RasGDP GTP
Gene expression
PI3 kinase
GSGS
Glycogen Synthesis
GSK 3
PP1
GLUT
GLUTPIPPIP2
Present Targets and their ValidationInsulin Trafficking
GLUT 4 RT-PCR, Western
GLUT 1 RT-PCR
PI3 Kinase RT-PCR
PKC RT-PCR
Insulin Sensitizer
PPAR RT-PCR
Glucose Storage
Glycogen Synthase RT-PCR
Potential future targetGSK-3 (Glycogen Synthase
Kinase- 3)RT-PCR
Plant MaterialHexane
DCM
Ethyl Acetate
Methanol
Active Extract
Bio Assay
Column Chromatography
Thin layer chromatograph
y
Pooling
Active Fraction
Bio Assay
Repeated till a single lead molecule is obtained
Lead Molecule
(Further Purification)HPLC
(Structure Elucidation)
NMR & Mass Spectroscopy
VALIDATIONRT-PCR, Western blotting
Incr
ea
sin
g P
ola
rity
Techniques Involved
Purification
(a) Extraction
3 days each
Hexane 0.0
Dichloromethane3.1
Ethyl Acetate4.4
Methanol5.1
(b) Chromatography
Solid – liquid Technique
2 phases Stationary phase
Mobile phasePrinciple:
Column Chromatography- POLARITY ADSORPTION
Thin Layer Chromatography CAPILLARY ACTION ADSORPTION
POLYMERASE CHAIN REACTION
CYCLING REACTIONS
Denaturation
Annealing Extension
n cycles
94 o C
72 o C
Standardized temperature
Agarose Gel Electrophoresis
TEB
Agarose
melt
Very small amount of Ethidium Bromide
1. Pour into Gel plate
2. Allow to solidify
+ -
3. Place the plate in the gel tank
Load samples mixed with Orange G
Set Voltage & Current
RUN THE GEL
Glucose uptake Assay
L 6 myotubes
Incubation ( 24 hr, 37oC, 5% CO2 )
Incubation ( 20 min, 37oC, 5% CO2 )
Termination of the Glucose Uptake
Spin [ 8000 rpm, 7 min ]
Spin [ 8000 rpm, 7 min ]
Incubate the lysate [Room Temp., overnight ]
Scintillation Count Plot the Graph
Dilutions of the plant extract
2 Deoxy D[3H] GlucoseDMEM medium
KRPH buffer
Ice cold KRPH buffer
buffer
STV buffer
KRPH
SDS
Supernatant
Supernatant