Mitochondrial Function In Type Research Articles

Identifying the mitochondrial metabolism network by integration of machine learning and explainable artificial intelligence in skeletal muscle in type 2 diabetes

Imbalance in glucose metabolism and insulin resistance are two primary features of type 2 diabetes/diabetes mellitus. Its etiology is linked to mitochondrial dysfunction in skeletal muscle tissue. The mitochondria are vital organelles involved in ATP synthesis and metabolism. The underlying biological pathways leading to mitochondrial dysfunction in type 2 diabetes can help us understand the pathophysiology of the disease. In this study, the mitochondrial gene expression dataset were retrieved from the GSE22309, GSE25462, and GSE18732 using Mitocarta 3.0, focusing specifically on genes that are associated with mitochondrial function in type 2 disease. Feature selection on the expression dataset of skeletal muscle tissue from 107 control patients and 70 type 2 diabetes patients using the XGBoost algorithm having the highest accuracy. For interpretation and analysis of results linked to the disease by examining the feature importance deduced from the model was done using SHAP (SHapley Additive exPlanations). Next, to comprehend the biological connections, study of protein-protien and mRNA-miRNA networks was conducted using String and Mienturnet respectively. The analysis revealed BDH1, YARS2, AKAP10, RARS2, MRPS31, were potential mitochondrial target genes among the other twenty genes. These genes are mainly involved in the transport and organization of mitochondria, regulation of its membrane potential, and intrinsic apoptotic signaling etc. mRNA-miRNA interaction network revealed a significant role of miR-375; miR-30a-5p; miR-16-5p; miR-129-5p; miR-1229-3p; and miR-1224-3p; in the regulation of mitochondrial function exhibited strong associations with type 2 diabetes. These results might aid in the creation of novel targets for therapy and type 2 diabetes biomarkers.

Aerobic Exercise Training Improves Skeletal Muscle Mitochondrial Function In Type 2 Diabetic Model

Type-2 diabetes mellitus (T2DM) severely affects skeletal muscle metabolism, especially the mitochondrial function. The mechanism of exercise-induced improvement in mitochondrial function underlying skeletal muscle-related T2DM changes is unclear. PURPOSE: To determine the effects of aerobic exercise training on exercise capacity and skeletal muscle mitochondrial function in the diabetes spontaneous mutation model (Leprdb). METHODS: 12-week-old male diabetic B6 db mice (db/db, n = 23) and control (db+/-, n = 23) were divided into four groups; db/+ CON, db/+ Ex, db/db CON, and db/db Ex. 8-weeks of exercise (5-times/week, 50 min/day, 12-15 m/min) was performed in db/+ Ex and db/db Ex. All groups were measured grip strength, muscle fatigue, and conducted rota-rod test and maximal-aerobic-running-capacity. After 8-weeks, the muscle fibers of soleus (SOL) and gastrocnemius (GAS) were collected and analyzed for mitochondrial O2, H2O2, and Ca2+ capacity. RESULTS: The SOL in the db/db Ex was higher in muscle mass than db/db CON (5.7 ± .4vs. 4.23 ± .2, p < .05). The GAS in the db/db Ex group was lower in muscle mass than db/+ CON (85.3 ± 3.6 vs.137.4 ± 4.4, p < .05). Grip strength (41.4 ± 1.5 vs. 31.8 ± 1.5, p < .05), muscle fatigue index (30.2 ± 1.3 vs. 39.7 ± 1.5, p < .05), rota-rod test (30.4 ± 1.8 vs. 19.6 ± 1.3, p < .05), and maximal running capacity (18.9 ± 1.4 vs. 12.6 ± 0.2, p < 0.05) in db/db Ex were higher than the db/db CON. Mitochondrial O2 consumption at ADP-stimulated maximal respiration of complex I substrates in both SOL (55.3 ± 2.6 vs. 24.8 ± 2.1, p < 0.05) and GAS (40.3 ± 1.7 vs. 24.1 ± 2.7, p < .05) in db/db Ex increased compared with the db/db CON. There were non-significant trends of mitochondrial H2O2 emission for db/db Ex in both SOL (43.07 ± 4.52 vs. 58.60 ± 2.66, p > 0.05) and GAS (33.9 ± 3.4 vs. 58.42 ± 3.7, p > .05) compared with db/db CON. The Ca2+ capacity increased in the db/db Ex at SOL muscle compared with db/db CON (1519 ± 83.3 vs. 972.8 ± 63.1, p < .05), and it only decreased in db/db CON at GAS muscle (929 ± 74.1 vs. 1518 ± 1291, p < 0.05) compared with db/+ CON. CONCLUSION: The 8-week exercise protects against T2DM-induced impairment in skeletal muscle mass, exercise capacity, and skeletal muscle mitochondrial function. Supported by the NRF-2018R1A2A3074577 and NRF-2019S1A5C2A03082727.

