Gene deletion of inositol hexakisphosphate kinase 1 reveals inositol pyrophosphate regulation of insulin secretion, growth, and spermiogenesis

Abstract

Inositol pyrophosphates, also designated inositol diphosphates, possess high-energy beta-phosphates that can pyrophosphorylate proteins and regulate various cellular processes. They are formed by a family of inositol hexakisphosphate kinases (IP6Ks). We have created mice with a targeted deletion of IP6K1 in which production of inositol pyrophosphates is markedly diminished. Defects in the mutants indicate important roles for IP6K1 and inositol pyrophosphates in several physiological functions. Male mutant mice are sterile with defects in spermiogenesis. Mutant mice are smaller than wild-type despite normal food intake. The mutants display markedly lower circulating insulin.

Fig. 1.

Generation and characterization of IP6K1 knockout mice. (a) Schematic representation of the mouse IP6K1 gene (Ihpk1) with coding regions shaded black, noncoding regions in white, and the exon number indicated on top. A close-up view of exons 5 and 6 in the wild-type, targeted and knockout alleles is shown. The knockout allele loses the splice site, coding region, and a portion of the 3′UTR of exon 6. (b) RT-PCR analysis of tissues from wild-type and IP6K1 knockout adult male mice. For both IP6K1 and IP6K2, the forward PCR primer was directed against exon 5 and the reverse primer against the coding region of exon 6. IP6K1 and IP6K2 cDNA in expression plasmids (pCMV) were used as positive controls. (c) (Upper) IP₆ kinase activity was assayed in extracts prepared from MEFs. Products (IP₇ and IP₈) derived from IP₆ are expressed as a percentage of IP₆ added to the reaction mix. Data are expressed as mean ± SE of duplicate determinations from two cell lines of each genotype. (Lower) To ensure that equal amounts of total protein were used for IP₆ kinase assays, the whole cell, nuclear, and cytosolic MEF extracts were quantified by Western blotting with the indicated antibodies. (d) IP₇ levels present in two MEF cell lines of each genotype were determined by equilibrium labeling with [³H]inositol and resolution of labeled inositol phosphates by HPLC. The HPLC fractions encompassing the IP₇ peak are shown. Levels of IP₇ in each cell line were normalized against the amount of [³H]inositol incorporated into the lipid fraction.

Fig. 2.

Spermiogenesis is impaired in male mice lacking IP6K1. Histological analysis of testes and epididymides from wild-type and IP6K1 knockout adult male mice are shown. Cross-sections of the testes [wild-type (a and b) and knockout (e and f)] and epididymides [wild-type (c and d) and knockout (g and h)] were imaged at ×40 and ×100 magnification as indicated.

Fig. 3.

IP6K1 knockout mice display growth retardation and altered insulin disposition. (a) The body weight of wild-type and IP6K1 knockout male littermates, measured once per week, is expressed as mean ± SE (n = 6–8). Two-way ANOVA shows significant difference between curves, P < 0.01. (b) The average daily food intake of 6-week-old male mice is expressed as mean ± SE (n = 6–8). There is no significant difference in food intake between wild-type and knockout mice (Student's t test, P > 0.1). (c) Serum insulin levels were measured in wild-type and IP6K1 knockout adult male mice, either after fasting for 4 or 16 h (overnight). Data are mean ± SE (n = 5–10). (d) Insulin tolerance test was performed by 0.75 unit/kg body weight i.p. insulin injection in adult male mice. Data are mean ± SE (n = 4). Two-way ANOVA indicates that the curves for insulin tolerance are significantly different (P < 0.05) between IP6K1 knockout and wild-type mice.

Fig. 4.

Mice lacking IP6K1 are not diabetic despite low insulin levels. (a) Tail vein blood glucose measured in ad libitum-fed or 16-hour-fasted adult male mice. Data are mean ± SE (n = 7 or 8). (b) Glucose tolerance test by 2 g/kg body weight i.p. glucose injection in adult male mice. Data are mean ± SE (n = 6 or 7). (c) Measurement of glycosylated hemoglobin (HbA1c) in whole blood from adult male and female mice. Data are mean ± SE (n = 5).