Tanycytic transcytosis inhibition disrupts energy balance, glucose homeostasis and cognitive function in male mice

Abstract

Objectives: In Western society, high-caloric diets rich in fats and sugars have fueled the obesity epidemic and its related disorders. Disruption of the body-brain communication, crucial for maintaining glucose and energy homeostasis, arises from both obesogenic and genetic factors, leading to metabolic disorders. Here, we investigate the role of hypothalamic tanycyte shuttles between the pituitary portal blood and the third ventricle cerebrospinal fluid in regulating energy balance.

Methods: We inhibited vesicle-associated membrane proteins (VAMP1-3)-mediated release in tanycytes by expressing the botulinum neurotoxin type B light chain (BoNT/B) in a Cre-dependent manner in tanycytes. This was achieved by injecting either TAT-Cre in the third ventricle or an AAV1/2 expressing Cre under the control of the tanycyte-specific promoter iodothyronine deiodinase 2 into the lateral ventricle of adult male mice.

Results: In male mice fed a standard diet, targeted expression of BoNT/B in adult tanycytes blocks leptin transport into the mediobasal hypothalamus and results in normal-weight central obesity, including increased food intake, abdominal fat deposition, and elevated leptin levels but no marked change in body weight. Furthermore, BoNT/B expression in adult tanycytes promotes fatty acid storage, leading to glucose intolerance and insulin resistance. Notably, these metabolic disturbances occur despite a compensatory increase in insulin secretion, observed both in response to exogenous glucose boluses in vivo and in isolated pancreatic islets. Intriguingly, these metabolic alterations are associated with impaired spatial memory in BoNT/B-expressing mice.

Conclusions: These findings underscore the central role of tanycytes in brain-periphery communication and highlight their potential implication in the age-related development of type 2 diabetes and cognitive decline. Our tanycytic BoNT/B mouse model provides a robust platform for studying how these conditions progress over time, from prediabetic states to full-blown metabolic and cognitive disorders, and the mechanistic contribution of tanycytes to their development. The recognition of the impact of tanycytic transcytosis on hormone transport opens new avenues for developing targeted therapies that could address both metabolic disorders and their associated cognitive comorbidities, which often emerge or worsen with advancing age.

Keywords: Blood-cerebrospinal fluid barrier; Blood–brain barrier; Hypothalamus; Normal-weight central obesity; Tanycytes; Transports.

