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. Author manuscript; available in PMC: 2011 Dec 10.
Published in final edited form as: Cell. 2010 Dec 10;143(6):897–910. doi: 10.1016/j.cell.2010.11.032

Inositol pyrophosphates inhibit Akt signaling, regulate insulin sensitivity and weight gain

Anutosh Chakraborty 1, Michael A Koldobskiy 1, Nicholas T Bello 2, Micah Maxwell 1, James J Potter 3, Krishna R Juluri 1, David Maag 1, Seyun Kim 1, Alex S Huang 1, Megan J Dailey 2, Masoumeh Saleh 1, Adele M Snowman 1, Timothy H Moran 2, Esteban Mezey 3, Solomon H Snyder 1,2,4
PMCID: PMC3052691  NIHMSID: NIHMS256478  PMID: 21145457

Summary

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 which regulates glucose homeostasis and protein translation respectively via the GSK3β and mTOR pathways. Thus Akt, mTOR and GSK3β signaling are dramatically augmented in skeletal muscle, white adipose tissue, and liver of mice with targeted deletion of IP6K1. IP7 impacts this pathway by potently inhibiting the PDK1 phosphorylation of Akt, preventing its activation and thereby impacting 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.

Introduction

Inositol phosphates are widely distributed in animal and plant tissues. Most studied is inositol 1,4,5-trisphosphate (IP3), which releases calcium from intracellular stores (Berridge MJ, 2000; Irvine RF, 2001). More recently, higher inositol phosphates with energetic pyrophosphate bonds have been described (Shears SB, 2007), which are synthesized by a family of three IP6 kinases (IP6Ks) (Saiardi A, 1999; Saiardi A, 2001). Best characterized is diphosphoinositol pentakisphosphate [5-PP-1(1,2,3,4,6)IP5], here designated IP7 (Barker CJ, 2009). In mammals IP7 modulates numerous physiologic functions including apoptosis (Chakraborty A, 2008; Morrison BH, 2001; Nagata E, 2005) and insulin secretion (Illies C, 2007), while in budding yeast it influences endocytosis (Saiardi A, 2002) and telomere length maintenance (Saiardi A, 2005; York SJ, 2005). Another isoform of IP7, identified as 1/3-PP-IP5, is formed by the Vip1 enzyme (Lin H, 2009; Mulugu S, 2007) and in yeast influences cell shape, growth and phosphate disposition (Lee YS, 2007).

IP6K1 depletion by RNA interference impairs insulin secretion by pancreatic beta cells (Illies C, 2007), and IP6K1 KO mice manifest reduced circulating insulin levels (Bhandari R, 2008). Despite low serum insulin, IP6K1 deleted (IP6K1 KO) mice display normal blood glucose levels and tolerance implying insulin hypersensitivity (Bhandari R, 2008).

IP7 can signal by physiologically pyrophosphorylating protein targets (Bhandari R, 2007; Saiardi A, 2004). In yeast 1/3-PP-IP5 binds the cyclin-cdk complex to regulate phosphate metabolism (Lee YS, 2007).

Pleckstrin homology domains (PH domains) (Haslam RJ, 1993; Lemmon MA., 2008), bind phospholipids such as phosphatidylinositol(3,4,5)-trisphosphate (PIP3) and phosphatidylinositol (4,5)-bisphosphate (PIP2) (Di Paolo G, 2006; Fruman DA, 1999) thereby recruiting signaling proteins to membranes. IP7 interferes with the binding of PIP3 to the PH domain of the Dictyostelium-specific cytosolic regulator of adenylyl cyclase (CRAC) to inhibit chemotaxis (Luo HR, 2003).

Akt (PKB), a PH domain containing serine/threonine kinase, regulates growth factor signaling (Chan TO, 1999; Fayard E, 2005; Taniguchi CM, 2006; Whiteman EL, 2002) to stimulate glucose uptake (Welsh GI, 2005), glycogen synthesis (Cross DA, 1995) and protein synthesis (Memmott RM, 2009; Ruggero D, 2005) by influencing glucose transporter 4 (GLUT4), glycogen synthase kinase 3 (GSK3)α/β and tuberous sclerosis complex 2 (TSC2)-mTOR signaling pathways.

