bims-kimdis 2023-08-20 papers

bims-kimdis

Biomed News

on Ketones, inflammation and mitochondria in disease

Issue of 2023–08–20
twenty-one papers selected by
Matías Javier Monsalves Álvarez, Universidad de O’Higgins

The proteomic effects of ketone bodies: implications for proteostasis and brain proteinopathies.
Eu- or hypoglycemic ketosis and ketoacidosis in children: a review.
Effects of ketogenic diet on the classification and functional composition of intestinal flora in children with mitochondrial epilepsy.
Ketone Ester Administration Improves Glycemia in Obese Mice.
Ketogenic diet: a potential adjunctive treatment for substance use disorders.
Ketonemia variability through menstrual cycle in patients undergoing classic ketogenic diet.
Nutritional Intervention Through Ketogenic Diet in GLUT1 Deficiency Syndrome.
Ketogenic diet ameliorates attention deficit hyperactivity disorder in rats via regulating gut microbiota.
Cardiovascular aging: the mitochondrial influence.
MARCH5-dependent NLRP3 ubiquitination is required for mitochondrial NLRP3-NEK7 complex formation and NLRP3 inflammasome activation.
Comprehensive interrogation of human skeletal muscle reveals a dissociation between insulin resistance and mitochondrial capacity.
The Whole of Diabetes Management: Low Carbohydrate Diet as an Opportunity for Remission.
Muscle mitochondrial transplantation can rescue and maintain cellular homeostasis.
Methods to Activate the NLRP3 Inflammasome.
Monitoring Mitochondria Function in Ferroptosis.
Resolvin D1 Attenuates Inflammation and Pelvic Pain Associated with EAP by Inhibiting Oxidative Stress and NLRP3 Inflammasome Activation via the Nrf2/HO-1 Pathway.
Canonical Inflammasomes.
NOX2 deficiency exacerbates diet-induced obesity and impairs molecular training adaptations in skeletal muscle.
Mitochondria and NLRP3 inflammasome in cardiac hypertrophy.
Structural mechanisms of the mTOR pathway.
Resistance exercise: a mighty tool that adapts, destroys, rebuilds and modulates the molecular and structural environment of skeletal muscle.

Front Mol Neurosci. 2023 ;16 1214092

The proteomic effects of ketone bodies: implications for proteostasis and brain proteinopathies.

Lizbeth García-Velázquez, Lourdes Massieu.

  A growing body of evidence supports the beneficial effects of the ketone bodies (KBs), acetoacetate and β-hydroxybutyrate (BHB), on diverse physiological processes and diseases. Hence, KBs have been suggested as therapeutic tools for neurodegenerative diseases. KBs are an alternative fuel during fasting and starvation as they can be converted to Ac-CoA to produce ATP. A ketogenic diet (KD), enriched in fats and low in carbohydrates, induces KB production in the liver and favors their use in the brain. BHB is the most abundant KB in the circulation; in addition to its role as energy fuel, it exerts many actions that impact the set of proteins in the cell and tissue. BHB can covalently bind to proteins in lysine residues as a new post-translational modification (PTM) named β-hydroxybutyrylation (Kbhb). Kbhb has been identified in many proteins where Kbhb sites can be critical for binding to other proteins or cofactors. Kbhb is mostly found in proteins involved in chromatin structure, DNA repair, regulation of spliceosome, transcription, and oxidative phosphorylation. Histones are the most studied family of proteins with this PTM, and H3K9bhb is the best studied histone mark. Their target genes are mainly related to cell metabolism, chromatin remodeling and the control of circadian rhythms. The role of Kbhb on physiological processes is poorly known, but it might link KB metabolism to cell signaling and genome regulation. BHB also impacts the proteome by influencing proteostasis. This KB can modulate the Unfolded Protein Response (UPR) and autophagy, two processes involved in the maintenance of protein homeostasis through the clearance of accumulated unfolded and damaged proteins. BHB can support proteostasis and regulate the UPR to promote metabolism adaptation in the liver and prevent cell damage in the brain. Also, BHB stimulates autophagy aiding to the degradation of accumulated proteins. Protein aggregation is common to proteinopathies like Alzheimer's (AD) and Parkinson's (PD) diseases, where the KD and BHB treatment have shown favorable effects. In the present review, the current literature supporting the effects of KBs on proteome conformation and proteostasis is discussed, as well as its possible impact on AD and PD.

Keywords:  autophagy; brain proteinopathies; ketone bodies; proteome adaptation; unfolded protein response; β-hydroxybutyrate; β-hydroxybutyrylation

DOI:  https://doi.org/10.3389/fnmol.2023.1214092
Pediatr Nephrol. 2023 Aug 16.

Eu- or hypoglycemic ketosis and ketoacidosis in children: a review.

