Research

Researcher: Ms Nikita Naicker

Designation: PhD study

Study: Trigonella foenum-graecum seed extract, 4-hydroxyisoleucine and metformin stimulate proximal insulin signaling and increases expression of glycogenic enzymes and GLUT2 in HepG2 cells

Summary: Fenugreek (Trigonella foenum-graecum) is globally recognized for its medicinal properties and hypoglycaemic effects. The seed extract as well as its active compound, 4-hydroxyisoleucine (4-OH-lle), have been shown to reduce hyperglycaemia insulin resistance. The mechanism by which this occurs has not been investigated in human liver cells (HepG2) in comparison to the anti-hyperglycaemic drug, metformin. We investigated the effect of fenugreek aqueous seed extract (FSE), 4-OH-lle and metformin in human hepatoma HepG2 cells relative to insulin as a positive control. Cells were treated with FSE and 4-OH-lle at 10ng/ml and 100ng/ml under normoglycaemic (5mM glucose) and hyperglycaemic (30mM glucose) conditions for 72h. Tyrosine phosphorylation of insulin receptor-ß (IR-ß), protein kinase B (Akt) and glycogen synthase kinase-3a/ß (GSK-3a/ß) was determined by western blotting. Gene expression of sterol regulatory element binding protein 1c (SREBP1c), glucose transporter 2 (GLUT2), glycogen synthase (GS) and glucokinase (GK) was evaluated by qPCR and supernatant glucose levels were measured using the Picollo Biochemistry Analyser. Under normo- and hyperglycaemic conditions, FSE, 4-OH-lle, insulin (100ng/ml) and metformin (2mM) caused a significant increase in tyrosine phosphorylation of IR-ß, Akt and GSK-3a/ß. Glucose uptake, however, was most significantly increased in FSE treated cells during normo-and hyperglycaemic conditions. FSE induced the most significant changes in downstream insulin signaling, GS, GK, SREBP1c and GLUT2 expression as compared to 4-OH-lle, metformin and insulin. Also, FSE significantly increased glucose uptake. Collectively, these findings provide a mechanism by which FSE exerts anti-hyperglycaemic effects similar to metformin and insulin occurs via enhanced insulin signaling, gene expression and increasing glucose uptake.

Researcher: Mr Naeem Sheik Abdul

Designation: Student

Study: Fusaric Acid Induces Mitochondrial Stress in Human Hepatocellular Carcinoma (HepG2) Cells

Summary: Mitochondria are attractive targets for cancer chemotherapy due to their role in apoptosis and endogenous reactive oxygen species (ROS) production. Fusaric acid (FA), a pyridine related compound and divalent ion chelator, is known to elevate levels of ROS and cause mitochondrial dysfunction. Silent information regulator 3 (SIRT3) is a NAD+ dependent deacytylase that localizes to the mitochondria where it deacytylates and activates proteins needed for ROS detoxification and mitochondrial maintenance. We investigated the effects of FA on mitochondrial stress and its ability to induce apoptosis in the HepG2 cell line. The effects of FA on SIRT3 activity and its substrates have yet to be evaluated in human cells.

FA cytotoxicity in HepG2 cells was evaluated (MTT assay; 24 h; [0-500 µg/ml]) to determine an IC50. ATP levels and caspase activity were measured using luminometry. Intracellular ROS and mitochondrial depolarization was evaluated by flow cytometry. Protein expression of Lon protease, NRF1, NRF2 and SIRT3 was quantified by western blot. qPCR was used to evaluate mitochondrial DNA viability and gene expression of OGG-1, Lon protease and TFAM.

FA caused a dose dependent decrease in viability of this cell line as well as changes to biochemical apoptotic markers, ROS levels, protein and gene expression profiles.

The results show that FA mediates mitochondrial stress and is cytotoxic to the cancerous liver (HepG2) cells.

Discipline Research