Increased protein translation following Akt activation elicits skeletal muscle hypertrophy (Lai KM, 2004) and augments hepatic fatty acid oxidation with reduced fat accumulation (Izumiya Y, 2008). GSK3β, which influences insulin resistance, is phosphorylated and inhibited by Akt (Cross DA, 1995). Akt and GSK3β activity are reciprocally regulated in insulin resistance and obesity. Akt/mTOR activity is decreased (Funai K, 2006; Shao J, 2000) and GSK3β increased (Kaidanovich O, 2002) in insulin resistant tissues of aging and obese mice.

The apparent insulin sensitivity of the IP6K1 KO mice prompted our interest in IP7 regulation of Akt and insulin signaling. We now show that IP7 is a physiologic inhibitor of Akt signaling acting at the enzyme's PH domain to block phosphorylation and activation by PDK1. Thus, IP6K1 KO mice display a very marked enhancement of Akt activity accompanied by augmented insulin sensitivity and resistance to weight gain.

Results

Growth factor induced IP7 formation inhibits Akt signaling

We monitored IP7 formation of serum starved MEFs in response to IGF-1 (Figures 1A and S1A). In WT MEFs serum starvation decreases IP7 formation more than 90%, while IGF-1 rapidly restores IP7 levels with complete restoration to WT values by 60 min. The stimulation of IP7 formation by IGF-1 is abolished in IP6K1 deleted MEFs. In WT MEFs serum deprivation reduces levels of IP6 much less than IP7, and IGF-1 enhances formation of IP6 much less than IP7 (Figure S1B). In hepatocellular carcinoma cell line HEPG2, insulin or IGF-1 treatment similarly stimulates IP7 formation (Figure S1C).

Figure 1. Growth factor induced IP7 regulates Akt activity.

Figure 1

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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.

Akt is activated by phosphorylation at T308 by PDK1 and at S473 by mTOR (Alessi DR, 1997; Sarbassov DD, 2005). IP6K1 KO MEFs display markedly augmented IGF-1 stimulated phosphorylation of Akt (T308/S473) (Figures 1B and 1C) without any alteration in tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1), an upstream activator of PI3 kinase. We also observe increased phosphorylation of Akt downstream effectors GSK3β (S9), TSC2 (T1462), S6K1 (T389) and S6 (S235/S236) in response to IGF-1 (Figure 1B). We detect similarly increased growth factor mediated signaling in a separate clone of IP6K1 KO MEFs (Figure S1D). To assess specificity, we monitored an atypical PKC, PKCζ which is a PH domain deficient PDK1 target (Figure 1B). PKCζ phosphorylation levels are the same in IP6K1 deleted and WT MEFs in the absence or presence of IGF-1. Phosphorylation of the growth factor stimulated kinase ERK and the PDK1 target PKCΔ are also unchanged (Figure S1D). Akt can be activated via a variety of mechanisms, especially those involving PI3 kinase and its generation of PIP3 (Alessi DR, 1997). We evaluated the formation of PIP3 in WT and IP6K1 KO MEFs (Figure 1D). Serum deprivation of WT MEFs markedly decreases PIP3 formation which is reversed by treatment with IGF-1. The effects of serum deprivation and IGF-1 treatment are the same in IP6K1 deleted as in WT MEFs. We also measured PI3 kinase catalytic activity and tyrosine phosphorylation status of its p85 subunit, which are unaltered in IP6K1 KO MEFs following IGF-1 treatment (Figures S1E and S1F). Basal PI3 kinase activity in WT and IP6K1 KO MEFs is also unaltered (data not shown). Thus, IP6K1 regulation of Akt is not due to alteration of PI3 kinase activity or PIP3 levels.

To examine insulin signaling in IP6K1 KO liver, we isolated primary hepatocytes which display ~60% reduction in IP7 with unaltered levels of IP6 relative to WT hepatocytes (Figures 1E, S1G and S1H). IP6K1 KO hepatocytes manifest elevated phosphorylation of Akt, GSK3β and S6 in response to insulin with no alteration in p-PKCζ/p-PKCΔ, other targets of PDK1 (Figures 1F and 1G).

Complementation of IP6K1-WT but not IP6K1-K/A restores physiological IP7 levels in IP6K1 KO MEFs (Figures 1H, S1I and S1J). Levels of p-Akt (T308/S473) and p-GSK3β are diminished in IGF-1 stimulated MEFs expressing IP6K1-WT but not in IP6K1-K/A clone (Figures 1I, 1J). Growth factor signaling is inhibited by S6K1 via phosphorylation of IRS1 at S636/639 residues (Um SH, 2004). We do not observe any change in phosphorylation status of IRS1 at S636/639 or at tyrosine residues (Figure 1I). We observe similar effects in complemented MEFs induced with serum (Figure S1K). IP6K1-WT overexpression lowers Akt and GSK3β phosphorylation levels in IGF-1 stimulated HEK293 cells (Figures 1K and 1L).