Graphical abstract

Figure 1

Alteration of vesicular trafficking in median eminence tanycytes promotes body weight gain. (A) Schematics for the Cre-dependent induction of BoNT/B in tanycytes after the injection of recombinant TAT-Cre or vehicle into the third ventricle (3V), or AAV1/2-Dio2-Cre or vehicle in the lateral ventricle (LV) of BoNT/B-EGFP^{loxP-STOP-loxP} mice. (B) Representative photomicrographs of EGFP immunopositive median eminence tanycytes in BoNT/B^Ctl and BoNT/B^Tan mice at 4 weeks (AAV1/2 -injected mice). Scale bar, 200 μm. (C) Diagram and gating strategy for the sorting of EGFP-positive putative tanycytes following AAV1/2-Dio2-Cre infusion into the LV of BoNT/B-EGFP^{loxP-STOP-loxP} mice. (D) mRNA levels of Botulinum neurotoxin light chain B mRNA in EGFP-positive FACS-sorted cells from ME and ARH BoNT/B^Tan and BoNT/B^Ctl explants at 4 weeks (AAV1/2- injected mice). One-tailed unpaired t-test; t = 2.051, df = 5, n = 4 and 3 mice. Botulinum neurotoxin light chain B mRNA was undetextale in EGFP-negative FACS-sorted cells. (E) Expression of VAMP1, VAMP2 and VAMP3 mRNA in EGFP-positive FACS-sorted cells of BoNT/B^Tan mice (n = 5) at 4 weeks (TAT-Cre or vehicle injected mice), normalized over the same mRNA expression (Value = 1) in Tomato-positive FACS-sorted cells of TdTomato^Tan mice (n = 5). Two-tailed unpaired t-test, VAMP1 t = 0.5214, df = 8, p = 0.6162; VAMP2 t = 0.8441, df = 8, p = 0.4231; VAMP3 t = 2.183, df = 8, p = 0.0606. (F) Expression of the tanycytic markers Ppp1r1b coding DARPP-32 and Dio2 in tomato-positive and negative cells isolated by FACS. Two-tailed unpaired t-test, Ppp1r1b t = 4.221, df = 8; Dio2 t = 3.204, df = 8. (G) Curves representing 24h of cumulative food intake in BoNT/B^Ctl (n = 7) and BoNT/B^Tan (n = 8) mice at 4 weeks (AAV1/2-injected mice). Two-way ANOVA, Interaction p = 0.6123, F_(23,299) = 0.8901; Time p < 0.0001, F (23,299) = 707.3; Subjects p = 0.0266, F _(1,13) = 6.250. LSD post-hoc test, 16.00h p = 0.0411; 17.00h p = 0.0414; 18.00h p = 0.0164; 19.00h p = 0.0159; 20.00h p = 0.0112; 21.00h p = 0.0324; 22.00h p = 0.0357; 4.00h p = 0.041; 5.00h p = 0.0361; 6.00h p = 0.013; 7.00h p = 0.0154; 8.00h p = 0.0103; 9.00h p = 0.0108; 10.00h p = 0.0095 and 11.00h p = 0.0275. (H) Body weight change of BoNT/B^Ctl (n = 9) and BoNT/B^Tan (n = 12) mice at 4 and 12 weeks after starting the experiments (AAV1/2-injected mice). Values are expressed in grams (gr).Two-way ANOVA, Time p = 0.0008, F _{(1, 16)} = 16.84; subjects p = 0.0041, F _{(1, 22)} = 10.29; Šídák's