The inositol pyrophosphate pathway in health and diseases

(a). Pleckstrin homology (PH)-domain interaction

PH-domains in proteins bind phosphoinositides (Lemmon, 2007). Growth factor-stimulated receptor tyrosine kinases promote class I PI3K-mediated conversion of membrane-bound PIP2 to phosphatidylinositol (3,4,5)-trisphosphate (PIP3). The protein kinase B (PKB or Akt) and phosphoinositide-dependent kinase-1 (PDK1) bind to PIP3 via their PH-domains. PDK1 then phosphorylates Akt at the threonine 308 residue, which activates the kinase. Studies have established that 5-IP7 reduces the phosphorylation, membrane translocation and activation of Akt (Luo et al., 2003; Chakraborty et al., 2010). 5-IP7 inhibits PDK1-mediated phosphorylation of Akt with half-maximal inhibitory concentration (IC₅₀) values of ~20–200 nM in vitro (Chakraborty et al., 2010; Wu et al., 2013). A stable version of 5-IP7, 5PCP-IP5 has an IC₅₀ of 129 nM (Wu et al., 2013). 1-IP7 and its analog 1PCP-IP5 also potently inhibit Akt phosphorylation (IC₅₀ of ~55 and 105 nM, respectively) (Wu et al., 2014).

PDK1-mediated phosphorylation of Akt is increased by PIP3 (Calleja et al., 2007). In the presence of 1 μM PIP3, 5-IP7 prevents phosphorylation of Akt (IC₅₀ ~1 μM), whereas IP6 shows a 10- to 15-fold lower efficiency. This inhibitory effect is observed when 5-IP7 and Akt are pre-incubated prior to PIP3 addition. When PIP3 is added before 5-IP7, the pyrophosphate is less effective (Downes et al., 2005; Chakraborty et al., 2010). Accordingly, IC₅₀ values for 5-IP7 and 1-IP7 analogs (5PCP-IP5 and 1PCP-IP5, respectively) on Akt phosphorylation are 129 and 105 nM, respectively, in the absence of PIP3, but increase to 4.5 and 3.6 μM if PIP3 is added simultaneously with the analogs (Wu et al., 2014). 5-IP7, IP6, 1-IP7 and 1,5-IP8 bind the Akt-PH-domain with IC₅₀ values of 7, 14, 45 and >50 μM, respectively (Gokhale et al., 2013). Thus, binding affinities of IPPs are higher than their inhibitory potencies, presumably due to differences in assay conditions.

In vivo, Akt activity is higher in IP6K1-deleted or IP6K-inhibited mice (Chakraborty et al., 2010; Prasad et al., 2011; Zhang et al., 2014; Sun et al., 2015; Zhu et al., 2016; Ghoshal et al., 2016). Furthermore, photouncaging-induced enhancement in intracellular 5-IP7 rapidly excludes the PH-domain of Akt from the plasma membrane (Pavlovic et al., 2016), whereas IP6 is ineffective (H. J. Jessen, personal communication). Although the precise molecular and structural details of this mechanism are yet to be understood, it is conceivable that ligand-induced conformational alterations of the PH-domain of Akt may contribute to its differential regulation by phosphoinositides and IPPs. In fact, the inactive (PH-in) conformation of Akt is inaccessible to PDK1. PIP3 binding converts Akt to the PDK1-accesible PH-out conformation (Calleja et al., 2007). 5-IP7 appears to stabilize PH-in. IPPs also interact with other mammalian PH-domain-containing proteins such as PI3-kinase enhancer (PIKE), T-lymphoma invasion and metastasis-inducing protein (TIAM), general receptor for phosphoinositides-1 (GRP1) and stress-activated protein kinase-interaction protein-1 (SIN1) in vitro (Luo et al., 2003; Gokhale et al., 2013), although the in vivo significance of these interactions is not known.

(b). C2-domain interaction

C2-domains, comprising about 130 residues, are found in various eukaryotic proteins. In classical PKCs, C2-domains are responsible for Ca²⁺-dependent membrane binding (Corbalan-Garcia & Gomez-Fernandez, 2014). The other class of C2-domain, containing the Ca²⁺-sensor protein synaptotagmin (Syt), regulates vesicle fusion and neurotransmitter release in the pre-synaptic terminal (Chapman, 2008). IPs such as IP4 and IP6 are known binders of the C2B domain of Syt (Mikoshiba et al., 1999; Yang et al., 2012). A recent study demonstrated that 5-IP7 displays a 45-fold higher binding affinity to Syt1 over IP6 (Lee et al., 2016). Molecular details of 5-IP7 and C2B domain interaction are not yet known.

(c). Other interactions that require catalytic activity and target-binding of IPP-metabolizing enzymes

IP6Ks interact with diverse proteins or protein complexes in an isoform-specific manner, followed by its catalytic activity-dependent modulation of the target proteins. For example, IP6K2/5-IP7 regulates the interaction of CK2 with telomere length regulation protein 2 (Tel2)-interacting proteins 1 and 2 (Tti1 and Tti2) complex (TTT) (Rao et al., 2014), tumour suppressor protein p53 (Koldobskiy et al., 2010) and liver kinase B1 (LKB1) (Rao et al., 2015), IP6K1/5-IP7 modulates Cullin-ring finger ligase-4 (CRL4) complex (Rao et al., 2014), α-actinin (Fu et al., 2017) and jumonji domain-containing 2C protein (JMJD2C) (Burton et al., 2013), whereas IP6K1/IP6 controls AMPK (Zhu et al., 2016). Details are described in Section VI.

(a). Extracelluar signal-regulated kinase (ERK) signalling

The Ras family of GTP-binding proteins transduces signals from several receptor tyrosine kinases, activating mitogen-activated protein kinases (MAPKs) like ERK. Ectopic expression of murine DIPP1 inhibits the ERK1/2 pathway. However, phosphohydrolase activity-deficient DIPP1 blocks ERK1/2 signalling with a greater efficiency, indicating that phosphohydrolase activity is not required by this process (Chu et al., 2004). The exact mechanism is not known.

(b). Cell migration

The 5′7-methyloguanosine cap and 3′-terminal poly(A) tract regulate the synthesis, translation, and degradation of mRNA. DIPP1 possesses mRNA-decapping activity, which modulates migration of the breast cancer cell line MCF-7. A reduction in DIPP1 level promotes migration of these cells, due to enhanced filopodial extension. The phenotype is reversed by complementation with the wild type, but not by the DIPP1 mutant that lacks mRNA-decapping activity. The impact of the inositol pyrophosphatase activity of DIPP1 is unknown (Grudzien-Nogalska et al., 2016).

