ABSTRACTAims/hypothesisRecurrent hypoglycaemia (RH) is a major side-effect of intensive insulin therapy for people with diabetes. Changes in hypoglycaemia sensing by the brain contribute to the development of impaired counterregulatory responses to and awareness of hypoglycaemia. Little is known about the intrinsic changes in human astrocytes in response to acute and recurrent low glucose (RLG) exposure.MethodsHuman primary astrocytes (HPA) were exposed to zero, one, three or four bouts of low glucose (0.1 mmol/l) for three hours per day for four days to mimic RH. On the fourth day, DNA and RNA were collected. Differential gene expression and ontology analyses were performed using DESeq2 and GOseq respectively. DNA methylation was assessed using the Infinium MethylationEPIC BeadChip platform.Results24 differentially expressed genes (DEGs) were detected (after correction for multiple comparisons). One bout of low glucose exposure had the largest effect on gene expression. Pathway analyses revealed that endoplasmic-reticulum (ER) stress-related genes such as HSPA5, XBP1, and MANF, involved in the unfolded protein response (UPR), were all significantly increased following LG exposure, which was diminished following RLG. There was little correlation between differentially methylated positions and changes in gene expression yet the number of bouts of LG exposure produced distinct methylation signatures.Conclusions/interpretationThese data suggest that exposure of human astrocytes to transient LG triggers activation of genes involved in the UPR linked to endoplasmic reticulum (ER) stress. Following RLG, the activation of UPR related genes was diminished, suggesting attenuated ER stress. This may be mediated by metabolic adaptations to better preserve intracellular and/or ER ATP levels, but this requires further investigation.

ABSTRACTAMP-activated protein kinase (AMPK) is a critical cellular and whole body energy sensor activated by energy stress, including hypoglycemia, which is frequently experienced by people with diabetes. Previous studies using direct delivery of an AMPK activator to the ventromedial hypothalamus (VMH) in rodents increased hepatic glucose production. Moreover, recurrent glucoprivation in the hypothalamus leads to blunted AMPK activation and defective hormonal responses to subsequent hypoglycemia. These data suggest that amplifying AMPK activation may prevent or reduce frequency hypoglycemia in diabetes. We used a novel brain-permeable AMPK activator, R481, which potently increased AMPK phosphorylation in vitro. R481 significantly increased peak glucose levels during glucose tolerance tests in rats, which were attenuated by treatment with AMPK inhibitor SBI-0206965 and completely abolished by blockade of the autonomic nervous system. This occurred without altering insulin sensitivity measured by hyperinsulinemic-euglycemic clamps. Endogenous insulin secretion was not altered by R481 treatment. During hyperinsulinemic-hypoglycemic clamp studies, R481 treatment reduced exogenous glucose requirements and amplified peak glucagon levels during hypoglycemia. These data demonstrate that peripheral administration of the brain permeable AMPK activator R481 amplifies the counterregulatory response to hypoglycemia in rats, which could have clinical relevance for prevention of hypoglycemia.

ABSTRACTHypoglycemia is a major barrier to good glucose control in type 1 diabetes and frequent exposure to hypoglycemia can impair awareness to subsequent bouts of hypoglycemia. The neural changes that occur to reduce a person’s awareness of hypoglycemia are poorly defined. Moreover, the molecular mechanisms by which glial cells contribute to hypoglycemia sensing and glucose counterregulation require further investigation. To test whether glia, specifically astrocytes, could detect changes in glucose, we utilized human primary astrocytes (HPA) and U373 astrocytoma cells and exposed them to recurrent low glucose (RLG) in vitro. This allowed measurement, with high specificity and sensitivity, of changes in cellular metabolism following RLG. We report that the AMP-activated protein kinase (AMPK) is activated over a pathophysiologically-relevant glucose concentration range. We observed an increased dependency on fatty acid oxidation for basal mitochondrial metabolism and 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, nor were glucose uptake or glycogen levels. Taken together, these data indicate that astrocyte mitochondria are dysfunctional following recurrent low glucose exposure, which could have implications for hypoglycemia glucose counterregulation and/or hypoglycemia awareness.

AbstractInflammation and metabolism are intrinsically linked with inflammatory stimuli inducing metabolic changes in cells and, in turn, metabolic capacity determining cellular inflammatory responses. Although well characterized in peripheral immune cells there is comparatively less known about these “immunometabolic” responses in astrocytes. In this study, we tested the hypothesis that the astrocytic inflammatory response driven by nuclear factor‐kappa B (NF‐κB) signaling is dependent on glycolytic metabolism. Using mouse primary cortical astrocyte cultures, we assessed changes in cellular metabolism after exposure to lipopolysaccharide (LPS), with cytokine ELISAs and immunoblotting being used to measure inflammatory responses. Results indicate temporally distinct metabolic adaptations to pro‐inflammatory stimulation in astrocytes: 3 hr LPS treatment increased glycolysis but did not alter mitochondrial metabolism, while following 24 hr of LPS treatment we observed increased oxidative phosphorylation, and decreased glycolytic capacity and glucose uptake, partly due to reduced glucose transporter 1 expression. Inhibition of NF‐κB signaling with the IKK‐beta inhibitor TPCA‐1 prevented the LPS induced changes to glycolysis and oxidative phosphorylation. Furthermore, TPCA‐1 treatment altered both glycolysis and oxidative phosphorylation independently from inflammatory stimulation, indicating a role for NF‐κB signaling in regulation of basal metabolism in astrocytes. Inhibition of glycolysis with 2‐deoxyglucose significantly attenuated LPS‐induced cytokine release and NF‐κB phosphorylation, indicating that intact glycolysis is required for the full inflammatory response to LPS. Together our data indicate that astrocytes display immunometabolic responses to acute LPS stimulation which may represent a potential therapeutic target for neuroinflammatory disorders.