The enhancement in Akt/mTOR signaling is accompanied by parallel changes in protein synthesis. Thus, IP6K1 KO MEFs manifest a 15% increase in protein translation (Figure S1L). Wortmannin and rapamycin each reduce wild-type protein translation about 20–25%, consistent with the Akt-mTOR pathway accounting for only about 20–25% of total protein synthesis (Holz MK, 2005). The increased protein translation of IP6K1 KO MEFs is reduced by about 25% following overexpression of IP6K1-WT but not IP6K1-K/A (Figure S1M). To ascertain whether IP6K1 regulates Akt/mTOR activation in intact organisms, we monitored phosphorylation of ribosomal protein S6 in the gastrocnemius muscle and liver of IP6K1 mutant mice and observe a pronounced enhancement (Figure S1N). IP6K1 deletion leads to decreased 4EBP1 binding to eIF4E (Holz MK, 2005) at the mRNA cap in insulin treated mice liver and gastrocnemius muscle (Figure S1O).

In summary, growth factor stimulation enhances IP7 formation which in turn inhibits Akt signaling. Accordingly, marked augmentation of Akt signaling is seen in IP6K1 deleted tissues.

IP7 inhibits Akt T308 phosphorylation and membrane translocation

In response to growth factors, PIP3 stimulates Akt at the membrane by facilitating its phosphorylation by PDK1 (Alessi DR, 1997). We monitored IGF-1 dependent membrane translocation of Akt in MEFs of WT and IP6K1 KO mice (Figures 2A and 2B). We observe increased membrane localization of total Akt and p-T308 Akt following IGF-1 treatment in IP6K1 deleted MEFs (Figure 2A). Membrane levels of Akt protein are markedly enhanced by IGF-1 in WT preparations with the enhancement increased in IP6K1 KO cells (Figures 2B and 2C). Membrane associated p-T308 Akt is also strikingly increased in IP6K1 KO preparations with some cytosolic increase as well, presumably reflecting movement of phosphorylated Akt from membrane to cytosol (Figures 2B and 2D). Complementation of IP6K1-WT markedly reduces IGF-1 elicited membrane translocation of Akt. Vector alone or kinase-dead IP6K1 (IP6K1-K/A) does not reduce membrane Akt (Figure 2E).

Figure 2. IP7 inhibits Akt T308 phosphorylation and membrane translocation.

Figure 2

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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.

The IP6K inhibitor TNP (10 μM) (Padmanabhan U, 2009) increases the IGF-1 elicited stimulation of T308 phosphorylation of Akt without influencing p-S473. (Figure S2A). The increased Akt signaling elicited by TNP is not evident in IP6K1 null cells (Figure S2B). TNP increases T308 Akt phosphorylation in both membrane and cytosol fractions (Figure S2C).

PDK1 mediated phosphorylation of Akt is dramatically increased by PIP3 binding to Akt's PH domain via presumed conformational alterations (Calleja V, 2007). We examined the influence of IP7 or IP6 upon PDK1 elicited phosphorylation of Akt in the presence of PIP3 in vitro (Figures 2F and 2G). IP7 inhibits phosphorylation of Akt at T308 about 50% at 1 μM, while IP6 does not. Interestingly, the IC50 for IP7 inhibition resembles the PIP3 concentration required for maximal activation. We observe the inhibitory effect only when IP7 and Akt are preincubated together at the same time. When PIP3 is preincubated with Akt prior to the addition of IP7, IP7's IC50 increases to 50 μM (data not shown), beyond its physiological range. This observation also fits with the prior reports that IP7 failed to release Akt pre-bound to PIP3 (Downes CP, 2005). Myristoylation anchors Akt to the plasma membrane and irreversibly activates it (Andjelković M, 1997). Thus, IP6K1-WT overexpression in HEK293 cells reduces T308 phosphorylation of WT-Akt but not of myristoylated Akt upon growth factor stimulation (data not shown).

In the absence of added PIP3, IP7 is substantially more potent, inhibiting PDK phosphorylation of Akt with an IC50 of about 20 nM (Figures 2H and 2I). Phosphorylation of overexpressed Akt immunoprecipitated from serum starved HEK293 cells by PDK1 in vitro is abolished by 1 μM IP7 with IP4 having no effect (Figure 2J). The inhibitory action of IP7 is selective with IP5 and IP6 exerting much less inhibition while IP3 and IP4 are inactive (Figure S2D).