(a). Type-1 interferon response

The retinoic acid-inducible gene-I (RIG-I) receptor facilitates type-I interferon production. A human genome-wide RNA interference (RNAi) screen identified PPIP5Ks to be positive regulators of this process, which is involved in cellular infection by Sendai and influenza A viruses (Pulloor et al., 2014). Overexpression of catalytically active IP5K, IP6Ks and PPIP5Ks induces interferon induction. Conversely, silencing of IP5K and PPIP5Ks but not IP6Ks has the opposite effect, indicating involvement of 1-IP7 in this process. PPIP5Ks mediate phosphorylation and activation of the interferon regulatory transcription factor 3 (IRF3) that promotes expression of type-1 interferon. The addition of purified IPPs to a cell-free reconstituted RIG-I signalling assay also showed 1-IP7 to be essential for IRF3 activation. 1-IP7 is thought to act by pyrophosphorylation, since its action was not mimicked by a synthetic phosphonoacetate analog, although no experimental evidence was presented (Pulloor et al., 2014). Intracellular 1-IP7 level is low and at times undetectable (Gu et al., 2016), so determining the concentration and regulation of 1-IP7 in primary macrophages and/or other relevant cells will be critical to evaluate the physiological relevance of this interesting finding.

(b). PIP3-mediated mammalian target of rapamycin complex-2 (mTORC2) signalling

In L6 myotubes, knockdown of PPIP5K1 impairs insulin-induced phosphorylation of S473 but not of T308 of Akt (Gokhale et al., 2013). The mTORC2 complex phosphorylates Akt at S473 and PPIP5K is proposed to activate this pathway. PPIP5K substrates (IP6/5-IP7) but not products (1-IP7/1,5-IP8) bind to the PH-domain of the mTORC2 regulator SIN1 in vitro (Section V.1a). In unstimulated cells, the PH-domain of SIN1 binds to and suppresses mTORC2 activity. Upon PI3K activation, SIN1 binds to PIP3 at the membrane, which activates mTORC2 (Yuan & Guan, 2015). Similar stimulation also promotes the binding of PPIP5K1 to PIP3 (Gokhale et al., 2011). Presumably, the catalytic activity of PPIP5K1 activates mTORC2 by a local reduction in IP6/5-IP7 level, which leads to increased SIN1–PIP3 and reduced SIN1–mTORC2 binding. PPIP5K1 activity seems specifically to regulate SIN1–PIP3 but not Akt–PIP3 binding, although the reason is unknown. Influences of various kinase, phosphatase and PBD mutants of PPIP5K1 on Akt phosphorylation should be assessed to determine the precise cellular mechanism involved. It is clear, however, that the IPP pathway regulates PI3K signalling at various levels.

(c). Cell growth and metabolism

Clustered regularly interspaced short palindromic repeats and its associated protein 9 (CRISPR-Cas9)-based genetic deletion of both PPIP5K1 and PPIP5K2 revealed impacts of 1,5-IP8 depletion on various phenotypes of the HCT116 colon cancer cell line. PPIP5K deletion abolishes 1,5-IP8, which causes a threefold increase in 5-IP7 levels in these cells. PPIP6K-KO cells display growth-inhibited and hypermetabolic phenotypes (Gu et al., 2017). Phenotypes of PPIP5K-KO cells are discussed and compared with IP6K1-or IP6K2-deleted cells in Sections VI.3 and 4.

(a). Vesicular trafficking

Vesicular trafficking includes endocytosis and exocytosis, by which vesicles transport materials among different cellular compartments and between a cell and its environment. Various studies demonstrate that IP6K1 exerts both stimulatory and inhibitory effects on vesicular trafficking.

IP6K1 stimulates insulin exocytosis. Pancreatic β-cells express IP6K1 and IP6K2 and maintain relatively high levels (~6 μM) of 5-IP7, which is further increased in hyperinsulinemic ob/ob mice (Section III) (Illies et al., 2007). Exogenous IPPs (5-IP7, 1-IP7, 3-IP7 and 4-IP7) enhance exocytosis in mouse β-cells. Overexpression of catalytically active IP6K1 or IP6K2 enhances exocytosis, whereas silencing of IP6K1 but not IP6K2 inhibits the process. Depletion of IP6K1 or IP6K2 in β-cells causes ~50 or 25% reduction in 5-IP7 levels, respectively. Presumably, an optimal concentration of IP6K1- but not IP6K2-generated 5-IP7 is critical for insulin exocytosis (Illies et al., 2007). A reduced serum insulin level in IP6K1-KO but not in IP6K2-KO mice supports this possibility (Bhandari et al., 2008; Morrison et al., 2009). The precise mechanism by which IP6K1/5-IP7 mediates insulin release is not known.

IP6K1 regulates neuroexocytosis via both catalytic activity-dependent and independent mechanisms. The nucleotide exchange factor GRAB inhibits exocytosis via its action on the GTPase Rab3A. Both catalytically active and inactive versions of IP6K1 interact (Section V.4) and compete with GRAB for binding to Rab3A, which stimulates dopamine or growth hormone release in PC12 cells (Luo et al., 2001). Conversely, IP6K1-generated 5-IP7 reduces Ca²⁺-mediated neuroexocytosis in PC12 and in primary hippocampal neuronal cells by interacting with the C2-domain of Syt1 (Section V.1b) (Lee et al., 2016). Overexpression of IP6K1 suppresses, whereas its depletion promotes, exocytosis. 5-IP7 inhibits vesicle fusion in vitro, at a 10-fold higher affinity than IP6 or 1-IP7 (Lee et al., 2016). 5-IP7-dependent reduction of synaptic vesicle fusion was abolished by increasing Ca²⁺ levels, suggesting competition between them. Ca²⁺-bound Syt directs vesicular fusion via macromolecular interactions of lipid membranes and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. IP6K1/5-IP7-dependent inhibition of synaptic vesicle fusion was abolished by increasing Ca²⁺ levels or by expressing the Ca²⁺-binding C2B domain of Syt1. Thus, binding of 5-IP7 to Syt1 interferes with the fusogenic activity of Ca²⁺. Whether 5-IP7 regulates other C2 domain-containing proteins, is not known.

