Don´t give up on mitochondria as a target for the treatment of diabetes and its complications

Abstract

In this editorial, we discuss an article by Wang et al, focusing on the role of mitochondria in peripheral insulin resistance and insulin secretion. Despite numerous in vitro and pre-clinical studies supporting the involvement of mitochondrial dysfunction and oxidative stress in the pathogenesis of diabetes and its complications, efforts to target mitochondria for glycemic control in diabetes using mitochondria-targeted antioxidants have produced inconsistent results. The intricate functionality of mitochondria is summarized to underscore the challenges it poses as a therapeutic target. While mitochondria-targeted antioxidants have demonstrated improvement in mitochondrial function and oxidative stress in pre-clinical diabetes models, the results regarding glycemic control have been mixed, and no studies have evaluated their hypoglycemic effects in diabetic patients. Nonetheless, pre-clinical trials have shown promising outcomes in ameliorating diabetes-related complications. Here, we review some reasons why mitochondria-targeted antioxidants may not function effectively in the context of mitochondrial dysfunction. We also highlight several alternative approaches under development that may enhance the targeting of mitochondria for diabetes treatment.

Key Words: Diabetes mellitus; Insulin resistance; Mitochondrion; Mitochondrial dysfunction; MitoQ; MitoTEMPO; SkQ; Elamipretide; Mitochondria transplantation; Glycemic control

Core Tip: Mitochondrial dysfunction and oxidative stress are closely linked to the development of diabetes and its complications. This has motivated the targeting of antioxidants to the mitochondria for diabetes treatment, which has generated in pre-clinical trials some encouraging results in diabetic complications, but inconsistent results in glycemic control. Moreover, there are very few studies with these molecules and only in healthy patients, with no encouraging results. There are several challenges to overcome to make mitochondria an efficient pharmacological target against diabetes, but recent developments such as mitochondrial transplantation, bioactive small peptides, and atomistic simulations could help to achieve this goal.

INTRODUCTION

The conventional view of mitochondria as solely energy-producing organelles is evolving, as these structures are now recognized to perform a diverse array of functions (Figure 1). These functions include the regulation of intracellular ion levels, which can be detrimental if concentrations exceed certain thresholds, such as iron[1], calcium[2], and copper[3]. Mitochondria are also implicated in thermoregulation through the activation of uncoupling proteins (UCPs), in the programmed cell death of defective cells via apoptosis, in the regulation of various physiological processes through the production of reactive oxygen species (ROS), and in the retrograde regulation of gene expression[4] (Figure 1). To integrate and regulate these functions, mitochondria respond to cellular energy levels by sensing the redox state of pyridine nucleotides (i.e., NADH/NAD⁺ ratios)[5] and the phosphorylation state of adenine nucleotides (i.e., ATP/AMP ratios)[6]. Similar to cytosolic processes, mitochondrial proteins involved in ATP synthesis, mitochondrial DNA (mtDNA) transcription and translation, the Krebs cycle, respiratory uncoupling, protein import, and apoptosis are believed to be regulated by phosphorylation/dephosphorylation cycles of kinases and phosphatases within the mitochondrion[7]. Furthermore, mitochondria are considered signaling organelles that modulate extracellular protein functions by activating cytosolic kinases through ROS or direct physical contact with other structures, such as the endoplasmic reticulum or cytoskeleton elements[8]. Additionally, the release of mtDNA from the matrix into the cytosol and extracellular space can induce inflammation[9] (Figure 1).

Figure 1 Complexity of mitochondrial functioning. Conventionally, mitochondria are recognized as central to oxidative metabolism for ATP production via the electron transport chain, regulated by mitochondrial energy levels and kinase/phosphatase systems. However, mitochondria also play roles in maintaining ionic homeostasis and various physiological functions. The physiological production of mitochondrial reactive oxygen species (ROS) modulates cell signaling and gene expression, while excessive ROS production can lead to detrimental processes such as inflammation and fibrosis. Uncontrolled opening of the mitochondrial permeability transition pore releases cytochrome c and mitochondrial DNA, promoting apoptosis and inflammation, respectively. These events are mitigated through processes like cell fission and mitophagy to prevent the transmission of mitochondrial DNA mutations to healthy mitochondria. Conversely, mitochondrial fusion into networks enhances mitochondrial function and reduces ROS production. ROS: Reactive oxygen species; Cyt C: Cytochrome c; UCPs: Uncoupling proteins.

