Inositol Hexakisphosphate Kinase 3 Regulates Metabolism and Lifespan in Mice

Abstract

Inositol hexakisphosphate kinase 3 (IP6K3) generates inositol pyrophosphates, which regulate diverse cellular functions. However, little is known about its own physiological role. Here, we show the roles of IP6K3 in metabolic regulation. We detected high levels of both mouse and human IP6K3 mRNA in myotubes and muscle tissues. In human myotubes, IP6K3 was upregulated by dexamethasone treatment, which is known to inhibit glucose metabolism. Furthermore, Ip6k3 expression was elevated under diabetic, fasting, and disuse conditions in mouse skeletal muscles. Ip6k3(-/-) mice demonstrated lower blood glucose, reduced circulating insulin, deceased fat mass, lower body weight, increased plasma lactate, enhanced glucose tolerance, lower glucose during an insulin tolerance test, and reduced muscle Pdk4 expression under normal diet conditions. Notably, Ip6k3 deletion extended animal lifespan with concomitant reduced phosphorylation of S6 ribosomal protein in the heart. In contrast, Ip6k3(-/-) mice showed unchanged skeletal muscle mass and no resistance to the effects of high fat diet. The current observations suggest novel roles of IP6K3 in cellular regulation, which impact metabolic control and lifespan.

Figure 1. Mouse Ip6k3 and human IP6K3 mRNA are highly expressed in skeletal muscles, and Ip6k3 is induced by myotube formation in C2C12 cells.

Tissue distribution of each mRNA in C57BL/6J mice (a) and humans (b). Expression of inositol phosphate kinase family genes in human skeletal muscle tissues (c). Expression profiles of each mRNA during C2C12 myotube formation (d). Mouse Ip6k3 and human IP6K3 were highly expressed in skeletal muscles. Among the inositol kinase family members tested, IP6K3 showed the highest mRNA expression in human skeletal muscle. Ip6k3 mRNA was induced during myotube formation in C2C12 cells. Means ± SD of four mice consisting of two technical replicates for (a), means of two technical replicates for human pooled samples in (b,c), and means ± SD of three independent samples consisting of two technical replicates for (d). ITPKA-C, inositol-trisphosphate 3-kinase A-C; ITPK1, inositol-tetrakisphosphate 1-kinase; IPMK, inositol polyphosphate multikinase; IPPK, inositol-pentakisphosphate 2-kinase; PPIP5K1&2, diphosphoinositol pentakisphosphate kinase 1&2.

Figure 2. Ip6k3 mRNA is induced in diabetic, fasting, and disused conditions in mice, and IP6K3 mRNA is elevated by dexamethasone in human primary myotubes.

Ip6k1, Ip6k2, and Ip6k3 mRNA expression in the skeletal muscle from ?/+ littermates and ob/ob mice (a), in the gastrocnemius muscle of 2 days-fasted C57BL/6J mice (b), and in the denervated gastrocnemius muscle in C57BL/6J mice (c). Human primary skeletal myoblast cells were treated with 2% horse serum containing medium with or without dexamethasone and the respective mRNA levels were measured (d). Ip6k3 expression was induced by fasting and altered in the skeletal muscles of diabetic ob/ob mice. In addition, elevation of Ip6k3 mRNA was observed in 2 day-fasted gastrocnemius muscles and denervated gastrocnemius muscles at 2 days after operation. When human primary myotubes were treated with dexamethasone, which inhibits glucose metabolism, IP6K3 mRNA expression was elevated. Means ± SD of three ?/+ and ob/ob mice consisting of two technical replicates for (a), means ± SD of three mice consisting of two technical replicates for (b), means ± SD of six sham-operated and denervated mice consisting of two technical replicates for (c), means and range of three independent samples consisting of two technical replicates for (d). For (d), the comparative C_T method was used and the ranges given were determined by evaluating the expression: 2^−ΔΔCT with ΔΔC_T + s and ΔΔC_T − s, where s = the standard deviation of the ΔΔC_T value. Dex, dexamethasone.