Two independent studies suggest that 5-IP7 reduces or facilitates trafficking by pyrophoshorylation (Section V.2) of distinct cytoskeletal proteins. Long-range intracellular transport occurs on the microtubule cytoskeleton and is driven by two classes of motor proteins: kinesins and cytoplasmic dynein (Vallee, McKenney & Ori-McKenney, 2012). In MEFs, 5-IP7 inhibits kinesin-induced exocytosis of viral particles (Azevedo et al., 2009). Interaction of AP3β1, a clathrin-associated protein complex with the kinesin family motor protein Kif3A is required for release of viral particles. Catalytically active IP6K1 or IP6K2 reduces AP3β1– Kif3A interaction and subsequent release of virus particles (Azevedo et al., 2009). Direct in vitro and indirect in vivo (back phosphorylation) studies demonstrated that 5-IP7 pyrophosphorylates AP3β1, which may regulate this process.

On the other hand, 5-IP7 facilitates dynein-mediated trafficking. One study employed the transferrin protein as a model to show that IP6K1-deleted MEFs display defective dynein-dependent trafficking pathways, including endosomal sorting, vesicle movement, and Golgi maintenance. Complementation of catalytically active IP6K1 reverses the phenotype. Accordingly, phagosomal motility is reduced in IP6K1-KO macrophages (see Section VI.3h for neutrophil phagocytosis). The interaction between dynein intermediate chain (DIC) and the p150^Glued subunit of dynactin is critical for the transport, which is reduced in IP6K1-KO cells. Direct in vitro and indirect in vivo (back phosphorylation) pyrophosphorylation, together with mutation studies, suggests that 5-IP7-mediated pyrophosphorylation of the serine 51 residue of DIC regulates its interaction with p150^Glued and presumably the transport process (Chanduri et al., 2016). Acquiring further evidence such as in vivo detection of pyrophosphorylation and impacts of pyrophosphorylation-deficient mutants on trafficking are critical to determine the molecular details of the mechanisms involved. IP6K1/5-IP7 regulates vesicle trafficking via diverse mechanisms, which can diminish or stimulate exocytosis depending on the cell/tissue and/or physiological context. The mechanisms and physiological relevance of insulin and neurotransmitter release are distinct, which explains differential role of IP6K1 in these processes. The activity-independent stimulatory and activity-dependent inhibitory actions of IP6K1 on neurotransmitter release require further study. Transgenic mouse models of active and inactive IP6K1 are necessary to distinguish these functions in vivo.

(b). Chromatin remodelling

Eukaryotic DNA is packaged in a complex manner, which hinders transcription and DNA repair. Coordinated remodelling of chromatin at specific sites is critical to initiate such processes. Various modifications including DNA and histone methylation regulate chromatin remodelling, and are modulated by IP6K1. DNA methylation leads to long-term gene repression (Cedar & Bergman, 2009; Rose & Klose, 2014). Nuclear-localized IP6K1 enhances DNA methylation in a catalytic activity-dependent manner, which inhibits expression of the inositol biosynthetic gene mIno1 (Yu, Ye & Greenberg, 2016).

Conversely, histone methylation can either activate or inhibit the transcription process. For histone H3, methylation of the lysine 4 residue activates, whereas similar modification at the lysine 9 residue blocks the transcription process (Kouzarides, 2002; Rice et al., 2003). The interaction of IP6K1 with the histone lysine demethylase JMJD2C dissociates the demethylase from the chromatin and causes a corresponding increase in trimethyl-histone H3 lysine 9 (H3K9me3) levels. Accordingly, IP6K1-KO MEFs display increased association of JMJD2C with the chromatin and reduced levels of H3K9me3, which is reversed by expression of catalytically active but not inactive IP6K1. Thus, IP6K1 inhibits transcription of the JMJD2C target gene, the E3 ubiquitin ligase MDM2 (Burton et al., 2013). The molecular mechanism by which 5-IP7 regulates DNA or histone methylation is not understood.

(c). DNA damage and repair

IP6K1-KO MEFs show decreased viability and reduced recovery after induction of DNA damage. Although homologous recombination repair is initiated, it is not completed in knockout cells. Expression of catalytically active but not inactive IP6K1 restores repair in these cells, implying a requirement for 5-IP7 in this process (Jadav et al., 2013), although the precise mechanism is not known.

A response to DNA damage (DDR)-induced stress is regulated by various pathways including the ubiquitin proteasome system. Cullin complex (CRL4) is a multi-protein complex primarily composed of DDB1, DDB2 (XPE), cullin-4A (Cul4A), regulator of Cullins-1 E3 (Roc1-E3) and the Cop9 de-ubiquitination signalosome (CSN) (Bergink, Jaspers & Vermeulen, 2007; Scrima et al., 2011). Following ultraviolet (UV) radiation (and other DNA-damaging agents), CSN is dissociated from the complex, leading to activation of the E3 ligase, which ubiquitinates several components of the nucleotide excision repair (NER) machinery, which is required to initiate NER. In MEF cells, IP6K1 forms a ternary complex with CSN and CRL4, in which both IP6K1 and CRL4 are inactive under basal conditions. DDB1 present in the complex inhibits IP6K1 (Section IV). UV dissociates IP6K1 from the complex, which activates the kinase. IP6K1-generated 5-IP7 disrupts the CSN–CRL4 complex, which activates CRL4-mediated NER. Accordingly, IP6K1-KO MEFs display enhanced UV-induced CRL4 activation, NER and reduced apoptosis. Thus, IP6K1 mediates UV-induced disassembly of the CRL4–CSN complex, which regulates NER and apoptosis (Rao et al., 2014). The molecular details of the process by which 5-IP7 dissociates the signalosome–Cullin complex are unclear.

(d). Haemostasis

Haemostasis is a physiological response to prevent blood loss following vascular injury. In aberrant haemostasis, the timing of blood clotting is altered. Slow blood clotting leads to diathesis, which is a condition of susceptibility to bleeding. Conversely, abnormally fast clotting leads to thromboembolism, which can cause fatal pathological conditions such as stroke, pulmonary embolism, deep vein thrombosis, and myocardial infarction. 5-IP7 is a key player in maintaining haemostasis (Ghosh et al., 2013). IP6K1-KO mice display a significant delay in platelet aggregation, lengthening plasma clotting time (Ghosh et al., 2013) and hence a longer tail bleeding time. As a result, IP6K1-KO mice are protected from ADP-induced pulmonary thromboembolism. Mechanistic studies reveal that levels of polyP, a critical clotting factor, are reduced in the platelets of IP6K1-KO mice. Addition of polyP but not 5-IP7 rescues clotting in sera derived from IP6K1-KO mice, indicating that 5-IP7 modulates the process in vivo indirectly by regulating polyP levels (Ghosh et al., 2013) (Section V.3).