The complexity of mitochondrial function increases when considering that mitochondria are not isolated organelles but form dynamic associations with other mitochondria through fusion and fission processes, known as mitochondrial dynamics. Mitochondrial dynamics significantly influence mitochondrial function, as mitochondria undergoing predominant fission and fragmentation exhibit impaired function, loss of membrane potential (ΔΨ), and increased ROS production, whereas the opposite occurs with predominant fusion and mitochondrial network formation[10]. Mitophagy is another process interconnected with mitochondrial dynamics and mitochondrial energization. Mitophagy serves as a quality control system that eliminates dysfunctional mitochondria in response to decreased mitochondrial ΔΨ, activating a series of proteins responsible for engulfing dysfunctional mitochondria in autophagosomal membranes for subsequent lysosomal degradation (Figure 1). This process is linked to mitochondrial dynamics, as mitochondrial fusion proteins Mitofusin 1 and 2 are degraded during mitophagy, preventing dysfunctional mitochondria from fusing and propagating defects, such as mtDNA mutations, to healthy mitochondria[11].

In a recent issue of the World Journal of Diabetes, Wang et al[12] reviewed evidence from animal and human models indicating that ATP production by oxidative phosphorylation and tight coupling of oxidative phosphorylation are crucial for maintaining insulin secretion in the pancreas and insulin sensitivity in peripheral tissues. They highlighted human studies showing that in obese patients with type 2 diabetes or elderly individuals with insulin resistance, there is an accumulation of fat in skeletal muscle and liver, decreased oxidative phosphorylation, reduced oxidative functions of mitochondria (e.g., NADH oxidation), and decreased size and number of mitochondria. In preclinical models of metabolic syndrome, these dysfunctions contribute to inefficient fatty acid oxidation and the accumulation of incomplete lipid oxidation products, such as diacylglycerol, which disrupts insulin receptor activation and leads to insulin resistance[12].

Correct functioning of the electron transport chain (ETC) is essential for the efficient oxidation of fatty acids and metabolic fuels, as NADH produced during mitochondrial β-oxidation is reoxidized in complex I of the ETC. If NADH is not reoxidized, fatty acid oxidation slows, leading to the accumulation of lipids such as diacylglycerol and ceramide, and resulting in reductive stress. This causes an increase in the NADH/NAD⁺ ratio and dysregulation of catabolic and antioxidant enzyme activity due to increased acetylation[13]. Slowing of electron transport in the ETC also increases the formation of ROS, which play a crucial role in the development of diabetic complications by activating processes of cell death, inflammation, and fibrosis (Figure 1), ultimately damaging target tissues such as the kidney and liver[14].

The hypothesis that mitochondrial ROS are responsible for the development of diabetic complications led to clinical trials with antioxidants to reduce these complications[15,16]. However, these results largely discouraged the idea that ROS is involved in the pathogenesis and complications of diabetes. The explanation for these disappointing results was that common antioxidants were unable to reach the sites of ROS production in the mitochondria and neutralize them. This is due to the large molecular size of most antioxidants and the relative impermeability of the inner mitochondrial membrane, preventing their passage into the mitochondrial matrix[17]. This led to the idea of conjugating antioxidants with Skulachev ions to promote their accumulation within mitochondria[18]. Skulachev ions, originally designed by Skulachev[19] in Russia to test the chemiosmotic hypothesis[19], are hydrophobic compounds that generally contain a positively charged phosphorus or nitrogen atom, allowing their penetration into the negatively charged mitochondrial matrix[19].

Thus, Skulachev[19] pioneered the study of the potential clinical utility of targeting antioxidants to the mitochondria for treating diseases involving excess ROS production. This research led to the design and synthesis of various mitochondria-targeted antioxidants, some of which have been tested for treating diabetes and its complications in both pre-clinical and clinical studies with mixed results. However, as discussed in the following sections, this marks only the beginning of an era of "mitochondrial medicine," which considers mitochondrial targets beyond ROS and even explores mitochondrial replacement strategies.