Martina Meoli, Sebastiano A G Lava, Gabriel Bronz, Barbara Goeggel-Simonetti, Giacomo D Simonetti, Ilaria Alberti, Carlo Agostoni, Mario G Bianchetti, Martin Scoglio, Stefano A Vismara, Gregorio P Milani.

  The last decade has been characterized by exciting findings on eu- or hypoglycemic ketosis and ketoacidosis. This review emphasizes the following five key points: 1. Since the traditional nitroprusside-glycine dipstick test for urinary ketones is often falsely negative, the blood determination of β-hydroxybutyrate, the predominant ketone body, is currently advised for a comprehensive assessment of ketone body status; 2. Fasting and infections predispose to relevant ketosis and ketoacidosis especially in newborns, infants, children 7 years or less of age, and pregnant, parturient, or lactating women; 3. Several forms of carbohydrate restriction (typically less than 20% of the daily caloric intake) are employed to induce ketosis. These ketogenic diets have achieved great interest as antiepileptic treatment, in the management of excessive body weight, diabetes mellitus, and in sport training; 4. Intermittent fasting is more and more popular because it might benefit against cardiovascular diseases, cancers, neurologic disorders, and aging; 5. Gliflozins, a new group of oral antidiabetics inhibiting the renal sodium-glucose transporter 2, are an emerging cause of eu- or hypoglycemic ketosis and ketoacidosis. In conclusion, the role of ketone bodies is increasingly recognized in several clinical conditions. In the context of acid-base balance evaluation, it is advisable to routinely integrate both the assessment of lactic acid and β-hydroxybutyrate.

Keywords:  Diet; Glucose; Ketone bodies; Ketosis; Metabolic acidosis; Protein

DOI:  https://doi.org/10.1007/s00467-023-06115-5
Front Neurol. 2023 ;14 1237255

Effects of ketogenic diet on the classification and functional composition of intestinal flora in children with mitochondrial epilepsy.

Jing Wang, LIjuan Huang, Hua Li, Guohong Chen, Liming Yang, Dong Wang, Hong Han, Guo Zheng, Xu Wang, Jianmin Liang, Weijie He, Fang Fang, Jianxiang Liao, Dan Sun.

  The ketogenic diet (KD) has shown excellent performance in the treatment of refractory epilepsy, but how it works is not yet fully understood. Gut microbiota is associated with various neurological disorders through the brain-gut axis. Different dietary patterns have different effects on the composition and function of gut microbiota. Here, by analyzing fecal samples from some patients with mitochondrial epilepsy before and after KD treatment through 16SrRNA sequencing, we found that KD intervention reduced the abundance of Firmicutes in the patient's gut, while the abundance of Bacteroidota increased in the KD group. LefSe analysis showed that Actinobacteriota, Phascolarctobacterium had significant advantages in the control group, while Bacteroides increased significantly after KD intervention, especially Bacteroides fragilis. Functional analysis showed that there were significant differences in 12 pathways in level 3. These changes suggest that KD can change the composition and diversity of the gut microbiota in patients and affect their function. Changes in specific bacterial groups in the gut may serve as biomarkers for the therapeutic effects of KD on epilepsy.

Keywords:  Bacteroides fragilis; gut microbiota; ketogenic diet; microbiota-gut-brain axis; mitochondrial epilepsy

DOI:  https://doi.org/10.3389/fneur.2023.1237255
Am J Physiol Cell Physiol. 2023 Aug 14.

Ketone Ester Administration Improves Glycemia in Obese Mice.

Sayed Amirhossein Tabatabaei Dakhili, Kunyan Yang, Cassandra A A Locatelli, Christina T Saed, Amanda A Greenwell, Jordan S F Chan, Jadin J Chahade, Jared Scharff, Shahad Al Imarah, Farah Eaton, Peter A Crawford, Keshav Gopal, Erin E Mulvihill, John R Ussher.

  During periods of prolonged fasting/starvation, the liver generates ketones (i.e., β-hydroxybutyrate [βOHB]) that primarily serve as alternative substrates for ATP production. Previous studies have demonstrated that elevations in skeletal muscle ketone oxidation contribute to obesity-related hyperglycemia, whereas inhibition of succinyl CoA:3-ketoacid CoA transferase (SCOT), the rate-limiting enzyme of ketone oxidation, can alleviate obesity-related hyperglycemia. As circulating ketone levels are a key determinant of ketone oxidation rates, we tested the hypothesis that increases in circulating ketone levels would worsen glucose homeostasis secondary to increases in muscle ketone oxidation. Accordingly, male C57BL/6J mice were subjected to high-fat diet-induced obesity, whereas their lean counterparts received a standard chow diet. Lean and obese mice were orally administered either a ketone ester (KE) or placebo, followed by a glucose tolerance test. In tandem, we conducted isolated islet perifusion experiments to quantify insulin secretion in response to ketones. We observed that exogenous KE administration robustly increases circulating βOHB levels, which was associated with an improvement in glucose tolerance only in obese mice. These observations were independent of muscle ketone oxidation, as they were replicated in mice with a skeletal muscle-specific SCOT deficiency. Furthermore, the R-isomer of βOHB produced greater increases in perifusion insulin levels versus the S-Isomer in isolated islets from obese mice. Taken together, acute elevations in circulating ketones promote glucose-lowering in obesity. Given that only the R-isomer of βOHB is oxidized, further studies are warranted to delineate the precise role of β-cell ketone oxidation in regulating insulin secretion.

