Adult female O. degus (1, 3, 5, and 6 years old; n = 4–5 at each age) were obtained from our colony at the Faculty of Biological Sciences, Pontificia Universidad Católica de Chile. These animals were all derived from wild-caught and captive-born degus. The degus were kept in pairs of related and unrelated females, housed in clear acrylic cages (length × height × depth: 50 cm × 35 cm × 23 cm) with hardwood chip bedding, and provided with water and food (rabbit commercial pellet; Champion, Santiago, Chile) ad libitum. Additionally, each cage contained one nest box made of clear acrylic (22 × 12 × 15 cm). The animals were kept in a ventilated room with a natural photoperiod and controlled temperature (yearly minimum = 13.4 ± 0.2°C; yearly maximum = 24.9 ± 0.2°C). All experiments followed the National Institutes of Health guidelines (NIH, Baltimore, MD). All procedures were approved by the Bioethical and Biosafety Committee of the Faculty of Biological Sciences of the Pontificia Universidad Católica de Chile (CBB-121-2013). All efforts were made to minimize the distress and suffering of animals as well as to reduce the number of animals used.

To study the uptake of glucose in degus in vivo, four animals of each age were injected with D-[1-¹⁴C] glucose via the tail vein. Briefly, the degus were anesthetized with isoflurane and injected intravenously via the tail with 50 μCi of tracer diluted to a final volume of 20 μl in isotonic saline. Following a 15-min uptake, the animals were killed, and tissues were collected. Tissue radioactivity was quantified by liquid scintillation. D-[1-¹⁴C] glucose levels were normalized to the weight of the resected tissue and expressed as the percent injected dose (Tsytsarev et al., 2012; Cox et al., 2014). We measured glucose uptake in the whole brain, hippocampus, and cortex as described previously (Cisternas et al., 2019).

Hippocampal slices were prepared as previously described (Cerpa et al., 2010). Briefly, transverse slices (350 μm) from the dorsal hippocampus were sectioned in cold artificial cerebrospinal fluid (ACSF) and incubated for 1 h at room temperature. After incubation, slices were treated to measure glucose metabolism for different lengths of time (0–60 min). The slices were then used in different glucose metabolism assays, namely, glucose uptakes, glycolytic rates, PPP, ATP/ADP levels, the NADPH/NADP+ ratios, and enzyme activity tests, such as HK and G6PDH.

Messenger ribonucleic acid was obtained from hippocampal tissue and used to generate cDNA. Quantitative real-time RT–PCR (qRT–PCR) was conducted using SYBR master mix and 18S mRNA as a control, according to the instructions of the manufacturer, as described previously (Cisternas et al., 2016). As a housekeeping gene, we used cyclophilin, and the values were calculated using the delta Ct and normalized to those of the control gene. Duplicate control reactions for every sample without reverse transcription were included to ensure that the PCR products were not due to amplification of contaminant genomic DNA. We used the following sets of primers: 18S gene, forward 5′-TCAACGAGGAATGCCTAGTAAGC-3′and reverse5′-ACAAAGGGCAGGGACGTAGTC-3′; cyclophilin, forward 5′-TGGAGATGAATCTGTAGGAGGAG-3′ and reverse 5′-TACCACATCCATGCCCTCTAGAA-3′; Glut1, forward 5′-ATGGATCCCAGCAGCAAGAAG-3′ and reverse 5′-AGAGACCAAAGCGTGGTGAG-3′; Glut3, forward 5′-GGATCCCTTG-3′ and reverse 5′-ACCAGTTCCCAATGCACACA-3′; Glut4, forward 5′-CGGCTCTGACGATGGGGAA-3′ and reverse 5′-TTGTGGGATGGAATCCGGTC-3′; hexokinase-1, forward 5′-GGATGGGAACTCTCCCCTG-3′ and reverse 5′-GCATACGTGCTGGACCGATA-3′; phosphofructokinase-1, forward 5′-AGGGCCTTGTCATCATTGGG-3′ and reverse 5′-ACTGCTTCCTGCCTTCCATC-3′.