(e). Spermatogenesis

IP6K1-KO male mice are sterile, with few advanced spermatids in the testes, and no sperm in the epididymis (Bhandari et al., 2008). IP6K1 is highly expressed in round spermatids and is enriched in a perinuclear ribonucleoprotein complex called the chromatoid body. Deletion of IP6K1 impairs formation of the chromatoid body, which causes defects in elongation and condensation of sperm nuclei that subsequently block differentiation of spermatids. During spermiogenesis, nucleosomal histones are replaced by transition proteins 1 and 2 (TNP1 and TNP2) and protamines (PRM1 and PRM2). Premature expression of these proteins is associated with defective spermatid formation and infertility in mice. Juvenile IP6K1-KO mice display premature synthesis of TNP2 and PRM2 in round spermatids, which correlates with this phenotype (Malla & Bhandari, 2017). Thus, IP6K1 mediates the post-transcriptional regulatory machinery of testicular germ cells via a hitherto unknown mechanism. However, pharmacological inhibtion of the relevant IPP pathway does not cause sterility (Ghoshal et al., 2016) (Section VII.2). The molecular details by which IP6K1 regulates TNP2 and PRM2 are not known.

(f). Cell migration-mediated neuronal development and cancer metastasis

Cell migration is critical in many physiological and pathological motility processes such as development, immunity and cancer metastasis. Two independent studies demonstrated that IP6K1 regulates cell migration. IP6K1-KO MEFs display impaired cell spreading and migration. The knockout cells also display reduced tyrosine phosphorylation and activity of the focal adhesion kinase (FAK), which diminishes cell migration (Jadav et al., 2016). Expression of catalytically active IP6K1 reverses migration defects in these cells suggesting involvement of 5-IP7 in this process. Actin cytoskeleton remodelling and cell migration support the ability of cancer cells to achieve their oncogenic potential. Accordingly, cancer cells like HeLa and HCT116 with depleted IP6K1 levels, display reduced migration, invasion, and anchorage-independent growth (Jadav et al., 2016). Subcutaneous injections of control and IP6K1-depleted HCT116 cells in nude mice form similar-sized tumours, yet, the invasive property of IP6K1-depleted cells is significantly less. Accordingly, IP6K1-KO mice fed the oral carcinogen 4-nitroquinoline-1-oxide (4-NQO) show reduced progression of invasive carcinoma (Jadav et al., 2016). This is discussed further in Sections VI.4b,c in the context of IP6K2.

IP6K1/5-IP7-mediated cell migration is also crucial for brain development. IP6K1-KO mice display abnormal brain development due to impaired neuronal migration. IP6K1 localizes to focal adhesion and interacts with the microfilament protein α-actinin. Experiments in MEF and HeLa cells revealed that IP6K1 binds α-actinin, which promotes its tyrosine phosphorylation by FAK, intracellular localization and function. These events are critical for stress fibre formation, cell migration and spreading. Catalytic activity of IP6K1 is not required for binding, but is indispensable in FAK-mediated phosphorylation of α-actinin. Accordingly, 5-IP7 promotes FAK autophosphorylation-mediated activation in vitro (Fu et al., 2017), which demonstrates the mechanism by which IP6K1/5-IP7 promotes cell migration (Jadav et al., 2016; Fu et al., 2017). The molecular details of 5-IP7-mediated FAK phosphorylation are unknown.

(g). Behavioural responses

IP6K1-KO mice display certain behavioural alterations such as reduced locomotor activity in novel environments and impaired social interactions (Chakraborty et al., 2014). Behavioural responses are results of complex biological actions and thus, may arise due to multiple mechanistic changes in the knockout mice. One such change is altered Akt/GSK3 signalling in the brain. IP6K1 activates the protein kinase GSK3 by: (i) 5-IP7-mediated inhibition of its negative regulator Akt (Chakraborty et al., 2010); and (ii) by direct protein interaction (Sections V.1a and 4) (Chakraborty et al., 2014). Consequently, IP6K1-KO mice display reduced GSK3 activity in diverse brain regions. However, the extent to which the catalytic or protein-interaction activity of IP6K1 contributes in vivo remains unclear. Defective brain development due to impaired neuronal migration (Section VI.3f) may also cause these alterations. Generation of brain-specific transgenic mice with catalytically active and inactive versions of IP6K1 may provide answers, but the above studies at least suggest that IP6K1 is essential for brain development. Conversely, targeting IP6K1 in the brain may have beneficial effects in psychiatric and neurodegenerative diseases, where abnormalities in insulin signalling are demonstrated (Chakraborty et al., 2014).

(h). Neutrophil functions

Neutrophils are critical players in innate immunity and host defence. However, excessive accumulation or hyper-responsiveness of neutrophils can be detrimental to the host, so neutrophils are under stringent regulation. IPPs are major regulators of neutrophil function in infection and inflammation (Luo & Mondal, 2015). PIP3-induced Akt activation plays a critical role in regulating neutrophil chemotaxis, phagocytosis, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-mediated superoxide production. IP6K1 regulates neutrophil function through 5-IP7-mediated Akt inhibition (Section V.1a). The chemoattractant fMLP stimulates Akt activity in neutrophils by reducing 5-IP7 levels (Section III). Accordingly, fMLP-induced Akt activation is significantly higher in IP6K1-KO neutrophils. Moreover, IP6K1-KO neutrophils display enhanced Akt-mediated transient superoxide production and bactericidal activity. However, unlike HCT116 or neuron cells, IP6K1-KO neutrophils do not display alterations in cell adhesion or migration (Prasad et al., 2011).

Abnormal accumulation of neutrophils in the lungs generates reactive oxygen species (ROS), which augments pathological phenotypes in chronic obstructive pulmonary disease (COPD) (Barnes, 2003). A 5-IP7-mediated decrease in Akt activity promotes spontaneous neutrophil death, which protects mice from nicotine-induced lung inflammation. Cigarette smoke extract or nicotine reduces 5-IP7 in aging neutrophils, which delays spontaneous neutrophil death and thus contributes to the pathogenesis of COPD. Accordingly, deletion of IP6K1 augments cigarette smoke-induced neutrophil accumulation and lung damage (Xu et al., 2013). Thus, 5-IP7-mediated Akt inhibition and neutrophil death exert differential effects on immunity and COPD. IP6K1-KO macrophages display reduced phagosomal motility (Chanduri et al., 2016), which may reduce phagocytosis. Genrally, neutrophils respond to acute, whereas macrophages function during chronic inflammation. Thus, IP6K1 may differentially regulate inflammation.