multiple comparisons test, BoNT/B^Ctl 4 weeks versus BoNT/B^Ctl 12 weeks p = 0.254. (I) Curves representing 24h of respiratory exchange ratio (RER) in BoNT/B^Ctl (n = 7) and BoNT/B^Tan (n = 8) mice at 4 weeks (AAV1/2 -injected mice). Two-way ANOVA, Interaction p = 0,5424, F _{(23, 299)} = 0.9417; Time p = <0.0001, F _{(23, 299)} = 69.28; Subjects p = 0.0927, F _{(1, 13)} = 3.293. LSD post-hoc test, 15.00h p = 0.0039; 16.00h p = 0.0081; 17.00h p = 0.0179. The area under the curve (AUC), one-tailed unpaired t-test, t = 1.797, df = 13. (J) Visceral fat mass expressed as a percentage of the body weight in BoNT/B^Ctl (n = 14) and BoNT/B^Tan (n = 12) at 12 weeks (AAV1/2- injected mice). Two-tailed Mann–Whitney test. Values are expressed as percentages calculated over the body weight. (K) Circulating leptin levels of BoNT/B^Ctl (n = 5) and BoNT/B^Tan (n = 7) mice at 12 weeks (TAT-Cre or vehicle infusion into the 3V). Two-tailed unpaired t-test, t = 2.422, df = 1. (L) Body weight follow-up in BoNT/B^Ctl (n = 6) and BoNT/B^Tan (n = 5) mice fed with a high-fat diet (HFD) for 4 weeks, at 4 weeks (AAV1/2-injected mice). Two-way ANOVA, Interaction p = 0.0821, F (4, 88) = 2.143; Time p < 0.0001, F (1,688, 37,13) = 73.62; Subjects p = 0.0228, F (1, 22) = 5.990. The two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli, at the start of the HFD, p = 0.2245; 1-week HFD p = 0.0153; 2-week HFD p = 0.0121; 3-week HFD p = 0.0292 and 4-week HFD p = 0.0994. (M) Curves representing 24h cumulative food intake in BoNT/B^Ctl (n = 6) and BoNT/B^Tan (n = 6) mice at 8 weeks after the beginning of the experiment (AAV1/2-injected mice) and 4 weeks of HFD. Two-way ANOVA, Interaction p < 0.0001, F _{(23, 230)} = 3.125; Time p < 0.0001, F _{(23, 230)} = 385.4; Subject p = 0.0672, F _{(1, 10)} = 4.213. LSD post-hoc test, 23.00h p = 0.0452; 1.00h p = 0.0276; 2.00h p = 0.0042; 3.00h p = 0.0022; 4.00h p = 0.0052; 5.00h p = 0.0034; 6.00h p = 0.0042; 7.00h p = 0.0064; 8.00h p = 0.0066; 9.00h p = 0.0111. (N) Curves representing 24h RER in BoNT/B^Ctl (n = 6) and BoNT/B^Tan (n = 6) mice at 8 weeks after the beginning of the experiment (AAV1/2-injected mice) and 4 weeks of HFD. Two-way ANOVA, Interaction p = 0.5955, F (23, 230) = 0.9024; Time p < 0.0001, F (23, 230) = 17,87; Subject p = 0.0169, F (1, 10) = 8,185. LSD post-hoc test, 14.00h p = 0.0442; 15.00h p = 0.0002; 17.00h p = 0.0067; 20.00h p = 0.0379; 2.00h p = 0.0432 and 3.00h p = 0.0229. Data are expressed as mean ± SEM. p < 0.05 values are indicated in the figures.

