Increased insulin sensitivity and reduced adiposity in phosphatidylinositol 5-phosphate 4-kinase beta-/- mice

Abstract

Phosphorylated derivatives of the lipid phosphatidylinositol are known to play critical roles in insulin response. Phosphatidylinositol 5-phosphate 4-kinases convert phosphatidylinositol 5-phosphate to phosphatidylinositol 4,5-bis-phosphate. To understand the physiological role of these kinases, we generated mice that do not express phosphatidylinositol 5-phosphate 4-kinase beta. These mice are hypersensitive to insulin and have reduced body weights compared to wild-type littermates. While adult male mice lacking phosphatidylinositol 5-phosphate 4-kinase beta have significantly less body fat than wild-type littermates, female mice lacking phosphatidylinositol 5-phosphate 4-kinase beta have increased insulin sensitivity in the presence of normal adiposity. Furthermore, in vivo insulin-induced activation of the protein kinase Akt is enhanced in skeletal muscle and liver from mice lacking phosphatidylinositol 5-phosphate 4-kinase beta. These results indicate that phosphatidylinositol 5-phosphate 4-kinase beta plays a role in determining insulin sensitivity and adiposity in vivo and suggest that inhibitors of this enzyme may be useful in the treatment of type 2 diabetes.

FIG. 1.

Generation of PI5P4Kβ^−/− mice. (A) Schematic representation of the PI5P4Kβ locus before and after insertion of the retroviral gene trap. Expression of the trapped gene is disrupted because the inserted sequence causes the endogenous transcript to be divided into two separate transcripts, neither of which can produce the endogenous protein. SA, splice acceptor sequence; βgeo, β-galactosidase and neomycin resistance cassette; SD, splice donor sequence. Primers used for genotyping by PCR are pkoF, pwtF, and pR. (B) PCR analysis of genomic DNA prepared from mouse tails. A 598-bp product (W) represents the wild-type allele; a 496-bp product (D) represents the disrupted allele. (C) RT-PCR analysis of RNA prepared from mouse brains. An 852-bp product (E) represents the endogenous transcript; a 613-bp product (H) represents the hybrid transcript formed by splicing of exon I to the gene trap sequence.

FIG. 2.

Loss of PI5P4Kβ protein in tissues. Western blots for PI5P4Kβ, PI5P4Kα, and PI5P4Kγ protein levels in skeletal muscle, heart, liver, brain, white adipose tissue (WAT), and brown adipose tissue (BAT) are shown.

FIG. 3.

PI5P4Kβ^−/− mice are hypersensitive to insulin. (A) Insulin tolerance tests in male and female wild-type (wt) and knockout (ko) mice. Results are means ± standard errors of the mean of the blood glucose/basal blood glucose ratio from at least nine animals of each gender and genotype. P values were calculated by repeated-measures analysis of variance. (B) Glucose tolerance test and insulin measurements during the test in 7-month-old male mice (seven wild-type and eight knockout mice) (*, P < 0.05, and **, P < 0.01, compared to the wild type).

FIG. 4.

Insulin-induced Akt activation is enhanced in skeletal muscle from PI5P4Kβ^−/− mice. (A) Akt phosphorylation at serine 473 (pS473) and threonine 308 (pT308) and total Akt protein levels (note that the antibody reacts more strongly with nonphosphorylated Akt) in skeletal muscles from 5-month-old female wild-type and PI5P4Kβ^−/− mice injected with saline or insulin. Each lane represents a single mouse. (B) Akt activity assays in tissues from wild-type and PI5P4Kβ^−/− mice injected with saline or insulin (two to six unstimulated wild-type and knockout mice; five to nine wild-type and knockout mice injected with insulin; * P < 0.05). Results are means ± standard errors of the means and represent one of two experiments.

FIG. 5.

PI5P4Kβ^−/− mice exhibit growth retardation. Growth curves for wild-type (black squares), heterozygous (gray squares) and knockout (white squares) male and female mice (5 to 10 mice per genotype and gender). The data are mean body weights ± standard errors of the mean. The insets show the period from 9 to 19 days of age, with the x axis marked in days.

FIG. 6.

Hypersensitivity to insulin is not caused by decreased adipose tissue content. Insulin tolerance tests and chemical carcass analysis were carried out for 20-week-old female mice and 11-week-old male mice. wt, wild-type mice; ko, PI5P4Kβ^−/− mice. All results are means ± standard errors of the means for at least 9 animals of each genotype and gender.

FIG. 7.

PI5P4Kβ^−/− mice produce normal amounts of leptin under fed and fasting conditions. Leptin was measured for 6-month-old female wild-type (filled circles) and PI5P4Kβ^−/− mice (open circles), after fasting or feeding, and reported as a function of body weight. Results are reported for at least seven mice of each genotype and condition.

FIG. 8.

PI5P4Kβ^−/− mice remain hypersensitive to insulin on a high-fat diet. (A) Growth curves for wild-type female mice on a high-fat diet (filled squares; n = 11), PI5P4Kβ^−/− female mice on a high-fat diet (open squares; n = 15), and PI5P4Kβ^−/− female mice on a regular chow diet (open circles; n = 6). (B) Insulin tolerance test for wild-type (filled circles; n = 6) and PI5P4Kβ^−/− (open circles, n = 11) 36-week-old female mice fed the high-fat diet continuously from the time of weaning. (C) Glucose tolerance test for wild-type (filled squares; n = 6) and PI5P4Kβ^−/− (open squares; n = 11) 36-week-old female mice fed the high-fat diet continuously from the time of weaning. All results are means ± standard errors of the means.