Corrigendum: Cysteine residues in signal transduction and its relevance in pancreatic beta cells.

[This corrects the article DOI: 10.3389/fendo.2023.1221520.].

NADPH oxidase 4 in mouse ß cells participates in inflammation on chronic nutrient overload.

OBJECTIVE: By exposing mice carrying a deletion of NADPH oxidase isoform 4, NOX4, specifically in pancreatic ß cells (ßNOX4-/-) to nutrient excess stimulated by a high-fat diet (HFD), this study aimed to elucidate the role of ß-cell redox status in the development of meta-inflammation within the diabetic phenotype. METHODS: The authors performed basic phenotyping of ßNOX4-/- mice on HFD involving insulin and glycemic analyses, histochemistry of adipocytes, indirect calorimetry, and cytokine analyses. To characterize local inflammation, the study used caspase-1 activity assay, interleukin-1ß immunochemistry, and real-time polymerase chain reaction during coculturing of ß cells with macrophages. RESULTS: The phenotype of ßNOX4-/- mice on HFD was not associated with hyperinsulinemia and hyperglycemia but showed accumulation of excessive lipids in epididymal fat and ß cells. Surprisingly, mice showed significantly reduced systemic inflammation. Decreased interleukin-1ß protein levels and downregulated NLRP3-inflammasome activity were observed on chronic glucose overload in ßNOX4-/- isolated islets and NOX4-silenced INS1-E cells resulting in attenuated proinflammatory polarization of macrophages/monocytes in vitro and in situ and reduced local islet inflammation. CONCLUSIONS: Experimental evidence suggests that NOX4 pro-oxidant activity in ß cells is involved in NLRP3-inflammasome activation during chronic nutrient overload and participates in local inflammatory signaling and perhaps toward peripheral tissues, contributing to a diabetic inflammatory phenotype.

The role of m6A and m6Am RNA modifications in the pathogenesis of diabetes mellitus.

The rapidly developing research field of epitranscriptomics has recently emerged into the spotlight of researchers due to its vast regulatory effects on gene expression and thereby cellular physiology and pathophysiology. N6-methyladenosine (m6A) and N6,2'-O-dimethyladenosine (m6Am) are among the most prevalent and well-characterized modified nucleosides in eukaryotic RNA. Both of these modifications are dynamically regulated by a complex set of epitranscriptomic regulators called writers, readers, and erasers. Altered levels of m6A and also several regulatory proteins were already associated with diabetic tissues. This review summarizes the current knowledge and gaps about m6A and m6Am modifications and their respective regulators in the pathophysiology of diabetes mellitus. It focuses mainly on the more prevalent type 2 diabetes mellitus (T2DM) and its treatment by metformin, the first-line antidiabetic agent. A better understanding of epitranscriptomic modifications in this highly prevalent disease deserves further investigation and might reveal clinically relevant discoveries in the future.

Microenvironmental regulation of T-cells in pulmonary hypertension.

Introduction: In pulmonary hypertension (PH), pulmonary arterial remodeling is often accompanied by perivascular inflammation. The inflammation is characterized by the accumulation of activated macrophages and lymphocytes within the adventitial stroma, which is comprised primarily of fibroblasts. The well-known ability of fibroblasts to secrete interleukins and chemokines has previously been implicated as contributing to this tissue-specific inflammation in PH vessels. We were interested if pulmonary fibroblasts from PH arteries contribute to microenvironmental changes that could activate and polarize T-cells in PH. Methods: We used single-cell RNA sequencing of intact bovine distal pulmonary arteries (dPAs) from PH and control animals and flow cytometry, mRNA expression analysis, and respirometry analysis of blood-derived bovine/human T-cells exposed to conditioned media obtained from pulmonary fibroblasts of PH/control animals and IPAH/control patients (CM-(h)PH Fibs vs CM-(h)CO Fibs). Results: Single-cell RNA sequencing of intact bovine dPAs from PH and control animals revealed a pro-inflammatory phenotype of CD4+ T-cells and simultaneous absence of regulatory T-cells (FoxP3+ Tregs). By exposing T-cells to CM-(h)PH Fibs we stimulated their proinflammatory differentiation documented by increased IFNγ and decreased IL4, IL10, and TGFß mRNA and protein expression. Interestingly, we demonstrated a reduction in the number of suppressive T-cell subsets, i.e., human/bovine Tregs and bovine γδ T-cells treated with CM-(h)PH-Fibs. We also noted inhibition of anti-inflammatory cytokine expression (IL10, TGFß, IL4). Pro-inflammatory polarization of bovine T-cells exposed to CM-PH Fibs correlated with metabolic shift to glycolysis and lactate production with increased prooxidant intracellular status as well as increased proliferation of T-cells. To determine whether metabolic reprogramming of PH-Fibs was directly contributing to the effects of PH-Fibs conditioned media on T-cell polarization, we treated PH-Fibs with the HDAC inhibitor SAHA, which was previously shown to normalize metabolic status and examined the effects of the conditioned media. We observed significant suppression of inflammatory polarization associated with decreased T-cell proliferation and recovery of mitochondrial energy metabolism. Conclusion: This study demonstrates how the pulmonary fibroblast-derived microenvironment can activate and differentiate T-cells to trigger local inflammation, which is part of the vascular wall remodeling process in PH.

