Type 2 diabetes mellitus (T2DM) is a complex metabolic disorder that is characterized by abnormal glucose and lipid metabolism. Hence, exploring the mechanisms of lipid metabolism is vital and may offer clues to the discovery of new strategies for diabetes mellitus. Lipin1, acts as the phosphatidate phosphates, play a key role in the regulation of lipid metabolism at multiple nodal points. Lipin1 usually locates in the cytosol and translocate to the endoplasmic reticulum (ER) to catalyze the conversion of PA to DAG, which is a key substrate for the synthesis of TAG, PE and PC. Lipin1 is highly expressed in liver, adipose, skeletal muscle, and is also present in endoneurium of peripheral nerve of adult mice (Nadra et al., 2008). Earlier studies from our laboratory have found that the expression of lipin1 in sciatic nerve and hippocampus were significantly decreased in diabetic neuropathy rat models (Xu et al, 2015(Shang et al., 2020)). Meanwhile, over-expression of lipin1 alleviated HG-induced peripheral neuropathy, emerged as enhanced motor nerve conduction velocity, improved nerve pathological morphology, and increased nerve fiber diameter and nerve fiber density (Xu et al, 2015). These observations were consistent with previous studies showing that lipin1 deficiency animals develop peripheral neuropathy, characterized by myelin degradation, decreased motor nerve conduction velocity and compound muscle action potentials (Nadra et al., 2008). Taken together, these observations demonstrated that lipin1 may have a relationship with the development of diabetic peripheral neuropathy.
Peripheral nerves are composed of three distinct tissue compartments: the epineurium, perineurium, and endoneurium. The outermost epineurium surrounds perineurium, which in turn surrounds the fascicle of axons, Schwann cells, and fibroblasts. Schwann cells, derived from the neural crest, are the most abundant cells of the endoneurium. In the peripheral neural system, the myelin sheath allows for efficient action potential transmission and provides trophic support for related axons(Nadra et al., 2008). Nerve myelin sheath is the extended plasma membrane of the Schwann cell, most of the myelin lipid is provided from Schwann cell. Therefore, Schwann cells viability could play a crucial role in the development of peripheral nerve growth. Since diabetic peripheral neuropathy is characterized by nerve demyelination and degeneration of nerve fibers in dorsal rot ganglion and peripheral nerves (Hong et al., 2008), the viability of Schwann cells is closely associated with diabetic peripheral neuropathy.
In our study, the cell viability of Schwann cells was significantly weakened with high glucose treatment or with lipin1 silencing. In contrast, over-expression of lipin1 ameliorated cell viability with HG stimulation. It suggested that the loss of lipin1 suppressed cell viability of Schwann cells, while lipin1 treatment could alleviate HG-aroused cell apoptosis. Our gain-of-function and loss-of-function studies uniformly revealed that lipin1 alleviated HG-aroused decline in cell viability of Schwann cells. However, the underling molecular mechanisms remain elusive.
Mitochondria are semi-autonomous organelles that maintain the bilateral structure of the membranes. Mitochondria, served as bioenergetic powerhouses, provide the cell with energy in the form of ATP generated by oxidative phosphorylation. Hence, mitochondria are the powerhouses of the cell (Spinelli and Haigis, 2018) and are critical for cells survival. Previous studies demonstrated that mitochondrial disorder have close relationship with DPN, presented as hyperglycemia decreased mitochondrial respiration and ATP production in cultured dorsal root ganglion neurons (DRGs) (Rumora et al., 2019). Peripheral nerves have long axons that are wrapped with myelin produced by Schwann cells. Because of its morphological complexity and rapid changes in metabolic requirements, neurons are critically dependent on energy metabolism, predominantly supplied with mitochondrial oxidative phosphorylation (Pellerin and Magistretti, 2003). Besides, due to the limited capacity for self-renewal, the nervous system has the lowest capacity to maintain healthy after impairment of mitochondrial bioenergetics (Sajic, 2014). Therefore, peripheral nerves are particularly dependent on effective mitochondrial function and distribution.