Keywords:  Glucose Tolerance; Insulin; Ketones; Obesity

DOI:  https://doi.org/10.1152/ajpcell.00300.2023
Front Nutr. 2023 ;10 1191903

Ketogenic diet: a potential adjunctive treatment for substance use disorders.

Deshenyue Kong, Jia-Xue Sun, Ji-Qun Yang, Yuan-Sen Li, Ke Bi, Zun-Yue Zhang, Kun-Hua Wang, Hua-You Luo, Mei Zhu, Yu Xu.

  Substance use disorders (SUD) can lead to serious health problems, and there is a great interest in developing new treatment methods to alleviate the impact of substance abuse. In recent years, the ketogenic diet (KD) has shown therapeutic benefits as a dietary therapy in a variety of neurological disorders. Recent studies suggest that KD can compensate for the glucose metabolism disorders caused by alcohol use disorder by increasing ketone metabolism, thereby reducing withdrawal symptoms and indicating the therapeutic potential of KD in SUD. Additionally, SUD often accompanies increased sugar intake, involving neural circuits and altered neuroplasticity similar to substance addiction, which may induce cross-sensitization and increased use of other abused substances. Reducing carbohydrate intake through KD may have a positive effect on this. Finally, SUD is often associated with mitochondrial damage, oxidative stress, inflammation, glia dysfunction, and gut microbial disorders, while KD may potentially reverse these abnormalities and serve a therapeutic role. Although there is much indirect evidence that KD has a positive effect on SUD, the small number of relevant studies and the fact that KD leads to side effects such as metabolic abnormalities, increased risk of malnutrition and gastrointestinal symptoms have led to the limitation of KD in the treatment of SUD. Here, we described the organismal disorders caused by SUD and the possible positive effects of KD, aiming to provide potential therapeutic directions for SUD.

Keywords:  addiction; gut microbiota; ketogenic diet; metabolism; neuroprotection; substance use disorders

DOI:  https://doi.org/10.3389/fnut.2023.1191903
Front Nutr. 2023 ;10 1188055

Ketonemia variability through menstrual cycle in patients undergoing classic ketogenic diet.

Ludovica Pasca, Cinzia Ferraris, Monica Guglielmetti, Costanza Varesio, Martina Totaro, Claudia Trentani, Claudia Marazzi, Ilaria Brambilla, Elena Ballante, Marisa Armeno, Gabriela Reyes Valenzuela, Roberto H Caraballo, Pierangelo Veggiotti, Anna Tagliabue, Valentina De Giorgis.

   Introduction: Ketogenic dietary therapies (KDT) are well-established, safe, non-pharmacologic treatments used for children and adults with drug-resistant epilepsy and other neurological disorders. Ketone bodies (KBs) levels are recognized as helpful to check compliance to the KDT and to attempt titration of the diet according to the individualized needs. KBs might undergo inter-individual and intra-individual variability and can be affected by several factors. Possible variations in glycemia and ketone bodies blood levels according to the menstrual cycle have not been systematically assessed yet, but this time window deserves special attention because of hormonal and metabolic related changes.
Methods: This study aims at searching for subtle changes in KBs blood level during menstrual cycle in female patients undergoing a stable ketogenic diet, by analyzing 3-months daily measurement of ketone bodies blood levels and glucose blood levels throughout the menstrual cycle.
Results: We report the preliminary results on six female patients affected by GLUT1DS or drug resistant epilepsy, undergoing a stable classic ketogenic diet. A significant increase in glucose blood levels during menstruation was found in the entire cohort. As far as the ketone bodies blood levels, an inversely proportional trend compared to glycemia was noted.
Conclusion: Exploring whether ketonemia variations might occur according to the menstrual cycle is relevant to determine the feasibility of transient preventive diet adjustments to assure a continuative treatment efficacy and to enhance dietary behavior support.
Clinical trial registration: clinicaltrials.gov, identifier NCT05234411.

Keywords:  GLUT1-DS; classic ketogenic diet; drug resistant epilepsy; epilepsy; ketogenic dietary therapies efficacy; ketonemia variability; menstrual cycle

DOI:  https://doi.org/10.3389/fnut.2023.1188055
Clin Nutr Res. 2023 Jul;12(3): 169-176

Nutritional Intervention Through Ketogenic Diet in GLUT1 Deficiency Syndrome.

Young-Sun Kim, Woojeong Kim, Ji-Hoon Na, Young-Mock Lee.