Cysteine residues in signal transduction and its relevance in pancreatic beta cells.

Cysteine is one of the least abundant but most conserved amino acid residues in proteins, playing a role in their structure, metal binding, catalysis, and redox chemistry. Thiols present in cysteines can be modified by post-translational modifications like sulfenylation, acylation, or glutathionylation, regulating protein activity and function and serving as signals. Their modification depends on their position in the structure, surrounding amino acids, solvent accessibility, pH, etc. The most studied modifications are the redox modifications by reactive oxygen, nitrogen, and sulfur species, leading to reversible changes that serve as cell signals or irreversible changes indicating oxidative stress and cell damage. Selected antioxidants undergoing reversible oxidative modifications like peroxiredoxin-thioredoxin system are involved in a redox-relay signaling that can propagate to target proteins. Cysteine thiols can also be modified by acyl moieties' addition (derived from lipid metabolism), resulting in protein functional modification or changes in protein anchoring in the membrane. In this review, we update the current knowledge on cysteine modifications and their consequences in pancreatic ß-cells. Because ß-cells exhibit well-balanced redox homeostasis, the redox modifications of cysteines here serve primarily for signaling purposes. Similarly, lipid metabolism provides regulatory intermediates that have been shown to be necessary in addition to redox modifications for proper ß-cell function and, in particular, for efficient insulin secretion. On the contrary, the excess of reactive oxygen, nitrogen, and sulfur species and the imbalance of lipids under pathological conditions cause irreversible changes and contribute to oxidative stress leading to cell failure and the development of type 2 diabetes.

SIRT3 Is a Critical Regulator of Mitochondrial Function of Fibroblasts in Pulmonary Hypertension.

Pulmonary hypertension (PH) is a heterogeneous and life-threatening cardiopulmonary disorder in which mitochondrial dysfunction is believed to drive pathogenesis, although the underlying mechanisms remain unclear. To determine if abnormal SIRT3 (sirtuin 3) activity is related to mitochondrial dysfunction in adventitial fibroblasts from patients with idiopathic pulmonary arterial hypertension (IPAH) and hypoxic PH calves (PH-Fibs) and whether SIRT3 could be a potential therapeutic target to improve mitochondrial function, SIRT3 concentrations in control fibroblasts, PH-Fibs, and lung tissues were determined using quantitative real-time PCR and western blot. SIRT3 deacetylase activity in cells and lung tissues was determined using western blot, immunohistochemistry staining, and immunoprecipitation. Glycolysis and mitochondrial function in fibroblasts were measured using respiratory analysis and fluorescence-lifetime imaging microscopy. The effects of restoring SIRT3 activity (by overexpression of SIRT3 with plasmid, activation SIRT3 with honokiol, and supplementation with the SIRT3 cofactor nicotinamide adenine dinucleotide [NAD+]) on mitochondrial protein acetylation, mitochondrial function, cell proliferation, and gene expression in PH-Fibs were also investigated. We found that SIRT3 concentrations were decreased in PH-Fibs and PH lung tissues, and its cofactor, NAD+, was also decreased in PH-Fibs. Increased acetylation in overall mitochondrial proteins and SIRT3-specific targets (MPC1 [mitochondrial pyruvate carrier 1] and MnSOD2 [mitochondrial superoxide dismutase]), as well as decreased MnSOD2 activity, was identified in PH-Fibs and PH lung tissues. Normalization of SIRT3 activity, by increasing its expression with plasmid or with honokiol and supplementation with its cofactor NAD+, reduced mitochondrial protein acetylation, improved mitochondrial function, inhibited proliferation, and induced apoptosis in PH-Fibs. Thus, our study demonstrated that restoration of SIRT3 activity in PH-Fibs can reduce mitochondrial protein acetylation and restore mitochondrial function and PH-Fib phenotype in PH.