In our study, we tested the mitochondrial membrane potential and ATP content in Schwann cells to assess mitochondrial metabolism. In our experiments, mitochondrial dysfunction occurs in Schwann cells insulted to high glucose and lipin1 silencing under normal glucose, presented as decreased levels of MMP and ATP content. By contrast, over-expression of lipin1 alleviate high glucose aroused mitochondrial dysfunction. In addition, in order to see if metabolic disorder induced by hyperglycemia are also reflected in the morphological changes, mitochondrial structures were also observed using electron microscopic. Mitochondrial after high glucose stimulation and lipin1 silencing exhibited morphological disorder, manifested as displayed irregularly and dense cristae, whereas lipin1 up-regulation reversed high glucose-aroused mitochondrial swelling and chromatin condensation. Our observations are consistent with previous reports that EDL muscle of lipin-1 deficiency fld mice presented impaired mitochondrial clearance and loss of membrane potential mitochondrial (Alshudukhi et al., 2018). Glycolysis muscle fibers in patients with heritable Lipin1-null mutations displayed mitochondrial aggregates and declined mitochondrial cytochrome c oxidase activity (Alshudukhi et al., 2018). Overall, these observations demonstrate that lipin1 reversed high glucose-induced mitochondrial dysfunction and morphological disorder. Lipin1 overexpression might improve cell viability through regulating mitochondrial functions in Schwann cells.
Mitochondria are highly dynamic organelle that constantly undergo fusion and fission, which are pivotal for the distribution of mtDNA and the maintenance of mitochondrial function (Friedman and Nunnari, 2014). Mitochondrial fusion is a process in which two separate mitochondria are fused into one (Sajic, 2014). Mitochondrial fusion mediates material mixing between damaged mitochondria and healthy mitochondria to ensure the integrity of the entire mitochondrial network. Mitochondrial fission is the division of one mitochondrial divide into two, which facilitates segregation of damaged mitochondria and enhance the number of mitochondria (Wu et al., 2016). It has previously been observed that the alterations of mitochondrial membrane lipid composition, such as CL and DAG, could change the balance of mitochondrial dynamics (Alshudukhi et al.,2017). Furthermore, mitochondrial dynamin-related family of large GTPases are all located at mitochondrial membranes, which are composed of proteins and lipids. Therefore, we speculate that lipin1, the key regulator of membrane lipid composition, maybe associated with mitochondrial dynamics by regulating phospholipid synthesis of the mitochondrial membrane (He et al., 2017).
The fusion of mitochondria is regulated by the proteins belonging to the dynamin-related family of large GTPases. MFN1 located in the outer membrane (OMM) and OPA1 located in the inner membrane (IMM). MFN1 serves as a tether between fusing mitochondria and contributed to artificial membrane clustering (Giacomello et al., 2020), OPA1 function as coordinate OMM and IMM, and remodel mitochondrial cristae (Giacomello et al., 2020). The fission of mitochondria is primarily regulated by DRP1, which translocate from the cytosol onto mitochondria outer membrane, more precisely onto ER-mitochondria contact sites (Lee and Yoon, 2016). This can be mediated by the OMM anchoring adaptors, including FIS1, MFF, Mid49 (Sabouny and Shutt, 2020). Although FIS1 is not essential for mitochondrial division, it could inhibit the mitochondrial fusion machinery and its over-expression leads to mitochondrial fragmentation (Giacomello et al., 2020). Studies have demonstrated that lipin1 catalytic domain co-localized with DRP1 at constricted regions of the tubules. Through the same localized domain, lipin1 could participate in the fission process and promotes fission in a PA-independent manner (Huang et al., 2011).
In our experiments, the levels of mitochondrial fission related proteins, including DRP1 and FIS1 were increased, whereas fusion related protein, such as MFN1 and OPA1 were decreased under high glucose conditions. The role of lipin1 silencing is similar to that under high glucose conditions. In contrast, lipin1 treatment inhibited DRP1 and FIS1 protein expression and promoted MFN1 and OPA1 protein expression. These findings suggested that lipin1 may have an antagonist effect to high glucose inducement on mitochondrial dynamic equilibrium of Schwann cells. Lipin1 treatment may protect against cells from mitochondrial dynamics imbalance induced by high glucose, and then keep mitochondria healthy and functional.
Our results are all based on in vitro experiments, which will be further verified by improving in vivo experiments in future studies. In addition, we only detected the influence of lipin1 on the expression of mitochondrial dynamic - related proteins, and lacked the analysis of changes in the lipid composition of mitochondrial membrane, which will be improved in the subsequent experiments.
Taken together, our study demonstrates for the first time that lipin1 could ameliorate cell viability of Schwann cells by affecting mitochondrial dynamics and functions,providing a novel approach for the prevention and treatment of DPN.