Switching harmful visceral fat to beneficial energy combustion improves metabolic dysfunctions

Figures

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  Figure 1. Food intake, body weight, BMI, browning of visceral WAT. (A) Food intake per week, body weight, and BMI with exposure to various temperatures (n = 20 mice per group, data represent mean ± SEM, one-way ANOVA). (B) Mouse morphology and epididymal WAT (eWAT) morphology. (C) Fat mass of eWAT, s.c. WAT, and interscapular brown adipose tissue (iBAT) (n = 15 mice per group, data represent mean ± SEM, one-way ANOVA). (D) Contingent survival and death of mice exposed to various temperatures (n = 20 mice per group). (E) H&E, UCP1, prohibitin, perilipin A, and endomucin staining of eWAT. Scale bar: 20 μm. (F) Quantification of adipocyte (AC) size and UCP1-, prohibitin-, and endomucin-positive signals of eWAT (50 random fields from 10 mice in each group). **P < 0.01; **P < 0.01; ***P < 0.001, one-way ANOVA. Box-and-whisker plots show median (line within box), upper and lower quartile (bounds of box), and minimum and maximum values (bars).

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  Figure 2. Genome-wide profiling, qPCR, and Western blot analyses of eWAT. (A–D) Genome-wide profiling of eWAT with exposure to various temperatures by heat map and volcano analyses (n = 3 samples per group). (E) Left panel: qPCR analysis of Ucp1 expression in eWAT with exposure to various temperatures (n = 6 samples per group). Right panel: qPCR and Western blot analyses of UCP1 protein expression in eWAT (n = 6 samples per group). (F) Quantification of Leptin, Adiponectin, and Resistin mRNA levels in s.c. WAT exposed to various temperatures (n = 6 samples per group). *P < 0.05; **P < 0.01; ***P < 0.001, one-way ANOVA. Box-and-whisker plots show median (line within box), upper and lower quartile (bounds of box), and minimum and maximum values (bars).

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  Figure 3. Food intake, body weight, BMI, and browning of visceral WAT of HFD-fed obese mice. (A) Food intake per week, body weight, and BMI of various temperature-exposed HFD-fed obese mice (n = 15 mice per group, data represent mean ± SEM, one-way ANOVA). (B) Mouse morphology and eWAT morphology. (C) Fat mass of eWAT, s.c. WAT, and iBAT of various HFD-fed obese mice (n = 15 mice per group, data represent mean ± SEM, one-way ANOVA). (D) Contingent survival and death of mice exposed to various temperatures (n = 20 mice per group). (E and F) H&E, UCP1, prohibitin, perilipin A, and endomucin staining of eWAT. Scale bars: 20 μm. (G) Quantification of AC size and UCP1-, prohibitin-, and endomucin-positive signals of eWAT (40 random fields from 8 mice in each group). *P < 0.05; **P < 0.01; ***P < 0.001, one-way ANOVA. Box-and-whisker plots show median (line within box), upper and lower quartile (bounds of box), and minimum and maximum values (bars).

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  Figure 4. Micro-PET imaging, nonshivering thermogenesis, lipolysis, blood lipid profiling, and glucose metabolism. (A) Micro-PET imaging of iBAT, s.c. WAT, and eWAT. (B) Metabolic rates of O2 consumption and CO2 production in response to norepinephrine (NE) (n = 5 mice per group, two-way ANOVA, data represent mean ± SEM). (C) Glycerol release from eWAT of various groups (n = 6 samples per group). One-way ANOVA. (D) Blood lipid profile of cholesterol, low-density lipoprotein cholesterol (LDL-C), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and nonesterified fatty acids (NEFAs) (n = 15 samples per group). One-way ANOVA. (E) Fasting glucose, fasting insulin, insulin-tolerance test, and glucose-tolerance test with exposure to various temperatures (n = 8–10 animals per group). AUC of insulin-tolerance test and glucose-tolerance test. One-way ANOVA. (F) Contingency survival and death of eWAT-less (eWL) and sham-operated (Sham) mice exposed to –10°C/–20°C (n = 20 animals per group, χ2 test). (G) Metabolic rates of O2 consumption and CO2 production of eWL and sham-operated mice in response to norepinephrine (NE) under –10°C/–20°C (n = 5 animals per group). Two-way ANOVA, data…