Contribution of Mitochondria to Insulin Secretion by Various Secretagogues.

Significance: Mitochondria determine glucose-stimulated insulin secretion (GSIS) in pancreatic ß-cells by elevating ATP synthesis. As the metabolic and redox hub, mitochondria provide numerous links to the plasma membrane channels, insulin granule vesicles (IGVs), cell redox, NADH, NADPH, and Ca2+ homeostasis, all affecting insulin secretion. Recent Advances: Mitochondrial redox signaling was implicated in several modes of insulin secretion (branched-chain ketoacid [BCKA]-, fatty acid [FA]-stimulated). Mitochondrial Ca2+ influx was found to enhance GSIS, reflecting cytosolic Ca2+ oscillations induced by action potential spikes (intermittent opening of voltage-dependent Ca2+ and K+ channels) or the superimposed Ca2+ release from the endoplasmic reticulum (ER). The ATPase inhibitory factor 1 (IF1) was reported to tune the glucose sensitivity range for GSIS. Mitochondrial protein kinase A was implicated in preventing the IF1-mediated inhibition of the ATP synthase. Critical Issues: It is unknown how the redox signal spreads up to the plasma membrane and what its targets are, what the differences in metabolic, redox, NADH/NADPH, and Ca2+ signaling, and homeostasis are between the first and second GSIS phase, and whether mitochondria can replace ER in the amplification of IGV exocytosis. Future Directions: Metabolomics studies performed to distinguish between the mitochondrial matrix and cytosolic metabolites will elucidate further details. Identifying the targets of cell signaling into mitochondria and of mitochondrial retrograde metabolic and redox signals to the cell will uncover further molecular mechanisms for insulin secretion stimulated by glucose, BCKAs, and FAs, and the amplification of secretion by glucagon-like peptide (GLP-1) and metabotropic receptors. They will identify the distinction between the hub ß-cells and their followers in intact and diabetic states. Antioxid. Redox Signal. 36, 920-952.

Redox Homeostasis in Pancreatic ß-Cells: From Development to Failure.

Redox status is a key determinant in the fate of ß-cell. These cells are not primarily detoxifying and thus do not possess extensive antioxidant defense machinery. However, they show a wide range of redox regulating proteins, such as peroxiredoxins, thioredoxins or thioredoxin reductases, etc., being functionally compartmentalized within the cells. They keep fragile redox homeostasis and serve as messengers and amplifiers of redox signaling. ß-cells require proper redox signaling already in cell ontogenesis during the development of mature ß-cells from their progenitors. We bring details about redox-regulated signaling pathways and transcription factors being essential for proper differentiation and maturation of functional ß-cells and their proliferation and insulin expression/maturation. We briefly highlight the targets of redox signaling in the insulin secretory pathway and focus more on possible targets of extracellular redox signaling through secreted thioredoxin1 and thioredoxin reductase1. Tuned redox homeostasis can switch upon chronic pathological insults towards the dysfunction of ß-cells and to glucose intolerance. These are characteristics of type 2 diabetes, which is often linked to chronic nutritional overload being nowadays a pandemic feature of lifestyle. Overcharged ß-cell metabolism causes pressure on proteostasis in the endoplasmic reticulum, mainly due to increased demand on insulin synthesis, which establishes unfolded protein response and insulin misfolding along with excessive hydrogen peroxide production. This together with redox dysbalance in cytoplasm and mitochondria due to enhanced nutritional pressure impact ß-cell redox homeostasis and establish prooxidative metabolism. This can further affect ß-cell communication in pancreatic islets through gap junctions. In parallel, peripheral tissues losing insulin sensitivity and overall impairment of glucose tolerance and gut microbiota establish local proinflammatory signaling and later systemic metainflammation, i.e., low chronic inflammation prooxidative properties, which target ß-cells leading to their dedifferentiation, dysfunction and eventually cell death.

The Pancreatic ß-Cell: The Perfect Redox System.