Because of the competition between IP7 and PIP3 for PH domain binding (Luo HR, 2003), we presume that the inhibitory effect of IP7 on Akt phosphorylation is primarily exerted via the PH domain. IP7 fails to inhibit PDK phosphorylation of Akt lacking its PH domain (Figure S2E). IP7 at 1 μM concentration does not inhibit S6K1 catalytic activity on peptide substrates in vitro (data not shown). IP7 binds to PDK1 (data not shown) but does not affect its catalytic activity on artificial peptide substrates, indicating that IP7 does not inhibit PDK1 activity in general (Figure S2F), consistent with an earlier report (Komander D, 2004). The PH domain of PDK1 occurs in the enzyme's C-terminus and does not influence its catalytic activity.

We presume that IP7 regulates Akt by binding directly to its PH domain. Previously we demonstrated that IP7 potently and selectively competes with PIP3 for binding to the PH domain of Akt as IP6 failed to inhibit binding except at very high concentrations (Luo HR, 2003). In the present study [3H]IP7 binds to full-length Akt with binding drastically reduced for Akt lacking the PH domain (Figure S2G). IP7 does not affect mTORC2 activity towards Akt-S473 in vitro (Figure S2H and S2I).

IP6K1 deleted mice display sustained insulin sensitivity

Six week old IP6K1 KO mice displayed reduced blood levels of insulin with normal plasma glucose implying insulin hypersensitivity (Bhandari R, 2008). Age induced insulin resistance is associated with decreased Akt activity (Funai K, 2006; Shay KP, 2009). Accordingly, we explored insulin sensitivity in terms of blood glucose levels in 10 month old mice (Figure 3A and 3B). These mice display significantly improved glucose tolerance following glucose infusion (Figure 3A). Following insulin administration, the IP6K1 KO mice display significantly lower blood levels of glucose than do WT mice (Figure 3B), establishing that older IP6K1 knockouts are indeed hypersensitive to insulin.

Figure 3. IP6K1 KO mice manifest sustained insulin sensitivity.

Figure 3

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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.

Increased insulin sensitivity should be associated with improved glucose uptake from plasma. To evaluate glucose utilization we employed hyperinsulinemic-euglycemic clamp studies (Figure 3C). The insulin sensitivity of the IP6K1 KO is more than double that of WT mice. We monitored the uptake of glucose into muscle and fat tissue in the clamp experiments (Figure 3D). In gastrocnemius muscle and epididymal white adipose tissues (EWAT), glucose uptake is approximately tripled in the mutant mice. We do not observe any significant change in liver glucose uptake (data not shown) presumably because uptake is largely mediated by GLUT4 in muscle and adipose tissue.

We monitored Akt signaling in response to acute insulin treatment (Figures 3E and 3F). In gastrocnemius muscle, levels of p-Akt (T308/S473) are markedly increased in IP6K1 KO mice as are levels of the Akt downstream target p-GSK3β. On the other hand, insulin receptor substrate (IRS1) phosphorylation is similar in KO and WT mice indicating that the insulin sensitivity is due to regulation of Akt/GSK3β downstream of IRS1. We do not observe any alteration in S6K1 mediated inhibitory phosphorylation of S636/S639 IRS1 under these conditions (data not shown). Increased insulin sensitivity is also observed in epididymal white adipose tissue (EWAT) of IP6K1 KO mice (Figure S3A). We detect enhancement in insulin mediated glycogen formation in the gastrocnemius muscle of IP6K1 KO mice (Figure 3G).

To explore relationships between age dependent Akt activity and IP7 levels, we measured inositol phosphates in 2 and 10 month old mice (Figures 3H, S3B and S3C). Both IP6 and IP7 levels are elevated in the older mice with greater augmentation in IP7, resulting in increased IP7/IP6 ratios. The knockout hepatocyte preparations display an enhancement in age dependent increase in p-T308 Akt suggesting that increases in IP7 levels with age interfere with Akt activation (Figure S3D and S3E).

In summary, in WT animals age dependent increases in IP7 formation accompany decreased insulin sensitivity, which may explain the increased insulin sensitivity in aged IP6K1 KO mice.

