Nearly two million Americans have type 1 diabetes, a condition in which the body does not produce enough insulin to effectively manage blood sugar, leading to high blood sugar levels, or hyperglycemia.
Persistent hyperglycemia puts people with diabetes at increased risk for heart disease, nerve damage, loss of sight, kidney disease and premature death. Controlling blood sugar through the use of insulin can prevent and delay these side effects of diabetes.
Now, scientists led by George King, MD, chief scientific officer and section head of vascular cell biology at Joslin Diabetes Center, have shed new light on exactly how hyperglycemia contributes to kidney disease and have also uncovered a potential therapeutic target. Their work, published in the Journal of Clinical Investigation, rests on a unique, long-running observational study of more than 1,000 people living with type 1 diabetes, known as the Medalist Study.
Since 2003, King and colleagues at Joslin have been studying people who have been living with the disease for 50 years or more—a feat that would have been considered highly unlikely at time of their diagnosis in the 1950s or earlier. The Medalist project has revealed surprising details about this remarkable cohort of participants.
For example, while as many as 30 percent of people with diabetes experience kidney disease, just 13 percent of Medalists – people given the moniker because they have received a medal from Joslin Diabetes Center to acknowledge their grit in managing diabetes for half a century or longer – do.
Prior studies of Medalists by King revealed that individuals who were protected from diabetic kidney disease showed elevated levels of glucose metabolizing enzymes compared to people with diabetes who developed diabetic kidney disease. King and colleagues suspected these enzymes – such as pyruvate kinase M2 (PKM2 – may play a protective role, neutralizing some of the damage hyperglycemia typically causes to the kidney.
“There is intense interest in identifying the mechanisms underlying the toxic effects of hyperglycemia,” said King, who is also a professor of medicine and ophthalmology at Harvard Medical School (HMS). “We have demonstrated at the molecular level that activation of PKM2 can increase an important protective factor for the kidney called vascular endothelial growth factor (VEGF) to normalize and maintain kidney function to prevent the dysfunction induced by chronic diabetes.”
Inspired by their previous findings, King and colleagues – including first author Jialin Fu, MD, Ph.D., senior postdoctoral fellow at Joslin -used mice genetically engineered to over-produce PKM2 to uncover its role in protecting the kidney from diabetes. After seven months, the researchers compared the kidneys of the engineered mice to those of wild type (non-engineered) mice with and without induced diabetes. The mice engineered to produce high levels of PKM2 demonstrated healthier kidneys than their wild type counterparts with diabetes.
“In this experiment, we made the surprising finding that overexpression of one enzyme in a subset of kidney cells can normalize kidney function and metabolism, even after seven months of elevated blood sugar levels,” said Fu, who is also a postdoctoral researcher at HMS. “These improvements of metabolism could benefit diabetic kidney disease, even in the presence of systemic inflammation, a common feature of diabetes.”
Specifically, the scientists demonstrated that the overexpression of PKM2 prevented multiple pathological changes to the cells of the kidney’s filtration system frequently seen in diabetes. The enzyme’s action preserved cell function and prevented disease progression. While further research is necessary, the findings support PKM2 as a potential therapeutic target for the prevention of diabetes-related kidney disease.
The results also suggest that monitoring PKM2 levels in the blood could also serve as a biomarker, allowing physicians to keep tabs on and predict the progression of diabetic kidney disease in patients with diabetes.
Co-authors included Takanori Shinjo, Qian Li, Ronald St-Louis, Kyoungmin Park, Marc Gregory Yu, Hisashi Yokomizo, Fabricio Simao, Qian Huang, I-Hsien Wu of Joslin Diabetes Center, Harvard Medical School.
PKM2: A Potential Therapeutic Target of Oxidative Stress and Inflammatory Damage
Oxidative stress is defined as the imbalance between the production of ROS and the endogenous antioxidant defence system. Oxidative stress has been viewed as one of the potential common aetiologies of many inflammatory diseases (169, 170). Studies have reported that dysregulated fatty acid metabolism is related to oxidative stress from the mitochondria, which drives chronic inflammation (171). Therefore, the relationship between oxidative stress and immunometabolism should be explored.