  Glucose transporter type 1 (GLUT1) deficiency syndrome (DS) is a metabolic brain disorder caused by a deficiency resulting from SLC2A1 gene mutation and is characterized by abnormal brain metabolism and associated metabolic encephalopathy. Reduced glucose supply to the brain leads to brain damage, resulting in delayed neurodevelopment in infancy and symptoms such as eye abnormalities, microcephaly, ataxia, and rigidity. Treatment options for GLUT1 DS include ketogenic diet (KD), pharmacotherapy, and rehabilitation therapy. Of these, KD is an essential and the most important treatment method as it promotes brain neurodevelopment by generating ketone bodies to produce energy. This case is a focused study on intensive KD nutritional intervention for an infant diagnosed with GLUT1 DS at Gangnam Severance Hospital from May 2022 to January 2023. During the initial hospitalization, nutritional intervention was performed to address poor intake via the use of concentrated formula and an attempt was made to introduce complementary feeding. After the second hospitalization and diagnosis of GLUT1 DS, positive effects on the infant's growth and development, nutritional status, and seizure control were achieved with minimal side effects by implementing KD nutritional intervention and adjusting the type and dosage of anticonvulsant medications. In conclusion, for patients with GLUT1 DS, it is important to implement a KD with an appropriate ratio of ketogenic to nonketogenic components to supply adequate energy. Furthermore, individualized and intensive nutritional management is necessary to improve growth, development, and nutritional status.

Keywords:  Diet, carbohydrate-restricted; Diet, ketogenic; Epilepsy; Glut1 deficiency syndrome; Seizures

DOI:  https://doi.org/10.7762/cnr.2023.12.3.169
PLoS One. 2023 ;18(8): e0289133

Ketogenic diet ameliorates attention deficit hyperactivity disorder in rats via regulating gut microbiota.

Yu Liu, Changhong Yang, Yingxue Meng, Yonghui Dang, Lin Yang.

Attention deficit hyperactivity disorder (ADHD) is a common mental behavioral disorder in children. Alterations in gut microbiota composition are associated with neurological disorders. We aimed to investigate whether a ketogenic diet (KD) can be an alternative therapy for ADHD by altering the gut microbiota. Male spontaneously hypertensive rats (SHR) and Wistar Kyoto (WKY) rats were randomly allocated to the normal diet (ND), methylphenidate (MPH), and KD groups. SHR in groups KD and MPH exhibited a significant increase in behavioral characteristics of ADHD, such as distance moved and immobility time. KD and MPH treatment led to a significant elevation in concentrations of 5-HT, AC, cAMP, and NE of brain tissue and the expression of DRD1, DAT, PKA, DARPP32, and cAMP at the protein level in WKY rats and SHR. KD and MPH significantly increased the richness and diversity of gut microbiota in SHR. The abundance of Ruminococcus_gauvreauii_group, Bacteroides, Bifidobacterium, and Blautia significantly increased, whereas that of Lactobacillus, Romboutsia, Facklamia, and Turicibacter significantly declined in the KD group compared with the ND group. The gut microbiota in the KD group of SHR mainly participated in amino acid metabolism- and sugar metabolism-related pathways. KD might alleviate behavioral disorders in ADHD by regulating gut microbiota. This study provides novel insights for the use of KD in treating ADHD.

DOI: https://doi.org/10.1371/journal.pone.0289133
J Cardiovasc Aging. 2023 Jul;pii: 33. [Epub ahead of print]3(3):

Cardiovascular aging: the mitochondrial influence.

Shakti Sagar, Asa B Gustafsson.

  Age-associated cardiovascular disease is becoming progressively prevalent due to the increased lifespan of the population. However, the fundamental mechanisms underlying the aging process and the corresponding decline in tissue functions are still poorly understood. The heart has a very high energy demand and the cellular energy needed to sustain contraction is primarily generated by mitochondrial oxidative phosphorylation. Mitochondria are also involved in supporting various metabolic processes, as well as activation of the innate immune response and cell death pathways. Given the central role of mitochondria in energy metabolism and cell survival, the heart is highly susceptible to the effects of mitochondrial dysfunction. These key organelles have been implicated as underlying drivers of cardiac aging. Here, we review the evidence demonstrating the mitochondrial contribution to the cardiac aging process and disease susceptibility. We also discuss the potential mechanisms responsible for the age-related decline in mitochondrial function.

Keywords:  Aging; heart disease; mitochondria

DOI:  https://doi.org/10.20517/jca.2023.22
EMBO J. 2023 Aug 14. e113481

MARCH5-dependent NLRP3 ubiquitination is required for mitochondrial NLRP3-NEK7 complex formation and NLRP3 inflammasome activation.

Yeon-Ji Park, Niranjan Dodantenna, Yonghyeon Kim, Tae-Hwan Kim, Ho-Soo Lee, Young-Suk Yoo, June Heo, Jae-Ho Lee, Myung-Hee Kwon, Ho Chul Kang, Jong-Soo Lee, Hyeseong Cho.