Figure 2

Blockade of exocytosis in ME tanycytes impairs leptin sensitivity and its transport into the medio basal hypothalamus. (A) Representative photomicrographs of phosphorylated STAT3 (p-STAT3) immunopositivity (white dots) in median eminence and medio basal hypothalamus cells and MECA32 immunopositive microvessel loops (green) in BoNT/B^Ctl and BoNT/B^Tan mice at 4 weeks after the beginning of the experiment (AAV1/2-injected mice). Yellow dotted insets one and two delimitate the ARH and ME respectively. Scale bar, 200 μm. (B,C) Quantification of p-STAT3 immunopositive cells in the ARH (B) and ME (C) of BoNT/B^Ctl (n = 3) and BoNT/B^Tan (n = 3) mice at 4 weeks (AAV1/2-injected mice). Two-tailed unpaired t-test. In ARH (B), t = 3.961, df = 4; ME (C), p = 0.0617, t = 2.574, df = 4). (D,E) Quantification of the number of MECA32 immunopositive microvessel loops in the ARH (D) and ME (E) in in BoNT/B^Ctl (n = 3) and BoNT/B^Tan (n = 3) mice 4 weeks after Cre-induced genetic recombination. Two-tailed unpaired t-test. In ARH (D), p = 0.3739, t = 1.000, df = 4; ME (E), p = 0.4165 t = 0.9054, df = 4. (F) Protocol of leptin tolerance test (i.p. 3 mg/kg or i.c.v. 1 mg/kg) in BoNT/B^Ctl and BoNT/B^Tan mice 12 weeks after the beginning of the experiment (TAT-Cre- or vehicle-injected mice). (G) Cumulative food intake 24 h after leptin or vehicle injection in BoNT/B^Ctl (n = 3) and BoNT/B^Tan (n = 3) mice. Two-way ANOVA, Interaction p = 0.0653, F _{(1, 8)} = 4.557; Treatment p = 0.2079, F _{(1, 8)} = 1.877; Subject p = 0.0053, F _{(1, 8)} = 14.38. LSD post hoc test, BoNT/B^Ctl_Veh vs. BoNT/B^Tan_Veh p = 0.2751 and BoNT/B^Tan_Veh vs. BoNT/B^Tan_Leptin p = 0.6034. (H) Cumulative food intake 12 h after leptin (i.c.v. 1 mg/kg) or vehicle injection in BoNT/B^Ctl (n = 8) and BoNT/B^Tan (n = 7). Two-way ANOVA, Interaction p = 0.9514 F _{(1, 24)} = 0.00379; Treatment p = 0.0006 F _{(1, 24)} = 15.34; Subject p = 0.6308 F _{(1, 24)} = 0.237. LSD post hoc test, BoNT/B^Ctl_Veh vs. BoNT/B^Tan_Veh p = 0.6908 and BoNT/B^Ctl_Leptin vs. BoNT/B^Tan_Leptin p = 0.7740. (I) Schematic diagram of the stereotactic implantation of the microdialysis probe in the mediobasal hypothalamus (MBH) and the protocol of the same experiment, that investigates exogenous leptin central transport in BoNT/B^Ctl and BoNT/B^Tan mice 4 weeks after the beginning of the experiment (AAV1/2-injected mice). (J) Leptin concentrations in the MBH extracellular fluid collected by microdialysis every 20 min following intraperitoneal leptin (t, 0 min) injection (3 mg/kg) in BoNT/B^Ctl (n = 5) and BoNT/B^Tan mice (n = 5). Mixed-effects analysis, Time p = 0.3901, F (0.8537, 5.265) = 0.7959; Subject p = 0.0423, F (1, 12) = 5.159; Interaction p = 0.1708, F (6, 37) = 1.614. LSD post hoc test, 40min p = 0.0387. AUC, two-tailed unpaired t-test t = 2.427, df = 10. Data are expressed as mean ± SEM. p < 0.05 values are indicated in the figures. (For interpretation of the references to color/colour in this figure legend, the reader is referred to the Web version of this article.)

