Metabolic learning and memory formation by the brain influence systemic metabolic homeostasis

Abstract

Metabolic homeostasis is regulated by the brain, but whether this regulation involves learning and memory of metabolic information remains unexplored. Here we use a calorie-based, taste-independent learning/memory paradigm to show that Drosophila form metabolic memories that help in balancing food choice with caloric intake; however, this metabolic learning or memory is lost under chronic high-calorie feeding. We show that loss of individual learning/memory-regulating genes causes a metabolic learning defect, leading to elevated trehalose and lipid levels. Importantly, this function of metabolic learning requires not only the mushroom body but also the hypothalamus-like pars intercerebralis, while NF-κB activation in the pars intercerebralis mimics chronic overnutrition in that it causes metabolic learning impairment and disorders. Finally, we evaluate this concept of metabolic learning/memory in mice, suggesting that the hypothalamus is involved in a form of nutritional learning and memory, which is critical for determining resistance or susceptibility to obesity. In conclusion, our data indicate that the brain, and potentially the hypothalamus, direct metabolic learning and the formation of memories, which contribute to the control of systemic metabolic homeostasis.

Figure 1. Food and metabolic control via metabolic learning and memory formation

(a) Adult CS flies received the indicated training cycles in associating conditioned odor stimuli with normal-calorie (50 g/L sorbitol) food vs. a high-calorie (200 or 300 g/L sorbitol) or a low-calorie (20 g/L sorbitol) food, and were tested for measuring the preference index (PI). n = 60–80 animals per sample, and n = 4–8 samples per group. (b&c) Adult CS flies received the indicated training cycles in associating conditioned odor stimuli with normal-calorie (50 g/L sorbitol) vs. high-calorie (200 g/L sorbitol) food, and were subjected to the test for the PI (b) or measured for 30-min food intake (c). n = 60–80 (b) or 10 (c) animals per sample, and n = 4–8 samples per group. (d&e) Adult CS flies were maintained on normal-calorie (50 g/L sorbitol) vs. high-calorie (200 or 300 g/L sorbitol) food for 3 weeks. These flies were determined for body’s contents of trehalose (d) and TG (e). n = 5 animals per sample, and n = 4–6 samples per group. (f) Adult CS flies were treated with normal-calorie (50 g/L sorbitol) vs. high-calorie (200 g/L sorbitol) food for 3 weeks. The mRNA levels of indicated learning/memory genes were determined. AU: arbitrary unit. n = 15 animals per sample, and n = 5–6 samples per group. (g&h) Adult CS flies were maintained on normal-calorie (50 g/L sorbitol) vs. high-calorie (200 or 300 g/L sorbitol) food. These flies were determined for lifespan (g) and fecundity (h). g: lifespan assay was performed using Kaplan–Meier survival analysis, and P values were determined by log-rank test. Maximum lifespan analysis was conducted on the longest-lived 10% of flies in each group. n = 30 (g) and 5 (h) animals per sample, and n = 3 (g) and 12 (h) samples per group. **P < 0.01, *P < 0.05 (a, b, d–h: ANOVA and Tukey; c&f: unpaired Student’s t-test); error bars reflect means ± s.e.m.

Figure 2. Requirement of rut gene in metabolic learning and memory formation

(a) Adult brains of CS flies were subjected to in situ hybridization for rutabaga mRNA (red) and immunostaning for dilp2 antibody (green). Arrows point to the mushroom body (MB) and the pars intercerebralis (PI) in the brain. Green fluorescence indicated the PI and was merged with red staining to show rutabaga mRNA in this region. DAPI staining (blue) revealed the brain in this staining. Control group was stained with the sense mRNA probe and showed the absence of red staining. Bar = 100 µm. (b–f) rut²⁰⁸⁰ flies and wild-type (wt) controls received 7 cycles of training in olfactory conditioning test, and analyzed for preference index (PI) (b–d) according to males (b), females (c) and both (d), and measured for body’s contents of trehalose (e) and TG (f). Basal controls were included based on the matched flies that were fed on the same normal-calorie diet for the same training duration (e&f). n = 60–80 (b–d) or 5 (e&f) animals per sample, and n = 4–6 samples per group. (g–i) Flies including rut²⁰⁸⁰, rut²⁰⁸⁰; elav-GAL4/UAS-rut⁺ and control elav-GAL4/+ received 7 cycles of training in olfactory conditioning test and analyzed for preference index (PI) (g–i) according to males (g), females (h) and both genders (i). n = 50–70 animals per sample, and n = 4–6 samples per group. **P < 0.01, *P < 0.05 (b–d: unpaired Student’s t-test; e–i: ANOVA and Tukey); error bars reflect means ± s.e.m.

Figure 3. Requirement of dnc gene in metabolic learning and memory formation

Dunce¹ and wt flies received 7 cycles of training in olfactory conditioning test, and then analyzed for preference index (PI) (a–c) according to males (a), females (b) and both (c), and measured for body’s contents of trehalose (d) and TG (e). Basal controls were included based on separate groups of these flies that were fed on the same normal-calorie diet for the same training duration (d, e). n = 50–70 (a–c) and 4–6 (d&e) animals per sample, and n = 4–6 samples per group. **P < 0.01, *P < 0.05 (a–c, unpaired Student’s t-test; d&e: ANOVA and Tukey); error bars reflect means ± s.e.m.

