Specific suppression of insulin sensitivity in growth hormone receptor gene-disrupted (GHR-KO) mice attenuates phenotypic features of slow aging

Abstract

In addition to their extended lifespans, slow-aging growth hormone receptor/binding protein gene-disrupted (knockout) (GHR-KO) mice are hypoinsulinemic and highly sensitive to the action of insulin. It has been proposed that this insulin sensitivity is important for their longevity and increased healthspan. We tested whether this insulin sensitivity of the GHR-KO mouse is necessary for its retarded aging by abrogating that sensitivity with a transgenic alteration that improves development and secretory function of pancreatic β-cells by expressing Igf-1 under the rat insulin promoter 1 (RIP::IGF-1). The RIP::IGF-1 transgene increased circulating insulin content in GHR-KO mice, and thusly fully normalized their insulin sensitivity, without affecting the proliferation of any non-β-cell cell types. Multiple (nonsurvivorship) longevity-associated physiological and endocrinological characteristics of these mice (namely beneficial blood glucose regulatory control, altered metabolism, and preservation of memory capabilities) were partially or completely normalized, thus supporting the causal role of insulin sensitivity for the decelerated senescence of GHR-KO mice. We conclude that a delayed onset and/or decreased pace of aging can be hormonally regulated.

Keywords: (neuro)endocrinology of senescence; endocrinology and metabolism; growth hormone hormonal signaling; insulin sensitivity; longevity regulation.

Figure 1

The RIP::IGF-1 Transgene Does Not Affect any of Multiple Measures of Proliferation or Growth within the GHR-KO Mouse. (A) Body weight measurements showed no effects of the RIP::IGF-1 transgene for either male (upper graph) or female (lower graph) mice of either Ghr/bp genotype. (B) Body length measurements revealed that the RIP::IGF-1 transgene had no effect on the length of female GHR-KO or GHR-N mice. (C) Measurement of body weight after extensive removal of adipose tissue documented that the RIP::IGF-1 transgene did not affect the combined weight of nonadipose tissues. (D) Biochemical analysis of plasma IGF-1 concentration confirmed that the RIP::IGF-1 transgene did not increase the circulating levels of IGF-1 in GHR-KO mutants or GHR-N littermates. (E) Adipose depot weight analysis showed no effect of the RIP::IGF-1 transgene on the weights of the following depots (relative to whole body weight): perigonadal, perinephric, retroperitoneal, mesenteric, total visceral adipose tissue (VAT), subcutaneous white adipose tissue (SWAT), brown adipose tissue (BAT), total subcutaneous adiposity, or total adiposity. All measures of central tendency are arithmetic means, and all depictions of variation (error bars) represent standard deviations (SD). (See also Figs S1–S2).

Figure 2

The RIP::IGF-1 Transgene Normalizes the Slow-Aging-Associated Insulin Sensitivity of the GHR-KO Mouse Specifically by Increasing Blood Insulin Concentration. (A) Plasma insulin concentration determination exhibited RIP::IGF-1 transgene-mediated increased insulin content in GHR-KO females. (B) Plasma glucagon concentration measurement showed no effect of the RIP::IGF-1 transgene on glucagon content. (C) Insulin tolerance testing (with statistical analysis table) revealed that female GHR-KO mice are more insulin-sensitive than their GHR-N littermate controls, but that GHR-KO;RIP::IGF-1 mice have the same degree of sensitivity to insulin as GHR-N mice. (D) Analysis of the plasma levels of the following proteins revealed no effect of the RIP::IGF-1 transgene in GHR-KO mice: adiponectin, leptin, resistin, monocyte chemo-attractant protein 1 (MCP-1), plasminogen activator inhibitor 1 (PAI-1), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6). (E) Assessment of the concentrations of the following lipid-associated energetic constituents documented the following RIP::IGF-1-mediated reductions in female GHR-KO mice: plasma triglycerides, plasma low-density lipoprotein (LDL)-cholesterol, and plasma nonesterified fatty acids (NEFAs); although there was no statistically significant outcome of the transgene on AL-fed or fasted blood β-hydroxybutyrate, fasted blood β-hydroxybutyrate concentration was greater in GHR-KO mice than in GHR-N controls (analysis grouped all GHR-KO mice together and all GHR-N mice together, regardless of the presence of the RIP::IGF-1 transgene, as the central tendency ± dispersion values within Ghr/bp genotype were very similar). All measures of central tendency are arithmetic means, and all depictions of variation (error bars) represent standard deviations (SD). (See also Fig. S3.).

Figure 3

The RIP::IGF-1 Transgene Normalizes the Slow-Aging-Associated Endocrinological Phenotypes of the Female GHR-KO Mouse. (A) AL-fed glucose tolerance testing (normalized values, with statistical analysis table). (B) Fasted glucose tolerance testing (normalized values, with statistical analysis table). (C) 0.75 USPU insulin tolerance testing (normalized values, with statistical analysis table). (D) 0.3 USPU insulin tolerance testing (normalized values, with statistical analysis table). (E) Gene expression analysis of insulin sensitivity-mediating genes in appendicular skeletal musculature and hepatic tissues. (F) Pyruvate conversion testing (normalized values, with statistical analysis table). (G) Gene expression analysis of hepatic gluconeogenesis-modulating genes in hepatic tissues. (H) AL-fed blood glucose concentration testing (with statistical analysis table). (I) Fasted blood glucose concentration testing (with statistical analysis table). All measures of central tendency are arithmetic means, and all depictions of variation (error bars) represent standard deviations (SD). (See also Figs S4–S15).

Figure 4

The RIP::IGF-1 Transgene Normalizes the Slow-Aging-Associated Body-Core Temperature of the Female GHR-KO Mouse. Body-core temperature measured at the end of the more active period (darkened) half of the subjects’ day confirmed that female GHR-KO mice consistently have a lower body-core temperature than their littermate controls and established that female GHR-KO;RIP::IGF-1 mice are indistinguishable from female GHR-N mice in this respect. All measures of central tendency are arithmetic means, and all depictions of variation (error bars) represent standard deviations (SD). (See also Figs S16–S22, and Table S4a–e).

Figure 5

The RIP::IGF-1 Transgene Normalizes the Slow-Aging-Associated Long-Term Memory Retention Capability of the Middle-aged Female GHR-KO Mouse. (A) Open-field assessment of anxiety detailed no effect of the RIP::IGF-1 transgene on anxiety, whether anxiety is defined as exploratory locomotion measured over 1, 2, or 5 min. (B) Open-field testing paradigm-based assessment of proximal (24-h delay) long-term memory performance documented greater memory capability in the middle-aged female GHR-KO mice relative to their GHR-N littermate controls, yet an invariant degree of capability between GHR-KO;RIP::IGF-1 mice and GHR-N controls. (C) Open-field testing paradigm-based assessment of proximal (24-h delay) long-term memory performance exhibited a great alteration of the proportional distribution of final location in middle-aged female GHR-KO mice, which was not documented in GHR-N littermate controls or GHR-KO;RIP::IGF-1 mice. (D) Passive avoidance chamber paradigm-based assessment of distal [(50 + 1)-day delay] long-term memory performance displayed that female GHR-KO;RIP::IGF-1 mice exhibit greater persistence of memory than GHR-KO mice, as considered by retention testing latency. All measures of central tendency are arithmetic means, and all depictions of variation (error bars) represent standard deviations (SD). (See also Figs S23–S24, and Table S5a–e.).