As previously described, the slices were washed with an incubation buffer (15-mM HEPES, 135-mM NaCl, 5-mM KCl, 1.8-mM CaCl₂, and 0.8-mM MgCl₂), supplemented with 0.5-mM glucose (Cisternas et al., 2014). Slices were then incubated for 0–60 min with 1–1.2 μCi 2-deoxy-D-[1,2-(N)3H] or glucose [(2-H3)-DG] (PerkinElmer, Waltham, MA, USA) at a final specific activity of 1–3 disintegrations/min/pmol (~1 mCi/mmol). Glucose uptake was arrested by washing the slices with ice-cold PBS, supplemented with 1-mM HgCl₂. The incorporated radioactivity was quantified by liquid scintillation counting.

Glycolytic rates were determined as previously described (Herrero-Mendez et al., 2009; Cisternas et al., 2014). Briefly, slices were placed in tubes containing 5-mM glucose and then washed two times in Krebs Henseleit solution, containing the appropriate concentration of glucose. After equilibration in 0.5 ml of Hank's balanced salt solution/glucose at 37°C for 10 min, 0.5 ml of Hank's balanced salt solution containing various concentrations of [3-³H] glucose was added, with a final specific activity of 1–3 disintegrations/min/pmol (~1 mCi/mmol). Aliquots of 100 μl were then transferred to another tube, placed inside a capped scintillation vial containing 0.5 ml of water and incubated at 45°C for 48 h. After this vapor-phase equilibration step, the tube was removed from the vial, a scintillation mixture was added, and the ³H₂O content was measured by counting over a 5 min period.

Glucose oxidation via the PPP was measured as previously described based on the difference in ¹⁴CO₂ production from [1-¹⁴C] glucose (decarboxylated in the 6-phosphogluconate dehydrogenase-catalyzed reaction and in the Krebs cycle) and [6-¹⁴C] glucose (decarboxylated only in the Krebs cycle) (Cisternas et al., 2014). Slices were washed with ice-cold PBS and collected by trypsinization. The tissue was then resuspended in an O₂-saturated Krebs Henseleit buffer, and 500 μl of this suspension (~10⁶ cells) was placed in Erlenmeyer flasks with another 0.5 ml of Krebs Henseleit solution containing 0.5 μCi D-[1-¹⁴C] glucose or 2 μCi D-[6-¹⁴C] glucose and 5.5-mM D-glucose (final concentration). The Erlenmeyer flasks were equipped with a central well-containing an Eppendorf tube with 500 μl of benzethonium hydroxide. The flasks were flushed with O₂ for 20 s, sealed with rubber caps, and incubated for 60 min in a 37°C water bath with shaking. The incubations were stopped by the addition of 0.2 ml of 1.75-M HClO₄ into the main well, and shaking was continued for another 20 min to facilitate the trapping of ¹⁴CO₂ by benzethonium hydroxide. Radioactivity was quantified as previously described by liquid scintillation spectrometry (Bolaños et al., 2008; Herrero-Mendez et al., 2009). Both [1-¹⁴C] glucose and [6-¹⁴C] glucose were purchased from PerkinElmer (Waltham, MA, USA).

Brain slices were washed with PBS, treated with trypsin/EDTA, and centrifuged at 500 × g for 5 min at 4°C. Then, the tissue was resuspended in isolation medium (250-mM sucrose, 20-mM HEPES, 10-mM KCl, 1.5-mM MgCl₂, 1-mM EDTA, 1-mM DTT, 2-mg/ml aprotinin, 1-mg/ml pepstatin A, and 2-mg/ml leupeptin) at a 1:3 dilution, sonicated at 4°C, and then centrifuged at 1,500 × g for 5 min at 4°C. The HK activity of the supernatant was quantified. For the assay, the purified fraction was mixed with the reaction medium (25-mM Tris-HCl, 1-mM DTT,0.5 mM NADP/Na⁺, 2 mM MgCl₂, 1 mM ATP, 2-U/ml G6PDH, and 10-mM glucose), and the mixture was incubated at 37°C for 30 min. The reaction was stopped by the addition of 10% trichloroacetic acid (TCA), and the generation of NADPH was measured at 340 nm (Cisternas et al., 2016).

The cellular ADP and ATP levels were measured in slices as previously described using an assay kit according to the instructions of the manufacturer (Cisternas et al., 2019). The NADPH/NADP⁺ ratio was measured using a commercial colorimetric assay, according to the instructions of the manufacturer, as described previously (Sun et al., 2017).

