Thermogenic Adipose ADH5 Counteracts Age-related Metabolic Decline

Abstract

Aging-associated decline in brown adipose tissue (BAT) function and mass contributes to energy and metabolic homeostasis disruption. Alcohol dehydrogenase 5 (ADH5) is a major denitrosylase that prevents cellular nitro-thiol redox imbalance, an essential feature of aging. However, the functional significance of BAT ADH5 in the context of aging is largely unknown. Here, we aimed to investigate the role of BAT ADH5 in protecting against age-related metabolic dysfunction. We show that aging promotes aberrant BAT protein S-nitrosylation modification and downregulates ADH5 in mice. Furthermore, BAT ADH5-deletion accelerates BAT senescence and aging-associated declines in metabolic homeostasis and cognition. Mechanistically, we found that aging inactivates BAT Adh5 by suppressing heat shock factor 1 (HSF1), a well-recognized proteostasis regulator. Moreover, pharmacologically enhancing HSF1 improved BAT senescence, metabolic decline, and cognitive dysfunction in aged mice. Together, these findings suggest that the BAT HSF1-ADH5 signaling cascade plays a key role in protecting against age-related systemic functional decline. Ultimately, unraveling the role of thermogenic adipose nitrosative signaling will provide novel insights into the interplay between BAT nitric oxide bioactivity and metabolism in the context of aging.

Keywords: Adipose tissue; Aging; Metabolic Dysfunction; Nitrosative Stress.

Figure 1.. Age-induced BAT ADH5 reduction contributes to BAT senescence.

A. Representative images (63X) of protein S-nitrosylation staining (SNO) in BAT from young (3-month) or old mice (12- and 22-month). –AS: no ascorbate; negative control for SNO staining. Scale bar: 10 μm. B. Representative Western and densitometric analysis (normalized to ACTB input) of SNO proteins in BAT from 3- or 22-month-old wild-type mice. C&D. Adh5 expression (normalized to Gapdh) in BAT and iWAT respectively of Adh5^fl and Adh5^BKO mice. N= 3-7 mice/group. E. Levels of mRNAs in BAT of young (3-month) Adh5^fl, Adh5^BKO, or WT mice compared to 22-month-old WT by RT-qPCR. N= 3-10 mice/group. F. Representative images of H&E, IF of p16^lNK4a, and β gal staining in BAT from age-matched Adh5^fl and Adh5^BKO mice. Scale bar: 10 μm in H&E and p16^lNK4a, 50 μm in β gal staining. Right panel: quantified fluorescence intensities of p16^lNK4a. 5 fields/mouse, n= 3 mice/group. Data are presented as means ± SEM. *indicates statistical significance relative to the 3-month groups, and ^#indicates genetic effects in mice at the same age; as determined by Student’s t-test in (B) and ANOVA followed by posthoc test in (C-E&F), p<0.05.

Figure 2.. BAT Adh5 deletion accelerates age-related functional declines.

A. Body weight, and B. Body composition of Adh5^fl and Adh5^BKO mice at indicated age (n= 5-22 mice/group). Inset: representative picture of Adh5^fl and Adh5^BKO mice at 12 months. C&D. Whole-body energy balance measured in Adh5^fl and Adh5^BKO mice housed within a metabolic cage (n= 5-7 mice/group). E-F. GTT and AUC from Adh5^fl and Adh5^BKO mice (n= 14-30 mice/group). G. Grip strength, H. Rotarod assessment, I. Barnes maze assessment of velocity, J. percent of time in target/nontarget zone and K. primary errors to escape hole in 19-month-old Adh5^fl and Adh5^BKO mice. N = 7-9 mice/group. L&M. Heart mass and left ventricle thickness (LV) from age-matched Adh5^fl and Adh5^BKO mice measured by echocardiography; n = 5-7 mice/group. Data are presented as means ± SEM. *indicates genetic effects in same-age mice, and #indicates age effects in the same type of mice; as determined by Student’s t-test (A, C, D, and G-M), and ANOVA in (B) or ANOVA of AUC in (F), p<0.05.

Figure 3.. BAT ADH5-deficiency augments age-associated BAT immuno-metabolic imbalances.

