Inositol pyrophosphates inhibit Akt signaling, thereby regulating insulin sensitivity and weight gain

Abstract

The inositol pyrophosphate IP7 (5-diphosphoinositolpentakisphosphate), formed by a family of three inositol hexakisphosphate kinases (IP6Ks), modulates diverse cellular activities. We now report that IP7 is a physiologic inhibitor of Akt, a serine/threonine kinase that regulates glucose homeostasis and protein translation, respectively, via the GSK3β and mTOR pathways. Thus, Akt and mTOR signaling are dramatically augmented and GSK3β signaling reduced in skeletal muscle, white adipose tissue, and liver of mice with targeted deletion of IP6K1. IP7 affects this pathway by potently inhibiting the PDK1 phosphorylation of Akt, preventing its activation and thereby affecting insulin signaling. IP6K1 knockout mice manifest insulin sensitivity and are resistant to obesity elicited by high-fat diet or aging. Inhibition of IP6K1 may afford a therapeutic approach to obesity and diabetes.

Figure 1. Growth factor induced IP7 regulates Akt activity

A: IGF-1 treatment enhances intracellular IP7 levels in WT but not in IP6K1 KO MEFs. B: IP6K1 KO MEFs exhibit increased phosphorylation of Akt and mTOR downstream targets GSK3β, TSC2 S6K1 and S6 after 15 min IGF-1 treatment. Tyrosine phosphorylation of IGF-1 induced upstream PI3 kinase activator IRS1 and PDK1 target PKCζ are unchanged. C: Densitometric analysis displays ~3 fold and ~1.75 fold enhancement respectively in T308 and S473 Akt phosphorylation of IP6K1 KO MEFs following IGF-1 treatment. D: Increased activation in IP6K1 KO MEFs is not due to elevated PI3 kinase signaling. Intracellular PIP3 levels are similar in WT and IP6K1 KO MEFs under basal and after 15 min IGF-1 treatment. E: IP6K1 is a primary source of IP7 synthesis in the liver. Primary hepatocytes isolated from 10 months old IP6K1 KO mice display ~60% reduction in the IP7 levels. F: Primary hepatocytes of 10 months old IP6K1 KO mice after insulin treatment manifest enhanced phosphorylation of Akt, GSK3β and S6 with unaltered phosphorylation status of PDK1 targets PKCΖ and PKCΔ. G: Densitometry reveals ~5 fold and 2 fold enhancement respectively in T308 and S473 phosphorylation levels of Akt in IP6K1 KO hepatocytes following insulin treatment for 30 min. H: Complementation of IP6K1-WT but not IP6K1-K/A restores physiological levels of IP7 in the IP6K1 KO MEFs. I: Complementation of IP6K1 KO MEFs with IP6K1-WT reduces phosphorylation of Akt and GSK3β with IP6K1-K/A having no effect. IGF-1 dependent tyrosine and S636/639 phosphorylation of upstream PI3 kinase activator IRS1 is unaltered. J: IP6K1-WT complementation elicits ~3 fold reduction in IGF-1 induced T308 and S473 Akt phosphorylation. IP6K1-K/A does not have any effect. K: Transient Myc-IP6K1 overexpression elicits decrease in IGF-1 dependent Akt and GSK3β phosphorylation in HEK 293 cells. L: Overexpression of IP6K1-WT reduces IGF-1 induced phosphorylation of T308 and S473 Akt to ~3 fold whereas IP6K1-K/A has much less effect. Each experiment was repeated at least three times. (***p <0.001, **p < 0.01, *p < 0.05). See also Figure S1.

