Growth Hormone Receptor Antagonist Transgenic Mice Have Increased Subcutaneous Adipose Tissue Mass, Altered Glucose Homeostasis and No Change in White Adipose Tissue Cellular Senescence

Abstract

Background: Growth hormone (GH)-resistant/deficient mice experience improved glucose homeostasis and substantially increased lifespan. Recent evidence suggests that long-lived GH-resistant/deficient mice are protected from white adipose tissue (WAT) dysfunction, including WAT cellular senescence, impaired adipogenesis and loss of subcutaneous WAT in old age. This preservation of WAT function has been suggested to be a potential mechanism for the extended lifespan of these mice.

Objective: The objective of this study was to examine WAT senescence, WAT distribution and glucose homeostasis in dwarf GH receptor antagonist (GHA) transgenic mice, a unique mouse strain having decreased GH action but normal longevity.

Methods: 18-month-old female GHA mice and wild-type (WT) littermate controls were used. Prior to dissection, body composition, fasting blood glucose as well as glucose and insulin tolerance tests were performed. WAT distribution was determined by weighing four distinct WAT depots at the time of dissection. Cellular senescence in four WAT depots was assessed using senescence-associated β-galactosidase staining to quantify the senescent cell burden, and real-time qPCR to quantify gene expression of senescence markers p16 and IL-6.

Results: GHA mice had a 22% reduction in total body weight, a 33% reduction in lean mass and a 10% increase in body fat percentage compared to WT controls. GHA mice had normal fasting blood glucose and improved insulin sensitivity; however, they exhibited impaired glucose tolerance. Moreover, GHA mice displayed enhanced lipid storage in the inguinal subcutaneous WAT depot (p < 0.05) and a 1.7-fold increase in extra-/intraperitoneal WAT ratio compared to controls (p < 0.05). Measurements of WAT cellular senescence showed no difference between GHA mice and WT controls.

Conclusions: Similar to other mice with decreased GH action, female GHA mice display reduced age-related lipid redistribution and improved insulin sensitivity, but no change in cellular senescence. The similar abundance of WAT senescent cells in GHA and control mice suggests that any protection against generation of senescent cells afforded by decreased GH action, low insulin-like growth factor 1 and/or improved insulin sensitivity in the GHA mice may be offset by their severe adiposity, since obesity is known to increase senescence.

Fig. 1

Body Composition and WAT distribution in 18mo female GHA (n=12) and WT (n=13) mice. Panel A: Comparison of absolute body weight. Panel B: Percent fat, lean, and fluid was determined by dividing fat, lean, and fluid mass by total body weight. Panel C: Relative WAT depot weight for the inguinal subcutaneous (Ing), paraovarian (Para), retroperitoneal (Retro), and mesenteric (Mes) depots was determined by dividing absolute depot weights by total body weight for each mouse. Panel D: The ratio of extra-peritoneal to intra-peritoneal WAT mass was determined by dividing the combined mass of the Ing and Retro depots by the combined mass of the Para and Mes depots. Data are expressed as mean ± SEM. *, significantly different from WT littermate controls (P<0.05).

Fig. 2

Glucose homeostasis in 18mo old female GHA (n=12) and WT (n=13) mice. Panel A: Comparison of plasma glucose levels after a 12h fast. Panel B: Comparison of circulating insulin levels after 12h fast. Panel C: Insulin tolerance tests showing glucose levels following intraperitoneal injection of 0.075u/ml insulin. Panel D: Glucose tolerance tests showing change in glucose levels following intraperitoneal injection of 10% glucose solution at 0.01 ml/g body weight. Panel E: Comparison of glucose tolerance test area under the curve after 1 hour and 2 hours. Data are expressed as mean ± SEM. *, significantly different from WT littermate controls (P<0.05).

Fig. 3

WAT senescence results for 18mo old female GHA (n=6) and WT (n=6) mice. Panel A: Representative images of Ing WAT from WT (top row) and GHA (bottom row) mice. Images on the left show nuclei stained with DAPI viewed under fluorescence settings (nuclei are light blue); images on the right show the same field viewed under phase contrast settings to visualize SA-βgal+ senescent cells. SA-βgal+ senescent cells give off a blue color when stained with X-galactose (indicated by black arrows). Percent of SA-βgal+ cells is calculated by dividing the number of SA-βgal+ cells by the number of nuclei in each image and the results represent the average of four images taken for each depot from each mouse. Panel B: Percent of SA-βgal+ cells in the inguinal subcutaneous (Ing), subscapular (Scap), paraovarian (Para), retroperitoneal (Retro), and mesenteric (Mes) WAT depots in 18mo old female GHA (n=6) and WT (n=6) mice. Superscript letters refer to the mean of the depot. Means shown with a common superscript letter are not significantly different, p>0.05. Data are expressed as mean ± SEM.

Fig. 4

Gene expression of senescent markers in 18mo female GHA (n=6) and WT (n=6) mice. Panel A: Comparison of p16 expression. Panel B: Comparison of IL6 expression. Data are expressed as mean ± SEM. Superscript letter refer to the mean of the depot. Means shown with a common superscript letter are not significantly different, p>0.05. Ing, inguinal subcutaneous; Scap, subscapular; Para, paraovarian; Retro, retroperitoneal; Mes, mesenteric; BAT, interscapular brown adipose tissue. N.S., no significant differences.