(i). Insulin signalling

Insulin induces 5-IP7, which in turn inhibits the hormone’s stimulatory action on Akt (Sections III and V.1a) (Manning, 2010; Chakraborty et al., 2011) implying that IP6K1 deletion will enhance insulin sensitivity. Indeed, mild insulin sensitivity is observed in young, chow-fed IP6K1-KO mice (Bhandari et al., 2008), and significant sensitivity in older (10-month old) knockouts (Chakraborty et al., 2010). In gastrocnemius muscle, epididymal WAT (EWAT) and primary hepatocytes of the older knockouts, insulin-induced Akt activity is markedly increased. These results, together with higher IP7 level in older wild-type hepatocytes compared to younger controls (Section III), suggest that age-induced insulin resistance is caused, at least in part, by 5-IP7-mediated Akt inhibition (Chakraborty et al., 2010). IP6K1 regulates insulin signalling in the adipose tissue, which in turn modulates global insulin sensitivity. As a result, both global (IP6K1-KO) and adipocyte-specific IP6K1-KO (AdKO) mice are also protected from high fat diet (HFD)-induced insulin resistance (Chakraborty et al., 2010; Zhu et al., 2016). HFD-fed AdKO mice display higher Akt activity in the EWAT, liver and gastrocnemius muscle. Thus, adipocyte-specific IP6K1 deletion alters expression/secretion of certain adipokines, enhancing insulin sensitivity in other tissues. Indeed, the plasma level of the insulin-sensitizing adipokine adiponectin (ADIPOQ) is higher in HFD-fed AdKO mice (Zhu et al., 2016). Serum insulin level is lower in HFD-fed AdKO mice compared to the corresponding wild-type, which suggests that adipocyte-specific IP6K1 deletion-mediated insulin hypersensitivity reduces insulin secretion indirectly (Zhu et al., 2016). Thus, IP6K1 regulates insulin secretion and signalling via pleiotropic mechanisms, which can be targeted in type-2 diabetes (T2D) (Chakraborty et al., 2011; Boucher, Kleinridders & Kahn, 2014; MacKenzie & Elliott, 2014; Zhang, Liu & Liu, 2017).

(j). Energy metabolism

IPPs are proposed to be metabolic messengers (Shears, 2009). Yet, the suggestion that IP6K1 inhibits energy expenditure (EE) emerged from the observation that IP6K1-KO mice are protected from HFD-induced weight gain despite eating the same amount of food (Chakraborty et al., 2010; Zhu et al., 2017). Furthermore, cold exposure, which enhances adipose tissue browning-mediated thermogenesis, reduces IP6K1 expression. These results suggest that IP6K1 deletion reduces weight gain by enhancing EE in adipocytes. Protection of AdKO mice from HFD-induced weight gain further supports this possibility (Zhu et al., 2016). Stored energy is expended by coupled respiration that generates ATP and/or by uncoupling protein-1 (UCP1)-mediated uncoupled respiration that releases heat. Brown adipocytes in brown adipose tissue (BAT) or beige adipocytes in WAT expend energy primarily by thermogenesis. Therefore, enhanced browning of WAT facilitates thermogenesis-mediated fat loss in rodents and humans (van Dam et al., 2015; Sidossis & Kajimura, 2015, and references therein). Indeed, both IP6K1-KO and AdKO mice expend more energy, at least in part, due to enhanced thermogenic EE (Chakraborty et al., 2010; Zhu et al., 2016, 2017). Stromal vascular fractions isolated from WAT display higher beige adipogenesis, further evidenced by enhanced expression of the EE machinery. Accordingly, mitochondrial oxygen consumption rate (OCR) is higher in AdKO beige adipocytes, whereas glycolysis is unaltered.

The protein kinase AMPK enhances EE (Zhu et al., 2016, and references therein). IP6K1 deletion augments EE by stimulating AMPK via a novel mechanism. IP6 stimulates LKB1-mediated stimulatory phosphorylation of the AMPKα (catalytic) subunit at the threonine 172 residue, whereas IP7 is ineffective. Catalytically active IP6K1 interferes with IP6-mediated AMPK activation. The amino acid residues 146– 205 in IP6K1 are required for its interaction with AMPKα (Fig. 3A). Presumably, IP6K1 interferes with IP6-mediated AMPKα activation by converting IP6 to 5-IP7 (or IP5*) (Fig. 2). Thus, dynamic alterations in IP6/5-IP7 ratio in a microenvironment also regulate protein targets (Zhu et al., 2016). However, the molecular mechanism by which IP6 enhances AMPK phosphorylation is not understood. IP6K1 may also indirectly inhibit intracellular AMPK. Similar to IP6K2 (Rao et al., 2015; Section VI.4c), IP6K1-generated 5-IP7 may promote nuclear localization of LKB1, which is known to perturb AMPK activation (Xie et al., 2009). Nevertheless, IP6K1-mediated inhibition of AMPK reduces adipocyte EE, promoting weight gain (Zhu et al., 2016). AMPK also displays insulin-sensitizing effects, which may contribute to insulin hypersensitivity in IP6K1-deleted mice. Although AMPK and Akt display synergistic effects on insulin sensitivity, they have differential roles in cell proliferation and survival. In general, Akt stimulates but AMPK inhibits these processes. Thus, in certain tissues/conditions, if IP6K1 inhibits one kinase, it may activate the other.

Moreover, IP6K1 regulates EE by modulating lipolysis via protein– protein interaction, which does not require catalytic activity of IP6K1 (Ghoshal et al., 2016). In lipolysis, lipases hydrolyse stored triglycerides (TAGs) that release free fatty acids (FFA) and glycerol. These products are utilized at various stages of the EE pathway. PLIN and comparative gene identification-58 (CGI-58) proteins regulate lipolysis by modulating activities of various lipases. PLIN1 binds and perturbs the stimulatory action of CGI-58 on adipose triglyceride lipase (ATGL) and thus, inhibits basal lipolysis (Ghoshal et al., 2016). Basal lipolysis is enhanced in IP6K1-KO mice. IP6K1 is a major PLIN1-interacting protein in the adipose tissue. Furthermore, PKA/PKC phosphorylation of IP6K1 at serine residues 118 and 121 (Fig. 3A) facilitates its binding to PLIN1, which modulates lipolysis (Section IV) (Ghoshal et al., 2016). Thus, IP6K1 regulates lipolysis and thermogenesis to promote fat accumulation.