Figure 4. Requirement of tequila gene in metabolic learning and memory formation

Teq⁻ and wt flies received 7 cycles of training in olfactory conditioning test, and analyzed for preference index (PI) (a–c) according to males (a), females (b) and both (c), and measured for body’s contents of trehalose (d) and TG (e). Basal controls were included based on separate groups of these flies that were fed on the same normal-calorie diet for the same training duration (d, e). n = 50–70 (a–c) and 4–6 (d&e) animals per sample, and n = 4–6 samples per group.**P < 0.01, *P < 0.05 (a–c: unpaired Student’s t-test; d&e: ANOVA and Tukey); error bars reflect means ± s.e.m.

Figure 5. The MB and dilp2-defined PI neurons in metabolic learning and memory formation

(a–c) Mutant flies including 17D-GAL4/UAS-rut^RNAi (a), 17D-GAL4/UAS-dunce^RNAi (b), 17D–GAL4/UAS-PKA^inh (c) and matched controls 17D-GAL4/+ received 6 cycles of training, and then analyzed for preference index (PI). n = 50–70 animals per sample, and n = 4–6 samples per group. (d–i) Flies including dilp2-GAL4/UAS-rut^RNAi (shown as dilp2/UAS-rut^RNAi) vs. matched control group dilp2-GAL4/+ (shown as dilp2/+) and dilp2-GAL4/UAS-PKA^inh(shown as dilp2/ UAS-PKA^inh) vs. matched control group dilp2-GAL4/+ received 6–9 cycles of training, and then analyzed for preference index (PI) (d&g) and body’s contents of trehalose (e&h) and TG (f&i). Data in (e, f, h, i) showed the change levels (Δ) after training cycles compared to the levels of basal controls in which flies were maintained on the same normal-calorie diet for the same training duration. n = 40–60 (d&g) or 5 (e, f, h, i) animals per sample, and n = 4–6 samples per group. **P < 0.01, *P < 0.05 (unpaired Student’s t-test); error bars reflect means ± s.e.m.

Figure 6. Impairment of metabolic learning and memory formation by NF-κB pathway

(a) Brain expression levels of inflammation related genes in the adult CS flies after 18 days normal-calorie (NC, 50g/L) or high-calorie (HC, 200g/L) food treatment. AU: arbitrary unit. n = 30 animals per sample, and n = 4 samples per group. (b–k) Flies elav-GAL4/UAS-dl (shown as elav/UAS-dl) vs. control elav-GAL4/+ (shown as elav/+) (b), dilp2-GAL4/UAS-dl (shown as dilp2/UAS-dl) vs. control dilp2-GAL4/+ (c–e), dilp2-GAL4/UAS-Rel (shown as dilp2/ UAS-Rel) vs. control dilp2-GAL4/+ (f–h), and dilp2-GAL4/UAS-cact (shown as dilp2/ UAS-cact vs. control dilp2-GAL4/+ (i–k) received indicated cycles of training, and then analyzed for preference index (PI) (b, c, f, i) and body’s contents of trehalose (d, g, j) and TG (e, h, k). Data in (d, e, g, h, j, k) showed the change levels (Δ) after indicated training cycles compared to the levels of basal controls in which flies were maintained on the same normal-calorie diet for the same training duration. n = 50–70 (b, c, f, i) or 5 (d, e, g, h, j, k) animals per sample, and n = 4–8 samples per group. **P < 0.01, *P < 0.05 (a–h, unpaired Student’s t-test; i–k: ANOVA and Tukey); error bars reflect means ± s.e.m.

Figure 7. Involvement of the hypothalamus in learning/memory-regulating genes of mice

(a&b) Hippocampal (a) and hypothalamic (b) levels of learning/memory genes in A/J and C57BL/6J mice (~ 3-month-old males) under basal and normal chow feeding conditions. (c&d) The mRNA levels of learning/memory genes were determined for the hypothalamus (b) and hippocampus (c) of adult male C57BL/6J mice that received 3-month normal diet (ND) vs. high-fat diet (HFD) feeding. **P < 0.01, *P < 0.05 (unpaired Student’s t-test), n = 4–6 mice per group; error bars reflect means ± s.e.m.

Figure 8. Different metabolic learning/memory profiles in A/J vs. C57BL/6J mice

(a&b) Preference Index (PI) of A/J (a) and C57BL/6J (b) mice following indicated training cycles. The PI was calculated as the time difference that mice spent in a cage of a conditioned stimulus associated with normal diet (ND) vs. high-fat diet (HFD) divided by the total time. (c&d) Percentage changes of ND intake (c) vs. HFD intake (d) in A/J and C57BL/6J mice at the indicated training cycles relative to food intake at training cycle 0. Statistics reflect comparison with 0 cycle in each strain. (e) Body weight gain of A/J and C57BL/6J mice at the indicated days during the training. Statistics reflect comparisons between C57 and A/J at matched time points. (f) Serum FFA in C57BL/6J and A/J prior to training (Pre) vs. post 21 training cycles (Post). (g) Calculated values for area under curve (AUC) during 120-min glucose tolerance test (GTT) of A/J and C57BL/6J mice prior to training (Pre) vs. post 21 training cycles (Post). n = 8–10 (a–e) and n = 5–8 mice (f, g) per group. **P<0.01, ##P < 0.01, *P < 0.05, #P < 0.05 (ANOVA and Tukey); error bars reflect means ± s.e.m.

Figure 9. Working model of metabolic learning and memory formation

Under an environmental condition containing normal and abnormal nutrition, Drosophila can develop a form of learning and memory of metabolic information, referred to as “metabolic learning & memory”, to mediate the central control of metabolic homeostasis, which requires cooperative actions of the MB and PI neurons. However, chronic overnutrition can employ NF-κB-driven neuroinflammation to impair this form of learning and memory, contributing to the development of overnutrition-induced metabolic disorders and diseases.