IP6K1 KO mice are resistant to obesity

IP6K1 knockout mice exhibit reduced body weight (Bhandari R, 2008) which is more prominent with age (Figure 4A). The reduced body weight primarily reflects reduced fat accumulation with decreased weight of epididymal adipose tissue (EWAT) (Figure 4B) as well as diminished weights of other visceral and subcutaneous fat (data not shown). Despite lower body weight, IP6K1 KO mice display increased gastrocnemius muscle mass (Figure S4A). These findings may be consistent with observations of Izumiya et al (Izumiya Y, 2008) that increased Akt signaling leads to muscle hypertrophy, enhanced insulin sensitivity and resistance to HFD-induced weight gain.

Figure 4. IP6K1 KO mice are resistant to obesity.

Figure 4

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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.

We examined body weight of IP6K1 KO mice under high fat diet (HFD) conditions. Six week old IP6K1 KO mice on control diet (CD) are slightly smaller than WT littermates (Figures 4C, 4E and 4F; orange and brown circles). However, when exposed to HFD, they display striking resistance to body weight gain (Figures 4D–4F light and dark green triangles) with less than a third of WT weight gain. WT mice on HFD display a 300% greater increase in body fat than IP6K1 KO mice (Figures 4G, 4H and S4B) as assessed by Echo-MRI analysis. With or without HFD, IP6K1 KO mice display markedly lower weight of diverse fat pads with unchanged brown fat (BAT) weight on control-diet (Figure 4I).

Serum leptin levels are markedly lower in KO mice on control or HFD (Figure 4J) consistent with their reduced fat mass and indicating increased leptin sensitivity (Myers MG, 2008).

The liver is the major organ responsible for metabolizing fat to generate energy. Aberrations in the process lead to fatty liver disease or hepatic steatosis (Reddy JK, 2006). IP6K1 KO mice display resistance to high-fat diet induced weight gain in the liver (Figure 4K). Lipid droplets visible in the WT liver on control or high-fat diet are absent in IP6K1 KO mice (Figure 4L and S4C). Thus, in the IP6K1 KO mice, resistance to weight gain is due to reduced fat accumulation. High fat diets cause increases in serum triglycerides, cholesterols, aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) (Hoffler U, 2009; Ito M, 2008). These substances are significantly lower in IP6K1 KO than WT mice (Figures S4D–4G).

IP6K1 deletion improves glucose homeostasis in high fat diet fed mice associated with increased Akt signaling

HFD induced weight gain impairs insulin sensitivity and glucose homeostasis (Kahn SE, 2006), while mice with insulin hypersensitivity resist the sequelae of HFD (Elchebly M, 1999; Izumiya Y, 2008). After 8 weeks on HFD, IP6K1 KO mice do not display the hyperglycemia evident in WT mice (Figures 5A and 5B). HFD in WT mice leads to prolonged elevations in blood glucose levels following a glucose injection (Figures 5C and S5). IP6K1 KO mice are protected from the impaired glucose tolerance. Insulin tolerance tests (ITT) reveal greater insulin-induced reductions of blood glucose in KO mice on HFD with no difference on regular-diet (Figure 5D). Serum insulin levels are significantly lower in IP6K1 KOs on regular-diet (Bhandari R, 2008) which is even more striking after high fat exposure when the WT insulin levels reach pathologic levels (Figure 5E). Under the same experimental conditions described in Figure 5E, we measured Akt signaling in 4 h fasted mice (Figure 5F). HFD elicits higher levels of phosphorylated Akt, GSK3β and S6 in IP6K1 KO mice than in WT. The mutant mice display similar insulin levels as WT mice on CD. Despite high insulin levels, WT mice on HFD do not exhibit increased Akt phosphorylation, consistent with insulin resistance. IP6K1 KO mice are protected from HFD induced insulin resistance. Thus, IP6K1 KO mice do not display the HFD induced insulin resistance associated with reductions in Akt signaling.

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

Figure 5

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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.

IP7 reduces fat breakdown and enhances adipogenesis

Besides altering insulin sensitivity, Akt and its downstream effectors can reduce fat accumulation by i), diminishing food intake via mTOR (Cota D, 2006), ii) increasing fat utilization or oxidation via Akt (Izumiya Y, 2008) and iii) reducing adipogenesis via GSK3β (Ross SE, 2000).