IP6K1 also inhibits EE in MEFs, albeit via reducing glycolysis (Szijgyarto et al., 2011). Under basal conditions, IP6K1-KO MEFs display higher ATP levels due to increased glycolysis, but mitochondrial activity is compromised. The mechanism of this phenotype is known in S. cerevisiae, which exhibit similar phenotypes in the absence of the IP6K ortholog KCS1. The interaction between glycolytic genes transcriptional activators 1 and 2 (GCR1 and GCR2) is essential for transcription of the glycolytic genes. The KCS1-KO strain displays increased GCR1–GCR2 interaction, which stimulates expression of glycolytic genes such as GAPDH and PGK1. In vitro pyrophosphorylation and mutation studies suggest that 5-IP7 pyrophosphorylates (Section V.2) GCR1 and destabilizes its interaction with GCR2, which subsequently reduces glycolysis (Szijgyarto et al., 2011).

Experiments in HCT116 cells suggest that the IPP 1,5-IP8 plays a role in regulating ATP level, although the precise mechanism by which 1,5-IP8 regulates this process is unknown. In these cells, PPIP5K deletion eliminates 1,5-IP8 (but increases 5-IP7) and elevates ATP levels. PPIP5K-KO cells accumulate more ATP due to increased EE caused by enhanced glycolysis and oxidative phosphorylation. Overexpressed active but not kinase-inactive PPIP5K1 restores 1,5-IP8 level and returns ATP to normal levels, whereas reduction of 5-IP7 does not (Gu et al., 2017). IP6K1 may also regulate EE partly via 1,5-IP8. Although the IPP pathway inhibits EE, the precise mechanism may vary depending on the specific enzyme and cell type/conditions as energy metabolism varies among cell types, conditions and proliferative nature (Bereiter-Hahn, Munnich & Woiteneck, 1998).

(k). Bone marrow-derived mesenchymal stem cell (BM-MSC) fitness and skeletal involution

During skeletal aging, oxidative stress reduces bone mineral density and increases marrow adiposity due to impaired osteogenesis and enhanced adipogenesis of bone marrow-resident MSCs. For this reason, obese and/or diabetic patients have an increased risk of fracture. Therefore, targeting pathways that prevent these dysfunctions have therapeutic potential in bone diseases. IP6K1-KO mice display an increased yield of BM-MSCs. Moreover, IP6K1-KO MSCs exhibit enhanced growth, survival, osteogenesis and haematopoiesis. Conversely, they show reduced adipogenesis, which further explains reduced adipose mass in these animals (Chakraborty et al., 2010). Unlike adipocytes (Section VI.3j), IP6K1-KO MSCs display unaltered OCR, yet a reduced ROS level. Oxidative stress induces p53-mediated growth arrest and apoptosis in murine MSCs. Accordingly, p53 protein level is reduced and its E3 ubiquitin ligase mouse double minute-2 homolog (MDM2) is increased in IP6K1-KO MSCs. IP6K1 inhibits the transcription of MDM2 by inhibiting the demethylase JMJD2C in MEFs (Section VI.3b), although this mechanism has not been tested in MSCs. In summary, IP6K1-mediated regulation of MSCs promotes fat accumulation but reduce bone formation (Boregowda et al., 2017). The therapeutic significance of this finding in osteoporosis is discussed in Section VII.1b.

IP6K1, which is abundant in brain and testis (Section II.1), is essential for development of these organs. Conversely, its role in metabolic and other tissues is regulatory, representing a possible therapeutic targeted in diet- or age-induced diseases.

(a). Apoptosis

IP7 level is increased during apoptosis (Section III). Several studies suggest an essential role of IP6K2-generated 5-IP7 in this process. An antisense screening study in OVCAR3 cells first identified IP6K2 as a mediator of IFNβ-induced apoptosis (Morrison et al., 2001). In addition to OVCAR3 cells, IP6K2 also sensitizes various other cells including HeLa, HEK293, PC12, Jurkat and HL60 to apoptotic actions of cisplatin, etoposide, hydrogen peroxide, staurosporine, hypoxia, γ-irradiation and mutant huntingtin expression (Morrison et al., 2002; Nagata et al., 2005, 2011). Consequently, IP6K2-KO mice display a better survival rate following total body irradiation (8–10 Gy; 1 Gy = 1J radiation/kg) (Morrison et al., 2002, 2009).

Catalytically active IP6K2 regulates the transcriptional function of p53 (Koldobskiy et al., 2010). In HEK293 cells, the chaperone HSP90 binds IP6K2 at the amino acid region 132– 143, which inhibits the kinase (Fig. 3B). Disruption of IP6K2–HSP90 binding promotes the activation and nuclear localization of IP6K2 (Chakraborty et al., 2008; Koldobskiy et al., 2010). Nuclear IP6K2 binds p53 via amino acids 1–67 (Fig. 3B), which suppresses p53-mediated transcription of the cell cycle arrest regulator p21. As a result, DNA repair is hampered, and the cells undergo apoptosis. Conversely, IP6K2 enhances the expression of one of p53’s apoptotic targets, phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1 or NOXA). Thus, IP6K2 reduces the cell cycle arrest functions of p53, whereas it enhances its apoptotic actions (Koldobskiy et al., 2010). Subsequent studies provided further details about the mechanism involved (Rao et al., 2014). The phosphoinositide-3-kinase-related kinases (PIKKs) DNA dependent protein kinase (DNA-PK) and ataxia–telangiectasia-mutated protein kinase (ATM) phosphorylate and activate p53. The TTT co-chaperone family of proteins stabilises PIKKs. The C-terminus of IP6K2 (amino acids 212–426, Fig. 3B) binds to the TTT complex through direct interactions with Tti1, which facilitates 5-IP7’s binding-mediated activation of CK2. 5-IP7 binds more potently than IP6 (IC₅₀ of 5-IP7 ~ 0.4 μM; IC₅₀ of IP6 ~14 μM) to the lysine-rich cluster of CK2α, which facilitates access of CK2’s substrate to the kinase. Activated CK2 stimulates phosphorylation of the TTT complex, thereby enhancing the ability of DNA-PK/ATM to activate p53. Thus, in IP6K2-KO HCT116 cells, DNA damage (etoposide)-mediated induction of p53 is reduced, attenuating expression of its apoptotic targets PUMA and NOXA. Even a reduced level of p53 is sufficient to elevate p21 in IP6K2-KO cells (Rao et al., 2014). Studies in PPIP5K-KO HCT116 cells further demonstrate the impacts of IPPs on cell proliferation. PPIP5K deletion increases levels of 5-IP7 (opposite effect to IP6K2-KO), decreases 1,5-IP8 (similar to IP6K2-KO), enhances p53 (opposite to IP6K2-KO) and p21 (similar to IP6K2-KO) and reduces proliferation (similar to IP6K2-KO) (Gu et al., 2017). Thus, although IP6K2 or PPIP5K deletion reduces cell proliferation, the mechanisms cannot be attributed solely to differences in IPPs. IP6K2 and PPIP5K may have distinct targets in this process.

