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Food odor perception promotes systemic lipid utilization

Nature Metabolism volume 4, pages 1514–1531 (2022)Cite this article

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Abstract

Food cues during fasting elicit Pavlovian conditioning to adapt for anticipated food intake. However, whether the olfactory system is involved in metabolic adaptations remains elusive. Here we show that food-odor perception promotes lipid metabolism in male mice. During fasting, food-odor stimulation is sufficient to increase serum free fatty acids via adipose tissue lipolysis in an olfactory-memory-dependent manner, which is mediated by the central melanocortin and sympathetic nervous systems. Additionally, stimulation with a food odor prior to refeeding leads to enhanced whole-body lipid utilization, which is associated with increased sensitivity of the central agouti-related peptide system, reduced sympathetic activity and peripheral tissue-specific metabolic alterations, such as an increase in gastrointestinal lipid absorption and hepatic cholesterol turnover. Finally, we show that intermittent fasting coupled with food-odor stimulation improves glycemic control and prevents insulin resistance in diet-induced obese mice. Thus, olfactory regulation is required for maintaining metabolic homeostasis in environments with either an energy deficit or energy surplus, which could be considered as part of dietary interventions against metabolic disorders.

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Fig. 1: Fasted mice explored foods on the basis of olfactory, but not visual, information.
Fig. 2: Olfactory perception of a food cue elicits lipid mobilization during fasting.
Fig. 3: Food-odor memory recall elicits lipid mobilization during fasting.
Fig. 4: Pharmacological profiles of serum NEFA mobilization by food-odor stimulation during fasting.
Fig. 5: Acute effects of food-odor stimulation on protein and gene expression in the peripheral tissues in the fasting state.
Fig. 6: Food-odor prestimulation promotes lipid absorption and utilization upon nutrient intake.
Fig. 7: Food-odor stimulation during fasting induces tissue-specific metabolic regulations for adapting to refeeding.
Fig. 8: Effects of food-odor stimulation on metabolic functions in diet-induced obesity.

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Data availability

RNA-seq datasets have been deposited in the DDBJ BioProject database with the BioProject accession number PRJDB14370 (PSUB018445). All other data that support the findings of this study are presented in figures or extended data figures in this paper. Source data are provided with this paper.

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Acknowledgements

This study was supported by JSPS KAKENHI (Grant Numbers JP18K08468, JP19H05011, JP19K08997, JP21K19704, and JP22H03506), Japan Diabetes Foundation (H. T. and T. S.), Smoking Research Foundation (H. T.), Toyama Pharmaceutical Valley Development Consortium (T. S.), Tamura Science and Technology Foundation (H. T.), First Bank of Toyama Scholarship Foundation Research Grant (H. T.), JST—the establishment of university fellowship towards the creation of science technology innovation (Grant Number JPMJFS2115, M. S.), and JST Moonshot R&D (Grant Number JPMJMS2021, T. S.).

Author information

Author notes

  1. These authors contributed equally: Hiroshi Tsuneki, Masanori Sugiyama, Toshihiro Ito.

Authors and Affiliations

  1. Department of Clinical Pharmacology, University of Toyama, Toyama, Japan

    Hiroshi Tsuneki, Masanori Sugiyama, Kiyofumi Sato, Hiroki Matsuda, Kengo Onishi, Koharu Yubune, Yukina Matsuoka, Sanaka Nagai, Towa Yamagishi, Takahiro Maeda, Kosuke Honda, Akira Okekawa, Tsutomu Wada & Toshiyasu Sasaoka

  2. Laboratory of Proteome Research, National Institutes of Biomedical Innovation, Health, and Nutrition, Osaka, Japan

    Toshihiro Ito

  3. Division of Nutritional Biochemistry, University of Toyama, Toyama, Japan

    Shiro Watanabe

  4. Department of Molecular and Medical Pharmacology, University of Toyama, Toyama, Japan

    Keisuke Yaku & Takashi Nakagawa

  5. Genome Information Research Center, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan

    Daisuke Okuzaki

  6. Laboratory of Infectious Diseases and Immunity, National Institutes of Biomedical Innovation, Health, and Nutrition, Osaka, Japan