Pancreatic ß-cell insulin secretion, which responds to various secretagogues and hormonal regulations, is reviewed here, emphasizing the fundamental redox signaling by NADPH oxidase 4- (NOX4-) mediated H2O2 production for glucose-stimulated insulin secretion (GSIS). There is a logical summation that integrates both metabolic plus redox homeostasis because the ATP-sensitive K+ channel (KATP) can only be closed when both ATP and H2O2 are elevated. Otherwise ATP would block KATP, while H2O2 would activate any of the redox-sensitive nonspecific calcium channels (NSCCs), such as TRPM2. Notably, a 100%-closed KATP ensemble is insufficient to reach the -50 mV threshold plasma membrane depolarization required for the activation of voltage-dependent Ca2+ channels. Open synergic NSCCs or Cl- channels have to act simultaneously to reach this threshold. The resulting intermittent cytosolic Ca2+-increases lead to the pulsatile exocytosis of insulin granule vesicles (IGVs). The incretin (e.g., GLP-1) amplification of GSIS stems from receptor signaling leading to activating the phosphorylation of TRPM channels and effects on other channels to intensify integral Ca2+-influx (fortified by endoplasmic reticulum Ca2+). ATP plus H2O2 are also required for branched-chain ketoacids (BCKAs); and partly for fatty acids (FAs) to secrete insulin, while BCKA or FA ß-oxidation provide redox signaling from mitochondria, which proceeds by H2O2 diffusion or hypothetical SH relay via peroxiredoxin "redox kiss" to target proteins.

Chronic n-3 fatty acid intake enhances insulin response to oral glucose and elevates GLP-1 in high-fat diet-fed obese mice.

n-3 polyunsaturated fatty acids (PUFA) can exert beneficial effects on glucose homeostasis, especially in obese rodents. Gut incretin hormones regulate glucose and lipid homeostasis, but their involvement in the above effects is not entirely clear. This study aims to assess the effects of chronic n-3 PUFA administration on the insulin and incretin responses in C57BL/6N obese male mice subjected to oral glucose tolerance test (oGTT) after 8 weeks of feeding a corn-oil-based high-fat diet (cHF). The weight gain and adiposity were partially reduced in mice fed cHF in which some of the corn oil was replaced with n-3 PUFA concentrate containing ∼60% DHA and EPA in a 3 : 1 ratio. In addition, these mice had improved glucose tolerance, which was consistent with an increased insulin response to oral glucose and plasma glucagon-like peptide-1 (GLP-1) levels. While the stimulatory effects of n-3 PUFA on GLP-1 levels could not be attributed to changes in intestinal or plasma dipeptidyl peptidase-4 activity, their beneficial effects on glucose tolerance were abolished when mice were pretreated with the GLP-1 receptor antagonist exendin 9-39. Moreover, chronic n-3 PUFA intake prevented the detrimental effects of cHF feeding on glucose-stimulated insulin secretion in the pancreatic islets. Collectively, our data suggest that n-3 PUFA may modulate postprandial glucose metabolism in obese mice through a GLP-1-based mechanism. The significance of these findings in terms of the effective DHA and EPA ratio of the n-3 PUFA concentrate as well as the effect of n-3 PUFA in humans requires further research.

Mitochondrial Superoxide Production Decreases on Glucose-Stimulated Insulin Secretion in Pancreatic ß Cells Due to Decreasing Mitochondrial Matrix NADH/NAD+ Ratio.

Aims: Glucose-stimulated insulin secretion (GSIS) in pancreatic ß cells was expected to enhance mitochondrial superoxide formation. Hence, we elucidated relevant redox equilibria. Results: Unexpectedly, INS-1E cells at transitions from 3 (11 mM; pancreatic islets from 5 mM) to 25 mM glucose decreased matrix superoxide release rates (MitoSOX Red monitoring validated by MitoB) and H2O2 (mitoHyPer, subtracting mitoSypHer emission). Novel double-channel fluorescence lifetime imaging, approximating free mitochondrial matrix NADHF, indicated its ∼20% decrease. Matrix NAD+F increased on GSIS, indicated by the FAD-emission lifetime decrease, reflecting higher quenching of FAD by NAD+F. The participation of pyruvate/malate and pyruvate/citrate redox shuttles, elevating cytosolic NADPHF (iNAP1 fluorescence monitoring) at the expense of matrix NADHF, was indicated, using citrate (2-oxoglutarate) carrier inhibitors and cytosolic malic enzyme silencing: All changes vanished on these manipulations. 13C-incorporation from 13C-L-glutamine into 13C-citrate reflected the pyruvate/isocitrate shuttle. Matrix NADPHF (iNAP3 monitored) decreased. With decreasing glucose, the suppressor of Complex III site Q electron leak (S3QEL) suppressor caused a higher Complex I IF site contribution, but a lower superoxide fraction ascribed to the Complex III site IIIQo. Thus, the diminished matrix NADHF/NAD+F decreased Complex I flavin site IF superoxide formation on GSIS. Innovation: Mutually validated methods showed decreasing superoxide release into the mitochondrial matrix in pancreatic ß cells on GSIS, due to the decreasing matrix NADHF/NAD+F (NADPHF/NADP+F) at increasing cytosolic NADPHF levels. The developed innovative methods enable real-time NADH/NAD+ and NADPH/NADP+ monitoring in any distinct cell compartment. Conclusion: The export of reducing equivalents from mitochondria adjusts lower mitochondrial superoxide production on GSIS, but it does not prevent oxidative stress in pancreatic ß cells.