A. Gene expression of measured markers in SVF from BAT of Adh5^fl or Adh5^BKO mice. N= 3-5 mice/group. B. Plasma levels of IL-1β in 3-month-old Adh5^fl or Adh5^BKO mice measured by ELISA. N = 5-6 mice/group. C. Representative images (63X) and quantification of IF of F4/80 staining in BAT from Adh5^fl or Adh5^BKO mice at indicated age. N= 3 mice/group, 4-8 fields/mouse. Scale bar: 1 μm. D. F4/80 (63X, scale bar: 1 μm) staining and quantification in iWAT from Adh5^fl or Adh5^BKO mice at the indicated age. N= 3 mice/group, 4-8 fields/mouse. E&F. Representative Western blot and quantification of ETC proteins in BAT of 3-month-old Adh5^fl or Adh5^BKO mice (n= 3 mice/group). G. Representative TEM images of BAT from 3-month-old Adh5^fl or Adh5^BKO mice. Red arrow, the mitochondrion. Scale bar: 5 μm (I), 2 μm (II), and 1 μm (III). H. Quantification of mitochondrial area in TEM images of BAT in mice in (C). Quantification was performed using ImageJ software. I. Schematic summary of the BAT metabolomic analysis in Adh5^fl or Adh5^BKO mice (3-month-old) analyzed by using Metaboanalyst online software; n = 3 age-matched mice per group. Downregulated metabolites by ADH5 deletion are depicted in red; upregulated metabolites are depicted in green. Data are presented as means ± SEM. * indicates genetic effects in same-age mice as determined by Student’s t-test (A, B, F and H), and ANOVA (C&D), p<0.05.

Figure 4.. HSF1 induces Adh5 and ameliorates senescence in BAT.

A. Hsf1 levels in BAT at indicated age (n= 6-12 mice/group) measured by qRT-PCR. Data were normalized to Gapdh. B. Left panel: representative HSF1 staining (63X, scale bar: 1 μm; zoomed images are on the right) from BAT of young and old wild-type mice. Right panel: quantification, n = 3-4 mice/age. 4-8 fields/mouse. C. Occupancy of Adh5 promoter regions by HSF1 from BAT treated with a pharmacological activator of HSF1 (HSF1A; Millipore, 20 μM, 24-hr) in controls or Adh5^BKO mice (n= 3 mice/group). D. Levels of mRNAs encoding the indicated genes of BAT explants treated ex vivo with HSF1A (20 μM, 24-hr) or HSF1 inhibitor (KRIBB11; 2 μm, 24-hr). Data were normalized to Gapdh. N=4 mice/group. E. Levels of mRNAs of the indicated genes in SVF from 3- or 5-month-old BAT pad isolated from wild-type mice followed by treatment of HSF1A (20 μM, 24-hr). N=4 mice/age group. Data were normalized to Gapdh. Data are presented as means ± SEM. *indicates statistical significance compared to the 3-month groups (A&B), to the Adh5^fl groups in (C), and to the control groups in (D-F) as determined by ANOVA in (A-D), and Student’s t-test in (E), p<0.05.

Figure 5.. BAT HSF1 mitigates age-associated functional decline.

A. Comparison of release curves between Collogen-NC-HSF1A vs Collagen-HSF1A. B. Body weight measurement over time in 12-month-old wild-type mice after interscapular injection with Collagen-NC-Vehicle (NC-Vehicle) or Collagen-NC-HSF1A (NC-HSF1A; 0.1 mg). C. Heat, VO₂, and energy expenditure (EE) in 12-month-old wild-type mice after one BAT injection of control (NC-Vehicle) or NC-HSF1A (0.1 mg; n= 5-6 mice/group). D. GTT and AUC of mice from (B). N= 19-37 mice/group. E. Latency (Barnes maze test) and F. AUC of escape measured from mice in (C) after one-month injection. N= 7-13 mice/group. G. Representative H&E (Scale bar: 20 or 10 μm) and IF of p16^lNK4a (scale bar: 10 μm) in BAT from mice in (C). H. Quantitation of IF staining of p16^lNK4a. N= 5 mice/group. 4-6 fields/images. I. Representative F4/80 staining of BAT from (C); quantitation is left of the image. Scale bar: 1 μm, n = 5 mice/group. 4-6 fields/images. J. Levels of mRNAs encoding genes of interest (normalized to Gapdh) in BAT from mice in (C). N = 3-9 mice/group. Data are presented as means ± SEM. *indicates statistical significance compared to the vehicle groups as determined by Student’s t-test, p<0.05.