Hydrogen sulfide attenuates lung ischemia–reperfusion injury through SIRT3-dependent regulation of mitochondrial function in type 2 diabetic rats

BackgroundLung ischemia–reperfusion injury is a complex pathophysiologic process associated with high morbidity and mortality. We have demonstrated elsewhere that diabetes mellitus aggravated ischemia-induced lung injury. Oxidative stress and mitochondrial dysfunction are drivers of diabetic lung ischemia-reperfusion injury; however, the pathways that mediate these events are unexplored. In this study using a high-fat diet–fed model of streptozotocin-induced type 2 diabetes in rats, we determined the effect of hydrogen sulfide on lung ischemia-reperfusion injury with a focus on Sirtuin3 signaling. MethodsRats with type 2 diabetes were exposed to GYY4137, a slow release donor of hydrogen sulfide with or without administration of the Sirtuin3 short hairpin ribonucleic acid plasmid, and then subjected to a surgical model of ischemia–reperfusion injury of the lung (n = 8). Lung function, oxidative stress, inflammation, cell apoptosis, and mitochondrial function were measured. ResultsCompared with nondiabetic rats, animals with type 2 diabetes at baseline exhibited significantly decreased Sirtuin3 signaling in lung tissue and increased oxidative stress, apoptosis, inflammation, and mitochondrial dysfunction (P < .05 each). In addition, further impairment in Sirtuin3 signaling was found in diabetic rats subjected to this model of lung ischemia–reperfusion. Simultaneously, the indexes showed further aggravation. Treatment with hydrogen sulfide restored Sirtuin3 expression and decreased lung ischemia–reperfusion injury in animals with type 2 diabetes mellitus by improving lung functional recovery, decreasing oxidative damage, suppressing inflammation, ameliorating cell apoptosis, and preserving mitochondrial function (P < .05). Conversely, these protective effects were largely reversed in Sirtuin3 knockdown rats. ConclusionImpaired lung Sirtuin3 signaling associated with type 2 diabetic conditions was further attenuated by an ischemia-reperfusion insult. Hydrogen sulfide ameliorated reperfusion-induced oxidative stress and mitochondrial dysfunction via activation of Sirtuin3 signaling, thereby decreasing lung ischemia-reperfusion damage in rats with a model of type II diabetes.

Effects on Insulin Sensitivity, but Not on Mitochondrial Function Are Dependent on Insulin Resistance Status after High Intensity Interval Training

High intensity interval training (HIIT) is a time-efficient training approach to stimulate biogenesis in healthy populations. We hypothesized that HIIT would increase skeletal muscle insulin sensitivity due to improved muscle mitochondrial function in type 2 diabetes (T2D) patients and age- and BMI-matched controls (CON). We examined 18 sedentary male patients with T2D and 23 healthy male CON (age: 58±5 vs. 57±4 years, BMI: 31.4±2.4 vs. 30.4±2.3 kg.m-2) that were enrolled in a 12-week HIIT cycling protocol. CON were further grouped in insulin-sensitive (IS) and -resistant (IR) (baseline M in mg.kg-1.min-1; 7.4 ± 1.3 vs. 4.2 ± 1.1, p&amp;lt;0.001). Two-step hyperinsulinemic-euglycemic clamps, skeletal muscle biopsies for high resolution respirometry and magnetic resonance spectroscopy for liver fat quantification were performed. After 12 weeks of HIIT, T2D and IR CON increased their insulin sensitivity (3.9±1.9 vs. baseline: 2.7±1.6, p=0.02 and 5.5±2.1 vs. 4.2 ± 1.1, p=0.03, respectively), which returned to baseline after detraining. T2D and IR CON increased suppression of endogenous glucose production (EGP) during the high-insulin clamp (T2D: 91 ± 14% vs. baseline: 76 ± 11%, p=0.003 and IR CON: 107 ± 13% vs. 94 ± 14%, p=0.04). IR CON also increased suppression of lipolysis during the low-insulin clamp (82 ± 9 vs. 84 ± 8%, p=0.04). In T2D liver fat was decreased by 17% and remained decreased after detraining. Liver fat content remained unchanged by HIIT training in both CON groups. Muscle maximal uncoupled respiration increased in T2D and CON by 44% and 48%, respectively, at 12 weeks and remained increased after detraining in all groups (p&amp;lt;0.01). We conclude that at the same level of insulin resistance HIIT training improved hepatic and peripheral insulin sensitivity in patients with T2D and insulin resistant controls whereas changes in mitochondrial function occurred irrespective of insulin resistance status. Disclosure M. Apostolopoulou: None. D. Pesta: None. Y. Karusheva: None. S. Gancheva: None. T. Jelenik: None. A. Bierwagen: None. K. M ssig: None. J. Szendroedi: None. M. Roden: Speaker's Bureau; Self; Boehringer Ingelheim GmbH. Research Support; Self; Boehringer Ingelheim GmbH. Consultant; Self; Poxel SA. Research Support; Self; Danone Nutricia Early Life Nutrition, GlaxoSmithKline plc., Nutricia Advanced Medical Nutrition, Sanofi.