Food intake of IP6K1 KOs does not differ from WT on control diet (Bhandari R, 2008) or HFD (Figure 6A). WT mice on HFD exhibit reduced oxygen consumption (VO2) and carbon dioxide release (VCO2) (Figures 6B and 6C). We assessed energy expenditure (EE) based on both fat and lean body mass, as fat mass also alters energy expenditure (Kaiyala KJ, 2010). WT on HFD display reduced EE presumably reflecting locomotor hypoactivity, similar to adipose tissue specific PPARγ knockout mice (Jones JR, 2005; Tou JC, 2002) (Figure 6D). IP6K1 KO mice on HFD are protected from reductions in VO2, VCO2 and energy expenditure resulting in an increase in energy expenditure in the knockouts (Figure 6D). Respiratory quotient (RQ), a reflection of carbohydrate and fat consumption, is decreased to a similar extent in WT and IP6K1 KO mice (Figure 6E).

Figure 6. IP7 reduces fat breakdown and enhances adipogenesis.

Figure 6

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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.

Increased fat oxidation in IP6K1 KO mice is confirmed by switching mice from high fat to control diet. The change in diet elicits decreased body weight to a much greater extent in IP6K1 mutants than WT mice (Figures 6F and 6G). Plasma ketone concentrations, which reflect hepatic fat oxidation, are significantly increased in IP6K1 KO mice on both control and high-fat diet (data not shown).

During adipogenic differentiation of NIH3T3-L1 cells, IP7 levels rise and are substantially reduced by the IP6K inhibitor TNP, whereas IP6 levels are unaffected (Figure 6H and S6A and S6B). IP6 levels are increased much less and are unaffected by TNP (Figure S6B). GSK3β, inhibited by Akt, inhibits adipogenesis (Ross SE, 2000). The GSK3β inhibitor SB21676 inhibits differentiation of NIH3T3-L1 cells (Tang QQ, 2005). We monitored differentiation of 3T3-L1 preadipocytes in the presence of IP6K and GSK3β inhibitors (Figures 6I and 6J). SB216763 completely blocks 3T3-L1 differentiation at 10 μM, whereas 1 μM drug elicits minimal effects. TNP (10 μM) inhibits differentiation ~20–25%. The combination of TNP (10 μM) and SB216763 (1 μM) virtually abolishes adipogenesis (Figures 6I and 6J). GSK3β facilitates adipogenesis through enhanced expression of the adipogenic transcription factor PPARγ (Farmer SR, 2005). PPARγ protein levels decline with co-treatment of IP6K and GSK3β inhibitors and in IP6K1 KO mice white adipose tissues (Figures S5C and S5D). These observations indicate that reduced fat accumulation in the IP6K1 KO mice is a result of sustained insulin sensitivity, increased fatty acid oxidation and reduced adipogenesis.

Discussion

In summary, IP7 generation by IP6K1 is enhanced by insulin. Moreover, IP7 is a physiologic inhibitor of Akt signaling, diminishing insulin sensitivity and protein translation via the GSK3β and mTOR signaling pathways which are associated with insulin resistance and weight gain (Figure 7). Insulin activation of Akt stimulates protein translation as well as glucose uptake and glycogen formation (Figure 7A). Aging or high fat diet increases IP7 levels which interfere with Akt activation leading to insulin resistance and weight gain (Figure 7B).

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

Figure 7

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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

IP7 inhibits Akt by acting at the PH domain of Akt to prevent its phosphorylation and activation by PDK1 both in vitro and in vivo. IP7's regulation of Akt phosphorylation by PDK1 is selective, as the catalytic activity of PDK1 toward artificial substrates is not affected by IP7. IP7 exerts this action with marked potency, with its IC50 of 20 nM being several orders of magnitude lower than the IC50 values for other reported actions of inositol pyrophosphates such as inhibition of cyclin-CDK activity by 1/3-IP7 (Lee YS, 2007) and similar to the Kd (35 nM) for PIP3 binding to the PH domain of Akt (Currie RA, 1999). Even in the presence of 1 μM PIP3, the physiologic activator of Akt, IP7 inhibits PDK1's influences on Akt at equimolar concentration, comparable to endogenous levels of IP7 (Bennett M, 2006). Effects of IP7 are highly selective with other inositol phosphates being substantially less potent. The diphosphate in IP7 differentiates it from IP6 and has been shown to alter the protonation state of the molecule (Hand CE, 2007). Thus, IP7 binds the clathrin assembly protein AP3 with 5–10 fold greater affinity than IP6 (Ye W, 1995).