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  Figure 5. Impacts of UCP1 knockdown on global metabolic functions under an extreme cold condition. (A) Immunohistological analysis of AAV transduction efficiency. AAV-Gfp– and AAV-shUcp1–transduced eWAT expressed GFP (green). The sections were counter-stained with DAPI (blue). Transduction efficiencies were quantified (n = 4 samples per group, 2-tailed t test). (B) Knockdown efficiency of endogenous UCP1 protein was analyzed by Western blot analysis (n = 6 samples per group, 2-tailed t test). (C) Contingent survival and death of AAV-Gfp– and AAV-shUcp1–transduced mice under –10°C/–20°C (n = 20 mice per group, χ2 test). (D) Metabolic rates of O2 consumption and CO2 production of AAV-Gfp– and AAV-shUcp1–transduced mice in response to norepinephrine (NE) under –10°C/–20°C (n = 5 mice per group). Two-way ANOVA, data represent mean ± SEM. (E) Fasting glucose, fasting insulin, and insulin-tolerance test of AAV-Gfp– and AAV-shUcp1–transduced mice under –10°C/–20°C (n = 10 mice per group). AUC of insulin-tolerance test. *P < 0.05; **P < 0.01; ***P < 0.001. 2-tailed t test for fasting glucose, fasting insulin, and AUC; two-way ANOVA for insulin-tolerance test. Data represent mean ± SEM.…

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  Figure 6. Nonshivering thermogenesis, lipolysis, blood lipid profiling, and glucose metabolism in HFD-fed obese mice. (A) Metabolic rates of O2 consumption and CO2 production of HFD-induced obese mice in response to NE (n = 5 mice per group). Two-way ANOVA. Data represent mean ± SEM. (B) Glycerol release from eWAT of various HFD-induced obese mice (n = 6 samples per group). One-way ANOVA. (C) Blood lipid profile of cholestero (TC), LDL-C, TG, HDL-C, and NEFAs of HFD-induced obese animals (n = 15 samples per group). One-way ANOVA. (D) Fasting glucose, fasting insulin, insulin-tolerance test, and glucose-tolerance test of HFD-induced obese mice exposed to various temperatures (n = 8 samples per group for 30°C; n = 11–15 samples per group for other groups). AUC of insulin-tolerance test and glucose-tolerance test. *P < 0.05; **P < 0.01; ***P < 0.001. One-way ANOVA for fasting glucose, fasting insulin, and AUC; two-way ANOVA for insulin-tolerance test and glucose-tolerance test. Data represent mean ± SEM. Box-and-whisker plots show median (line within box), upper and lower quartile (bounds of box), and minimum and maximum values (bars).

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  Figure 7. Liver steatosis in HFD-fed obese mice. (A–C) Weight, H&E staining, and Oil Red O staining of liver tissues from HFD-induced obese mice exposed to various temperatures (n = 8 animals per group). Scale bar: 50 μm. Oil Red O–positive signals were quantified from 20 random fields. *P < 0.05; ***P < 0.001. One-way ANOVA.

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  Figure 8. Schematic diagram of successive activation of defensive mechanisms against decreasing environmental cold. Under mild cold such as 4°C exposure, activation of the BAT-nonshivering thermogenesis and modest browning of s.c. WAT are sufficient to maintain core body temperature. However, visceral WAT remains thermogenically inactive. Further decreased environmental temperature — to –10°C, for example — enhances browning of s.c. WAT and triggers modest browning of visceral fat to generate nonshivering heat. With extreme cold such as –10°C/–20°C, browning of visceral fat markedly contributes to nonshivering thermogenesis to main core body temperature. Additionally, browning of visceral fat increases insulin sensitivity and improves liver steatosis in obese mice.