1. “Investigation of the role of thrombin in insulin resistance”
Ali Al-Bayati, Dhanisha Lukka, Audrey Brown and Mark Walker
Diabetes Research Group, Institute of Cellular Medicine, Faculty of Medical Science, Newcastle University
Thrombin increases the glucose uptake both basal and in response
to insulin in both myoblast and myotubes n=5
Introduction:
Thrombin, serine protease enzyme, is a major substrate in the blood coagulation process with other vital cellular functions. Thrombin acts on skeletal muscle cells via cell surface receptors named as PARs (protease activated receptors).
In type 2 diabetes, patients seem to be a hyper-coagulable (elevated thrombin) with high risk of thrombotic and vascular events.
A strong association between metabolic abnormalities such as hyperglycaemia and hyperinsulinemia was discovered with hyper-coagulability.
Thrombin shown to increase glucose transporters (GLUT1 and GLUT4)expression in cultured muscle cells **
Thrombin formation and action.
Hypothesis and Aims:
Our hypothesis was that Thrombin impairs insulin action in human skeletal muscle cells and the enhanced thrombin action in diabetic muscles might play a significant role in insulin resistant.
PKC inhibition shown to increase Akt phosphorylation in cultured muscle cells
The targets of the study were to explore the impact of thrombin on insulin action and glucose uptake and discovering its pathway in cultured human skeletal muscle cells, hoping to highlight the link between hypercoagulation and insulin resistance.
Methodology:
Western blotting for protein detection.
rPCR : for GLUT1 and GLUT 4 expression.
Glucose Uptake for the absorption of radio-labelled 2-deoxyglucose (2-DG) with tritium (3H).
Thrombin dose determination:
(1, 5 and 10units/mL) for either (6 or 24) hours incubation then measuring changes morphology and LDH cytotoxicity 6 hours of
5U (5µl)/ml of thrombin is the ideal dosage.
P-Akt
Treatment of cells with (1nM, 10 nM and 100 nM) of PKC general inhibitor (GF109203X ) then western blotting to show the dose effects of this inhibitor on insulin signaling represented by Akt phosphorylation .
T-Akt
Insulin
Discussion :
Results:
Data presented as mean ± SEM *= P. value < 0.05
Thrombin decreased activation of Akt and
decreased insulin signalling.
Thrombin increased absolute rates of glucose
uptake, associated with increased expression of
GLUT1 and GLUT4.
Effects of thrombin on Akt reversed by general PKC inhibition (GF109203X ).
Thrombin decrease Akt phosphorylation in response to insulin
in both myoblast and myotubes.
*careful examination of literatures revealed that different PKC isoforms have variable effects on insulin signaling pathway .
Conclusions:
Thrombin decreases insulin sensitivity by inhibiting Akt related pathways but not affects glucose uptake that could be due to different signalling pathway for example, (PKC related).
Further research needed to explore the exact PKC isoform role in potentiating thrombin actions.
P-Akt
T-Akt
Thrombin
Insulin