The physiologic relevance of these findings is buttressed by the increased Akt signaling, decreased GSK3β phosphorylation and augmented protein translation in IP6K1 knockouts. Phosphorylation of GSK3β inhibits its catalytic activity leading to increased glycogen levels and reduced adipogenesis (Kaidanovich O, 2002) predicting that deletion of IP6K1 should lead to insulin hypersensitivity, as observed in IP6K1 KO mice. Insulin hypersensitivity of IP6K1 KO mice protects them from the impaired glucose tolerance and hyperinsulinemia associated with age or high fat diet consumption. Thus, IP7 synthesized by IP6K1 appears to mediate obesity and insulin resistance in mice at least in part by inhibiting Akt and increasing GSK3β activity.

Genetic models of insulin hypersensitivity, such as murine mutants of protein phosphatase 1B, PPARγ, S6K1, and JNK mutants, are resistant to HFD-induced obesity (Elchebly M, 1999; Hirosumi J, 2002; Izumiya Y, 2008; Jones JR, 2005 ; Um SH, 2004). Akt activation is a common feature of these diverse models of increased insulin sensitivity. These models support the notion that the sustained insulin sensitivity of IP6K1 KO mice conveys resistance to weight gain. Both reduced obesity and increased Akt signaling may elicit the improved glucose tolerance and insulin sensitivity of the IP6K1 mutants.

Akt has lipogenic effects. Akt 1 and Akt 2 double knockout mice display reduced adipose mass and skeletal muscle atrophy (Peng XD, 2003). Akt 2 deletion in ob/ob mice reduces fat accumulation with insulin resistance and hyperglycemia (Leavens KF, 2009). On the other hand, high fat diet induced hepatic steatosis is correlated with decreased Akt phosphorylation upon insulin treatment (Pinto Lde F, 2010). Skeletal muscle specific overexpression of Akt 1 reduces fat accumulation, while increasing fatty acid oxidation in the liver with less steatosis (Izumiya Y, 2008). Akt/mTOR mediated skeletal muscle hypertrophy (Rommel C, 2001) leading to increased insulin sensitivity (Harrison BC, 2008; Izumiya Y, 2008) may be physiologically associated with the alterations in insulin sensitivity of IP6K1 deleted mice. Moreover, GSK3β is adipogenic so that its inhibition in IP6K1 mutants may contribute to their leanness (Ross SE, 2000). Thus, the role of Akt in lipogenesis is complex and may reflect isoform and tissue specific effects.

Overexpression of Akt can be tumorigenic (Manning BD, 2007). IP6K1 knockouts do not display spontaneous tumors in their lifetime (data not shown), though we have not exhaustively explored possible tumorigenicity.

We observe increased IP6K activity in the skeletal muscle of HFD mice and older mice. Moreover, leptin receptor deficient obese `pound mice' display increased IP6K protein levels (Chakraborty and Snyder unpublished observation). These findings are consistent with age dependent increases in IP7 levels leading to insulin resistance and obesity.

Our findings imply that selective inhibitors of IP6K1 will have therapeutic potential in treating Type-2 diabetes associated with obesity and insulin resistance. The risk of adverse effects from such treatment can be inferred from the phenotype of IP6K1 knockouts. IP6K1 mutants weigh about 15% less than controls due to less fat deposition, but otherwise appear normal. Males manifest decreased sperm formation, but potential infertility of males may not represent a major problem in typical elderly Type-2 diabetics.

Experimental procedures

Detection of intracellular inositol phosphates

The cells were plated at 60% density and incubated with 100 μCi [3H]myoinositol for 3 days. For IGF-1 treatment, on the 3rd day, cells were incubated overnight with serum free media containing 100 μCi [3H]myoinositol. Next morning, cells were harvested after indicated IGF-1 treatment and were processed for inositol phosphate dectection by HPLC. For details please see supplemental method section.

IGF-1, insulin and serum treatment of mouse embryonic fibroblasts, primary hepatocytes and HEK293 cells

Unless otherwise stated, cells were starved overnight and then treated with media containing one of the following i) 10 nM IGF-1, ii) 10% FBS or iii) 10–20 ng/ml insulin for indicated time periods.

Membrane isolation

Membrane isolation employed a standard protocol using a Biovision cell fractionation kit. Caveolin1 or cadherin and lactate dehydrogenase were used as membrane and cytosolic markers respectively. Cytosolic contamination of the membrane preparations were checked by blotting with cytosolic markers, which showed negative results.

Membrane isolation of TNP treated HEK293 cells employed the above protocol after 10 μM TNP treatment of serum starved cells for indicated time periods. Cells were fractionated 15 min after IGF-1 treatment.

