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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 20, 2005 as doi:10.1096/fj.05-4192fje. |
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Institute of Molecular Biology, The University of Hong Kong, Hong Kong, SAR, China
2 Correspondence: Institute of Molecular Biology, The University of Hong Kong, 8/F Kadoorie Biological Science Building, Pokfulam, Hong Kong, SAR, China. E-mail: S.S.M.C, smchung{at}hkucc.hku.hk; S.K.C., skchung{at}hkucc.hku.hk
SPECIFIC AIMS
We developed sodium/myo-inositol cotransporter-1 (SMIT-1) gene knockout mice to assess the contribution of this transporter to the cellular level of myo-inositol (MI) in different tissues and to determine the physiological consequences of MI depletion.
PRINCIPAL FINDINGS
1. SMIT-1 null mice died of respiratory failure soon after birth
MI is a precursor for a class of signal transduction molecules, the phosphatidylinositol (PI) and its derivatives, that regulates a number of physiological functions. In some tissues, it also serves as an organic osmolyte. Cellular MI is thought to originate from three sources: de novo synthesis, recycling of PIs, and uptake from extracellular fluid. Dietary MI is an important source of MI. MI in the circulating fluid is taken up by cells mainly through the membrane-associated transporters. SMIT-1 is thought to be the major MI transporter. How much of the cellular MI in different tissues is imported via SMIT-1 is not clear and the consequences of lacking MI are not known. We therefore developed SMIT-1 gene knockout mice to investigate these issues.
Two lines of SMIT-1 knockout mice were generated from two independent ES cell clones (4038 and 4050). Homozygous gene knockout mice from both lines were not found at weaning when the genotypes of the offspring were determined. Line 4038 mice did yield 
20% of the expected homozygous SMIT-1 null mice in the early breeding, but none were found in subsequent breeding, suggesting that lethality of SMIT-1 deficiency may be modulated by other genes. The ratio of the wild-type (SMIT+/+): heterozygous (SMIT+/–): homozygous (SMIT–/–) 18.5-day-old (E18.5) fetuses, was close to the expected 1:2:1, indicating that the SMIT–/– mice were able to survive throughout the entire period of embryonic development, and they probably died soon after birth. A similar result was observed with line 4050 mice. Close monitoring of the delivery of the pups revealed that soon after birth some of them appeared to have difficulty breathing. Their skin turned cyanotic color and they died within a few minutes. Eight dead neonates were collected and they were found to be all of the SMIT–/– genotype, suggesting that SMIT-1 deficiency causes respiratory dysfunction and neonatal lethality.
2. Neonatal lethality of the SMIT-null mice was prevented by prenatal maternal MI supplement
Since there are other transporters that can facilitate cellular uptake of MI, we determined whether maternal dietary MI supplement can prevent neonatal lethality of the SMIT-1 null mice. The drinking water of SMIT+/– females, mated with SMIT+/– males, was supplemented with MI (1% w/v) starting from the day of impregnation (E0.5) until weaning. At the time of weaning, the ratio of SMIT+/+: SMIT+/–: SMIT–/– genotypes was close to 1:2:1 for both lines of knockout mice, indicating that prenatal maternal MI supplement prevented neonatal lethality of the SMIT–/– mice. MI supplement started at E9.5 also prevented the death of SMIT–/– neonates. However, there was no beneficial effect when the treatment began at E15.5, indicating that E9.5 to E15.5 is the period of development that required high level of MI.
3. SMIT-1 deficiency impaired the development of peripheral nerves
Light microscopic examination of the lung sections showed that the structure of the lungs of the dead SMIT–/– neonates and E18.5 SMIT–/– fetuses appeared normal except that the air sacs were closed. Moreover, their lung surfactant proteins A, C, and D mRNA levels also appeared normal, suggesting that SMIT-1 deficiency did not significantly affect lung development. Defect in neural control of respiration was suspected.
To facilitate the visualization of nerve fibers in the mouse embryos, the thy1-YFP transgene that specifically express the yellow fluorescence protein in neurons was introduced into line 4038 SMIT-1 null mice. The expression of YFP made the nerve fibers visible under fluorescent microscope without staining for nerve-specific proteins. Examination of the E18.5 fetuses revealed that the peripheral nerves of SMIT–/– fetuses, including the brachial plexus, facial, vagus, intercostal, and phrenic nerve that innervates the diaphragm (Fig. 1
), all showed abnormal development. In some cases, the nerve fibers were absent, and in others the nerve fibers were much thinner and with fewer branches. Examination of the E18.5 SMIT–/– fetuses from mothers receiving MI supplement showed normal development in all the above-mentioned nerves. These results indicate that MI is essential for the development of peripheral nerves and that SMIT-1 deficiency leads to insufficient uptake of MI to support their development.