  The NLRP3 inflammasome plays a key role in responding to pathogens, and endogenous damage and mitochondria are intensively involved in inflammasome activation. The NLRP3 inflammasome forms multiprotein complexes and its sequential assembly is important for its activation. Here, we show that NLRP3 is ubiquitinated by the mitochondria-associated E3 ligase, MARCH5. Myeloid cell-specific March5 conditional knockout (March5 cKO) mice failed to secrete IL-1β and IL-18 and exhibited an attenuated mortality rate upon LPS or Pseudomonas aeruginosa challenge. Macrophages derived from March5 cKO mice also did not produce IL-1β and IL-18 after microbial infection. Mechanistically, MARCH5 interacts with the NACHT domain of NLRP3 and promotes K27-linked polyubiquitination on K324 and K430 residues of NLRP3. Ubiquitination-defective NLRP3 mutants on K324 and K430 residues are not able to bind to NEK7, nor form NLRP3 oligomers leading to abortive ASC speck formation and diminished IL-1β production. Thus, MARCH5-dependent NLRP3 ubiquitination on the mitochondria is required for NLRP3-NEK7 complex formation and NLRP3 oligomerization. We propose that the E3 ligase MARCH5 is a regulator of NLRP3 inflammasome activation on the mitochondria.

Keywords:  MARCH5; NEK7; NLRP3 inflammasome; mitochondria; ubiquitination

DOI:  https://doi.org/10.15252/embj.2023113481
Am J Physiol Endocrinol Metab. 2023 Aug 16.

Comprehensive interrogation of human skeletal muscle reveals a dissociation between insulin resistance and mitochondrial capacity.

Katie L Whytock, Maria F Pino, Yifei Sun, Gongxin Yu, Flavia G De Carvalho, Reichelle X Yeo, Rick B Vega, Parmar Gaganvir, Adeline Divoux, Nidhi Kapoor, Fanchao Yi, Heather H Cornnell, David A Patten, Mary-Ellen Harper, Stephen J Gardell, Steven R Smith, Martin J Walsh, Lauren M Sparks.

  Insulin resistance and blunted mitochondrial capacity in skeletal muscle are often synonymous; however, this association remains controversial. The aim of this study was to perform an in-depth multi-factorial comparison of skeletal muscle mitochondrial capacity between individuals who were lean and active (Active- n = 9), individuals with obesity (Obese- n = 9) and individuals with Obesity, insulin resistance and type 2 diabetes (T2D- n = 22). Mitochondrial capacity was assessed by ex vivo mitochondrial respiration with fatty-acid and glycolytic supported protocols adjusted for mitochondrial content (mtDNA and citrate synthase activity). Supercomplex assembly was measured by BN-PAGE and immunoblot. TCA cycle intermediates were assessed with targeted metabolomics. Exploratory transcriptomics and DNA methylation analyses were performed to uncover molecular differences affecting mitochondrial function among the three groups. We reveal no discernable differences in skeletal muscle mitochondrial content, mitochondrial capacity, supercomplex assembly, TCA cycle intermediates and mitochondrial molecular profiles between obese individuals with and without T2D that had comparable levels of confounding factors (BMI, age, aerobic capacity). We highlight that lean, active individuals have greater; mitochondrial content, mitochondrial capacity, supercomplex assembly and TCA cycle intermediates. These phenotypical changes are reflected at the level of DNA methylation and gene transcription. The collective observation of comparable muscle mitochondrial capacity in individuals with obesity and T2D (vs. individuals without T2D) underscores a dissociation from skeletal muscle insulin resistance.

Keywords:  Insulin Resistance; Mitochondrial Capacity; Skeletal Muscle; Transcriptomics; Type 2 Diabetes

DOI:  https://doi.org/10.1152/ajpendo.00143.2023
Holist Nurs Pract. 2023 Sep-Oct 01;37(5):37(5): 301-303

The Whole of Diabetes Management: Low Carbohydrate Diet as an Opportunity for Remission.

Barbara Osborne, Patti Rager Zuzelo.

DOI: https://doi.org/10.1097/HNP.0000000000000605
Am J Physiol Cell Physiol. 2023 Aug 14.

Muscle mitochondrial transplantation can rescue and maintain cellular homeostasis.

Debasmita Bhattacharya, Mikhaela B Slavin, David A Hood.