PKM2 Directly Induces Release of Proinflammatory Cytokines
Cytokines are key modulators of immunity. Cytokines are involved in every facet of immunity and inflammation, including innate immunity, antigen presentation, bone marrow differentiation, cellular recruitment and activation, and expression of adhesion molecules (172). High levels of proinflammatory cytokines and hyperactivation of immune cells leads to a life-threatening systemic inflammatory syndrome, which is also known as cytokine storm (1).
Several studies have shown that PKM2-mediated immunometabolic reprogramming promotes the secretion of proinflammatory cytokines. PKM2 forms a complex with HIF-1α in LPS-stimulated macrophages then the complex directly binds to the IL-1β promoter, and can be inhibited by PKM2 activators such as DASA-58 and TEPP-46 (10). The activation of PKM2 by both compounds inhibits glycolytic reprogramming and succinate production.
In addition, activation of PKM2 by TEPP-46 in vivo blocks IL-1β production induced by LPS and Salmonella, whereas production of the anti-inflammatory IL-10 is increased. PKM2 mediates production of early proinflammatory mediators, and promotes secretion of late proinflammatory mediators. PKM2 interacts with HIF-1α and activates HIF-1α-dependent transcription of enzymes involved in the Warburg effect in macrophages. PKM2-mediated Warburg effect then regulates the release of HMGB1 (4). PKM2 knockdown and pharmacological inhibition reduces serum lactate and HMGB1 levels. Shikonin protects mice from lethal endotoxemia and sepsis and other inflammatory diseases by inhibiting PKM2 expression.
In addition, studies have shown that the levels of proinflammatory cytokine in injured lungs induced by mechanical ventilation significantly increased whereas CXCL14 level decreased and PKM2 expression was increased (173). During this process, overexpression of CXCL14 can alleviate ventilator-induced lung injury and inhibition of pulmonary inflammation through downregulation of PKM2-mediated cytokine production. Previous studies have reported that shikonin, metformin, vitamin K (VK)3/5 exhibit significant inhibitory effects on PKM2 expression. Moreover, these agents exhibit an anti-inflammation effect that protects lungs against sepsis-associated lung injury (174–176).
Therefore, novel agents targeting PKM2 can be explored to inhibit proinflammatory cytokine production and alleviate organ failure caused by the cytokine storm. Furthermore, PKM2 plays an important role in pathogenesis of allergic airways disease partly through mediating phosphorylation of STAT3, thus increasing IL-1β–induced proinflammatory signalling (177). In addition, the nuclear PKM2-STAT3 pathway is implicated in LPS-induced lung injury (178). These findings indicate that PKM2 exhibits a proinflammatory effect by phosphorylating STAT3. The PKM2 activator, TEPP-46 exerts an anti-inflammatory effect and low activation of STAT3 (177).
Therefore, PKM2 promotes the production of proinflammatory cytokines, and expression of PKM2 significantly promotes inflammatory responses. Shikonin and other PKM2 inhibitors exert an anti-inflammatory effect by preventing the production of proinflammatory cytokines. Hence, the design of drugs targeting PKM2 inhibition can reduce the release of pro-inflammatory cytokines and alleviate cytokines storms.
Bidirectional Role of PKM2 in Maintenance of Cellular Redox Homeostasis
Oxidative stress is an imbalance between production of free radicals and ROS, and their elimination by protective mechanisms, such as antioxidants. This imbalance can lead to the damage of important biomolecules and organs, with potential effects on the whole organism (179). Several studies have explored the mechanism through which continued oxidative stress can lead to chronic inflammation and further cause chronic diseases. Oxidative stress can activate various transcription factors such as NF-κB, HIF-1α, β-catenin and Nuclear factor E2-related factor (Nrf-2), resulting in the expression of over 500 different genes, including inflammatory cytokines and chemokines (180). Therefore, oxidative stress and inflammation are closely linked.