Figure 3

BoNT/B expression in tanycytes reduces lipid mobilization in adipose tissue. (A) Curves representing 24h fatty acid oxidation (FAO) in BoNT/B^Ctl (n = 7) and BoNT/B^Tan (n = 8) mice, 4 weeks after the beginning of the experiment (AAV1/2-injected mice). Two-way ANOVA, Interaction p = 0.3634, F_(23,322) = 1.082; Time p < 0.0001, F _{(4.788, 67.03)} = 35.71; Subjects p = 0.8217, F _(1,14) = 0.05273. LSD post-hoc test, 17h p = 0.0483). AUC, one-tailed unpaired t-test, p = 0.4069, t = 0.2400, df = 14. (B–D) Circulating levels of triglycerides (B), cholesterol (C) and non-esterified fatty acids (NEFAs, D) of BoNT/B^Ctl (n = 4) and BoNT/B^Tan (n = 5) mice at 12 weeks (TAT-Cre- or vehicle-injected mice). Two-tailed unpaired t-test; triglycerides, p = 0.1832, t = 1.477, df = 7; cholesterol, p = 0.3396, t-test, t = 1.025, df = 7; NEFAs, p = 0.626, t test, t = 0.5096, df = 7. (E–G) Production levels of selected proteins involved in fatty acid (FA) synthesis in liver (E), carnitine-palmitoyl transferase A (CPT1A) (F) and lipoprotein lipase (LPL) (G), from BoNT/B^Ctl (n = 10) and BoNT/B^Tan (n = 10) mice at 12 weeks (TAT-Cre- or vehicle-injected mice). In E, one-way ANOVA, p = 0.0005, F = 5.803. Šídák's multiple comparisons test ACCa BoNT/B^Ctl vs. ACCa BoNT/B^Tan p = 0.9638, t = 0.5811; pACCa BoNT/BCtl vs. pACCa BoNT/B^Tan p = 0.9721 t = 0.5405; pACCa/ACCa BoNT/BCtl vs. pACCa/ACCa BoNT/B^Tan p = 0.9227 t = 0.7239 and FAS BoNT/B^Ctl vs. FAS BoNT/B^Tan p = 0.9456 t = 0.6528. In F, two-tailed unpaired t-test, p = 0.6178, t = 0.5078, df = 18). In G, two-tailed unpaired t-test, t = 2.250, df = 12). (H) Representative immunoblots of proteins from panels E–G. (I–M) Expression levels of selected proteins involved in FA synthesis in adipose tissue (I), CPT1A (J), LPL (K) and proteins involved in lipolysis (M), from BoNT/B^Ctl (n = 7) and BoNT/B^Tan (n = 7) mice at 12 weeks (TAT-Cre- or vehicle-injected mice). In I, one-way ANOVA, p = 0.0007, F = 4.431. Šídák's multiple comparisons test ACCa BoNT/B^Ctl vs. ACCa BoNT/B^Tan t = 05776, p = 0.9646; pACCa BoNT/B^Ctl vs. pACCa BoNT/B^Tan p = 0.0927, t = 2.331; pACCa/ACCa BoNT/B^Ctl vs. pACCa/ACCa BoNT/B^Tan t = 3.705; and FAS BoNT/B^Ctl vs. FAS BoNT/B^Tan p = 0.5632, t = 1.338. In J, two-tailed unpaired p = 0.2124, t test, t = 1.293, df = 18. In K), two-tailed Mann Whitney test, p = 0.535). In M, one-way ANOVA, p = 0.0005. Šídák's multiple comparisons test, HSL BoNT/B^Ctl vs. HSL BoNT/B^Tan p = 0.9995, t = 0.1026; pHSL BoNT/B^Ctl vs. pHSL BoNT/B^Tan t = 3.214 and pHSL/HSL BoNT/B^Ctl vs. pHSL/HSL BoNT/B^Tan t = 3.329. (L) Representative immunoblots of proteins in (I–K, M). (N) Circulating noradrenaline levels of BoNT/B^Ctl (n = 8) and BoNT/B^Tan (n = 8) mice at 12 weeks (TAT-Cre- or vehicle-injected mice). One-tailed Mann Whitney test. Data are expressed as mean ± SEM. p < 0.05 values are indicated in the figures.