Glucose-Stimulated Insulin Secretion Fundamentally Requires H2O2 Signaling by NADPH Oxidase 4.

NADPH facilitates glucose-stimulated insulin secretion (GSIS) in pancreatic islets (PIs) of ß-cells through an as yet unknown mechanism. We found NADPH oxidase isoform 4 (NOX4) to be the main producer of cytosolic H2O2, which is essential for GSIS; an increase in ATP alone was insufficient for GSIS. The fast GSIS phase was absent from PIs from NOX4-null, ß-cell-specific knockout mice (NOX4ßKO) (though not from NOX2 knockout mice) and from NOX4-silenced or catalase-overexpressing INS-1E cells. Lentiviral NOX4 overexpression or H2O2 rescued GSIS in PIs from NOX4ßKO mice. NOX4 silencing suppressed Ca2+ oscillations, and the patch-clamped KATP channel opened more frequently when glucose was high. Mitochondrial H2O2, decreasing upon GSIS, provided alternative redox signaling when 2-oxo-isocaproate or fatty acid oxidation formed superoxides through electron-transfer flavoprotein:Q-oxidoreductase. Unlike GSIS, such insulin secretion was blocked with mitochondrial antioxidant SkQ1. Both NOX4 knockout and NOX4ßKO mice exhibited impaired glucose tolerance and peripheral insulin resistance. Thus, the redox signaling previously suggested to cause ß-cells to self-check hypothetically induces insulin resistance when it is absent. In conclusion, increases in ATP and H2O2 constitute an essential signal that switches on insulin exocytosis for glucose and branched-chain oxoacids as secretagogues (it does so partially for fatty acids). Redox signaling could be impaired by cytosolic antioxidants; hence, those targeting mitochondria should be preferred for clinical applications to treat (pre)diabetes at any stage.

Redox Signaling from Mitochondria: Signal Propagation and Its Targets.

Progress in mass spectroscopy of posttranslational oxidative modifications has enabled researchers to experimentally verify the concept of redox signaling. We focus here on redox signaling originating from mitochondria under physiological situations, discussing mechanisms of transient redox burst in mitochondria, as well as the possible ways to transfer such redox signals to specific extramitochondrial targets. A role of peroxiredoxins is described which enables redox relay to other targets. Examples of mitochondrial redox signaling are discussed: initiation of hypoxia-inducible factor (HIF) responses; retrograde redox signaling to PGC1α during exercise in skeletal muscle; redox signaling in innate immune cells; redox stimulation of insulin secretion, and other physiological situations.

Mitochondrial cristae narrowing upon higher 2-oxoglutarate load.

Hypoxia causes mitochondrial cristae widening, enabled by the ~20% degradation of Mic60/mitofilin, with concomitant clustering of the MICOS complex, reflecting the widening of crista junctions (outlets) (Plecitá-Hlavatá et al. FASEB J., 2016 30:1941-1957). Attempting to accelerate metabolism by the addition of membrane-permeant dimethyl-2-oxoglutarate (dm2OG) to HepG2 cells pre-adapted to hypoxia, we found cristae narrowing by transmission electron microscopy. Glycolytic HepG2 cells, which downregulate hypoxic respiration, instantly increased respiration with dm2OG. Changes in intracristal space (ICS) morphology were also revealed by 3D super-resolution microscopy using Eos-conjugated ICS-located lactamase-ß. Cristae topology was resolved in detail by focused-ion beam/scanning electron microscopy (FIB/SEM). The spatial relocations of key cristae-shaping proteins were indicated by immunocytochemical stochastic 3D super-resolution microscopy (dSTORM), while analyzing inter-antibody-distance histograms: i) ATP-synthase dimers exhibited a higher fraction of shorter inter-distances between bound F1-α primary Alexa-Fluor-647-conjugated antibodies, indicating cristae narrowing. ii) Mic60/mitofilin clusters (established upon hypoxia) decayed, restoring isotropic random Mic60/mitofilin distribution (a signature of normoxia). iii) outer membrane SAMM50 formed more focused clusters. Less abundant fractions of higher ATP-synthase oligomers of hypoxic samples on blue-native electrophoresis became more abundant fractions at the high dm2OG load and at normoxia. This indicates more labile ATP-synthase dimeric rows established at crista rims upon hypoxia, strengthened at normoxia or dm2OG-substrate load. Hypothetically, the increased Krebs substrate load stimulates the cross-linking/strengthening of rows of ATP-synthase dimers at the crista rims, making them sharper. Crista narrowing ensures a more efficient coupling of proton pumping to ATP synthesis. We demonstrated that cristae morphology changes even within minutes.