Slices were washed with PBS, collected by trypsinization [0.25% trypsin-0.2% EDTA (w/v)], and pelleted. The tissue was then resuspended in isolation medium (250-mM sucrose, 20-mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1-mM DTT, 2 mg/ml aprotinin, 1-mg/ml pepstatin A, and 2 mg/ml leupeptin) at a 1:3 dilution, sonicated at 4°C, and centrifuged for 5 min at 1,500 × g at 4°C. Subsequently, the pellet was discarded, and the supernatant was further separated by centrifugation at 13,000 × g for 30 min at 4°C. Finally, the G6PDH activity of the supernatant was quantified in a reaction buffer, containing 1-mM ATP and 10 mM glucose-6-phosphate (G6P) for 30 min at 37°C. The reaction was stopped by the addition of 10% TCA. The generation of NADPH was measured at 340 nm, as described previously (Tsai and Chen, 1998).

All experiments were performed four to five times (biological replicates), with triplicates of each condition in each experimental run. The results are expressed as the mean ± SEM. The data were analyzed by one-way or two-way analysis of variance (ANOVA) and adjusted for multiple comparisons using Bonferroni's post-hoc test; ^*p ≤ 0.05, ^**p ≤ 0.01, and ^***p ≤ 0.001 were considered indicative of significant differences. Statistical analyses were performed using Prism software (GraphPad, La Jolla, CA).

To evaluate whether degus exhibit alterations in brain glucose metabolism, we first injected animals with D-[¹⁴C]-glucose (¹⁴C-G) and followed its uptake in the brain. Our initial analysis showed no differences in whole brain ¹⁴C-G uptake in 3-year-old animals compared to that in 1-year-old control degus. However, 5-year-old animals exhibited a significant decrease in ¹⁴C-G uptake, which was further reduced in 6-year-old animals (one-way ANOVA F_(3,12) = 55.17, p < 0.0001, followed by Bonferroni's post-hoc test, WT vs. 5 years old, p = 0.0004 and WT vs. 6 years old, p < 0.0001) (Figure 1A). Importantly, ¹⁴C-G uptake in the hippocampus and cortex was also observed, with 58 and 60% reductions in 6-year-old degus relative to the control group, respectively (one-way ANOVA F_(3,16) = 11, p < 0.0001 and F_(3,16) = 79.41, p < 0.0001, followed by Bonferroni's post-hoc test, WT vs. 6 years old, p < 0.0001 and WT vs. 6 years old, p < 0.0001 for hippocampus and cortex, respectively) (Figures 1B,C). To evaluate whether the observed disturbances in glucose uptake are caused by alterations in Gluts, we measured the mRNA expression of hippocampal Glut 1, 3, and 4. No differences in Glut1 expression were observed in any of the groups (one-way ANOVA F_(3,16) = 0.1298, p = 0.9410) (Figure 2A). However, Glut3 expression decreased in 5- and 6-year-old degus relative to 1-year-old control animals (One-way ANOVA F_(3,16) = 43.68, p < 0.0001, followed by Bonferroni's post-hoc test, WT vs. 5 years old, p = 0.0007 and WT vs. 6 years old, p < 0.0001) (Figure 2B). The expression of Glut4, on the other hand, showed a significant decrease only in 6-year-old animals compared with 1-year-old animals (one-way ANOVA F_(3,16) = 23.79, p < 0.0001, followed by Bonferroni's post-hoc test, WT vs. 6 years old, p < 0.0001) (Figure 2C). To gain further insight into glucose metabolism alterations in degus, we measured the expression of two key glycolytic enzymes, phosphofructokinase 1 (PFK1) and hexokinase (HK). HK, the initial enzyme of glycolysis, phosphorylates glucose to glucose 6-phosphate, whereas PFK1 converts fructose 6-phosphate to fructose 1,6-biphosphate. Interestingly, levels of both enzymes were significantly reduced in both 5- and 6-year-old animals for PFK1 and only in 6-year-old animals for HK (one-way ANOVA F_(3,17) = 110, p < 0.0001 and F_(3,16) = 18.94, p < 0.0001, followed by Bonferroni's post-hoc test, WT vs. 5 years old, p = 0.0022 and WT vs. 6 years old, p < 0.0001 for PFK1 and HK, respectively) (Figures 2D,E). Given the reduction in HK expression, we also measured HK activity. Similar to our expression results, HK activity was significantly decreased in 5- and 6-year-old animals relative to controls (one-way ANOVA F _(3,16) = 72.41, p < 0.0001, followed by Bonferroni's post-hoc test, WT vs. 5 years old, p = 0.0011 and WT vs. 6 years old, p < 0.0001) (Figure 2F).