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4. SMIT-1 deficiency reduced tissue MI content
To determine the effect of SMIT-1 deficiency and MI supplement on tissue MI content, MI levels in several tissues from the 10-wk-old mice were determined. These mice received prenatal and postnatal maternal MI supplement. After weaning (at 4 wk old), one group continued to receive MI supplement via their drinking water while another group received no MI supplement. For the wild-type mice where MI supplement was terminated at weaning, the MI levels in their sciatic nerve, brain, kidney, skeletal muscle, and liver were 5.64 ± 0.23, 6.77 ± 0.26, 6.21 ± 0.15, 0.26 ± 0.016, and 0.34 ± 0.0085 nmol/mg wet weight, respectively. In the SMIT-1 null mice, MI contents in these tissues were reduced to 0.56 ± 0.026, 2.63 ± 0.11, 1.39 ± 0.034, 0.062 ± 0.0056, and 0.13 ± 0.0053 nmol/mg wet weight, respectively. The dramatic decrease of MI indicates that import from the circulating fluid is the major source of intracellular MI in these tissues. In the SMIT-1 null mice that continued to receive MI supplement after weaning, the MI levels in the sciatic nerve, brain, kidney, skeletal muscle, and liver were 0.7 ± 0.036, 2.74 ± 0.11, 1.50 ± 0.044, 0.305 ± 0.026, and 0.22 ± 0.021 nmol/mg wet weight, respectively.
5. SMIT-1 deficiency impaired nerve conduction velocity and reduced PKC activity of the sciatic nerve
To determine whether MI depletion affects the function of the peripheral nerves, nerve conduction velocity (NCV) of the sciatic nerve of the 10-wk-old SMIT–/– and SMIT+/+ mice was measured. One group of mice received postweaning MI supplement and one group did not. NCV in SMIT–/– mice was dramatically reduced, and MI supplement partially restored it (Fig. 2
). SMIT-1 deficiency also reduced the PKC activity in the sciatic nerve. MI supplement increased the PKC activity slightly, but the difference was not statistically significant (Fig. 2)
.
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CONCLUSIONS AND SIGNIFICANCE
The SMIT–/– neonates died of respiratory failure soon after birth. The development of peripheral nerves, including the phrenic nerve, which is crucial for regulating movement of the diaphragm, was severely impaired by SMIT-1 deficiency, indicating that MI is important for the development of these nerves, and that respiratory failure is due to abnormal development of the nerves controlling breathing.
Prevention of neonatal lethality of the SMIT-1 null mice by prenatal maternal MI supplement indicates that MI can be transferred across the placenta, and that other transporters can facilitate the uptake of MI to different tissues when the plasma level of MI is high. The 1% dietary MI supplement has been shown to increase plasma MI level by 3- to 5.5-fold in rats. In mammalian tissues, three different MI cotransporters were identified, namely, SMIT-1, SMIT-2, and HMIT (H+/MI symporter). The relative contributions of these MI transporters to the uptake of MI in various tissues are not clear. In the wild-type mice, sciatic nerve, brain, and kidney MI levels are >10-fold higher than that in skeletal muscle and liver, suggesting greater need of this metabolite in these tissues. SMIT-1 deficiency reduced the MI levels in the sciatic nerve, brain, and kidney by 90, 60, and 78%, respectively, indicating these tissues are most dependent on SMIT-1 for their cellular MI content. It is difficult to obtain a sufficient amount of embryonic peripheral nerves to determine their MI content. If they have a dependency on SMIT-1 similar to that of the adult sciatic nerve, it might explain why development of peripheral nerves is most sensitive to SMIT-1 deficiency (Fig. 3
).
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Postweaning MI supplement increased the MI level in the sciatic nerve from 10% to 12.5% that of the wild-type, indicating that other transporters contribute only a small amount of MI uptake in this tissue. If this is also true in embryonic peripheral nerves, it would suggest that the MI level in the peripheral nerves of the SMIT–/– fetuses is only marginally below the minimum level required for their development, and only a small increase in MI level will normalize their development. This might explain the observation that in the early breeding of line 4038 mice, 20% of the SMIT–/– mice survived to adulthood without prenatal MI supplement. This implies that other "modifying genes" that modestly affect the endogenous synthesis or uptake of MI could affect the peripheral nerve development and survival. The wide range of severity in the malformation of the peripheral nerves in the SMIT–/– embryos also agrees with this notion.
Depletion of MI leading to reduction of PKC and Na+/K+-ATPase activity has been postulated as the cause of NCV reduction in diabetic neuropathy. MI is a precursor for the synthesis of PI, which in turn, is a precursor of IP3 and DAG. DAG is required for the activation of PKC (Fig. 3)
. Therefore, reduction of MI would lead to reduction of PKC activity. However, this is a controversial hypothesis because in diabetic patients and mice, neuropathy is not always accompanied by nerve MI depletion. In the SMIT-1 null mice we found that MI depletion indeed leads to reduced PKC activity and slowing of NCV. Whether this pathogenic mechanism is generally applicable to diabetic neuropathy is not clear.
Another line of SMIT-1 gene knockout mice has been reported earlier. These mice also exhibit neonatal lethality, but respiratory failure in these mice was attributed to malfunction in the control of breathing at the brainstem. We found no obvious structural abnormality in the brain or brainstem of the E18.5 SMIT–/– fetuses. Although we showed that severe defects in the development of peripheral nerves most likely lead to respiratory failure, we cannot rule out functional abnormalities or subtle developmental irregularities in the brainstem that may also contribute to this lethal consequence of SMIT-1 deficiency.
FOOTNOTES
1 These authors contributed equally to this work. 
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4192fje;
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