Senolytic drugs, dasatinib and quercetin, attenuate adipose tissue inflammation, and ameliorate metabolic function in old age

Abstract

Aging results in an elevated burden of senescent cells, senescence-associated secretory phenotype (SASP), and tissue infiltration of immune cells contributing to chronic low-grade inflammation and a host of age-related diseases. Recent evidence suggests that the clearance of senescent cells alleviates chronic inflammation and its associated dysfunction and diseases. However, the effect of this intervention on metabolic function in old age remains poorly understood. Here, we demonstrate that dasatinib and quercetin (D&Q) have senolytic effects, reducing age-related increase in senescence-associated β-galactosidase, expression of p16 and p21 gene and P16 protein in perigonadal white adipose tissue (pgWAT; all p ≤ 0.04). This treatment also suppressed age-related increase in the expression of a subset of pro-inflammatory SASP genes (mcp1, tnf-α, il-1α, il-1β, il-6, cxcl2, and cxcl10), crown-like structures, abundance of T cells and macrophages in pgWAT (all p ≤ 0.04). In the liver and skeletal muscle, we did not find a robust effect of D&Q on senescence and inflammatory SASP markers. Although we did not observe an age-related difference in glucose tolerance, D&Q treatment improved fasting blood glucose (p = 0.001) and glucose tolerance (p = 0.007) in old mice that was concomitant with lower hepatic gluconeogenesis. Additionally, D&Q improved insulin-stimulated suppression of plasma NEFAs (p = 0.01), reduced fed and fasted plasma triglycerides (both p ≤ 0.04), and improved systemic lipid tolerance (p = 0.006). Collectively, results from this study suggest that D&Q attenuates adipose tissue inflammation and improves systemic metabolic function in old age. These findings have implications for the development of therapeutic agents to combat metabolic dysfunction and diseases in old age.

Keywords: aging; dasatinib; immune cells; inflammation; metabolic function; quercetin; senescence; senolytics.

FIGURE 1

Administration of D&Q suppresses age‐related increases in senescence and inflammatory markers in pgWAT. (a) Schematic representation of the dasatinib and quercetin (D&Q) treatment and the non‐terminal experiments such as glucose‐, insulin‐, pyruvate‐, and intralipid‐tolerance tests (GTT, ITT, PTT, and LTT), insulin secretion during GTT, insulin‐stimulated free fatty acid suppression (Ins‐FFA) measurement, (b) representative H&E images of perigonadal white adipose tissue (pgWAT) after staining with senescence‐associated β‐galactosidase (SA β‐gal), blue arrowheads indicate SA β‐gal+ cells and red arrowheads indicate crown‐like structures (CLS) (c and d) quantification of SA β‐gal+ cells and CLS, (e) p16 and p21 gene expression relative to 18s in pgWAT, (f and g) Western blot images and quantification of P16 protein expression relative to vinculin, (h) mcp1, tnf‐α, il‐1α, il‐1β, il‐6, cxcl2, and cxcl10 gene expression relative to 18s in pgWAT. Data are shown as mean ± SEM with individual data points in the bar graphs. N = 5–8/group. *Denote p ≤ 0.04 versus Y Ctrl, ^†denote p ≤ 0.04 versus O Ctrl. Group differences were assessed by one‐way ANOVA with Tukey's post hoc tests.

FIGURE 2

Suppressed senescence and inflammatory burden is concomitant to reduced T cells and macrophages in pgWAT. (a and b) representative images and quantification of total T cells (CD45+, CD3+) in perigonadal white adipose tissue (pgWAT), (c) CD3e and FoxP3 gene expression relative to 18s in pgWAT, (d–g) representative images and quantification of total, M1 (CD206−) and M2 (CD206+) macrophages in pgWAT. Data are shown as mean ± SEM with individual data points in the bar graphs. N = 7/group. *Denote p ≤ 0.01 versus Y Ctrl, ^†denote p ≤ 0.04 versus O Ctrl. Group differences were assessed by one‐way ANOVA with Tukey's post hoc tests.

FIGURE 3

Administration of D&Q ameliorates glucose tolerance and insulin‐stimulated NEFA suppression independent of the Akt signaling. (a and b) Blood glucose response curves and area under the curves (AUC) during glucose tolerance test (GTT: 2 g/kg, ip), (c) plasma insulin at baseline and during GTT, (d) blood glucose response during insulin tolerance test (ITT: 1 U/kg, ip), (e) plasma non‐esterified fatty acids (NEFA) at baseline and during ITT, (f) irs‐1 and cpt‐1α gene expression relative to 18s in pgWAT, (g–j) Western blot images and quantification for total Akt, phosphorylated Akt and vinculin form the liver and pgWAT. Data in the time response curves are shown as mean ± SEM. Other data are shown as mean ± SEM with individual data points in the bar graphs. N = 7–12/group. *Denote p ≤ 0.03 versus O Ctrl, ^†denote p ≤ 0.02 versus baseline. Group differences in GTT, ITT and plasma insulin at baseline and during GTT as well as plasma NEFA at baseline and during ITT were assessed by two‐way repeated measure ANOVA with Tukey's post hoc tests. Unpaired Student's t tests were used when comparing two groups.

FIGURE 4

Improvement in glucose tolerance is accompanied by attenuated hepatic gluconeogenesis and collagen content. (a) Blood glucose response during pyruvate tolerance test (PTT: 2 g/kg, ip), (b) gluconeogenic gene (pck1, pck2, fbp2, and g6pc) expression relative to 18s in liver, (c) representative Western blot images of phosphorylated‐ and total‐cAMP‐response element‐binding protein (CREB) as well as vinculin in liver, (d) densitometric quantification of p‐CREB and CREB, (e) fgf21 and pparα gene expression relative to 18s in liver, (f) representative hematoxylin and eosin (H&E) and picrosirius red (PSR) staining of liver, (g) quantification of the pixel positivity for collagen in the liver. Data in the time response curves are shown as mean ± SEM. Other data are shown as mean ± SEM with individual data points in the bar graphs. N = 5–12/group. *Denote p ≤ 0.05. Group difference in PTT was assessed by two‐way repeated measure ANOVA with Tukey's post hoc tests. Unpaired Student's t tests were used when comparing two groups.

FIGURE 5

Administration of D&Q ameliorates plasma triglycerides and lipid tolerance but does not alter plasma cholesterols. (a) Fed plasma triglycerides, (b and c) plasma triglycerides response curves and area under the curves (AUC) during an intralipid‐tolerance test (LTT: 15 μl/g, oral gavage), (d) plasma cholesterol, (e) plasma low‐density and very low‐density lipoprotein, (f) plasma high‐density lipoprotein. Data in the time response curves are shown as mean ± SEM. Other data are shown as mean ± SEM with individual data points in the bar graphs. N = 8–10/group. *Denote p ≤ 0.01. Group difference in LTT was assessed by two repeated measure ANOVA with Tukey's post hoc tests. Other differences were assessed by unpaired Student's t test.