Once inside the cells, glucose undergoes phosphorylation to form G6P, which can be metabolized by glycolysis to produce ATP and NADH or by the PPP, the main source of NADPH synthesis. To determine whether age can shift G6P utilization in degus, we measured glucose uptake and followed both the PPP and glycolytic fluxes in hippocampal slices. Similar to our in vivo results, we found a significant glucose uptake reduction in hippocampal slices from 5- and 6-year-old degus after 60 min compared to those from 1-year-old controls (Two-way ANOVA F_(9,38) = 4.69, P = 0.0003, followed by Bonferroni's post-hoc test, WT vs. 6 years old p < 0.0001 for Figure 3A. One-way ANOVA F_(3,16) = 79.71, p < 0.0001, followed by Bonferroni's post-hoc test, WT vs. 5 years old p = 0.0078 and WT vs. 6 years old p < 0.0001 for Figure 3B) (Figures 3A,B). Interestingly, the rate of glycolysis was significantly lower in 5-year-old animals than in control animals and further decreased at 6 years; however, no differences were observed in the 3-year-old group (Two-way ANOVA F_(9,48) = 9.93, p < 0.0001, followed by Bonferroni's post-hoc test, WT vs. 3 years old, p = 0.0253, WT vs. 5 years old, p < 0.0001 and WT vs. 6 years old, p < 0.0001 for Figure 3C. One-way ANOVA F_(3,16) = 90.41, p < 0.0001, followed by Bonferroni's post-hoc test, WT vs. 5 and 6 years old, p < 0.0001 for Figure 3D) (Figures 3C,D). Similarly, the rate of glucose oxidized through the PPP in 5- and 6-year-old degus was reduced by ~27 and 59%, respectively, relative to the control (one-way ANOVA F_(3,16) = 67.05, p < 0.0001, followed by Bonferroni's post-hoc test, WT vs. 5 years old, p = 0.0005 and WT vs. 6 years old, p < 0.0001) (Figure 3E). To further confirm the drop in the PPP flux, we also measured the NADPH/NADP⁺ ratio and the activity of glucose-6-phosphate dehydrogenase (G6PDH), the first and rate-limiting enzyme of the PPP. Our results showed an important drop in the NADPH/NADP⁺ ratio of both 5- and 6-year-old animals compared with that of 1-year-old controls (one-way ANOVA F_(3,16) = 69.86, p < 0.0001, followed by Bonferroni's post-hoc test, both WT vs. 5 years old and WT vs. 6 years old p < 0.0001) (Figure 4A). Moreover, and in accordance with our results, we observed a significant reduction in G6PDH activity in both 5- and 6-year-old degus but not in 3-year-old animals, suggesting an overall decrease in PPP flux (One-way ANOVA F_(3,16) = 25.06, p < 0.0001, followed by Bonferroni's post-hoc test, WT vs. 5 years old, p = 0.0035 and WT vs. 6 years old, p < 0.0001) (Figure 4B). Finally, to determine whether the decrease in glucose uptake has an effect on ATP synthesis, we quantified ATP levels in hippocampal slices. As expected, we observed a significant 20 and 50.8% reduction in ATP levels in 5- and 6-year-old animals compared with those in 1-year-old controls, respectively (one-way ANOVA F_(3,16) = 105.6, p < 0.0001, followed by Bonferroni's post-hoc test, WT vs. 5 years old, p = 0.0002 and WT vs. 6 years old, p < 0.0001) (Figure 4C). To further investigate whether the drop in ATP levels is caused by a reduction in ATP release or by a decrease in ATP production, we also measured ADP levels and calculated the ATP/ADP ratio accordingly. Interestingly, we found a significant reduction in both 5- and 6-year-old animals relative to controls, suggesting a reduction in ATP synthesis (one-way ANOVA F_(3,16) = 72.69, p < 0.0001, followed by Bonferroni's post-hoc test, both WT vs. 5 and 6 years old p < 0.0001) (Figure 4D).