    Ryota Otsubo & Teruhito Yasui

  7. Laboratory of Immunobiologics Evaluation, Center for Vaccine and Adjuvant Research, National Institutes of Biomedical Innovation, Health, and Nutrition, Osaka, Japan

    Ryota Otsubo & Teruhito Yasui

  8. Department of Biochemistry, University of Toyama, Toyama, Japan

    Masanori Nomoto & Kaoru Inokuchi

  9. Research Centre for Idling Brain Science, University of Toyama, Toyama, Japan

    Masanori Nomoto & Kaoru Inokuchi

  10. Laboratory of Pharmaceutical Integrated Omics, Department of Pharmaceutical Engineering, Facility of Engineering, Toyama Prefectural University, Toyama, Japan

    Teruhito Yasui

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  1. Hiroshi Tsuneki
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Contributions

H. T. designed the experiments. M. S., T. I., K. S., H. M., K. O., K. Yubune, Y. M., S. N., T. Yamagishi, T. M., K. H., A. O., S. W., K. Yaku, D. O. and R. O. performed the experiments. H. T., M. N., K. I., T. N., T. W., T. Yasui and T. S. provided supervision. H. T. and T. S. wrote the manuscript with editing by all authors.

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Correspondence to Hiroshi Tsuneki, Teruhito Yasui or Toshiyasu Sasaoka.

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Nature Metabolism thanks the anonymous reviewers for their contribution to the peer review of this work. Primary Handling editor: Ashley Castellanos-Jankiewicz, in collaboration with the Nature Metabolism team.

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Extended data

Extended Data Fig. 1 Increase in serum NEFA by food odor-stimulation in a fasting duration-dependent manner.

a,b, Influence of the fasting duration (a, 6 h vs. 24 h fasting; b, 16 h and 24 h fasting) on the olfactory exploratory behaviors of C57BL/6 J mice in the SOS4 cage with tubes releasing the already- known odors of normal chow diet (NCD), high fat diet (HFD) and high sucrose diet (HSD), and an empty tube (None). The time spent exploring each tube was evaluated. n = 10 biological replicates. Different letters (A, B, and C) represent significant difference based on two-way ANOVA followed by an ANOVA with Tukey’s test in a and by Bonferroni’s test in b. c, Five independent tests to analyze the changes in serum NEFA levels by NCD-odor stimulation in C57BL/6 J mice fasted for 24 h. The respective data are expressed as changes in absolute NEFA levels over time (upper), absolute difference (∆) in NEFA levels from baseline (middle), and percentage changes in NEFA levels from baseline (lower). n = 6–12 biological replicates (as indicated). Overall averages and the standard errors of these five results (same dataset) are shown in Fig. 2b. d,e, Serum NEFA levels during stimulation with or without NCD odor in the 6 h (d) and 16 h fasting state (e) in C57BL/6 J mice. n = 6 biological replicates. f, Apparatus used for forced olfactory stimulation (FOS1) equipped with an air-pump. The tube contained either NCD, HFD, HSD, or none. The odor release from the tube was assisted by an air-pump. g, Serum levels of NEFA (left panel) and β-hydroxybutyrate (right panel, β-OHB) in C57BL/6 J mice that were fasted for 24 h and stimulated with NCD odor in the FOS1 cage. n = 11 (None) and n = 12 (Odor) biological replicates. *P < 0.05, **P < 0.01 in c-g determined by unpaired two-tailed t-test. Data are presented as mean ± s.e.m.

Source data

Extended Data Fig. 2 The composition of serum NEFA during food-odor stimulation in the fasting state.

C57BL/6 J mice were fasted for 24 h, and stimulated with NCD odor for 0, 30, and 60 min in the SOS1 cage. The composition of serum NEFA was analyzed by gas-liquid chromatography. a, Time course of changes in the levels of palmitic acid, palmitoleic acid, stearic acid, vaccenic acid, oleic acid, linoleic acid, α-linoleic acid, arachidonic acid, and docosahexaenoic acid during the olfactory stimulation. Values represent differences from the basal concentration (that is, 0-min level) of each free fatty acid. b, Ratios of palmitic acid (PA) to oleic acid (OA), and those of stearic acid (SA) to OA. The ratio was normalized to the value at 0 min in each mouse, and averaged. n = 6 biological replicates. *P < 0.05, **P < 0.01 determined by unpaired two-tailed t-test. Data are presented as mean ± s.e.m.