Exercise-induced alterations in pancreatic oxidative stress and mitochondrial function in type 2 diabetic Goto-Kakizaki rats.

Progressive metabolic complications accompanied by oxidative stress are the hallmarks of type 2 diabetes. The precise molecular mechanisms of the disease complications, however, remain elusive. Exercise‐induced nontherapeutic management of type 2 diabetes is the first line of choice to control hyperglycemia and diabetes associated complications. In this study, using 11‐month‐old type 2 Goto‐Kakizaki (GK) rats, we have investigated the effects of exercise on mitochondrial metabolic and oxidative stress in the pancreas. Our results showed an increase in the NADPH oxidase enzyme activity and reactive oxygen species (ROS) production in GK rats, which was inhibited after exercise. Increased lipid peroxidation and protein carbonylation and SOD activity were also inhibited after exercise. Interestingly, glutathione (GSH) level was markedly high in the pancreas of GK diabetic rats even after exercise. However, GSH‐peroxidase and GSH‐reductase activities were significantly reduced. Exercise also induced energy metabolism as observed by increased hexokinase and glutamate dehydrogenase activities. A significant decrease in the activities of mitochondrial Complexes II/III and IV were observed in the GK rats. Exercise improved only Complex IV activity suggesting increased utilization of oxygen. We also observed increased activities of cytochrome P450s in the pancreas of GK rats which was reduced significantly after exercise. SDS‐PAGE results have shown a decreased expression of NF‐κB, Glut‐2, and PPAR‐ϒ in GK rats which was markedly increased after exercise. These results suggest differential oxidative stress and antioxidant defense responses after exercise. Our results also suggest improved mitochondrial function and energy utilization in the pancreas of exercising GK rats.

Delayed skeletal muscle mitochondrial ADP recovery in youth with type 1 diabetes relates to muscle insulin resistance.

Insulin resistance (IR) increases cardiovascular morbidity and is associated with mitochondrial dysfunction. IR is now recognized to be present in type 1 diabetes; however, its relationship with mitochondrial function is unknown. We determined the relationship between IR and muscle mitochondrial function in type 1 diabetes using the hyperinsulinemic-euglycemic clamp and 31P-MRS before, during, and after near-maximal isometric calf exercise. Volunteers included 21 nonobese adolescents with type 1 diabetes and 17 nondiabetic control subjects with similar age, sex, BMI, Tanner stage, and activity levels. We found that youths with type 1 diabetes were more insulin resistant (median glucose infusion rate 10.1 vs. 18.9 mg/kglean/min; P < 0.0001) and had a longer time constant of the curve of ADP conversion to ATP (23.4 ± 5.3 vs. 18.8 ± 3.9 s, P < 0.001) and a lower rate of oxidative phosphorylation (median 0.09 vs. 0.21 mmol/L/s, P < 0.001). The ADP time constant (β = −0.36, P = 0.026) and oxidative phosphorylation (β = 0.02, P < 0.038) were related to IR but not HbA1c. Normal-weight youths with type 1 diabetes demonstrated slowed postexercise ATP resynthesis and were more insulin resistant than control subjects. The correlation between skeletal muscle mitochondrial dysfunction in type 1 diabetes and IR suggests a relationship between mitochondrial dysfunction and IR in type 1 diabetes.