Some cells highly depend on PKM2 whereas some do not, therefore, their regulation of redox state through PKM2 modulation occurs through opposite mechanisms. Redox-regulated PKM2 directly binds to p53 and the redox status of cysteine-423 of PKM2 tetramer is important for differential regulation of p53 transcriptional activity. The PKM2 tetramer suppresses p53 transcriptional activity and apoptosis in a high oxidation state but enhances these two processes in a low oxidation state (181).
TEPP-46 alleviates oxidative stress in cardiomyocytes and suppresses apoptosis of cardiomyocytes, thus preventing cardiac dysfunction, whereas it exacerbates oxidative stress and promotes apoptosis in lung cancer cells. These findings indicate the redox-dependent differences expressed in different tissues. In endothelial cells, cancer cells and neuronal cells, cellular metabolism is highly dependent on PKM2 expression and it’s mediated by aerobic glycolysis.
Therefore, expression of PKM2 plays a protective role against oxidative stress in these cells. Studies have reported that PKM2 is a crucial neuroprotective target against oxidative stress (182). PKM2 drives GSH biosynthesis which is an antioxidant intermediate, thus protecting neurons from oxidative damage and conferring neuroprotective effects. In endothelial cells, active endothelial NO synthase (eNOS) interacts with PKM2 and S-nitrosates PKM2, thus reducing PKM2 activity. PKM2 inhibition increases substrate flux through the pentose phosphate pathway to generate reducing equivalents, including NADPH and GSH, thus protecting cells against oxidative stress (183).
Moreover, PKM2 translocates to mitochondria under oxidative stress (184). In the mitochondria, PKM2 interacts with Bcl2 and phosphorylates it at threonine (T) 69, thus preventing Cul3-RBX1 ligase-mediated degradation of Bcl2, ultimately enhancing apoptosis resistance of tumour cells. ROS inhibits PKM2 activity thus contributes to cellular antioxidant responses (34). The inhibition of PKM2 activity diverts glucose flux into PPP to generate sufficient reducing potential for detoxification of ROS.
PKM2 expression supports the survival of cancers under acute oxidative stress by promoting anti-oxidant responses, whereas PKM2 activators compromise both pro-anabolic and anti-oxidant functions of cancer cells by increasing PKM2 activity thus interfering with its metabolism. 3,3′,5-triiodothyroxine (T3), a hormone secreted from the thyroid gland protects cells by improving the redox state and regulates the expression of PKM (185).
T3 can inhibit apoptosis and oxidative stress in human myocardial cells by upregulating the PKM2/PKM1 ratio. In addition, Nrf-2 regulates expression of several genes responsible for cellular detoxification, antioxidant function, anti-inflammatory effect, drug/xenobiotic transportation, and stress-related factors (186). Astrocytic DRD2 induces GSH synthesis through PKM2-mediated Nrf2 transactivation (187).
PKM2 expression plays different roles in oxidative stress in cells of different tissues. Selective protection against oxidative damage can be achieved by activating or inhibiting PKM2 activity. Although inhibition of PKM2 can significantly alleviate inflammation, complete knockdown of PKM2 is not feasible as it causes damage to cells that are highly dependent on PKM2, such as endothelial cells. Therefore, selective modulation of PKM2 is important to reduce the secretion of inflammatory cytokines and suppression of cytokine storm through alleviation of oxidative stress.
Alleviating Inflammatory Damage by Pharmacologically Targeting PKM2
PKM2 forms a bridge between immunometabolic reprogramming and inflammatory dysfunction, therefore, PKM2 is a potential therapeutic target for the treatment of inflammatory disease. Several agents have been reported that improve inflammatory damage by inhibiting PKM2 expression. Increased glucose uptake and glycolytic flux promotes generation of mitochondrial ROS in monocytes and macrophages of patients with atherosclerotic coronary artery disease (CAD), which in turn promotes PKM2 dimerization thus inducing its nuclear translocation (9).