Figure 2. IP7 inhibits Akt T308 phosphorylation and membrane translocation

A: Immunofluorescence analysis of IGF-1 induced T308 phosphorylation and membrane translocation of Akt in absence of IP6K1. IGF-1 treated IP6K1 KO MEFs display enhanced T308 phosphorylation of Akt and augmented membrane translocation. Green and red represent total and p-T308 Akt respectively whereas yellow is the merged color for total and p-T308 Akt. B, C and D: Western blot analysis demonstrates increased T308 phosphorylation and membrane localization of Akt in IP6K1 KO MEFs after IGF-1 treatment. We also observe an increase in cytosolic p-T308 Akt levels in the IP6K1 KO MEFs. E: Complementation of IP6K1 KO MEFs with IP6K1-WT causes a delay in Akt translocation to the plasma membrane whereas IP6K1-K/A does not show this effect. F and G: PIP3 (1 μM) induced Akt-T308 phosphorylation is inhibited by IP7 with an IC₅₀ of ~1 μM in vitro. H and I: IP7 inhibits PDK1 dependent Akt phosphorylation at T308 in vitro with an IC₅₀ value of 20 nM. J: IP7 Inhibition of PDK1 dependent phosphorylation of overexpressed Akt immunoprecipitated from serum starved HEK293 cells. PDK1 increases Akt phosphorylation in vitro which is abolished by IP7. IP4 does not have any significant effect. Each experiment was repeated at least three times. (***p <0.001, **p < 0.01, *p < 0.05). See also Figure S2.

Figure 3. IP6K1 KO mice manifest sustained insulin sensitivity

A: Glucose tolerance test (GTT): IP6K1 KO mice display improved glucose tolerance than WT (male, n=5, each set). B: Insulin tolerance test (ITT): In response to insulin, IP6K1 KO mice display a greater glucose removal rate than WT littermates (male n=5, each set). C: Hyperinsulinemic-euglycemic clamp studies: Glucose infusion rates (GIR) display ~3 fold increase in IP6K1 KO mice than WT littermates (male, n=4, each set). D: Glucose uptake in gastrocnemius muscle and in epididymal white adipose tissue (WAT) is significantly enhanced in IP6K1 KO mice (male, n=4, each set). E: Acute insulin sensitivity in IP6K1 KO mice. Insulin treatment causes enhanced p-Akt and p-GSK3β levels downstream of IRS-1 phosphorylation in the gastrocnemius muscles of IP6K1 KO mice. F: Acute insulin treatment leads to ~2-, 2.5 and 4 fold increase in phosphorylation status of T308, S473 of Akt and S9 of GSKβ respectively. G: Increased glycogen content in gastrocnemius muscle of IP6K1 KO mice after 30 min insulin treatment of 16 h fasted mice (n=3, each set). H: IP7 levels in young and old hepatocytes. IP7 levels increase significantly with age in the WT mice (n=3, each set). (***p <0.001, **p < 0.01, *p < 0.05). See also Figure S3.

Figure 4. IP6K1 KO mice are resistant to obesity

A: IP6K1 KO mice display significant reduction in body weight compared to WT littermates at the age of 10 months (male, n=5, each set). B: Reduced body weight in IP6K1 KO mice reflects less fat accumulation. Epididymal white adipose tissue (EWAT) weight is significantly less in 10 months old IP6K1 KO mice than WT littermates (male, n=5, each set). C: Six week old WT and IP6K1 KO mice under control diet (CD) conditions. D: IP6K1 KO mice are resistant to weight gain following high fat diet (HFD) exposure. Six week old IP6K1 KO and their WT littermates (males and females) were exposed to HFD for 15 weeks. E and F: Time dependent increase in body weight of IP6K1 KO and WT littermate males (E) and females (F) upon exposure to control and high fat diet (***p<0.001, n=8, each set). G and H: Echo-MRI analysis for body fat quantification in IP6K1 KO mice after 8 weeks of HFD exposure (male, n=5, each set). IP6K1 KO mice display significantly less deposition of total fat (G) and percent fat/lean mass (H). I: Weights of epididymal (E), retroperitoneal (R), dorso-subcutaneous (D), inguinal (I) white adipose tissues (WAT) and brown adipose tissue (BAT) isolated from WT and IP6K1 KO mice on CD and on HFD for 8 weeks (male, n=3, each set). IP6K1 KO display reduced WAT mass under both the diet conditions. BAT mass is similar in mice on CD but is increased at a lower rate in the IP6K1 KO on HFD. J: IP6K1 KO mice display low serum leptin levels and are resistant to HFD induced hyperleptinemia (male, n=6, each set). K: IP6K1 KO mice are protected from high fat diet induced enhancement in liver weight (male, n=3, each set). Mice were exposed to CD or HFD for 8 weeks. L: Oil Red `O' staining of lipid droplets in the livers of WT and IP6K1 KO mice on CD or HFD. Magnification 20×, scale bar-30 μM. (***p <0.001, **p < 0.01, *p < 0.05). See also Figure S4.