  Mitochondria control cellular functions through their metabolic role. Recent research that has gained considerable attention is their ability to transfer between cells. This has the potential of improving cellular functions in pathological or energy deficit conditions, but little is known about the role of mitochondrial transfer in sustaining cellular homeostasis. Few studies have investigated the potential of skeletal muscle as a source of healthy mitochondria that can be transferred to other cell types. Thus, we isolated intermyofibrillar mitochondria from murine skeletal muscle and incubated them with host cells. We observed dose- and time-dependent increases in mitochondrial incorporation into myoblasts. This resulted in elongated mitochondrial networks and an enhancement of bioenergetic profile of the host cells. Mitochondrial donation also rejuvenated the functional capacities of the myoblasts when respiration efficiency and lysosomal function were inhibited by complex I inhibitor rotenone and bafilomycin A, respectively. Mitochondrial transfer was accomplished via tunneling nanotubes, extracellular vesicles, gap junctions and by macropinocytosis internalization. Murine muscle mitochondria were also effectively transferred to human fibroblast cells having mitochondrial DNA mutations, resulting in augmented mitochondrial dynamics and metabolic functions. This improved cell function by diminishing ROS emission in the diseased cells. Our findings suggest that mitochondria from donor skeletal muscle can be integrated in both healthy and functionally compromised host cells leading to mitochondrial structural refinement and respiratory boost. This mitochondrial trafficking and bioenergetic reprogramming to maintain and revitalise tissue homeostasis could be a useful therapeutic strategy in treating diseases.

Keywords:  Lysosome; Mitochondrial DNA Defects; Mitochondrial Dynamics; Mitochondrial Transplantation; Oxygen Consumption

DOI:  https://doi.org/10.1152/ajpcell.00212.2023
Methods Mol Biol. 2023 ;2696 169-197

Methods to Activate the NLRP3 Inflammasome.

Benedikt S Saller, Emilia Neuwirt, Olaf Groß.

  The inflammasome-nucleating cytoplasmic sensor protein NLRP3 (NACHT-, LRR, and PYD domains-containing protein 3, also known as NOD-like receptor pyrin domain-containing 3, NALP3, or cryopyrin) is triggered by a broad spectrum of sterile endogenous danger signals and environmental irritants. Upon activation, NLRP3 engages the adapter protein ASC that in turn recruits the third inflammasome component, the protease caspase-1. Subsequent caspase-1 activation leads to its auto-processing and maturation of the leaderless IL-1 family cytokines IL-1β and IL-18 as well as cleavage of the pore-forming protein Gasdermin D (GSDMD). GSDMD plasma membrane pores, formed by its N-terminus, facilitate IL-1 release and, typically, subsequent cell lysis (pyroptosis). This protocol explains standard methods, which are routinely used in our laboratory to study NLRP3 inflammasome biology in vitro. It includes experimental approaches using primary murine bone marrow-derived macrophages (BMDMs) and bone marrow-derived dendritic cells (BMDCs), human peripheral blood mononuclear cells (PBMCs), as well as inflammasome-competent cell lines (HoxB8 and THP-1 cells). The protocol covers the use of a broad spectrum of established NLRP3 activators and outlines the use of common inhibitors blocking NLRP3 itself or its upstream triggering events. We also provide guidelines for experimental set-up and crucial experimental controls to investigate NLRP3 inflammasome signaling or study new activators and inhibitors.

Keywords:  Bone marrow-derived macrophages and dendritic cells; HoxB8; Inflammasome; NLRP3 activators and inhibitors; Peripheral blood mononuclear cells; Priming; THP-1

DOI:  https://doi.org/10.1007/978-1-0716-3350-2_12
Methods Mol Biol. 2023 ;2712 103-115

Monitoring Mitochondria Function in Ferroptosis.

Fangquan Chen, Jiao Liu, Daolin Tang, Rui Kang.

  Ferroptosis is a type of regulated necrosis driven by uncontrolled membrane lipid peroxidation. Mitochondria, which are membrane-bound organelles present in almost all eukaryotic cells and play a central role in energy metabolism and various types of cell death, have a complicated role in ferroptosis. On one hand, mitochondrial-derived iron metabolism and reactive oxygen species (ROS) production may promote ferroptosis. On the other hand, mitochondria also possess a dihydroorotate dehydrogenase (DHODH)-dependent antioxidant system that detoxifies lipid peroxides. This chapter summarizes several methods, such as western blotting, immunofluorescence, cell viability assays, mitochondrial fluorescent probes, adenosine 5'-triphosphate (ATP) assay kits, mitochondrial respiration, and mitophagy tests, that may enable researchers to gain a deeper understanding of the dual role of mitochondria in ferroptosis.

Keywords:  Ferroptosis; Methods; Mitochondria

DOI:  https://doi.org/10.1007/978-1-0716-3433-2_10
J Inflamm Res. 2023 ;16 3365-3379

Resolvin D1 Attenuates Inflammation and Pelvic Pain Associated with EAP by Inhibiting Oxidative Stress and NLRP3 Inflammasome Activation via the Nrf2/HO-1 Pathway.

Jiong Zhang, Juan Chen, Qing Jiang, Rui Feng, Xiaohu Zhao, Haolin Li, Cheng Yang, Xiaoliang Hua.

