Abstract
Aims/hypothesisHypoglycaemia is a major barrier to good glucose control in type 1 diabetes. Frequent hypoglycaemic episodes impair awareness of subsequent hypoglycaemic bouts. Neural changes underpinning awareness of hypoglycaemia are poorly defined and molecular mechanisms by which glial cells contribute to hypoglycaemia sensing and glucose counterregulation require further investigation. The aim of the current study was to examine whether, and by what mechanism, human primary astrocyte (HPA) function was altered by acute and recurrent low glucose (RLG).MethodsTo test whether glia, specifically astrocytes, could detect changes in glucose, we utilised HPA and U373 astrocytoma cells and exposed them to RLG in vitro. This allowed measurement, with high specificity and sensitivity, of RLG-associated changes in cellular metabolism. We examined changes in protein phosphorylation/expression using western blotting. Metabolic function was assessed using a Seahorse extracellular flux analyser. Immunofluorescent imaging was used to examine cell morphology and enzymatic assays were used to measure lactate release, glycogen content, intracellular ATP and nucleotide ratios.ResultsAMP-activated protein kinase (AMPK) was activated over a pathophysiologically relevant glucose concentration range. RLG produced an increased dependency on fatty acid oxidation for basal mitochondrial metabolism and exhibited hallmarks of mitochondrial stress, including increased proton leak and reduced coupling efficiency. Relative to glucose availability, lactate release increased during low glucose but this was not modified by RLG. Basal glucose uptake was not modified by RLG and glycogen levels were similar in control and RLG-treated cells. Mitochondrial adaptations to RLG were partially recovered by maintaining euglycaemic levels of glucose following RLG exposure.Conclusions/interpretationTaken together, these data indicate that HPA mitochondria are altered following RLG, with a metabolic switch towards increased fatty acid oxidation, suggesting glial adaptations to RLG involve altered mitochondrial metabolism that could contribute to defective glucose counterregulation to hypoglycaemia in diabetes.
Highlights
Hypoglycaemia remains a major concern for people with type 1 diabetes and advanced insulin-treated type 2 diabetes
AMPK is activated by acute low glucose in human astrocytes AMPK activation in neurons is required for sensing hypoglycaemia [25], though it is not known whether astrocytic AMPK reacts to the same energy stress
In cells exposed to low glucose for 30 min, we noted, in both the human primary astrocyte (HPA) (Fig. 1a, c) and U373 cells (Fig. 1b, d), an increased phosphorylation of AMPK at threonine 172, a site which must be phosphorylated for full kinase activation
Summary
Hypoglycaemia remains a major concern for people with type 1 diabetes and advanced insulin-treated type 2 diabetes. Hypoglycaemia is a major barrier to enjoying the benefits of exercise [1, 2] and is increasingly thought to increase the risk of cardiovascular events in both type 1 and type 2 diabetes [3, 4]. The largest group is the glial cells, the largest subpopulation of which are astrocytes. As well as exhibiting a range of cell-to-cell signalling pathways, astrocytes are increasingly recognised as important players in central nervous system-based diseases, such as obesity [8], Alzheimer’s disease [9], Parkinson’s disease [10] and motor neuron disease [11]
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