Nuclear PKM2 functions as a protein kinase and phosphorylates STAT3 and further promotes the production of IL-6 and IL-1β, resulting in systemic and tissue inflammation. PKM2 serves as a molecular integrator of metabolic dysfunction, oxidative stress, and tissue inflammation, representing a novel therapeutic target for treatment of cardiovascular disease and other inflammatory diseases. Notably, PKM2-mediated glycolysis is significantly increased in the complete Freund’s adjuvant-induced astrocyte activation, and platelet-rich plasma effectively inhibits inflammatory response and HMGB1 expression by regulating PKM2-mediated glycolysis and STAT3 signalling-mediated astrocyte activation (188). PKM2-mediated aerobic glycolysis and protein kinase interactions play important roles in cytokine production and inflammation and provide a basis for the development of PKM2-targeted anti-inflammatory drugs.
Activators of PKM2 activity and PKM2 dimer inhibitors can suppress cytokine production and abrogate inflammatory diseases by mediating immunometabolic reprogramming. In addition, several pharmacological active ingredients have been reported that target PKM2 to modulate inflammatory responses. Plumbagin (5-hydroxy-2-methyl-1, 4-naphthoquinone) is a quinone isolated from the roots of Plumbago zeylanica. Previous studies reported that plumbagin exerts an anti-inflammatory effect by regulating pro-inflammatory signalling (189–191).
Plumbagin was shown to protect mice from lethal endotoxemia and polymicrobial sepsis induced by cecal ligation and puncture (CLP) by inhibiting the NADPH oxidase 4 (NOX4)/PKM2-dependent immunometabolism pathway, which inhibits LPS-induced PKM2 expression, lactate production and subsequent proinflammatory cytokine (IL-1β and HMGB1) release in macrophages (192). These findings indicate that plumbagin downregulates PKM2 expression to control proinflammatory cytokine production.
The traditional Chinese medicine formula Xijiao Dihuang decoction (XJDHT) was first reported in the medical classic “Beiji Qianjin Yaofang” and TCM doctors utilize XJDHT to improve prognosis of patients with sepsis. The main components of XJDHT include Rehmannia, Peony, Cortex Moudan and Cornu Bubali. Studies have shown that XJDHT improves survival of rats with sepsis by modulating the HIF-1α signalling pathway and inhibiting the release of inflammatory cytokine, such as IL-6 (193).
Recent studies reported that XJDHT alleviates sepsis by reducing the release of proinflammatory cytokines. XJDHT can improve sepsis by suppressing aerobic glycolysis through downregulation of the TLR4/HIF-1α/PKM2 signalling pathway (194). Therefore, the treatment effect of XJDHT on sepsis indicates that cytokine release can be inhibited by modulating PKM2. However, some agents reduce ROS production and alleviate oxidative stress by upregulating PKM2 in PKM2-dependent cells such as endothelial cells.
Qiliqiangxin is a traditional Chinese medicine preparation, comprising 11 Chinese herbal medicines, including astragalus, ginseng and aconite. A multicentre, randomized, double-blind, parallel-group, placebo-controlled study reported effective treatment of 512 chronic heart failure patients using qiliqiangxin (195). The therapeutic effect of qiliqiangxin is through improving cardiomyocyte metabolism and inhibiting cardiomyocyte apoptosis (196–199).
Qiliqiangxin improves glucose utilization and metabolism and increases ATP production by upregulating HIF-1α and several glycolysis-related enzymes, including PKM2, thus protecting cardiac microvascular endothelial cells against hypoxia injury (15). In addition, the effects of Chinese medicine in controlling macrophage polarization by modulating PKM2 have been reported. Lycium barbarum polysaccharide is the main bioactive component of Chinese wolfberry, and this compound inhibits PKM2 ubiquitination by upregulating the expression of ubiquitin ligases, thus suppressing LPS-induced inflammation by modulating glycolysis and M1 macrophage polarization (200). Therefore, targeting PKM2 to inhibit glycolysis in an overactive macrophage can provide a new therapeutic option for the treatment of inflammatory diseases.
Several drugs that inhibit cytokine release and improve inflammatory damage by regulating PKM2 expression have been reported. However, effective pharmacological interventions for effective treatment of cytokine storms have not been designed. Therefore, drugs that target and modulate PKM2 to inhibit inflammatory storms should be designed.
