In this study we report the first cloning and sequencing of the α and β splice variants of lipin 1 from Chinese hamster cells. These hamster isoforms were similar to previously cloned lipin 1α and lipin 1β from rat [10
] and mouse [11
] in sequence and in cellular localization for wild-type cells. Overexpression of either form of hamster lipin 1 in CHO cells resulted in an increase in a Mg2+
-dependent PAP activity, indicating that these cloned sequences indeed encode an active PAP enzyme. It is interesting that, while retroviral-mediated expression of lipin 1α and lipin 1β in the GPI-deficient cells increased in-vitro
PAP activity, it failed to restore TG accumulation and phospholipid biosynthesis in this mutant cell line. (). This confirms a pleiotropic effect of the GPI mutation or loss of GPI activity on PAP cellular activity. We have ruled out that the nature of this effect on the suppression of glycerolipid biosynthesis is related to an impairment of glycolysis, per se, since we have previously reported no reduction in ATP levels or glycerol-3-phosphate levels in the GPI mutant cell line [5
]. Furthermore, we have shown that levels of the glycolytic metabolite F6P and phospho AMPK, a kinase that responds to changes in the ATP/AMP ratio [28
], are unaltered in the mutant GroD1 when compared to the parent cell line, ZR-82.
Our evidence suggests that the reduced glycerolipid biosynthesis is a result of an abnormal activation of the nutrient signaling mTOR pathway. In order for PAP/lipin 1 to be lipogenically active, it must associate with membranes since this is where its substrate, PA, is located [12
]. It has been previously demonstrated that activation of mTOR results in phosphorylation of lipin 1 [10
], resulting in the sequestration of lipin 1 in the cytosol [12
], thus preventing PAP access to the nucleus and membranes. Additionally, inhibition of the mTOR pathway results in membrane-association and nuclear localization of PAP/Lipin 1 [12
]. Abnormal activation of the mTOR pathway would explain the altered subcellular distribution of lipin 1α and reduced glycerolipid biosynthesis in GroD1, as well as the observed increase in phosphorylated mTOR targets, S6K(Ser 389) and Akt(Ser 473)[26
]. Furthermore, treatment of mutant cells with rapamycin, a well established inhibitor of the mTOR complex,[10
] reverts the nuclear localization of lipin 1α. This is consistent with the notion that altered subcellular localization is a consequence of the overactivated mTOR signaling.
While lipin 1α appears to function in the nucleus as a transcription factor it is lipin 1β that is thought to be involved in glycerolipid synthesis functioning as a PAP [29
]. Association with the membrane (e.g. the endoplasmic reticulum) would be required for effectiveness of this protein. Consistent with this are reports that PAP activity has been found to associate with membranes under conditions that increase glycerolipid synthesis [34
]. Although the fluorescence associated with the lipin 1β/GFP fusion construct appeared to have a diffuse distribution pattern in both the parent strain and GroD1, the fluorescence in the ZR-82 cells appeared to be more localized in the perinuclear region, consistent with an association with the endoplasmic reticulum. Consistently, we did find evidence that lipin 1β is hyperphosphorylated in the mutant cells.
The GPI-deficiency resulted in accumulation of high levels of G6P, a metabolite described as an activator of the mTOR signaling pathway in isolated cardiomiocytes [36
]. In addition, growth of GroD1 cells in reduced glucose medium or retroviral expression of wild-type GPI, two situations that would lower cellular levels of G6P, result in reversion of the lipogenic deficient phenotype and the abnormal subcellular localization of lipin 1α. Hence, an activated mTOR nutrient-signaling pathway, due to the accumulation of G6P, might explain the observed phenotype in GroD1 cells. Additionally, the GPI deficiency in these cells increased PA levels [5
]. PA is a well established activator the mTOR signaling pathway [37
]. Thus, the initial effects of G6P on mTOR activity would be exacerbated as glycerolipid biosynthesis is impaired and PA accumulates in these cells ().
Schematic diagram depicting the possible pathophysiological state of GPI-deficient GroD1 cells
These results have relevance to symptoms associated with human GPI deficiency. Patients with GPI deficiency present different degrees of non-spherocytic hemolytic anemia (NSHA). In some cases, these patients manifest neuromuscular dysfunctions characterized by muscle weakness and mental retardation [2
]. The molecular basis for these symptoms has not been established [2
]. There are 29 identified GPI mutations that cause NSHA. In all cases in which G6P levels were assayed, increased levels of this metabolite were found in red blood cells as well as in muscle and brain tissue [1
Different NSHA patients present different degrees of hemolytic anemia [1
]. However, the degree of the GPI deficiency does not correlate well with the severity of the anemia. For example, some patients with 60% of normal GPI activity display severe anemia [1
] while patients with only 9% residual activity present with a mild to moderate anemia [44
]. Interestingly, homozygous patients with the same mutation, display the same reduction in GPI activity, but display different severities of anemia [1
] as well as varying degrees of neurological and muscular dysfunction [3
]. Hence, it has been proposed that there must be either secondary genetic influences or environmental factors involved in the severity of the symptoms associated with NSHA disease [1
]. Perhaps our observation with respect to the glucose-dependent growth inhibition and the observed G6P accumulation may help to explain the differences in the severity in human patients and might warrant future in vivo
studies to test the hypothesis that altering metabolite levels in patients with inherited GPI deficiency could alleviate the severity of symptoms.
It is interesting to note that similar symptoms are associated with GPI deficiencies and lipin mutations. For instance, GPI-deficient patients can present with mental retardation and neurological impairment that affects the brain and the spinal cord [38
]. Similarly, lipin 1 mutant mice present peripheral neuropathies accompanied by demyelination, mediated by PA accumulation in Schwann cells [14
]. Additionally, while human patients with lipin 1 mutations manifest generalized muscular hypotonia due to an alteration in the phospholipid composition of the skeletal muscle fibers [16
], similar symptoms are present in human patients with the “GPI Homburg” mutation, which results in muscle weakness with histologically disproportioned muscle fibers [38
]. Finally, “Majeed syndrome”, which is due to mutations in lipin 2, is characterized by recurrent anemia, due to inefficiency of the bone marrow to produce rapidly dividing erythrocyte progenitor cells [17
]. Abnormalities in the red blood cell membrane lipid composition have been reported in inherited erythrocyte metabolic disorders [46
]. Alteration in the membrane lipid composition should compromise cell permeability and viability. Hence, it is conceivable that the traits observed in our GPI-deficient cells might help explain some of the manifestations observed in GPI deficient mice and human patients. The neuromuscular abnormalities and the anemia could be caused by dysfunction in lipid biosynthesis, a hypothesis that needs to be examined in the future.