Figure 5. IP6K1 deletion improves glucose homeostasis under high fat conditions

A and B: IP6K1 KO mice are significantly resistant to hyperglycemia induced by 8 weeks exposure to HFD (male, n=8, each set). C: Glucose tolerance test (GTT) in mice after CD and HFD exposure for 8 weeks (male, n=5, each set). IP6K1 KO mice on HFD display more efficient glucose removal from serum than WT. Same aged IP6K1 KO and WT mice have similar glucose tolerance on CD. D: Insulin tolerance test (ITT) at 8 weeks of CD or HFD exposure in mice (male, n=5, each set). In response to insulin, IP6K1 KO mice display a greater glucose disposal rate than WT littermates on HFD with no difference on control diet. E: IP6K1 KO mice display reduced serum insulin under control diet conditions and do not display the hyperinsulinemia of WT mice at 8 weeks of HFD exposure (male, n=6, each set). F: Representative western blot of 4 h fasted IP6K1 KO mice (as described in Figure 5E) do not display insulin resistance of WT mice. Knockouts on HFD exhibit increased Akt signaling in skeletal muscle. (***p <0.001, **p < 0.01, *p < 0.05). Also see Figure S5.

Figure 6. IP7 reduces fat breakdown and enhances adipogenesis

A: IP6K1 KO mice and WT littermates consume high-fat diets similarly (male, n=4, each set). B–E: Whole body oxygen consumption (VO₂), carbon dioxide release (VCO₂), energy expenditure (EE) and respiratory exchange ratio (RER) in IP6K1 KO mice on control and high fat diet (male, n=4, each set). IP6K1 KO mice do not display high fat diet induced hypoactivity elicited by WT littermates resulting in increased VO₂ and EE in the knockouts. F and G: Increased fat breakdown in IP6K1 KO mice. Mice on HFD for 25 weeks were switched to regular diet for the indicated time periods. IP6K1 KO mice display significantly greater decreases in body weight compared to WT littermates (male, n=3, each set). H: Enhancement in IP7 levels during differentiation of NIH3T3-L1 cells. Inositol phosphate levels were detected in undifferentiated and 3 days post differentiated cells. TNP reduces IP7 levels under both the conditions (n=3). I and J: IP7 regulates adipogenesis through GSK3β pathway. TNP, 10 µM and SB216763, 1 μM in conjunction completely block differentiation of NIH3T3-L1 cells with minimal effect when treated alone (n=3). Also see Figure S6.

Figure 7. Model depicting insulin and IP6K1 regulation of Akt and sequelae

A: Basal signaling. Insulin stimulates IP7 formation. IP7 inhibits Akt activity and its downstream targets. Akt physiologically stimulates mTOR while inhibiting GSK3β. B: Signaling in insulin resistant tissues. In aging tissues that manifest insulin resistance, insulin stimulation of IP7 formation is augmented leading to pronounced inhibition of Akt with associated lessening of mTOR activation and GSK3β inhibition. Arrows: Green: Activation, Red: Inhibiton, Bold: Increased, Regular: Decreased, Dotted: Unknown mechanism. Boxes: Large: Active; Small: Less active