Enzymatic synthesis of radiolabeled IP7 by IP6K1

Purified recombinant 6XHis-IP6K1 was used in the reaction containing 500 μM cold IP6, 85 nCi of [3H]IP6 (total 8×104 cpm). IP7 was purified based on standard procedures (Saiardi A, 2004).

PDK1 activity assay on Akt T308 site in vitro

Purified recombinant, inactive unphosphorylated Akt at 20 nM final concentration (unless otherwise stated) was incubated with 100 μM ATP and indicated concentrations of inositol polyphosphates for 10 min in a reaction buffer containing 50 mM Tris, 100 mM NaCl and 1 mM DTT. PDK1, final concentration 20 nM, was added, and the mixture incubated at 30°C for 30 min. Samples were then boiled with LDS-buffer, run on SDS-PAGE and detected with α-p-T308 antibody. Bands were quantified using `ImageJ' software. Data from three independent experiments were plotted using `Sigmaplot' software. Details are in the supplemental procedure section.

Metabolic measurements

Metabolic parameters were measured in 10 month old mice ad libitum-fed or 4 h/16 h fasted mice. Blood glucose levels were measured from tail vein bleedings of mice using an Ascensia Contour blood glucose meter and test strips (Bayer). Ultrasensitive mouse insulin ELISA kit (Alpco Diagnostics) and mouse leptin ELISA kit (Millipore) were used to measure insulin and leptin respectively.

Glucose tolerance test (GTT) was performed on 16 h fasted mice injected i.p. with D-glucose (2 g/kg body weight). Blood glucose level was monitored by tail bleeding immediately before and at indicated time points after injection (Bhandari R, 2008). For insulin tolerance tests, mice were fasted 4 h and were given 0.75 units/kg body weight human recombinant insulin (Invitrogen) i.p. Blood glucose measurements were obtained from tail veins at indicated time points post-injection (Bhandari R, 2008).

Hyperinsulinemic-euglycemic clamp study and tissue glucose uptake analysis

Ten month old mice were used in the study. Details are in the supplemental information section.

Acute insulin treatment in mice

Ten month old mice, after 4 h fast, were anaesthetized and 25 mU/kg insulin (Invitrogen) or equal volumes of vehicle were administered through the portal vein. Gastrocnemius muscle, epididymal white adipose tissue (EWAT) and liver were collected 120 s after the injection and immediately stored in liquid nitrogen. Protein extracts from the tissue samples were prepared and run on SDS-PAGE. For detection of tyrosine phosphorylation on IRS1, IRS1 was immunoprecipitated from 1 mg total cell lysate and was blotted with α-p-tyrosine and α-IRS1 antibody.

Indirect calorimetry

Indirect calorimetry was conducted in an open-flow indirect calorimeter (Oxymax Equal Flow System; Columbus Instruments, Columbus, OH) at the Center for Metabolism and Obesity Research (Johns Hopkins University School of Medicine). Energy expenditure (EE) was calculated based on total body mass (fat mass+lean mass) (Kaiyala KJ, 2010). Details are in the supplemental information section.

Adipocyte differentiation studies

NIH3T3-L1 preadipocyte cells were cultured and differentiated following standard protocol (ZenBio). Briefly, preadipocytes were maintained in preadipocyte media (PM-1-L1), differentiated for 3 days with differentiation media (DM-2-L1). After 3 days of differentiation, cells were maintained for another 7 days in adipocyte maintenance media (AM-1-L1). See details in the supplemental information section.

Statistical analysis

All results are presented as the mean and standard error of at least three independent experiments. Statistical significance was calculated by Student's t-test using the `Sigmaplot software' (***p <0.001, **p < 0.01, *p < 0.05).

Supplementary Material

01
02

Acknowledgement

We thank Robert Luo for providing the pCDNATOPO-V5/His full length and ΔPH Akt constructs, Susan Aja for the Oxymax experiments, Cory Brayton for histological analysis, Molee Chakraborty, Nadine Forbes, Kent Werner and Gary Ho for technical support. This work was supported by U.S. Public Health Service Grants MH18501 and DA-000266, and Research Scientist Award DA00074 (to S.H.S.).

Footnotes

Author information The authors declare no financial interests.

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Supplemental information: Supplemental information includes Extended Experimental Procedures and six figures.

None of the authors of this manuscript have a financial interest related to this work.

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NIHMS256478-supplement-01.doc (81KB, doc)
02
NIHMS256478-supplement-02.pdf (632.9KB, pdf)

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