Long-term p110α PI3K inactivation exerts a beneficial effect on metabolism

Abstract

The insulin/insulin-like growth factor-1 signalling (IIS) pathway regulates cellular and organismal metabolism and controls the rate of aging. Gain-of-function mutations in p110α, the principal mammalian IIS-responsive isoform of PI 3-kinase (PI3K), promote cancer. In contrast, loss-of-function mutations in p110α impair insulin signalling and cause insulin resistance, inducing a pre-diabetic state. It remains unknown if long-term p110α inactivation induces further metabolic deterioration over time, leading to overt unsustainable pathology. Surprisingly, we find that chronic p110α partial inactivation in mice protects from age-related reduction in insulin sensitivity, glucose tolerance and fat accumulation, and extends the lifespan of male mice. This beneficial effect of p110α inactivation derives in part from a suppressed down-regulation of insulin receptor substrate (IRS) protein levels induced by age-related hyperinsulinemia, and correlates with enhanced insulin-induced Akt signalling in aged p110α-deficient mice. This temporal metabolic plasticity upon p110α inactivation indicates that prolonged PI3K inhibition, as intended in human cancer treatment, might not negatively impact on organismal metabolism.

Figure 1

Middle-aged p110α^D933A/WT male mice are resistant to age-related fat accumulation and display better glucose homeostasis than WT littermates. Parameters were assessed in approximately 500-day-old male mice. Bars represent mean ± standard error. Statistical comparisons were performed with Student's t-test. A. Body weight (n = 17 per genotype). B. Epididymal fat pad weight (n = 3 per genotype). C. Cumulative food intake over 10 days (left panel), expressed per kg of body weight (right panel; n = 9 p110α^D933A/WT–7 WT). D. Fasting plasma insulin (n = 5 per genotype). E. Fasting plasma glucose (n = 11 p110α^D933A/WT–12 WT). F. Glucose tolerance injected intraperitoneally with 2 g glucose/kg body weight (n = 11 p110α^WT/D933A–12 WT).

Figure 2

Middle-aged p110α^D933/WT mice fed a high fat diet maintain better metabolic profiles than WT littermates. Male mice were fed a high fat diet (45% calories derived from fat) at 66 weeks of age and for 14 weeks and were subsequently metabolically phenotyped as follows: A. Intraperitoneal insulin (2 U/kg) tolerance test (n = 6 per genotype). B. Intraperitoneal glucose (1 g/kg) tolerance tests (n = 6 per genotype). C. Insulin-stimulated Akt phosphorylation. Mice were injected with insulin (0.25 mU/g) via the inferior vena cava and Akt phosphorylation was assessed by quantitative immunoblot analysis in tissue homogenates [liver, gastrocnemius muscle, and white adipose tissue (WAT)]. Each lane represents an individual mouse (n = 4 per genotype). Signal intensity was quantified and data (mean ± standard deviation) are shown in the graphs below the respective blots.

Figure 3

Chronic p110α inhibition promotes IRS-dependent signalling. A. p110α inhibition prevents IRS downregulation induced by chronic insulin treatment in C2C12 myotubes. C2C12 myotubes (cultured in medium with 5.5 mM glucose) were pre-treated for 16 h with insulin (10 nM) in the presence or absence of A66 (2 µM) or TGX-221 (1 µM). Monolayers were then rinsed with phosphate-buffered saline and lysed either directly for detection of IRS and S6K T389 phosphorylation or after 1 h culture in serum-free medium and re-stimulation with insulin (10 nM for 10 min; referred to in the figure as ‘acute insulin’) for detection of Akt T308 phosphorylation. Lysates were processed for immunoblot analysis with the indicated antibodies. Panels have been rearranged for clarity, but they are from the same immunoblots. Signal intensity was quantified and normalized to that of vinculin, used as a loading control in all immunoblots. B. Quantitative data (mean ± standard deviation) from three independent experiments performed as in A. Statistical comparisons were performed with Student's t-test. C. IRS protein levels (mean ± standard deviation) as determined by immunoblot analysis in epididymal fat pads [white adipose tissue (WAT)] and gastrocnemius muscle homogenates of 80 week old male p110α^D933A/WT mice and WT littermates (n = 4 per genotype).

Figure 4

Extension of lifespan of male p110α^D933A/WT mice. A. Survival curves of p110α^D933A/WT mice. Cohorts of p110α^D933A/WT (n = 26) and WT (n = 42) male mice were monitored from birth to death as described in Materials and Methods. Median lifespan was 818 days (p110α^WT/D933A) versus 799 days (WT). The difference between the curves is marginally statistically significant (log-rank test, p = 0.0545, x² = 3.697). B. Survival analysis limited to mice surviving to 500 days of age (n = 21 for p110α^WT/D933A and n = 40 for WT). Median life span was 852 days (p110α^WT/D933A) versus 805 days (WT). The difference between the curves is statistically significant (log-rank test, p = 0.0094, x² = 6.739).

Figure 5

Schematic overview of the beneficial impact of chronic p110α inactivation on age-related deterioration of insulin sensitivity. The levels of IRS are key to insulin signalling. Chronic stimulation with insulin, as occurs in hyperinsulinemia, downregulates IRS via a negative feedback loop that involves IRS phosphorylation by S6K, exacerbating age-related insulin resistance. Reduced p110α activity as a consequence of loss-of-function mutation (D933A) or pharmacologic inhibition (e.g. A66) attenuates S6K activity, protecting against chronic insulin-induced IRS depletion, thus promoting insulin sensitivity.