ANTIOXIDANTS CONJUGATED WITH TRIPHENYLPHOSPHONIUM CATIONS AND THEIR EFFECTS ON DIABETES

One of the most studied antioxidants derived from the conjugation of a triphenylphosphonium cation (i.e., Skulachev ion) to an antioxidant is mitoquinone (MitoQ). MitoQ contains a triphenylphosphonium cation attached to ubiquinone-10 via a hydrophobic linker. MitoQ has shown mixed results in lowering blood hyperglycemia in preclinical models of diabetes or obesity. In a model of accelerated metabolic syndrome development, MitoQ decreased fasting blood glucose, insulin, cholesterol, and triglyceride levels and normalized glucose tolerance during a 7-week administration following a high-fat diet[20]. MitoQ has been shown to increase insulin secretion in pancreatic beta cells grown in hyperglycemic media, simulating hyperglycemic conditions in humans[21]. However, in two recent studies in rats with type 2 diabetes induced by a high-fat diet and streptozotocin, MitoQ failed to lower blood glucose levels. The rationale for this result is that diabetes-induced in this manner is a more severe and advanced form of diabetes than in humans[22,23]. Similarly, using Akita (Ins2⁺/^-AkitaJ) mice, a model of type 1 diabetes, it was found that MitoQ administration did not lower blood glucose levels, which reached values of 681.78 mg/dL[24]. Despite these discouraging results regarding blood glucose, MitoQ showed promising results by protecting against diabetic kidney damage in at least two studies[24,25], improving diabetic neuropathy[23], and protecting against the development of hepatic steatosis[20,22].

Another mitochondria-targeted antioxidant tested in pre-clinical trials against hyperglycemia is SkQ1. SkQ1 is similar to MitoQ, except that the antioxidant portion containing ubiquinone-10 is replaced by a plastoquinone molecule, which is present in chloroplasts and provides SkQ1 with a wider antioxidant window than MitoQ[26]. In rats with alloxan-induced type 1 diabetes, administration of SkQ1 seven days before diabetes induction normalized blood glucose levels[27]. In contrast, in a study using db/db mice, a model of type 2 diabetes, administration of SkQ1 for 12 weeks did not reduce blood glucose or glycosylated hemoglobin levels[28]. In the case of alloxan-induced diabetes, it can be inferred that SkQ1 inhibits the development of diabetes due to its antioxidant properties, as alloxan induces diabetes by destroying pancreatic beta cells via oxidative damage[29]. Therefore, to demonstrate that SkQ1 has a hypoglycemic effect once diabetes is established, it is necessary to test the effect of SkQ1 once diabetes is established with alloxan or to test its effects in a model of diabetes that better mimics the development of diabetes in humans. Despite the mixed results of SkQ1 in hyperglycemia, it is noteworthy that its administration in diabetic rats significantly improved wound healing regardless of the absence of a hypoglycemic effect. This is a promising result given the relatively high incidence of defective wound healing in diabetic patients, the high proportion of patients suffering from diabetic foot ulceration, and the very high costs and dramatic impairment to the quality of life of those who suffer amputations[30].

Regarding human studies on antioxidants targeting mitochondria via a triphenylphosphonium cation, a recent systematic review and meta-analysis analyzed the results of 19 randomized controlled trials (RCTs). Some of these studies used MitoQ and MitoTEMPO, in which the ubiquinone-10 portion of MitoQ was substituted with the antioxidant piperidine nitroxide (TEMPO). This analysis revealed that only two studies in healthy patients showed no effect of MitoQ supplementation for 4 or 6 weeks on glycosylated hemoglobin and fasting blood glucose levels[31]. Importantly, there are no RCTs on the effects of mitochondria-targeted antioxidants in patients with impaired glucose metabolism. Therefore, it was concluded that there is limited evidence from RCTs to support the use of mitochondria-targeted antioxidants for the management of glycemic control[31].

SOME CONSIDERATIONS ON THE INCONSISTENCY OF THE OUTCOMES OF MITOCHONDRIA-TARGETED ANTIOXIDANTS IN THE CONTROL OF HYPERGLYCEMIA IN DIABETES

The inconsistency in the results obtained with antioxidants targeting mitochondria with a triphenylphosphonium cation could be due to several reasons. If the entry of conjugated antioxidants to a positively charged triphenylphosphonium cation is dependent on mitochondrial Δψ, will the accumulation of antioxidants in the mitochondrial matrix be possible when there is a dissipation of Δψ? The Δψ can decrease for different reasons, including an increase in UCP expression, uncontrolled opening of the mitochondrial permeability transition pore (mPTP) by an increase in calcium levels, or mitochondrial fatty acid transport[32]. These events also occur in diabetes, with increased UCP2 expression in pancreatic beta cells in type 2 diabetes[33], increased fatty acid transport in the mitochondria due to increased blood levels of free fatty acids in obesity and diabetes[34], and increased induction of mPTP in the liver[35]. Therefore, one or several of these events occurring simultaneously could limit the entry of MitoQ, MitoTEMPO, or SkQ1 into the matrix by dissipating the Δψ, thus decreasing their efficacy in diabetes. It has also been argued that MitoQ has a narrow window of anti- and pro-oxidant concentrations, which limits its efficacy in human clinical trials of neurodegenerative diseases[36]. Other considerations that could limit the action of mitochondria-targeted antioxidants include their lack of specificity for a particular oxidizing species, the influence of the physicochemical environment surrounding the mitochondrial inner membrane on the reactivity of the antioxidants with their targets, among other factors previously discussed[37].