Potential of Mitochondria-Targeted Antioxidants to Prevent Oxidative Stress in Pancreatic ß-cells.

Pancreatic ß-cells are vulnerable to oxidative stress due to their low content of redox buffers, such as glutathione, but possess a rich content of thioredoxin, peroxiredoxin, and other proteins capable of redox relay, transferring redox signaling. Consequently, it may be predicted that cytosolic antioxidants could interfere with the cytosolic redox signaling and should not be recommended for any potential therapy. In contrast, mitochondrial matrix-targeted antioxidants could prevent the primary oxidative stress arising from the primary superoxide sources within the mitochondrial matrix, such as at the flavin (IF) and ubiquinone (IQ) sites of superoxide formation within respiratory chain complex I and the outer ubiquinone site (IIIQ) of complex III. Therefore, using time-resolved confocal fluorescence monitoring with MitoSOX Red, we investigated various effects of mitochondria-targeted antioxidants in model pancreatic ß-cells (insulinoma INS-1E cells) and pancreatic islets. Both SkQ1 (a mitochondria-targeted plastoquinone) and a suppressor of complex III site Q electron leak (S3QEL) prevented superoxide production released to the mitochondrial matrix in INS-1E cells with stimulatory glucose, where SkQ1 also exhibited an antioxidant role for UCP2-silenced cells. SkQ1 acted similarly at nonstimulatory glucose but not in UCP2-silenced cells. Thus, UCP2 can facilitate the antioxidant mechanism based on SkQ1+ fatty acid anion- pairing. The elevated superoxide formation induced by antimycin A was largely prevented by S3QEL, and that induced by rotenone was decreased by SkQ1 and S3QEL and slightly by S1QEL, acting at complex I site Q. Similar results were obtained with the MitoB probe, for the LC-MS-based assessment of the 4 hr accumulation of reactive oxygen species within the mitochondrial matrix but for isolated pancreatic islets. For 2 hr INS-1E incubations, some samples were influenced by the cell death during the experiment. Due to the frequent dependency of antioxidant effects on metabolic modes, we suggest a potential use of mitochondria-targeted antioxidants for the treatment of prediabetic states after cautious nutrition-controlled tests. Their targeted delivery might eventually attenuate the vicious spiral leading to type 2 diabetes.

Contribution of Oxidative Stress and Impaired Biogenesis of Pancreatic ß-Cells to Type 2 Diabetes.

Significance: Type 2 diabetes development involves multiple changes in ß-cells, related to the oxidative stress and impaired redox signaling, beginning frequently by sustained overfeeding due to the resulting lipotoxicity and glucotoxicity. Uncovering relationships among the dysregulated metabolism, impaired ß-cell "well-being," biogenesis, or cross talk with peripheral insulin resistance is required for elucidation of type 2 diabetes etiology. Recent Advances: It has been recognized that the oxidative stress, lipotoxicity, and glucotoxicity cannot be separated from numerous other cell pathology events, such as the attempted compensation of ß-cell for the increased insulin demand and dynamics of ß-cell biogenesis and its "reversal" at dedifferentiation, that is, from the concomitantly decreasing islet ß-cell mass (also due to transdifferentiation) and low-grade islet or systemic inflammation. Critical Issues: At prediabetes, the compensation responses of ß-cells, attempting to delay the pathology progression-when exaggerated-set a new state, in which a self-checking redox signaling related to the expression of Ins gene expression is impaired. The resulting altered redox signaling, diminished insulin secretion responses to various secretagogues including glucose, may lead to excretion of cytokines or chemokines by ß-cells or excretion of endosomes. They could substantiate putative stress signals to the periphery. Subsequent changes and lasting glucolipotoxicity promote islet inflammatory responses and further pathology spiral. Future Directions: Should bring an understanding of the ß-cell self-checking and related redox signaling, including the putative stress signal to periphery. Strategies to cure or prevent type 2 diabetes could be based on the substitution of the "wrong" signal by the "correct" self-checking signal.