Relationships between Mitochondrial Function and Metabolic Flexibility in Type 2 Diabetes Mellitus

IntroductionMitochondrial dysfunction, lipid accumulation, insulin resistance and metabolic inflexibility have been implicated in the etiology of type 2 diabetes (T2D), yet their interrelationship remains speculative. We investigated these interrelationships in a group of T2D and obese normoglycemic control subjects.Methods49 non-insulin dependent male T2D patients and 54 male control subjects were enrolled, and a hyperinsulinemic-euglycemic clamp and indirect calorimetry were performed. A muscle biopsy was taken and intramyocellular lipid (IMCL) was measured. In vivo mitochondrial function was measured by PCr recovery in 30 T2D patients and 31 control subjects.ResultsFasting NEFA levels were significantly elevated in T2D patients compared with controls, but IMCL was not different. Mitochondrial function in T2D patients was compromised by 12.5% (p<0.01). Whole body glucose disposal (WGD) was higher at baseline and lower after insulin stimulation. Metabolic flexibility (ΔRER) was lower in the type 2 diabetic patients (0.050±0.033 vs. 0.093±0.050, p<0.01). Mitochondrial function was the sole predictor of basal respiratory exchange ratio (RER) (R2 = 0.18, p<0.05); whereas WGD predicted both insulin-stimulated RER (R2 = 0.29, p<0.001) and metabolic flexibility (R2 = 0.40, p<0.001).ConclusionsThese results indicate that defects in skeletal muscle in vivo mitochondrial function in type 2 diabetic patients are only reflected in basal substrate oxidation and highlight the importance of glucose disposal rate as a determinant of substrate utilization in response to insulin.

Insulin Resistance and Type 2 Diabetes

For well over half a century, the link between insulin resistance and type 2 diabetes has been recognized. Insulin resistance is important. Not only is it the most powerful predictor of future development of type 2 diabetes, it is also a therapeutic target once hyperglycemia is present. In this issue of Diabetes , Morino et al. (1) report a series of studies that provide evidence of a genetic mechanism linking expression of lipoprotein lipase (LPL) to peroxisome proliferator–activated receptor (PPAR)-δ expression and mitochondrial function. This is likely to contribute to the muscle insulin resistance that predisposes to type 2 diabetes. Observation of abnormal mitochondrial function in vitro in type 2 diabetes (2) was soon followed by in vivo demonstration of this abnormality in insulin-resistant, first-degree relatives of people with type 2 diabetes (3). Further reports of a modest defect in muscle mitochondrial function in type 2 diabetes were published shortly thereafter (4,5). These studies raised the question of whether type 2 diabetes could be a primary disorder of the mitochondria. However, the study of first-degree relatives tended to be misinterpreted as having shown a major defect in mitochondrial function in type 2 diabetes, although it had studied nondiabetic groups from the opposite ends of the insulin resistance–sensitivity spectrum. Indeed, other studies showed no defect in mitochondrial function in type 2 diabetes (6,7), which led to further confusion. Mitochondrial function was then shown to be acutely modifiable by changing fatty acid availability (8) and that it …

Restoration of Muscle Mitochondrial Function and Metabolic Flexibility in Type 2 Diabetes by Exercise Training Is Paralleled by Increased Myocellular Fat Storage and Improved Insulin Sensitivity

OBJECTIVEMitochondrial dysfunction and fat accumulation in skeletal muscle (increased intramyocellular lipid [IMCL]) have been linked to development of type 2 diabetes. We examined whether exercise training could restore mitochondrial function and insulin sensitivity in patients with type 2 diabetes.RESEARCH DESIGN AND METHODSEighteen male type 2 diabetic and 20 healthy male control subjects of comparable body weight, BMI, age, and Vo2max participated in a 12-week combined progressive training program (three times per week and 45 min per session). In vivo mitochondrial function (assessed via magnetic resonance spectroscopy), insulin sensitivity (clamp), metabolic flexibility (indirect calorimetry), and IMCL content (histochemically) were measured before and after training.RESULTSMitochondrial function was lower in type 2 diabetic compared with control subjects (P = 0.03), improved by training in control subjects (28% increase; P = 0.02), and restored to control values in type 2 diabetic subjects (48% increase; P < 0.01). Insulin sensitivity tended to improve in control subjects (delta Rd 8% increase; P = 0.08) and improved significantly in type 2 diabetic subjects (delta Rd 63% increase; P < 0.01). Suppression of insulin-stimulated endogenous glucose production improved in both groups (−64%; P < 0.01 in control subjects and −52% in diabetic subjects; P < 0.01). After training, metabolic flexibility in type 2 diabetic subjects was restored (delta respiratory exchange ratio 63% increase; P = 0.01) but was unchanged in control subjects (delta respiratory exchange ratio 7% increase; P = 0.22). Starting with comparable pretraining IMCL levels, training tended to increase IMCL content in type 2 diabetic subjects (27% increase; P = 0.10), especially in type 2 muscle fibers.CONCLUSIONSExercise training restored in vivo mitochondrial function in type 2 diabetic subjects. Insulin-mediated glucose disposal and metabolic flexibility improved in type 2 diabetic subjects in the face of near–significantly increased IMCL content. This indicates that increased capacity to store IMCL and restoration of improved mitochondrial function contribute to improved muscle insulin sensitivity.