Figure 4

Expression of BoNT/B in tanycytes impairs glucose metabolism triggering compensatory mechanisms in pancreatic beta cells. (A) Curve representing glycaemia during a glucose tolerance test in BoNT/B^Ctl (n = 7) and BoNT/B^Tan (n = 8) mice at 12 weeks (TAT-Cre- or vehicle-injected mice). Two-way ANOVA, Interaction p = 0.2853, F _(7,112) 1.243; Time p = <0.0001, F _{(7, 112)} = 249.5; Treatment p = 0,0167, F _{(1, 16)} = 7.136. Bonferroni's multiple comparisons test, 45′ p = 0,0006, 120′ p = 0,0376). AUC, two-tailed unpaired t-test, t = 2.693, df = 16. (B) Serum insulin levels 0, 15 and 30 min after intraperitoneal delivery of glucose (2 g/kG) during the glucose tolerance test in A. Two-way ANOVA, Interaction p = 0.1142, F _(2,28) = 2.347; Time p = 0.0138, F _{(2, 28)} = 5.011; Treatment p = 0.0364, F _(1,14) = 5.351. Šídák's multiple comparisons test, 0min p = 0.8174; 15min p = 0.1925. AUC, two-tailed unpaired t-test, t = 2.286, df = 14. (C) Glycaemia during an insulin tolerance test in BoNT/B^Ctl (n = 7) and BoNT/B^Tan (n = 8) mice at 12 weeks (TAT-Cre- or vehicle-injected mice). Two-way ANOVA; Time p = <0.0001, F _{(6, 78)} = 26.36; Subject p = 0.0864, F _{(1, 13)} = 3.441. Uncorrected Fisher's LSD, 15min p = 0.0377; 30min p = 0.0150. AUC, one-tailed unpaired t-test, t = 1.955, df = 13. (D) Homa-IR, two-tailed unpaired t-test, t = 2.417, df = 10). (E) Insulin secretion in a static incubation of pancreatic islets isolated from BoNT/BCtl (n = 4) and BoNT/B^Tan (n = 4) mice, at 12 weeks (TAT-Cre- or vehicle-injected mice). Two-way ANOVA, Interaction p = 0.0091, F _(1,12) = 9,645; Glucose concentration p = <0.0001, F _{(1, 12)} = 121.9; Subject p = 0,0034, F _{(1, 12)} = 13.21. Šídák's multiple comparisons test, Low glucose _{(2,8 mM):}BoNT/B^Ctl vs. Low glucose _{(2,8 mM)}:BoNT/B^Tan p = 0.9995; Low glucose _{(2,8 mM)}:BoNT/B^Ctl vs. High glucose _{(16,7 mM)}:BoNT/B^Ctl p = 0.0007; Low glucose _{(2,8 mM)}:BoNT/B^Tan vs. High glucose _{(16,7 mM)}:BoNT/B^Tan p = <0.0001. (F) Intracellular insulin content of pancreatic islets isolated from BoNT/B^Ctl (n = 4) and BoNT/B^Tan (n = 4) mice at 12 weeks (TAT-Cre- or vehicle-injected mice). Two-tailed unpaired t-test, p = 0.5756, t = 0.5918, df = 6. (G,H) Relative mRNA expression levels of β-cell function and identity (G) and endoplasmic reticulum (ER) stress markers (H) in pancreatic islets isolated from BoNT/B^Ctl (n = 4) and BoNT/B^Tan (n = 4) mice at 12 weeks (TAT-Cre- or vehicle-injected mice). Unpaired t test, two-stage step-up (Benjamini, Krieger, and Yekutieli). In G, Gck p = 0.8919; Kcnj11 p = 0.7457; Ins1 p = 0.3688; Pcsk1 p = 0.07637; Nkx2.2 p = 0.0555 and Ucn3 p = 0.8395. In H, Xbp1t p = 0.1444; Xbp1s p = 0.2788. (I) Ratio between insulin-positive and glucagon-positive area to total islet surface area in BoNT/B^Ctl (n = 4) and BoNT/B^Tan (n = 4) mice at 12 weeks (TAT-Cre- or vehicle-injected mice). Two-way ANOVA, Interaction p = 0.516, F _{(1, 12)} = 0,4799; Insulin/Glucagon p = < 0.0001, F _{(1, 12)} = 4700; Subjects p = 0.9949, F _{(1, 12)} = 4.304e-005. Šídák's multiple comparisons test, Insulin-positive:BoNT/B^Ctl vs. Insulin-positive:BoNT/B^Tan p = 0.9609 and Glucagon-positive:BoNT/B^Ctl vs. Glucagon-positive:BoNT/B^Tan p = 0.9588. (J) Average surface area of pancreatic islets from I. Two-tailed unpaired t-test, p = 0.9182, t = 0.1071, df = 6. (K) Representative photomicrographs representing nuclei (blue), insulin (red) and glucagon (green) in isolated pancreatic islets from BoNT/B^Ctl and BoNT/B^Tan mice at 4 weeks (TAT-Cre- or vehicle-injected mice) in I and J. Scale bar, 50 μm. Data are expressed as mean ± SEM. p < 0.05 values are indicated in the figures. (For interpretation of the references to color/colour in this figure legend, the reader is referred to the Web version of this article.)

Figure 5

BoNT/B expression in tanycytes impairs spatial working memory. (A) Diagram representing the Y-maze test. In phase one, only one arm is accessible for the mice to explore. In phase 2, both arms of the maze are accessible, challenging the spatial memory of the mice by quantifying the time they spent in each arm. (B) Percentage of time that BoNT/B^Ctl (n = 4) and BoNT/B^Tan (n = 6) spent in the arms during phase 2 of the Y-maze test, at 12 weeks (AAV1/2 -infused mice) in the lateral ventricle. One-tailed unpaired t-test; BoNT/B^Ctl old-arm vs BoNT/B^Ctl new-arm, t = 2.140, df = 6; BoNT/B^Tan old-arm vs BoNT/B^Tan new-arm, t = 0.05559, df = 10, p = 0.2952. Two-way ANOVA, Interaction p = 0.0384, F (1,16) = 5.092; Space p = 0.2131, F (1,16) = 1.682; Genptype P > 0,9999, F (1,16) = 0. Uncorrected Fisher's LSD, BoNT/BTanOld vs BoNT/BTan Novel p = 0.4590. Data are expressed as mean ± SEM. p < 0.05 values are indicated in the figures.