Aglycemic HepG2 Cells Switch From Aminotransferase Glutaminolytic Pathway of Pyruvate Utilization to Complete Krebs Cycle at Hypoxia.

Human hepatocellular carcinoma HepG2 cells are forced to oxidative phosphorylation (OXPHOS), when cultured in aglycemic conditions at galactose and glutamine. These Oxphos cells represent a prototype of cancer cell bioenergetics with mixed aerobic glycolysis and OXPHOS. We aimed to determine fractions of (i) glutaminolytic pathway involving aminotransferase reaction supplying 2-oxoglutarate (2OG) to the Krebs cycle vs. (ii) active segment of the Krebs cycle with aconitase and isocitrate dehydrogenase-3 (ACO-IDH3), which is typically inactive in cancer cells due to the citrate export from mitochondria. At normoxia, Oxphos cell respiration was decreased down to ~15 and ~10% by the aminotransferase inhibitor aminooxyacetate (AOA) or with AOA plus the glutamate-dehydrogenase inhibitor bithionol, respectively. Phosphorylating to non-phosphorylating respiration ratios dropped from >6.5 to 1.9 with AOA and to zero with AOA plus bithionol. Thus, normoxic Oxphos HepG2 cells rely predominantly on glutaminolysis. Addition of membrane-permeant dimethyl-2-oxoglutarate (dm2OG) to inhibited cells instantly partially restored respiration, evidencing the lack of 2OG-dehydrogenase substrate upon aminotransferase inhibition. Surprisingly, after 72 hr of 5% O2 hypoxia, the AOA (bithionol) inhibition ceased and respiration was completely restored. Thus in aglycemic HepG2 cells, the hypoxia-induced factor (HIF) upregulation of glycolytic enzymes enabled acceleration of glycolysis pathway, preceded by galactolysis (Leloir pathway), redirecting pyruvate via still incompletely blocked pyruvate dehydrogenase toward the ACO-IDH3. Glycolytic flux upregulation at hypoxia was evidently matched by a higher activity of the Leloir pathway in Oxphos cells. Hypoxic Oxphos cells increased 2-fold the NADPH oxidase activity, whereas hypoxic glycolytic cells decreased it. Oxphos cells and glycolytic cells at 5 mM glucose decreased their reduced glutathione fraction. In contrast to aglycemic cells, glycolytic HepG2 cells decreased their respiration at hypoxia despite the dm2OG presence, i.e., even at unlimited respiratory substrate availability for 72 hr at 5% O2, exhibiting the canonical HIF-mediated adaptation. Nevertheless, their ATP content was much higher with dm2OG as compared to its absence during hypoxic adaptation. Thus, the metabolic plasticity of cancer cells is illustrated under conditions frequently established for solid tumors in vivo, such as aglycemia plus hypoxia. Consequently, a wide acceptance of the irreversible and exclusive Warburg phenotype in cancer cells is incorrect.

Fatty Acid-Stimulated Insulin Secretion vs. Lipotoxicity.

Fatty acid (FA)-stimulated insulin secretion (FASIS) is reviewed here in contrast to type 2 diabetes etiology, resulting from FA overload, oxidative stress, intermediate hyperinsulinemia, and inflammation, all converging into insulin resistance. Focusing on pancreatic islet ß-cells, we compare the physiological FA roles with the pathological ones. Considering FAs not as mere amplifiers of glucose-stimulated insulin secretion (GSIS), but as parallel insulin granule exocytosis inductors, partly independent of the KATP channel closure, we describe the FA initiating roles in the prediabetic state that is induced by retardations in the glycerol-3-phosphate (glucose)-promoted glycerol/FA cycle and by the impaired GPR40/FFA1 (free FA1) receptor pathway, specifically in its amplification by the redox-activated mitochondrial phospholipase, iPLA2γ. Also, excessive dietary FAs stimulate intestine enterocyte incretin secretion, further elevating GSIS, even at low glucose levels, thus contributing to diabetic hyperinsulinemia. With overnutrition and obesity, the FA overload causes impaired GSIS by metabolic dysbalance, paralleled by oxidative and metabolic stress, endoplasmic reticulum stress and numerous pro-apoptotic signaling, all leading to decreased ß-cell survival. Lipotoxicity is exerted by saturated FAs, whereas ω-3 polyunsaturated FAs frequently exert antilipotoxic effects. FA-facilitated inflammation upon the recruitment of excess M1 macrophages into islets (over resolving M2 type), amplified by cytokine and chemokine secretion by ß-cells, leads to an inevitable failure of pancreatic ß-cells.