Catalytic activity-independent apoptotic action of IP6K2 has also been reported. IP6K2 interacts with TRAF2, which interferes with transforming growth factor beta-activated kinase-1 (TAK1) phosphorylation, nuclear factor kappa B (NF-κB) signalling, and apoptosis. Overexpression of the TRAF2-binding deficient IP6K2 (S347A/S359A) (Fig. 3B) mutant in OVCAR3 cells enhances NF-κB signalling, which partly impairs tumour necrosis factor alpha (TNFα)-induced apoptosis (Morrison et al., 2007). On the other hand, CK2, which mediates cell survival, degrades IP6K2 by phosphorylating it at residues S347 and S356 (Fig. 3B) (Section IV) (Chakraborty et al., 2011). Further studies are required to determine the precise effects of these IP6K2 residues in stressor-mediated apoptosis. Although the apoptotic function of IP6K2 is established, to what extent its subcellular distribution regulates this process remains unclear. Besides a nuclear role (Morrison et al., 2005; Koldobskiy et al., 2010), apoptotic functions of mitochondrial (Nagata et al., 2005) and cytosolic (Nagata et al., 2011, 2016; Moriya et al., 2017) IP6K2 have also been reported, which require elucidation.

(b). Chemical-induced carcinogenesis

IP6K2-KO mice display normal embryogenesis, development, growth, blood parameters, serum insulin level and fertility (Morrison et al., 2009). Although these mice do not develop spontaneous tumours, they do show accelerated development of aerodigestive tract carcinoma when exposed to the UV-mimetic chemical carcinogen 4-NQO (Morrison et al., 2009). Enhanced susceptibility of IP6K2-KO mice to this chemical carcinogen is associated with upregulation of the transcription termination factor 1 (TTF1) and the twist family of basic helix-loop-helix transcription factor neighbour protein (TWISTNB) that may have implications in cancer (Morrison et al., 2009). Conversely, downregulation of putative tumor suppressor genes like DUSP16 and EXT2 are observed in IP6K2-KO mice. IP6K2 promotes the apoptosis actions of p53 (Koldobskiy et al., 2010; Rao et al., 2014). Transgenic mice with a dominant negative p53 mutation are also susceptible to 4-NQO-induced oral cancer (Zhang et al., 2006). Therefore, susceptibility of IP6K2-KO mice to 4-NQO-induced cancer may involve p53 activity. IP6K2, but not IP6K1, exhibits apoptotic functions, which may partly explain why IP6K1 deletion protects, whereas IP6K2 deletion augments chemical carcinogenesis.

(c). Cell migration and cancer metastasis

IP6K2 enhances cell–matrix adhesion, but reduces cell–cell adhesion (Rao et al., 2015). IP6K2-KO HCT116 cells exhibit delayed growth, reduced cell–matrix adhesion, spreading and tyrosine phosphorylation of FAK (see Section VI.3f). Conversely, cell–cell adhesion and level of the adhesion-promoting protein zona occludin-1 (Zo-1) are increased in knockout cells. As a result, these cells display reduced anchorage-independent growth and migration. In vitro tumour invasion, monitored by tumour growth in a collagen invasion assay, is greatly reduced by IP6K2 deletion. Moreover, subcutaneous xenograft tumours grown from IP6K2-KO HCT116 cells are reduced in size. Knockout xenografts, when implanted into the caecum of nude mice, display reduced growth and diminished metastasis to liver. Overexpression of catalytically active but not inactive IP6K2 restores adhesion and associated features in the knockout cells. A complex mechanism is suggested. The tumour suppressor LKB1 inhibits cell migration and metastasis via activation of cytosolic tyrosine phosphatases that dephosphorylate FAK. The catalytic activity of IP6K2 is not required for binding, but is critical for nuclear sequestration of LKB1, which inhibits the phosphatases. Accordingly, IP6K2-KO cells accumulate cytosolic LKB1 and display higher tyrosine phosphatase activity, which facilitates FAK dephosphorylation and cell migration. However, direct evidence of enhanced LKB1 phosphorylation-dependent activation of phosphatases in IP6K2-KO cells is lacking. Nevertheless, results obtained in two different studies indicate that IP6K1- or IP6K2-generated 5-IP7 promotes cell migration via: (i) direct stimulation of FAK autophosphorylation (Fu et al., 2017); and/or (ii) LKB1–tyrosine phosphatase-mediated indirect regulation of FAK (Rao et al., 2015).

Based on the studies discussed here and in Sections VI.1b and 3f, it is clear that deletion of IP6K1 or IP6K2 reduces cell migration, whereas a reduction of DIPP1 level has the opposite effects (Grudzien-Nogalska et al., 2016). Conversely, deletion of IP6K2 but not IP6K1 reduces tumour volume. This phenotypic variation may arise due to: (i) distinct methods of disruption of these genes; (ii) differential expression patterns of these isoforms; or (iii) additional, yet to be identified mechanisms. Understandably, 5-IP7 levels in these two systems are different. IP6K2-KO HCT116 cells display an almost total loss, whereas IP6K1-depleted HCT116 cells show a modest decrease in IPP levels (Koldobskiy et al., 2010; Jadav et al., 2016). Perhaps cell migration but not tumour growth is susceptible to a smaller decrease in IPP levels (Jadav et al., 2016). Tissue-specific IP6K-KO mice or isoform-selective inhibitors may allow the roles of these isoforms in the initiation, progression and metastasis of cancer to be clarified.

(a). Synapse formation and motor function

Although IP6K3-KO mice do not display gross abnormalities in brain structure, their motor learning and coordination are disturbed, which suggests Purkinje cell dysfunction. IP6K3 is highly expressed in Purkinje cells. Moreover, IP6K3-KO mice display alterations in Purkinje cell structure with withered dendritic trees. The cell size and spine density of mutant Purkinje cells are also decreased in these knockouts. Both inhibitory symmetric and excitatory asymmetric synapses in the cerebellar molecular layer of IP6K3-KO mice are reduced. IP6K3-KO cerebella display reduced levels of the γ-aminobutyric acid (GABA)-synthesizing enzyme glutamic acid decarboxylase (GAD65), which is a marker of GABAnergic inhibitory neurons. Whether IP6K3 directly influences GAD65, is not known. The shape of Purkinje cell dendritic spines is determined by the arrangements and attachments of their cytoskeletal proteins such as adducin and spectrin. IP6K3 binds to the calponin homology (CH) domain of adducin and spectrin, whose mutual interactions are perturbed in IP6K3-KO mice. Catalytic activity of IP6K3 is not required to maintain β-adducin–β2-spectrin binding (Fu et al., 2015).

