Cell proliferation under hyperglycemia.

<p>Cells were cultured in three concentrations of glucose including 5(normal) and 10 and 25 mM (high glucose). Changes in cell proliferation were assessed via MTS assay at 24, 48, and 72 hr in estrogen-receptor positive breast cancer cells (A), triple negative breast cancer cells (B), and non-tumorigenic breast epithelial cells (C). In each case, results were first normalized to baseline growth at 24 hr for each condition and expressed as fold change in cell proliferation above the 24 hr baseline. Results shown here are representative of at least five separate experiments. Statistical significance was calculated using unpaired Student's t-test. Results are expressed as mean±SEM. ns, not statistically significant *, p-value≤0.05 **, p-value≤0.01 ***, p-value≤0.001.</p

Proposed model of hyperglycemia's protumorigenic effects.

<p>Under normal glucose levels or normoglycemia, glucose uptake and leptin/IGF1R signaling remain at relatively low basal levels. Under hyperglycemia, cells respond by increasing glucose uptake through GLUT receptors and this in turn activates metabolic pathways that not only respond to increased glucose levels but also have mitogenic effects on breast epithelial cells. Two key metabolic pathways that can have this effect include the leptin and IGF1R signaling pathways. Breast epithelial cells respond to increased glucose levels by increasing production of leptin and IGF1, which in turn increase the expression and activation of leptin and IGF1 receptors at the cell surface. Together, leptin and IGF1 act synergistically to enhance AKT/mTOR signaling leading to increased cell proliferation, in part, through modulation of PKC proteins and cell cycle-associated proteins.</p

Cell cycle proteins increase with hyperglycemia.

<p>Cells were cultured 24 and 72(N) and high glucose (HG). Levels of CDK2 and Cyclin D1, which enhance progression through the G1 and S phases of the cell cycle, were assessed by Western blot. Levels of these proteins were assessed in estrogen-receptor positive breast cancer cells (A), triple-negative breast cancer cells (B), and non-tumorigenic breast epithelial cells (C). Ten micrograms of protein was loaded in each lane. Results shown are representative of two separate experiments.</p

Expression and activation of AKT and mTOR.

<p>Changes in the phosphorylated and total form of AKT (left panel) and mTOR (right panel) were assessed via Western blot in estrogen-receptor positive MCF7 cells (A), triple-negative MDA-231 cells (B), and non-tumorigenic MCF10A cells (C) exposed to either normal (5 mM) or high glucose (10 mM) levels. Twenty micrograms of total protein was loaded for each sample and changes in protein levels were determined at 24 hr and 72 hr. Results are representative of at least three separate experiments.</p

Expression and activation of IGF1R/IGF-1 signaling.

<p>Cells were cultured in normal (N) and high glucose (HG) for 24 and 72 hr. Using Western blot, changes in the phosphorylated and total form of IGF1R were determined for normal and diabetic conditions (left panel). Likewise, differences in IGF1 production in normoglycemia vs. hyperglycemia were assessed via Western blot (right panel, D). At least three separate experiments were performed and representative images are shown here.</p

Expression and activation of IRS1/IRS2.

<p>MCF7 (A), MDA-231 (B), and MCF10A cells (C) were cultured in 5 mM (N) or 10 mM glucose (HG). After 24 and 72 hr, cells were harvested and whole cell lysates were subjected to Western blot analysis for IRS1 (left panel) and IRS2 (right panel). A total of 10 µg of protein was loaded per sample and results shown are representative of three replicate experiments.</p

Leptin receptor expression.

<p>Cells were cultured in normal (N) and high glucose (HG) for 24 and 72 hr. Changes in leptin receptor expression were assessed via Western blot for estrogen-receptor positive breast cancer cells (A), triple-negative breast cancer cells (B), and non-tumorigenic breast epithelial cells (C). A total of 10 µg of protein was loaded for each sample. Results are representative of at least three separate experiments. ‘OB-Rs’ is the short isoform of leptin receptor, ‘OB-Rl’ is the long isoform of leptin receptor.</p

Expression and activation of STAT3.

<p>Cells were cultured in normal (N) and high glucose (HG) levels for 24 and 72 hr. Changes in the phosphorylated and total forms of STAT3 were detected via Western blot in MCF7 cells (A), MDA-231 cells (B), and non-tumorigenic MCF10A cells (C). Fifteen micrograms of protein was loaded for each sample. Data shown is representative of at least three separate experiments.</p