Supplementary Figure 1. The expression of Botulinum toxin type B light chain (BoNT/B) in tanycytes does not induce tanycytic cell death. (A) Representative images from three BoNT/B^loxP/loxP and BoNT/B^Tan littermates are presented, captured 12 weeks after mock or Cre-mediated genetic recombination. The top subpanels depict higher magnification of the framed regions in the corresponding bottom panels. (B–C) Representative cleaved caspase-3 immunoreactivity in frontal sections of the tuberal region of the hypothalamus from BoNT/B^loxP/loxP and six BoNT/B^Tan littermates, captured at 4 weeks (B, n = 3 per genotype) and 12 weeks (C, n = 4 and 6, respectively) after mock or Cre-mediated genetic recombination. Note the absence of cleaved caspase 3 immunoreactivity in cell nuclei lining the floor and wall of the 3V. In C, the inset shows a cleaved caspase 3 immunoreactive cell in the parenchyma. Scale bars, 50 μm in B and C (10 μm in inset).

Supplemenatry figure 2. Energy expenditure and ambulatory activity in mice fed on chow or high-fat diet. (A) Body weight of BoNT/B^Ctl (n = 9) and BoNT/B^Tan (n = 12) littermates, 4 and 12 weeks after Cre-induced genetic recombination by TAT-Cre infusion into the third ventricle. Values are expressed in grams (gr). Two-way ANOVA, Genotype x time p = 0.0019, F2, 38 = 7.405, Time p < 0.0001, F1, 19 = 0.1438; subjects p < 0.0001, F (19, 38) = 14; Tukey's multiple comparisons test. (B–C) Curves representing 24h energy expenditure B and ambulatory activity C in BoNT/B^Ctl (n = 7) and BoNT/B^Tan (n = 8) mice 4 weeks after the beginning of the experiment (AAV1/2-injected mice) Cre-induced genetic recombination. Two-way ANOVA. In B, Interaction p = 0.6239 F _{(23, 322)} = 0.8816; Time p < 0.0001, F _{(23, 322)} = 58.66; Subject p = 0.1029, F _{(1, 14)} = 3,045. LSD post hoc, 18h p = 0.0164; 5h p = 0.0486 and 6h p = 0.0475. AUC, one-tailed Mann Whitney t = 1.789, df = 14. In C, Interaction p = 0.0868, F _{(23, 299)} = 1.449; Time p < 0.0001, F _{(23, 299)} = 22.06; Subject p = 0.1383 F _{(1, 13)} = 2.493. LSD post-hoc test, 20h p = 0.0481; 5h p = 0.0018; 6h p = 0.0025 and 7h p = 0.0182. AUC, one-tailed Mann Whitney. (D) Curves representing 24 h EE in BoNT/B^Ctl (n = 6) and BoNT/B^Tan (n = 6) mice at 8 weeks (AAV1/2-injected mice) and 4 weeks of HFD. Two-way ANOVA, Interaction p = 0.9293, F _{(23, 230)} = 1.814; Time p < 0.0001, F _{(23, 230)} = 38.70; Subject p = 0.2078, F _{(1, 10)} = 1.814. LSD post-hoc test. (E) Curves representing 24h ambulatory activity in BoNT/B^Ctl (n = 6) and BoNT/B^Tan (n = 6) mice at 8 weeks (AAV1/2-injected mice) and 4 weeks of HFD. Two-way ANOVA, Interaction p = 0.2022, F _{(23, 230)} = 1.253; Time p < 0.0001, F _{(23, 230)} = 14.16; Subject p = 0.8224, F _{(1, 10)} = 0.05313. LSD post-hoc test, 2.00 p = 0.0064, 5.00 p = 0.0075. Data are expressed as mean ± SEM. p < 0.05 values are indicated in the figures.