Muscle Mitochondrial Function in Patients with Type 2 Diabetes Mellitus and Peripheral Arterial Disease: Implications in Vascular Surgery

(1) To review the available information on mitochondrial function in type 2 diabetes mellitus (T2DM) and peripheral arterial disease (PAD) obtained by non-invasive phosphorus magnetic resonance spectroscopy ((31)PMRS), near-infrared spectroscopy (NIRS) in vivo and respirometry on mitochondria isolated from muscle biopsies in vitro (2) to evaluate the usefulness of such data in the diagnosis, treatment and prognosis of these patients. Review. PubMed (http://www.ncbi.nlm.nih.gov/PubMed) and manual literature search. Fifty-three articles were retrieved, which included (31)PMRS, 15, NIRS, 11, Combined, 1 and Respirometry, 2 and background literature, 24. Muscle mitochondrial function is impaired in both T2DM and PAD patients, but differently. Patients suffering from both pathological conditions will display more serious impairment of the mitochondrial function. Mitochondrial function and the degree of ischaemic disease as evaluated by (31)PMRS and NIRS are well correlated. The NIRS technique appears to determine the degree of PAD better than (31)PMRS. It is argued that systematic testing of mitochondrial function may be a useful prognostic tool with PAD and T2DM, but clinical studies are needed.

Increased Daily Walking Improves Lipid Oxidation Without Changes in Mitochondrial Function in Type 2 Diabetes

OBJECTIVE—To determine whether increased daily physical activity improves mitochondrial function and/or lipid oxidation in type 2 diabetes.RESEARCH DESIGN AND METHODS—Volunteers with (n = 10) and without (n = 10) type 2 diabetes were matched for habitual physical activity, age, sex, and weight. Basal and maximal mitochondrial activity, physical activity, and resting substrate oxidation were measured at baseline and after 2 and 8 weeks of increased physical activity.RESULTS—Baseline physical activity (6,450 ± 851 vs. 7,638 ± 741 steps/day), basal ATP use (12 ± 1 vs. 12 ± 1 μmol · ml−1 · min−1), phosphocreatine recovery from exercise (31 ± 5 vs. 29 ± 3 s), and basal lipid oxidation (0.57 ± 0.07 vs. 0.65 ± 0.06 mg · kg body wt−1 · min−1) were similar in people with and without type 2 diabetes. There was a significant increase in physical activity after 8 weeks (12,322 ± 1,979 vs. 9,187 ± 1,159 steps/day, respectively). Following increased physical activity, there were no changes in basal ATP use or phosphocreatine recovery after exercise in either group. Basal lipid oxidation increased after 8 weeks of increased physical activity in people with type 2 diabetes (0.79 ± 0.08 mg · kg−1 · min−1) but not people without (0.68 ± 0.13 mg · kg body wt−1 · min−1).CONCLUSIONS—Resting and maximal ATP turnover are not impaired in people with well-controlled type 2 diabetes compared with control subjects matched for physical activity as well as age and weight. Increased unsupervised daily physical activity is sustainable and improves lipid oxidation independent of change in mitochondrial activity in people with type 2 diabetes.