   Background: Resolvin D1 (RvD1), a member of the specialized pro-resolving lipid mediators family, has a potent anti-inflammatory effect and alleviates tissue damage. The purpose of the current research was to study the effect of RvD1 on CP/CPPS and the underlying mechanisms using a mouse model of experimental autoimmune prostatitis (EAP) mice.
Materials and Methods: The EAP mouse model was successfully established, and was used to test the therapeutic effect of RvD1. Hematoxylin-eosin staining and dihydroethidium staining were used to evaluate the histological changes and oxidative stress levels of prostate tissues. Chronic pelvic pain was assessed by applying von Frey filaments to the lower abdomen. The superoxide dismutase enzyme and malondialdehyde levels were detected using enzyme-linked immunosorbent assay (ELISA). The levels of inflammation-related cytokines, including IL-1β, IL-6, and TNF-α were detected by ELISA.
Results: RvD1 treatment ameliorated prostatic inflammation and the pelvic pain of EAP mice. RvD1 treatment could inhibit activation of the NLRP3 inflammasome and oxidative stress. RvD1 treatment could activate Nrf2/HO-1 signaling in mice with EAP. Blockade of Nrf2/HO-1 signaling abolished the RvD1-mediated inhibition of oxidative stress, NLRP3 inflammasome activation and the anti-inflammatory effect of RvD1 in EAP.
Conclusion: RvD1 treatment can reduce inflammatory cell infiltration in prostate tissue and attenuate pelvic pain associated with EAP by inhibiting oxidative stress and NLRP3 inflammasome activation via the Nrf2/HO-1 pathway. These results provide new insights that RvD1 has the potential as an effective agent in the treatment of EAP.

Keywords:  CP/CPPS; NLRP3 inflammasome; chronic prostatitis and chronic pelvic pain syndrome; inflammation; oxidative stress; resolvin D1

DOI:  https://doi.org/10.2147/JIR.S408111
Methods Mol Biol. 2023 ;2696 1-27

Canonical Inflammasomes.

Vinicius Nunes Cordeiro Leal, Alessandra Pontillo.

  The innate immune response represents the first line of host defense, and it is able to detect pathogen- and damage-associated molecular patterns (PAMPs and DAMPs, respectively) through a variety of pattern recognition receptors (PRRs). Among these PRRs, certain cytosolic receptors of the NLRs family (specifically NLRP1, NLRP3, NLRC4, and NAIP) or those containing at least a pyrin domain (PYD) such as pyrin and AIM2, activate the multimeric complex known as inflammasome, and its effector enzyme caspase-1. The caspase-1 induces the proteolytic maturation of the pro-inflammatory cytokines IL-1ß and IL-18, as well as the pore-forming protein gasdermin D (GSDMD). GSDMD is responsible for the release of the two cytokines and the induction of lytic and inflammatory cell death known as pyroptosis. Each inflammasome receptor detects specific stimuli, either directly or indirectly, thereby enhancing the cell's ability to sense infections or homeostatic disturbances. In this chapter, we present the activation mechanism of the so-called "canonical" inflammasomes.

Keywords:  Canonical activation; Inflammasome; PRRs

DOI:  https://doi.org/10.1007/978-1-0716-3350-2_1
Redox Biol. 2023 Aug 06. pii: S2213-2317(23)00243-4. [Epub ahead of print]65 102842

NOX2 deficiency exacerbates diet-induced obesity and impairs molecular training adaptations in skeletal muscle.

Carlos Henriquez-Olguin, Roberto Meneses-Valdes, Steffen H Raun, Samantha Gallero, Jonas R Knudsen, Zhencheng Li, Jingwen Li, Lykke Sylow, Enrique Jaimovich, Thomas E Jensen.

  The production of reactive oxygen species (ROS) by NADPH oxidase (NOX) 2 has been linked to both insulin resistance and exercise training adaptations in skeletal muscle. This study explores the previously unexamined role of NOX2 in the interplay between diet-induced insulin resistance and exercise training (ET). Using a mouse model that harbors a point mutation in the essential NOX2 regulatory subunit, p47phox (Ncf1*), we investigated the impact of this mutation on various metabolic adaptations. Wild-type (WT) and Ncf1* mice were assigned to three groups: chow diet, 60% energy fat diet (HFD), and HFD with access to running wheels (HFD + E). After a 16-week intervention, a comprehensive phenotypic assessment was performed, including body composition, glucose tolerance, energy intake, muscle insulin signaling, redox-related proteins, and mitochondrial adaptations. The results revealed that NOX2 deficiency exacerbated the impact of HFD on body weight, body composition, and glucose intolerance. Moreover, in Ncf1* mice, ET did not improve glucose tolerance or increase muscle cross-sectional area. ET normalized body fat independently of genotype. The lack of NOX2 activity during ET reduced several metabolic adaptations in skeletal muscle, including insulin signaling and expression of Hexokinase II and oxidative phosphorylation complexes. In conclusion, these findings suggest that NOX2 mediates key beneficial effects of exercise training in the context of diet-induced obesity.