Figure 3. Lower body weight, reduced fat mass, decreased blood glucose, higher plasma lactate, and reduced insulin in Ip6k3^−/− mice.

Body weight (a), lean mass (b), fat mass (c), blood glucose (d), plasma lactate (e), and plasma insulin (f) of Ip6k3^−/− mice and their wild-type littermates at the indicated ages. The observations became evident with ageing. B6.Cg-Ip6k3^−/− and wild-type littermates were used for the analysis. The mean values and individual values are shown. *P < 0.05 and **P < 0.01 by the Student’s t-test, and ^†P < 0.05 and ^††P < 0.01 by the Aspin-Welch test. n = 30 for 8 and 12 weeks old wild-type and Ip6k3^−/− mice, n = 24 for 24 weeks old wild-type and Ip6k3^−/− mice, n = 27 and 28 for 51 weeks old wild-type and Ip6k3^−/− mice, n = 28 and 29 for 1.5 years old wild-type and Ip6k3^−/− mice.

Figure 4. Lower glucose levels in glucose and insulin tolerance testing of Ip6k3^−/− mice.

An oral glucose tolerance test was performed in 15 weeks old mice (a). Plasma insulin levels at 15 min after glucose loading are shown (b). Insulin (0.75 U/kg) was injected to 12 weeks old mice (c). B6;129-Ip6k3^−/− and wild-type littermates were used for the analysis. Values represent the means ± SD. n = 8. *P < 0.05 by the Student’s t-test.

Figure 5. Extended lifespan of Ip6k3^−/− mice.

Kaplan-Meier survival curves show a significant (log-rank χ² = 4.2587 *P < 0.05) lifespan extension in Ip6k3^−/− mice ((a) n = 30 for each group). Western blot analysis of signalling proteins and quantitation of proteins in the hearts and the skeletal muscles at the end of the lifespan study (b,c). Decreased phospho-S6 ribosomal protein was observed in the heart. B6.Cg-Ip6k3^−/− and wild-type littermates were used for the analysis. The mean values and individual values are shown. **P < 0.01 by the Student’s t-test

Figure 6. Ip6k3^−/− mice show no resistance to high-fat diet feeding.

Ip6k3^−/− mice were fed a high fat diet (45% kcal as fat) from 7 to 15 weeks old. Ip6k3^−/− mice showed similar levels of the indicated parameters compared to wild-type littermates during the 8-week high fat diet feeding period. B6;129-Ip6k3^−/− and wild-type littermates were used for the analysis. Values represent the means ± SD. n = 8.

Figure 7. Ip6k3^−/− mice have no impact on basal skeletal muscle mass and denervation-induced skeletal muscle atrophy but exhibit reduced levels of Pdk4.

Body, gastrocnemius muscle, and soleus weights (a), and gene expression in the gastrocnemius muscles (b) and soleus (c) after the denervation operation. Ip6k3 deletion had no impact on basal muscle mass and denervation-induced muscle loss, which was measured 14 days following the operation. Pdk4 was down-regulated in the muscles of Ip6k3^−/− mice. B6;129-Ip6k3^−/− and wild-type littermates were used for the analysis. *P < 0.05 by the Student’s t-test. Means ± SD of 6 mice for (a). Means and range are shown for (b,c). n = 4 and 6 for wild-type and Ip6k3^−/− mice at day 0, n = 4 and 5 for wild-type and Ip6k3^−/− mice at day 2, n = 6 for wild-type and Ip6k3^−/− mice at day 14. For calculating gene expression in (b,c), the comparative C_T method was used and the ranges given were determined by evaluating the expression: 2^−ΔΔCT with ΔΔC_T + s and ΔΔC_T − s, where s = the standard deviation of the ΔΔC_T value. Two technical replicates were performed per sample.