DON’T GIVE UP ON MITOCHONDRIA FOR THE TREATMENT OF DIABETES AND ITS COMPLICATIONS!

Based on the information presented in the previous section, it appears that treatment with mitochondria-targeted antioxidants is not promising for the management of glycemic control in patients with diabetes. However, these studies have demonstrated the need, as mentioned by Wang et al[12] in their article in the World Journal of Diabetes[12], to develop new models of diabetes that more closely mimic the features of human diabetes and to foster a collaborative venture involving academia and industry to enable better and faster development of drugs for diabetes management[12], including mitochondria-targeted molecules such as MitoQ, MitoTEMPO, and SkQ. Notably, these molecules already show promising beneficial effects on diabetes complications independent of a decrease in hyperglycemia[20,22-25,30].

In addition to mitochondria-targeted antioxidants, other therapeutic strategies do not depend on the mitochondrial membrane potential to act within this organelle. For example, Elamipretide is a tetrapeptide (H-D-Arg-Dmt-Lys-Phe-NH₂) that selectively binds cardiolipin. This phospholipid is essential for the structure and function of several mito-chondrial inner membrane proteins, including the ETC complexes. Elamipretide increases electron flow through complex IV, which enhances mitochondrial respiration, ATP synthesis, Δψ, and decreases ROS formation. Additionally, it inhibits the interaction of cytochrome c with cardiolipin that activates its peroxidase function, preventing peroxidative damage to cardiolipin and detachment of cytochrome c from the mitochondria, thus preventing apoptosis[38]. Similar to antioxidants containing a triphenylphosphonium cation, Elamipretide protects against the development of diabetic nephropathy in diabetic db/db mice without improving glycemic control[39].

Finally, mitochondrial transfer between different cells has attracted significant attention. This phenomenon has been found to occur actively in vivo via extracellular vesicles and nanotubes and is postulated to play physiological and pathological roles[40]. This inspired the development of therapeutic strategies involving the transplantation of healthy mitochondria into diseased tissues with dysfunctional mitochondria. In the case of diabetes, the transfer of mitochondria from mesenchymal stromal cells to damaged pancreatic beta cells has been successfully tested in cell culture, improving the bioenergetics of damaged cells and, thus, insulin secretion[41]. Therefore, further research is warranted to develop the technology required for mitochondrial transplantation to either increase insulin production in the pancreas or decrease insulin resistance in peripheral tissues.

CONCLUSION

The mitochondrion is an extraordinarily complex organelle that functions as a hub for integrating oxidative metabolism, cell signaling, and death signals, protecting surrounding cells from the spread of cellular defects. Cells can transfer mitochondria to each other to improve the function of neighboring cells that are damaged or exhibit mitochondrial dysfunction. Moreover, mitochondria can fuse to enhance their function and undergo fission when mitochondrial function is defective. Under these circumstances, mitochondria undergo a process of self-destruction by mitophagy to discard those with defects in their mtDNA or bioenergetics. Additionally, the mitochondrion is a signaling organelle that, through the emission of different stimuli, regulates gene expression in the nucleus, kinase activity in the cytosol, as well as immunity and inflammation. Given their multiple layers of complexity, it is not surprising that mitochondria are a difficult therapeutic target, which is reflected by inconsistent results regarding glycemic control in pre-clinical and, in some cases, human clinical trials with mitochondria-targeted antioxidants or peptides. However, these molecules have been successfully used in pre-clinical models of diabetic complications, including diabetic nephropathy, diabetic wound healing, hepatic steatosis, and diabetic neuropathy. This suggests that mitochondria remain a promising therapeutic target for treating diabetic complications and highlights the need for further development of molecules targeting other aspects of mitochondria, such as mitophagy, mitochondrial dynamics, cation overload, and channels such as mPTP or UCPs. Computational developments such as molecular docking and molecular dynamics simulations could improve the design and selection of highly specific molecules for their targets. As discussed by Wang et al[12], a collaborative venture involving academia and industry is necessary for better and faster development of new drugs targeting mitochondria for treating diabetes and its complications or improving existing drugs.

ACKNOWLEDGEMENTS

Manuel Vargas-Vargas receives a postdoctoral fellowship from the National Council for Humanities, Sciences and Technologies (CONAHCYT), Mexico.