Keywords:  Exercise; Insulin sensitivity; Metabolism

DOI:  https://doi.org/10.1016/j.redox.2023.102842
Mol Cell Biochem. 2023 Aug 17.

Mitochondria and NLRP3 inflammasome in cardiac hypertrophy.

Ruyu Yan, Yuxin Sun, Yifan Yang, Rongchao Zhang, Yujiao Jiang, Yan Meng.

  Cardiac hypertrophy is the main adaptive response of the heart to chronic loads; however, prolonged or excessive hypertrophy promotes myocardial interstitial fibrosis, systolic dysfunction, and cardiomyocyte death, especially aseptic inflammation mediated by NLRP3 inflammasome, which can aggravate ventricular remodeling and myocardial damage, which is an important mechanism for the progression of heart failure. Various cardiac overloads can cause mitochondrial damage. In recent years, the mitochondria have been demonstrated to be involved in the inflammatory response during the development of cardiac hypertrophy in vitro and in vivo. As the NLRP3 inflammasome and mitochondria are regulators of inflammation and cardiac hypertrophy, we explored the potential functions of the NLRP3 inflammasome and mitochondrial dysfunction in cardiac hypertrophy. In particular, we proposed that the induction of mitochondrial dysfunction in cardiomyocytes may promote NLRP3-dependent inflammation during myocardial hypertrophy. Further in-depth studies could prompt valuable discoveries regarding the underlying molecular mechanisms of cardiac hypertrophy, reveal novel anti-inflammatory therapies for cardiac hypertrophy, and provide more desirable therapeutic outcomes for patients with cardiac hypertrophy.

Keywords:  Cardiac hypertrophy; Inflammation; Mitochondrial dysfunction; NLRP3 inflammasome

DOI:  https://doi.org/10.1007/s11010-023-04812-1
Curr Opin Struct Biol. 2023 Aug 10. pii: S0959-440X(23)00137-9. [Epub ahead of print]82 102663

Structural mechanisms of the mTOR pathway.

Karen Y Linde-Garelli, Kacper B Rogala.

The mTOR signaling pathway is essential for regulating cell growth and mammalian metabolism. The mTOR kinase forms two complexes, mTORC1 and mTORC2, which respond to external stimuli and regulate differential downstream targets. Cellular membrane-associated translocation mediates function and assembly of the mTOR complexes, and recent structural studies have begun uncovering the molecular basis by which the mTOR pathway (1) regulates signaling inputs, (2) recruits substrates, (3) localizes to biological membranes, and (4) becomes activated. Moreover, indications of dysregulated mTOR signaling are implicated in a wide range of diseases and an increasingly comprehensive understanding of structural mechanisms is driving novel translational development.

DOI: https://doi.org/10.1016/j.sbi.2023.102663
Phys Act Nutr. 2023 Jun;27(2): 78-95

Resistance exercise: a mighty tool that adapts, destroys, rebuilds and modulates the molecular and structural environment of skeletal muscle.

Käthe Bersiner, So-Young Park, Kirill Schaaf, Woo-Hwi Yang, Christian Theis, Daniel Jacko, Sebastian Gehlert.

   PURPOSE: Skeletal muscle regulates health and performance by maintaining or increasing strength and muscle mass. Although the molecular mechanisms in response to resistance exercise (RE) significantly target the activation of protein synthesis, a plethora of other mechanisms and structures must be involved in orchestrating the communication, repair, and restoration of homeostasis after RE stimulation. In practice, RE can be modulated by variations in intensity, continuity and volume, which affect molecular responses and skeletal muscle adaptation. Knowledge of these aspects is important with respect to planning of training programs and assessing the impact of RE training on skeletal muscle.
METHODS: In this narrative review, we introduce general aspects of skeletal muscle substructures that adapt in response to RE. We further highlighted the molecular mechanisms that control human skeletal muscle anabolism, degradation, repair and memory in response to acute and repeated RE and linked these aspects to major training variables.
RESULTS: Although RE is a key stimulus for the activation of skeletal muscle anabolism, it also induces myofibrillar damage. Nevertheless, to increase muscle mass accompanied by a corresponding adaptation of the essential substructures of the sarcomeric environment, RE must be continuously repeated. This requires the permanent engagement of molecular mechanisms that re-establish skeletal muscle integrity after each RE-induced muscle damage.
CONCLUSION: Various molecular regulators coordinately control the adaptation of skeletal muscle after acute and repeated RE and expand their actions far beyond muscle growth. Variations of key resistance training variables likely affect these mechanisms without affecting muscle growth.

Keywords:  adaptation; hypertrophy; mTOR signaling; muscle damage; proteostasis; resistance exercise; skeletal muscle

DOI:  https://doi.org/10.20463/pan.2023.0021