Hyperglycemia Alters Astrocyte Metabolism and Inhibits Astrocyte Proliferation
Li Wenjun1, Roy Choudhury Gourav1, Winters Ali1, Prah Jude1, Lin Wenping1,2, Liu Ran1, Yang Shao-Hua1,*
1Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, TX 76107, USA 2Department of Orthopedic Surgery, The Second Affiliated Hospital, Fujian Medical University, Fujian Province, 362000, China
Diabetes milieu is a complex metabolic disease that has been known to associate with high risk of various neurological disorders. Hyperglycemia in diabetes could dramatically increase neuronal glucose levels which leads to neuronal damage, a phenomenon referred to as glucose neurotoxicity. On the other hand, the impact of hyperglycemia on astrocytes has been less explored. Astrocytes play important roles in brain energy metabolism through neuron-astrocyte coupling. As the component of blood brain barrier, glucose might be primarily transported into astrocytes, hence, impose direct impact on astrocyte metabolism and function. In the present study, we determined the effect of high glucose on the energy metabolism and function of primary astrocytes. Hyperglycemia level glucose (25 mM) induced cell cycle arrest and inhibited proliferation and migration of primary astrocytes. Consistently, high glucose decreased cyclin D1 and D3 expression. High glucose enhanced glycolytic metabolism, increased ATP and glycogen content in primary astrocytes. In addition, high glucose activated AMP-activated protein kinase (AMPK) signaling pathway in astrocytes. In summary, our in vitro study indicated that hyperglycemia might impact astrocyte energy metabolism and function phenotype. Our study provides a potential mechanism which may underlie the diabetic cerebral neuropathy and warrant further in vivo study to determine the effect of hyperglycemia on astrocyte metabolism and function.
Figure 1. High glucose inhibited astrocyte proliferation
A) Growth curve assay of primary astrocytes cultured in normal (5.5 mM) and high glucose (11 or 25 mM) for 2 weeks. 40,000 cells per well were seeded in 6-well plates (n=6) and cells were counted at 1 to 5 days after culture at each condition. High glucose at 25 mM significantly inhibited primary astrocyte proliferation (* p < 0.05, n=6). B) Growth curve of astrocytes with medium switched on day 2 (n=3-6). Switch normal glucose medium (5.5 mM) dramatically inhibited astrocyte proliferation. Switch high glucose medium (25 mM) to normal glucose medium had no impact on astrocyte proliferation. C) Normal glucose cultured astrocytes were seeded and cultured for 2 days before medium was replaced with normal glucose medium (5.5 mM), normal glucose medium with mannitol (Mannitol), and high glucose medium (25 mM) (n=4). Osmolarity adjusted by mannitol has no impact on growth curve assay of primary astrocyte culture. D) Normal (5.5 mM) and high glucose (25 mM) cultured astrocytes were seeded in 96-well plates and cultured for 3 days and viability was analyzed by calcein AM. High glucose significant inhibited astrocyte proliferation (* p<0.05, n=10).
Figure 2. High glucose induced astrocyte cell cycle arrest without increasing apoptosis
A) Flow cytometry analysis of Annexin-V and PI staining of astrocytes cultured in normal (5.5 mM) and high glucose (25 mM) medium for 3 days (n=4). B) TUNEL staining (green) of astrocytes cultured in normal (5.5 mM) and high glucose (25 mM) for 3 days. Astrocytes were treated with 50 μM H2O2 for ~12 hours as positive control. Cells were counter stained with DAPI (blue). C) Astrocytes were cultured in normal (5.5 mM) and high glucose (25 mM) for 3 days. Then astrocytes were stained with PI and analysis by flow cytometry (* p<0.05 vs 5.5 mM, n=4). D) Real-time rtPCR analysis of cyclin expression in astrocytes cultured in normal (5.5 mM) and high glucose (25 mM) medium for 2 days (* p<0.05 vs 5.5 mM, n=4).
Figure 3. High glucose altered astrocyte morphology and inhibited cell migration
A) GFAP staining of astrocytes cultured in normal (5.5 mM) and high glucose (25 mM) for 3 days. Nucleus was stained by DAPI. B) Western blot analysis of GFAP expression in astrocytes cultured in normal (5.5 mM) and high glucose (25 mM) for 2 weeks (n=4). C) Astrocytes were maintained in normal (5.5 mM) and high glucose (25 mM) medium for 2 weeks and then seeded for scratch assay. Cells were staining with calcein AM and fluorescent images were obtained.
Figure 4. High glucose enhanced astrocyte glycolysis
Seahorse extracellular flux analysis of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) after 6-hour (A) and 24-hour culture (B) in normal (5.5 mM) and high glucose (25 mM) (* p<0.05 vs 5.5 mM, n=5-6). C) Real-time rtPCR analysis of HEKs and MCT1 expression of astrocytes after 24-hour culture in normal (5.5 mM) and high glucose (25 mM) (* p<0.05 vs 5.5 mM, n=6-8).
Figure 5. High glucose increased ATP content and glycogen storage
A) Total ATP level of astrocytes after 24- and 48-hour culture in normal (5.5 mM) and high glucose (25 mM) (*p<0.05 vs 5.5 mM, n=4). B) Cellular ATP level of astrocytes after 72-hour culture in normal (5.5 mM) and high glucose (25 mM). Oligomycin was added to astrocyte culture 2 hours before ATP assay (*p<0.05 vs 5.5 mM, n=3). C) Glycogen assay of astrocytes cultured for 72 hours in normal (5.5 mM) and high glucose (25 mM) (*p<0.05 vs 5.5 mM, n=4).
Figure 6. High glucose activated AMPK pathway
A) Western blots and statistical analysis of AMPK and mTOR pathway in astrocytes after 4-day culture in normal (5.5 mM) and high glucose (25 mM) (*p<0.05 vs 5.5 mM, n=3). B) Western blot and statistical analysis of mTOR, ACC and GS activation after 2-week culture in normal (5.5 mM) and high glucose (25 mM) (*p<0.05 vs 5.5 mM, n=3).
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