Hallmarks of Pulmonary Hypertension: Mesenchymal and Inflammatory Cell Metabolic Reprogramming.

SIGNIFICANCE: The molecular events that promote the development of pulmonary hypertension (PH) are complex and incompletely understood. The complex interplay between the pulmonary vasculature and its immediate microenvironment involving cells of immune system (i.e., macrophages) promotes a persistent inflammatory state, pathological angiogenesis, and fibrosis that are driven by metabolic reprogramming of mesenchymal and immune cells. Recent Advancements: Consistent with previous findings in the field of cancer metabolism, increased glycolytic rates, incomplete glucose and glutamine oxidation to support anabolism and anaplerosis, altered lipid synthesis/oxidation ratios, increased one-carbon metabolism, and activation of the pentose phosphate pathway to support nucleoside synthesis are but some of the key metabolic signatures of vascular cells in PH. In addition, metabolic reprogramming of macrophages is observed in PH and is characterized by distinct features, such as the induction of specific activation or polarization states that enable their participation in the vascular remodeling process. CRITICAL ISSUES: Accumulation of reducing equivalents, such as NAD(P)H in PH cells, also contributes to their altered phenotype both directly and indirectly by regulating the activity of the transcriptional co-repressor C-terminal-binding protein 1 to control the proliferative/inflammatory gene expression in resident and immune cells. Further, similar to the role of anomalous metabolism in mitochondria in cancer, in PH short-term hypoxia-dependent and long-term hypoxia-independent alterations of mitochondrial activity, in the absence of genetic mutation of key mitochondrial enzymes, have been observed and explored as potential therapeutic targets. FUTURE DIRECTIONS: For the foreseeable future, short- and long-term metabolic reprogramming will become a candidate druggable target in the treatment of PH. Antioxid. Redox Signal. 28, 230-250.

Metabolic Reprogramming and Redox Signaling in Pulmonary Hypertension.

Pulmonary hypertension is a complex disease of the pulmonary vasculature, which in severe cases terminates in right heart failure. Complex remodeling of pulmonary arteries comprises the central issue of its pathology. This includes extensive proliferation, apoptotic resistance and inflammation. As such, the molecular and cellular features of pulmonary hypertension resemble hallmark characteristics of cancer cell behavior. The vascular remodeling derives from significant metabolic changes in resident cells, which we describe in detail. It affects not only cells of pulmonary artery wall, but also its immediate microenvironment involving cells of immune system (i.e., macrophages). Thus aberrant metabolism constitutes principle component of the cancer-like theory of pulmonary hypertension. The metabolic changes in pulmonary artery cells resemble the cancer associated Warburg effect, involving incomplete glucose oxidation through aerobic glycolysis with depressed mitochondrial catabolism enabling the fueling of anabolic reactions with amino acids, nucleotides and lipids to sustain proliferation. Macrophages also undergo overlapping but distinct metabolic reprogramming inducing specific activation or polarization states that enable their participation in the vascular remodeling process. Such metabolic synergy drives chronic inflammation further contributing to remodeling. Enhanced glycolytic flux together with suppressed mitochondrial bioenergetics promotes the accumulation of reducing equivalents, NAD(P)H. We discuss the enzymes and reactions involved. The reducing equivalents modulate the regulation of proteins using NAD(P)H as the transcriptional co-repressor C-terminal binding protein 1 cofactor and significantly impact redox status (through GSH, NAD(P)H oxidases, etc.), which together act to control the phenotype of the cells of pulmonary arteries. The altered mitochondrial metabolism changes its redox poise, which together with enhanced NAD(P)H oxidase activity and reduced enzymatic antioxidant activity promotes a pro-oxidative cellular status. Herein we discuss all described metabolic changes along with resultant alterations in redox status, which result in excessive proliferation, apoptotic resistance, and inflammation, further leading to pulmonary arterial wall remodeling and thus establishing pulmonary artery hypertension pathology.