APOΕ4 lowers energy expenditure in females and impairs glucose oxidation by increasing flux through aerobic glycolysis.

BACKGROUND: Cerebral glucose hypometabolism is consistently observed in individuals with Alzheimer's disease (AD), as well as in young cognitively normal carriers of the Ε4 allele of Apolipoprotein E (APOE), the strongest genetic predictor of late-onset AD. While this clinical feature has been described for over two decades, the mechanism underlying these changes in cerebral glucose metabolism remains a critical knowledge gap in the field. METHODS: Here, we undertook a multi-omic approach by combining single-cell RNA sequencing (scRNAseq) and stable isotope resolved metabolomics (SIRM) to define a metabolic rewiring across astrocytes, brain tissue, mice, and human subjects expressing APOE4. RESULTS: Single-cell analysis of brain tissue from mice expressing human APOE revealed E4-associated decreases in genes related to oxidative phosphorylation, particularly in astrocytes. This shift was confirmed on a metabolic level with isotopic tracing of 13C-glucose in E4 mice and astrocytes, which showed decreased pyruvate entry into the TCA cycle and increased lactate synthesis. Metabolic phenotyping of E4 astrocytes showed elevated glycolytic activity, decreased oxygen consumption, blunted oxidative flexibility, and a lower rate of glucose oxidation in the presence of lactate. Together, these cellular findings suggest an E4-associated increase in aerobic glycolysis (i.e. the Warburg effect). To test whether this phenomenon translated to APOE4 humans, we analyzed the plasma metabolome of young and middle-aged human participants with and without the Ε4 allele, and used indirect calorimetry to measure whole body oxygen consumption and energy expenditure. In line with data from E4-expressing female mice, a subgroup analysis revealed that young female E4 carriers showed a striking decrease in energy expenditure compared to non-carriers. This decrease in energy expenditure was primarily driven by a lower rate of oxygen consumption, and was exaggerated following a dietary glucose challenge. Further, the stunted oxygen consumption was accompanied by markedly increased lactate in the plasma of E4 carriers, and a pathway analysis of the plasma metabolome suggested an increase in aerobic glycolysis. CONCLUSIONS: Together, these results suggest astrocyte, brain and system-level metabolic reprogramming in the presence of APOE4, a 'Warburg like' endophenotype that is observable in young females decades prior to clinically manifest AD.

Oral Gavage Delivery of Stable Isotope Tracer for In Vivo Metabolomics.

Stable isotope-resolved metabolomics (SIRM) is a powerful tool for understanding disease. Advances in SIRM techniques have improved isotopic delivery and expanded the workflow from exclusively in vitro applications to in vivo methodologies to study systemic metabolism. Here, we report a simple, minimally-invasive and cost-effective method of tracer delivery to study SIRM in vivo in laboratory mice. Following a brief fasting period, we orally administered a solution of [U-13C] glucose through a blunt gavage needle without anesthesia, at a physiological dose commonly used for glucose tolerance tests (2 g/kg bodyweight). We defined isotopic enrichment in plasma and tissue at 15, 30, 120, and 240 min post-gavage. 13C-labeled glucose peaked in plasma around 15 min post-gavage, followed by period of metabolic decay and clearance until 4 h. We demonstrate robust enrichment of a variety of central carbon metabolites in the plasma, brain and liver of C57/BL6 mice, including amino acids, neurotransmitters, and glycolytic and tricarboxylic acid (TCA) cycle intermediates. We then applied this method to study in vivo metabolism in two distinct mouse models of diseases known to involve dysregulation of glucose metabolism: Alzheimer's disease and type II diabetes. By delivering [U-13C] glucose via oral gavage to the 5XFAD Alzheimer's disease model and the Lepob/ob type II diabetes model, we were able to resolve significant differences in multiple central carbon pathways in both model systems, thus providing evidence of the utility of this method to study diseases with metabolic components. Together, these data clearly demonstrate the efficacy and efficiency of an oral gavage delivery method, and present a clear time course for 13C enrichment in plasma, liver and brain of mice following oral gavage of [U-13C] glucose-data we hope will aid other researchers in their own 13C-glucose metabolomics study design.

APOE alters glucose flux through central carbon pathways in astrocytes.

The Apolipoprotein E (APOE) gene is a major genetic risk factor associated with Alzheimer's disease (AD). APOE encodes for three main isoforms in humans (E2, E3, and E4). Homozygous E4 individuals have more than a 10-fold higher risk for developing late-onset AD, while E2 carriers are protected. A hallmark of AD is a reduction in cerebral glucose metabolism, alluding to a strong metabolic component in disease onset and progression. Interestingly, E4 individuals display a similar regional pattern of cerebral glucose hypometabolism decades prior to disease onset. Mapping this metabolic landscape may help elucidate the underlying biological mechanism of APOE-associated risk for AD. Efficient metabolic coupling of neurons and glia is necessary for proper neuronal function, and disruption in glial energy distribution has been proposed to contribute to neuronal cell death and AD pathology. One important function of astrocytes - canonically the primary source of apolipoprotein E in the brain - is to provide metabolic substrates (lactate, lipids, amino acids and neurotransmitters) to neurons. Here we investigate the effects of APOE on astrocyte glucose metabolism in vitro utilizing scintillation proximity assays, stable isotope tracer metabolomics, and gene expression analyses. Glucose uptake is impaired in E4 astrocytes relative to E2 or E3 with specific alterations in central carbon metabolism. Using stable isotope labeled glucose [U-13C] allowed analyses of astrocyte-specific deep metabolic networks affected by APOE, and provided insight to the effects downstream of glucose uptake. Enrichment of 13C in early steps of glycolysis was lowest in E4 astrocytes (highest in E2), while synthesis of lactate from glucose was highest in E4 astrocytes (lowest in E2). We observed an increase in glucose flux through the pentose phosphate pathway (PPP), with downstream increases in gluconeogenesis, lipid, and de novo nucleotide biosynthesis in E4 astrocytes. There was also a marked increase in 13C enrichment in the TCA cycle of E4 astrocytes - whose substrates were also incorporated into biosynthetic pathways at a higher rate. Pyruvate carboxylase (PC) and pyruvate dehydrogenase (PDH) are the two main enzymes controlling pyruvate entry to the TCA cycle. PC gene expression is increased in E4 astrocytes and the activity relative to PDH was also increased, compared to E2 or E3. Decreased enrichment in the TCA cycle of E2 and E3 astrocytes is suggestive of increased oxidation and non-glucose derived anaplerosis, which could be fueling mitochondrial ATP production. Conversely, E4 astrocytes appear to increase carbon flux into the TCA cycle to fuel cataplerosis. Together, these data demonstrate clear APOE isoform-specific effects on glucose utilization in astrocytes, including E4-associated increases in lactate synthesis, PPP flux, and de novo biosynthesis pathways.