Protective Role of PKM2 in Specific Cells
PKM2 is highly expressed in rapidly dividing cells whereas its PK activity is significantly decreased. Notably, inhibition of PKM2 by cell growth signals can uncouple the ability of cells to assimilate nutrients into biosynthetic pathways from production of ATP (12). Therefore, PKM2 is an important regulator of cancer cell metabolism, and inhibition of PKM2 can block cancer cell proliferation and excessive immune cell responses (2).
However, endothelial cells and a few other types of cells display similar rapid proliferation characteristics such as cancer cells. Therefore, metabolism is a key regulator of growth of endothelial cells, in particular angiogenesis (201, 202). Glycolysis is a driving force for endothelial cell proliferation, whereas the Warburg effect has been reported in endothelial cells (203). Studies have shown that endothelial cells express PKM2 almost exclusively over PKM1, which is significant for maintaining their tight junctions and barrier function (204). PKM2 is required for the suppression of p53 and for maintaining cell cycle progression in proliferating endothelial cells.
On the contrary, PKM2 regulates vascular barrier function by suppressing NF-κB and its downstream target, the vascular permeability factor angiopoietin 2 in quiescent endothelial cells. Notably, the loss of PKM2 in endothelial cells results in TCA cycle dysfunction, changes in mitochondrial substrate utilization, thus impairing endothelial cell proliferation and migration (205). In addition, the loss of PKM2 can impair S-adenosylmethionine (SAM) synthesis, causing DNA hypomethylation, de-repression of endogenous retroviral elements and autoimmune activation.
Moreover, infiltrated/activated neutrophils at wound site release PKM2 during early stages of wound repair, which facilitates early wound healing by promoting angiogenesis at the wound site (206). Furthermore, PKM2 activates the STAT3 pathway which in turn promotes tissue repair and functional recovery by enhancing neuroblast migration, neurogenesis, and angiogenesis after stroke (207). PKM2 has been proposed as a new myokine, which contributes to axonal extension and functional motor recovery in spinal cord injured mice (208). PKM2 targets valosin-containing protein (VCP) to increase the density of axons and motor function which are mediated by extracellular PKM2-VCP-driven ATPase activity (209). The expression of PKM2 dimer plays an important role in repairing injury by promoting endothelial cell proliferation.
The potential effects of systemic pharmaceutical PKM2 inhibition on normal cells have not been fully elucidated, thus potential on-target side effects of PKM2 modulation should be explored. Targeting PKM2 as an adjuvant antineoplastic therapy can damage the vascular barrier and increase permeability of the vascular endothelium, thus leading to sepsis in immuno-compromised patients. Therefore, it is necessary to be cautious when developing therapies against cancer or inflammation that target PKM2.
PKM2 exerts an antioxidant effect thus protecting PKM2-dependent cells, such as endothelial cells, from oxidative stress. This protective effect of PKM2 on endothelial alleviates kidney injury (210). Endothelial nitric oxide synthase (eNOS) plays a protective role against kidney injury. S-nitroso-CoA (SNO-CoA)–aldoketo reductase family member (AKR1A1) is highly expressed in renal proximal tubules, where it promotes the activity of eNOS in reprogramming intermediary metabolism which is regulated by PKM2, thus protecting kidneys against acute kidney injury.
In addition, podocyte-specific PKM2-knockdown mice with diabetes present with worse albuminuria and glomerular pathology. PKM2 activation protects against diabetic nephropathy by increasing glucose metabolic flux, inhibiting production of toxic glucose metabolites and by inducing mitochondrial biogenesis to restore mitochondrial function (211). Therefore, PKM2 protects organ injury by abrogating oxidative stress in specific cells. Specificity of agents targeting PKM2 should be explored to avoid adverse side effects, however, PKM2 is an attractive target.
reference link :https://www.ncbi.nlm.nih.gov/labs/pmc/articles/PMC8572858/
More information: Jialin Fu et al, Regeneration of glomerular metabolism and function by podocyte pyruvate kinase M2 in diabetic nephropathy, JCI Insight (2022). DOI: 10.1172/jci.insight.155260