Source data

Extended Data Fig. 3 DREADD-mediated derangement of the olfactory memory system impairs food odor-induced lipid mobilization during fasting.

a, Total cell numbers and infection success rates of pAAV-CaMKIIa-hM4Di-mCherry within layer I, II, and III in the piriform cortex (PC) of C57BL/6 J mice. Open columns indicate total numbers of Hoechst positive cells in each layer, whereas black columns indicate the numbers of mCherry positive cells. *P < 0.05 vs. total cell numbers in the layer II. #P < 0.05 vs. mCherry+ cell numbers in the layer II. Statistical significance was determined by one-way ANOVA with Dunnett’s test. n = 6 biological replicates. b, cFos expression after treatment with saline (SAL) or CNO followed by intermittent NCD-odor stimulations in the PC of fasted mice expressing hM4Di-mCherry. Left, representative images of cFos-like immunoreactivity (green), mCherry (violet) and Hoechst (blue) at 10X magnification. Scale bar = 200 μm. Inset, higher (63X) magnification images. White arrow head: cFos+mCherry+ cells. Black arrow head: cFos+mCherry− cells. Scale bar = 50 μm. Right, probability for observing mCherry and cFos double positive cells (Prob. of cFos+mCherry+, closed column) compared to chance level (open column). *P < 0.05 vs. chance level, determined by two-sided Mann–Whitney U test. n = 4 biological replicates. c-f, Influence of DREADD-mediated derangement of the olfactory memory-processing system on food-searching behavior (c and e) and serum NEFA levels (d and f) during fasting in C57BL/6 J mice (12 months old) expressing hM4Di in the PC (c and d) and sham-operated controls (e and f). The effects of saline and CNO on the time spent exploring tubes which were releasing none, NCD, or already-known HFD odor in the SOS4 cage (c), and on the NEFA levels during exposure to HFD odor in the FOS1 cage (d) were compared. Different letters (A, B, and C) in c and e represent significant difference based on two-way ANOVA followed by an ANOVA with Tukey’s test in c and Bonferroni’s test in e. **P < 0.01 in d determined by paired two-tailed t-test. n = 12 biological replicates (c and d). n = 9 biological replicates (e and f). g, No correlation between the mCherry positive areas in the PC and the NEFA levels after 60 min of treatment with saline (upper) or CNO (lower) in mice expressing hM4Di-mCherry, determined by two-sided Pearson correlation coefficient test. n = 12 biological replicates. h-j, No influence of CNO on behaviors in the novel object recognition test (h, NOR), Y-maze test (i), and elevated plus maze test (j, EPM). n = 11 biological replicates. *P < 0.05 in h determined by one-way ANOVA with Dunnett’s test. Data are presented as mean ± s.e.m.

Source data

Extended Data Fig. 4 RNA-seq analysis of gene expression profiles in mice stimulated with food odor during fasting.

C57BL/6 J mice fasted for 24 h were stimulated with or without NCD odor for 1 h in the SOS1 cage. Gene expression profiles were determined by RNA-seq data analysis. a, Principal component analysis (PCA) of RNA-seq data for the hypothalamus, epididymal white adipose tissue (eWAT), small intestine (gut), soleus muscle, liver, and pancreas in odor-stimulated and control mice (n = 1 for each). b, Lists of DEGs in the transcriptome data of the soleus muscle and eWAT between the odor-stimulated and control mice. These DEGs were identified at a false discovery rate (FDR) < 0.05 and fold change > 1.5 (n = 3 biological replicates). The same DEGs between the soleus muscle and eWAT are highlighted in orange. c, Parametric analysis of gene set enrichment (PGGE) for group-on-group comparison into one-on-one comparison between DEGs and the related Gene Ontology (GO) biological process annotations of eWAT, pancreas (Panc), and soleus muscle in the odor-stimulated and control mice. The significantly different GO terms (P < 0.05) in terms of eWAT, pancreas, and soleus muscle transcriptome data between the odor-stimulated and control mice are labeled with †, §, and #, respectively.

