The microbiota-gut-brain interaction in regulating host metabolic adaptation to cold in male Brandt's voles (Lasiopodomys brandtii)

Abstract

Gut microbiota play a critical role in orchestrating metabolic homeostasis of the host. However, the crosstalk between host and microbial symbionts in small mammals are rarely illustrated. We used male Brandt's voles (Lasiopodomys brandtii) to test the hypothesis that gut microbiota and host neurotransmitters, such as norepinephrine (NE), interact to regulate energetics and thermogenesis during cold acclimation. We found that increases in food intake and thermogenesis were associated with increased monoamine neurotransmitters, ghrelin, short-chain fatty acids, and altered cecal microbiota during cold acclimation. Further, our pair-fed study showed that cold temperature can alter the cecal microbiota independently of overfeeding. Using cecal microbiota transplant along with β3-adrenoceptor antagonism and PKA inhibition, we confirmed that transplant of cold-acclimated microbiota increased thermogenesis through activation of cAMP-PKA-pCREB signaling. In addition, NE manipulation induced a long-term alteration in gut microbiota structure. These data demonstrate that gut microbiota-NE crosstalk via cAMP signaling regulates energetics and thermogenesis during cold acclimation in male Brandt's voles.

Fig. 1

Cold and rewarming induce alterations in metabolic phenotypes and monoamine neurotransmitters. a The design paradigm. b Changes of food intake with time of acclimation (repeated measures ANOVA). c Uncoupling protein 1 (UCP1) expression in interscapular brown adipose tissue (BAT) (UCP1 and others, independent t-test between W4 and C4, W4W4 and C4W4). d The ratio of UCP1-positive expression in white adipose tissue (WAT). e Ghrelin level in serum. f T₃/T₄ ratio in serum. g–i Tyrosine hydrogenase (TH) expression in small intestine, BAT and WAT. W4, warm condition for 4 weeks; C4, cold acclimation for 4 weeks; W4W4, warm condition for 8 weeks; C4W4, cold acclimation for 4 weeks and then back to warm condition for 4 weeks. Data are means ± SEM (n = 8 per group). *p < 0.05, **p < 0.01, and ***p < 0.001

Fig. 2

Cold and rewarming shape diversity and composition of cecal microbiota. a Alpha diversity (PD_whole tree) of bacterial communities across groups (n = 8 per group, independent t-test). b NMDS plot based on unweighted UniFrac distance metrics representing the differences in fecal microbial community structure in different groups (ANOSIM). c LDA scores of the differentially abundant taxa enriched in microbiota from all the groups (taxa with LDA score >2 and a significance of a < 0.05 are shown). d Abundance represented as the proportions of OTUs classified at the phylum rank. e Heatmap showing the correlation between specific OTUs and physiological measurements. The OTU IDs with only numbers were from Greengenes database, whereas those with the letters of OTU were clustered into de novo OTUs. NE norepinephrine, SI small intestine, TH tyrosine hydroxylase, UCP1 uncoupling protein 1, BAT brown adipose tissue, WAT white adipose tissue. Data are means ± SEM (n = 7 per group). *p < 0.05, **p < 0.01

Fig. 3

Cold temperature rather than overfeeding alters bacterial diversity and composition. a The design paradigm. b Body mass gain after acclimation. c Resting metabolic rate (RMR). d Nonshivering thermogenesis (NST). e Alpha diversity measurement (Observed OTUs) of bacterial communities across groups. f, g NMDS and PCoA plots based on weighted UniFrac distance metrics analysis of OTUs (ANOSIM). Each symbol represents a single sample of cecal contents. Data are means ± SEM (n = 6 per group, two-way ANOVA with Tukey’s post hoc tests except β diversity). *p < 0.05, **p < 0.01

Fig. 4

Cecal microbiota transplant alters metabolic phenotypes, neurotransmitters, and bacterial diversity and composition. a The design paradigm. b Food intake, c serum ghrelin, d resting metabolic rate (RMR), and e uncoupling protein 1 (UCP1) expression in brown adipose tissue (BAT) in antibiotic (Ab), recipients with cold microbiota (R-C4), recipients with warm microbiota (R-W4), and control groups. f Norepinephrine (NE) concentration in BAT, g small intestine, and h hypothalamus (hypo). i The cAMP-activated PKA expression in the small intestine. j Phospho-CREB/CREB in the small intestine. k The FFAR2 expression in the small intestine and l BAT. m The comparison of alpha diversity measurement (PD_whole tree) of bacterial communities across groups. n NMDS plot based on unweighted UniFrac distance metrics shows the microbial community structure of samples from different groups (ANOSIM). o The concentrations of six short-chain fatty acids (SCFAs) in cecal contents. Data are means ± SEM (n = 6 per group, one-way ANOVA with Tukey’s post hoc tests except β diversity). *p < 0.05, **p < 0.01

Fig. 5

Cold microbiota promotes thermogenesis via the cAMP–PKA–pCREB pathway. a The design paradigm. b Nonshivering thermogenesis (NST). c Uncoupling protein 1 (UCP1) expression in brown adipose tissue (BAT). d cAMP-activated PKA expression in BAT and e small intestine. f Phospho-CREB/CREB in BAT and g small intestine. Data are means ± SEM (n = 8 per group, except phospho-CREB/CREB (n = 7 per group), one-way ANOVA with Tukey’s post hoc tests). Different superscript letters indicate significant differences among different groups (p < 0.05)

Fig. 6

Norepinephrine (NE) manipulation affects energy metabolism and gut microbiota. a The design paradigm. b Body mass. c Food intake. d Body temperature. e Uncoupling protein 1 (UCP1) expression in brown adipose tissue (BAT). f The cAMP-activated PKA expression in the small intestine. g Phospho-CREB/CREB in the small intestine. h The comparison of alpha diversity measurement (PD_whole tree) of bacterial communities among groups. i NMDS plot based on unweighted UniFrac distance representing the differences in the microbial community structures of samples from different groups. j Differential bacterial taxonomy selected by LEfSe analysis with LDA score > 2 in microbiota. k Abundance represented as the proportions of OTUs classified at the phylum level. Data are means ± SEM (n = 8 per group). Body mass, food intake, and body temperature were analyzed by repeated measures ANOVA, β diversity by ANOSIM, and others by one-way ANOVA with Tukey’s post hoc tests. NE, NE manipulation for 7 days; NE-R7, NE manipulation for 7 days and then recover for 7 days; NE-R21, NE manipulation for 21 days and then recover for 21 days. Different superscript letters indicate significant differences among different groups (p < 0.05). *P < 0.05

Fig. 7

The paradigm summarizing the crosstalk between gut microbiota and norepinephrine (NE) via the cAMP signaling pathway in mediating host energetics and thermogenesis during cold acclimation. BAT brown adipose tissue, cAMP cyclic adenosine monophosphate, FFAR2 free fatty acid receptor 2, p-CREB phosphorylation of cAMP-response elementbinding protein, PKA protein kinase A, SCFAs short-chain fatty acids, UCP1 uncoupling protein 1, WAT white adipose tissue