(b). Metabolism and aging

IP6K3 is highly expressed in both mouse and human myotubes and muscle tissues (Moritoh et al., 2016). Furthermore, IP6K3 expression is elevated in skeletal muscle under diabetic, fasting, and disuse conditions. Chow-fed, aged (1.5-year old) IP6K3-KO mice display reduced body mass and accumulate less fat, although they eat a similar amount of food. Moreover, aged IP6K3-KO mice exhibit lower blood glucose and reduced circulating insulin levels. These mice also exhibit increased plasma lactate levels, suggesting that glycolysis is increased in the knockouts. IP6K3-KO mice (15-weeks old) also display enhanced glucose removal during glucose- and insulin-tolerance tests. The knockouts exhibit reduced expression of the muscle pyruvate dehydrogenase kinase-4 (PDK4) enzyme. PDK4 inhibits pyruvate to acetyl coenzyme A (acetyl-CoA) conversion and thus inhibits glucose oxidation. Thus, reduced PDK4 expression may also increase glucose oxidation, which may reduce lipogenesis-mediated fat accumulation in these mice. Published results (Chakraborty et al., 2010) and recent experiments (S. Ghoshal & A. Chakraborty, unpublished data) suggest that IP6K1-KO mice are also protected from age-induced weight gain and insulin resistance. However, unlike IP6K1-KO, IP6K3-KO mice are not protected against diet-induced obesity (DIO) (Moritoh et al., 2016).

IP6K3-KO mice exhibit an increased survival rate, although the lifespan experiment was not conducted to completion (Moritoh et al., 2016). Phosphorylation of the mTORC1 effector S6 ribosomal protein is reduced in heart but not in skeletal muscle of IP6K3-KO mice, although stimulatory phosphorylation of the direct mTORC1 target S6 kinase-1 (S6K1) and the mTORC2 target Akt are unchanged (Moritoh et al., 2016). Although mTOR activity is associated with aging (Kennedy & Lamming, 2016), it is not clear whether the same mechanism is operative in IP6K3-KO mice. Although the impact of IP6K1 on mTOR pathway in the heart is unknown, in various IP6K1-KO cells and in the skeletal muscle of IP6K1-KO HFD-fed mice, it is upregulated along with the Akt pathway (Chakraborty et al., 2010). Further studies are needed to distinguish tissue-specific functions of IP6K1 and IP6K3 in metabolism and aging.

(a). TNP ameliorates DIO and insulin resistance

Similar to IP6K1 deletion (Sections VI.3i,j) (Chakraborty et al., 2010; Zhu et al., 2016, 2017), TNP protects mice from DIO and associated metabolic abnormalities (Ghoshal et al., 2016). Both short-term (2 weeks) and long-term (10 weeks) TNP treatments (10–20 mg/kg daily, intraperitoneal) reduce fat mass, reduce fatty liver, enhance insulin sensitivity and restore metabolic homeostasis in DIO mice without altering food intake. TNP decelerates both initiation and progression of DIO and insulin resistance in mice (Zhu et al., 2016; Ghoshal et al., 2016). These effects are abolished in IP6K1-KO mice, indicating that at least part of the beneficial effects of TNP on metabolism act via inhibition of IP6K1. TNP enhances insulin sensitivity in DIO mice via Akt activation (Ghoshal et al., 2016). Moreover, TNP reduces weight gain by enhancing AMPK-mediated browning and EE in the adipose tissue and adipocytes (Zhu et al., 2016). Thus, TNP has strong anti-obesity and anti-diabetic effects.

(b). TNP attenuates HFD-induced loss of trabecular bone volume

IP6K1-KO mice display increased MSC fitness and osteogenesis (Section VI.3k). Accordingly, long-term TNP treatment (Ghoshal et al., 2016) increases yield and colony-formation capacity of MSCs in DIO mice (Boregowda et al., 2017). TNP conserved trabecular bone, but decreased marrow adiposity. Therefore, in addition to its beneficial effects on obesity and T2D, TNP also protects mice from HFD-induced MSC dysfunctions in the marrow (Boregowda et al., 2017). Insulin sensitizers like peroxisome proliferator-activated receptor gamma (PPARγ) antagonists increase obesity and fracture risk. Thus, IP6K1 inhibitors may have greater acceptability as anti-obesity and anti-diabetic drugs due to their beneficial effects on bone properties.

(c). TNP protects against cardiac I/R injury in leptin receptor mutant db/db mice

The cytokine OSM, which works via activation of the OSM receptor, alleviates cardiac ischaemic/reperfusion (I/R) injury by inhibiting cardiomyocyte apoptosis in leptin receptor-deficient obese and diabetic db/db mice (Sun et al., 2015). OSM enhances mitochondrial biogenesis and function, whereas it reduces IP7 levels in db/db mice subjected to cardiac I/R injury (Section III). Conversely, OSM receptor knockout increases IP7, impairs mitochondrial biogenesis, glucose homeostasis and insulin sensitivity and augments cardiomyocyte apoptosis, together aggravating cardiac I/R injury. Short-term TNP treatment (10 mg/kg daily, tail vein injection) mimics the effects of OSM in db/db mice (Sun et al., 2015). OSM exerts pleiotropic signalling effects, which may regulate 5-IP7 production, although the precise mechanism is not clear.

(d). TNP improves aging effects of BM-MSCs and their therapeutic efficacy for myocardial infarction (MI)

Decreased viability and impaired function of aged BM-MSCs hamper their therapeutic efficacy. Aged (18-month old) BM-MSCs exhibit higher 5-IP7 levels compared to young cells (Section III). In addition, Akt activity is decreased, whereas apoptosis is increased in aged BM-MSCs. TNP treatment (10 μM, 2 h) reduces 5-IP7 level and apoptosis, whereas it enhances Akt activity in aged BM-MSCs. Levels of angiogenic factors such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), IGF-1 and hepatocyte growth factor (HGF), which are decreased in aged BM-MSCs, are restored by TNP treatment (Zhang et al., 2014). Moreover, TNP-treated BM-MSCs, when transplanted into infarcted hearts, display enhanced survival, which promotes their anti-apoptotic and pro-angiogenic efficacy in vivo. Thus, TNP-treated BM-MSC therapy decreases fibrosis and preserves heart function (Zhang et al., 2014).