Extended Data Fig. 5 Effects of non-food odors on serum NEFA levels.

a-d, Effects of attractive (muscone) and aversive odorants (PEA and TMT) on serum NEFA levels during fasting in C57BL/6 J mice. a, Timeline of the experimental protocol in b-d. Mice were fasted for 24 h, acclimated (Acc.) to the test cage in the absence of olfactory cue, and exposed to non-food odors in the FOS1 cage for 1 h. b, The effect of muscone. n = 6 biological replicates. c, The effect of PEA. n = 6 biological replicates. d, The effect of TMT. n = 6 biological replicates. e,f, Analysis of aversive behaviors in mice exposed to PEA and TMT during fasting in C57BL/6 J mice. e, The SOS2 apparatus used for the analysis of aversive behaviors. Two dishes with small holes were diagonally placed in the SOS2 cage. Non-food odorant was applied to a soft paper in one dish, and none was applied to another dish (a negative control). Mice fed ad libitum (AL) before the start of the tests were placed inside the SOS2 cage for 3 min, and time staining on half side with non-food-odorant was compared with that on the other half side with none. f, Upper: timeline of the experimental protocol. Lower: the effects of PEA (left, n = 8 biological replicates) and TMT (right, n = 6 biological replicates) on place preference between the non-food-odorant and control side. *P < 0.05 determined by unpaired two-tailed t-test. Data are presented as mean ± s.e.m.

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Extended Data Fig. 6 Influences of food-odor stimulation during fasting on glucose and energy metabolism and gallbladder function in C57BL/6 J mice.

a, The gallbladder volumes 15 min (left) or 30 min (right) after oral fat administration. C57BL/6 J mice fasted for 24 h were prestimulated with NCD odor or none for 1 h, and then administered with corn oil or saline (SAL, 10 mL/kg). n = 7 (None-SAL) and n = 8 (None-OIL and Odor-OIL) biological replicates (in left). n = 10 biological replicates (in right). *P < 0.05 determined by one-way ANOVA with Dunnett’s test. b,c, Pyruvate tolerance test (b, PTT) and oral glucose tolerance test (c, OGTT) after food-odor stimulation during fasting. C57BL/6 J mice previously challenged with HFD were fasted for 24 h, and stimulated with or without HFD odor for 1 h in the SOS1 cage. Pyruvate (2 g/kg, i,p., n = 8 biological replicates) or glucose [3 g/kg, n = 7 (None) and n = 8 (Odor) biological replicates] was administered at 0 min. d,e, Serum levels of insulin (d) and adiponectin (e) in C57BL/6 J mice fasted for 24 h, stimulated with or without NCD odor in the SOS1 cage for 1 h, and then refed NCD. N.D.: not detected. n = 11 (None) and n = 13 (Odor) biological replicates. f,g, Energy expenditure (f) and locomotor activity (g) in saline-treated mice expressing hM4Di in the PC during fasting, stimulation with NCD odor, and subsequent refeeding of NCD. These data were obtained simultaneously with RQ in Fig. 6g. n = 8 biological replicates. h, i, Food intake in mice expressing hM4Di in the PC, that were fasted for 24 h, pretreated with saline (h, SAL, n = 8 biological replicates) or CNO (i, n = 7 (CNO-None) and n = 5 (CNO-Odor) biological replicates), stimulated with NCD odor for 1 h, and then refed NCD. The amounts of food intake in the refeeding state are shown. *P < 0.05 in b-i determined by unpaired two-tailed t-test. Data are presented as mean ± s.e.m.

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Extended Data Fig. 7 Influences of food-odor stimulation during fasting on postprandial substrate utilization in orexin knockout and dopamine D2 receptor knockout mice.

a,b, Respiratory quotient (RQ) in orexin knockout (Orexin-/-) mice and their wild-type (Orexin+/+) mice (a) or dopamine D2 receptor knockout (D2R-/-) and their wild-type (D2R+/+) mice (b) that were fasted for 24 h, stimulated with the already-known food odor for 1 h, and then refed. n = 3 biological replicates. *P < 0.05 determined by unpaired two-tailed t-test. Data are presented as mean ± s.e.m.

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Extended Data Fig. 8 Shift in balance of AgRP and Pomc gene expression in the hypothalamus of mice stimulated with food odor during fasting.

C57BL/6 J mice were fasted for 24 h, and stimulated with or without NCD odor for 1 h in the SOS1 cage. The expression levels of AgRP and Pomc mRNAs in the hypothalamus were measured by RT-PCR. a, Relative expression levels of AgRP and Pomc mRNAs compared to 18 S rRNA. b, The AgRP/Pomc expression ratio. c, The mRNA levels of hypothalamic neuropeptides, Orexin (Hypocretin), melanin-concentrating hormone (Mch), and thyrotropin-releasing hormone (Trh), compared to 18 S rRNA. n = 12 biological replicates. *P < 0.05, **P < 0.01 determined by unpaired two-tailed t-test. Data are presented as mean ± s.e.m.

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Extended Data Fig. 9 Negligible effects of food-odor stimulation during fasting on metabolic activity in the adipose tissues and brain and gene expression in the liver after refeeding.

C57BL/6 J mice fasted for 24 h were stimulated with NCD odor or none for 1 h, and then refed NCD for 3 h. a, Metabolomic profiles in the epididymal white adipose tissue (eWAT), brown adipose tissue (BAT), and whole brain under the 3-h refeeding condition. OAA: oxaloacetic acid, α-KG: α-ketoglutarate. n = 8 (None) and n = 9 (Odor) biological replicates. b, Insulin tolerance test (ITT) under the 3-h refeeding condition. Insulin (0.5 unit/kg, i.p.) was injected at 0 min. n = 7 biological replicates. c, Expression levels of genes related to de novo lipogenesis and cholesterol synthesis in the liver under the 3-h refeeding condition. Srebp1c: sterol regulatory element-binding protein 1c, Acc: acetyl-CoA carboxylase, Fasn: fatty acid synthase, Mtp: microsomal triglyceride transfer protein, Atgl: adipose triglyceride lipase. n = 16 (None) and n = 18 (Odor) biological replicates. **P < 0.01 determined by unpaired two-tailed t-test. Data are presented as mean ± s.e.m.

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Extended Data Fig. 10 Plausible mechanism underlying the link between food-odor perception and lipid metabolism.

Food-odor perception during fasting elicits biphasic regulations of lipid metabolism, as follows: Food-odor information is transmitted to the olfactory bulb-piriform cortex pathway, and processed for evaluating the valence, based on its memory. If the odor valence is found to be attractive during fasting, lipid mobilization was elicited as an acute-phase response through a rapid activation of hypothalamic POMC and sympathetic nervous system that promotes lipolysis in the white adipose tissue (WAT). Such lipid mobilization is considered to be beneficial for survival during starvation. Then, if the food is not immediately available, the late-phase responses are elicited through sustained activation of the hypothalamic AgRP signaling associated with reduction of sympathetic tones that can promote whole-body lipid utilization upon subsequent refeeding. Tissue-specific regulations of lipid metabolism, including an increase in the gastrointestinal (GI) lipid absorption, increase in the hepatic cholesterol turnover, decrease in the WAT lipolysis, and decrease in the skeletal muscle (SM) lipid metabolism seem to underlie the olfactory system-induced lipid partitioning. In particular, a liver-specific increase in metabolic flow and reduction of hepatic ER stress could contribute to the maintenance of metabolic health. NEFA: non-esterified fatty acid, CM: chylomicron, VLDL: very low-density lipoprotein.

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Tsuneki, H., Sugiyama, M., Ito, T. et al. Food odor perception promotes systemic lipid utilization. Nat Metab 4, 1514–1531 (2022). https://doi.org/10.1038/s42255-022-00673-y

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