31P MR spectroscopy and in vitro markers of oxidative capacity in type 2 diabetes patients

Skeletal muscle mitochondrial function in type 2 diabetes (T2D) is currently being studied intensively. In vivo (31)P magnetic resonance spectroscopy ((31)P MRS) is a noninvasive tool used to measure mitochondrial respiratory function (MIFU) in skeletal muscle tissue. However, microvascular co-morbidity in long-standing T2D can interfere with the (31)P MRS methodology. To compare (31)P MRS-derived parameters describing in vivo MIFU with an in vitro assessment of muscle respiratory capacity and muscle fiber-type composition in T2D patients. (31)P MRS was applied in long-standing, insulin-treated T2D patients. (31)P MRS markers of MIFU were measured in the M. vastus lateralis. Muscle biopsy samples were collected from the same muscle and analyzed for succinate dehydrogenase activity (SDH) and fiber-type distribution. Several (31)P MRS parameters of MIFU showed moderate to good correlations with the percentage of type I fibers and type I fiber-specific SDH activity (Pearson's R between 0.70 and 0.75). In vivo and in vitro parameters of local mitochondrial respiration also correlated well with whole-body fitness levels (VO (2peak)) in these patients (Pearson's R between 0.62 and 0.90). Good correlations exist between in vivo and in vitro measurements of MIFU in long-standing insulin-treated T2D subjects, which are qualitatively and quantitatively consistent with previous results measured in healthy subjects. This justifies the use of (31)P MRS to measure MIFU in relation to T2D.

Mitochondrial oxidative function and type 2 diabetes

The cause of insulin resistance and type 2 diabetes is unknown. The major part of insulin-mediated glucose disposal takes place in the skeletal muscle, and increased amounts of intramyocellular lipid has been associated with insulin resistance and linked to decreased activity of mitochondrial oxidative phosphorylation. This review will cover the present knowledge and literature on the topics of the activity of oxidative enzymes and the electron transport chain (ETC) in skeletal muscle of patients with type 2 diabetes. Different methods of studying mitochondrial function are described, including biochemical measurements of oxidative enzyme and electron transport activity, isolation of mitochondria for measurements of respiration, and ATP production and indirect measurements of ATP production using nuclear magnetic resonance (NMR) - spectroscopy. Biochemical markers of mitochondrial content are also discussed. Several studies show reduced activity of oxidative enzymes in skeletal muscle of type 2 diabetics. The reductions are independent of muscle fiber type, and are accompanied by visual evidence of damaged mitochondria. In most studies, the reduced oxidative enzyme activity is explained by decreases in mitochondrial content; thus, evidence of a functional impairment in mitochondria in type 2 diabetes is not convincing. These impairments in oxidative function and mitochondrial morphology could reflect the sedentary lifestyle of the diabetic subjects, and the influence of physical activity on oxidative activity and mitochondrial function is discussed. The studies on insulin-resistant offspring of type 2 diabetic parents have provided important insights in the earliest metabolic defects in type 2 diabetes. These defects include reductions in basal ATP production and an attenuated response to insulin stimulation. The decreased basal ATP production does not affect overall lipid or glucose oxidation, and no studies linking changes in oxidative activity and insulin sensitivity in type 2 diabetes have been published. It is concluded that evidence of a functional impairment in mitochondria in type 2 diabetes is not convincing, and that intervention studies describing the correlation between changes in insulin resistance and mitochondrial function in type 2 diabetes are lacking. Specific effects of regular physical training and muscular work on mitochondrial function and plasticity in type 2 diabetes remain an important area of research.

The effect of mitochondrial inhibitors on membrane currents in isolated neonatal rat carotid body type I cells

Inhibitors of mitochondrial energy metabolism have long been known to be potent stimulants of the carotid body, yet their mechanism of action remains obscure. We have therefore investigated the effects of rotenone, myxothiazol, antimycin A, cyanide (CN(-)) and oligomycin on isolated carotid body type I cells. All five compounds caused a rapid rise in intracellular Ca(2+), which was inhibited on removal of extracellular Ca(2+). Under current clamp conditions rotenone and CN(-) caused a rapid membrane depolarization and elevation of [Ca(2+)](i). Voltage clamping cells to -70 mV substantially attenuated this rise in [Ca(2+)](i). Rotenone, cyanide, myxothiazol and oligomycin significantly inhibited resting background K(+) currents. Thus rotenone, myxothiazol, cyanide and oligomycin mimic the effects of hypoxia in that they all inhibit background K(+) current leading to membrane depolarization and voltage-gated calcium entry. Hypoxia, however, failed to have any additional effect upon membrane currents in the presence of CN(-) or rotenone or the mitochondrial uncoupler p-trifluoromethoxyphenyl hydrazone (FCCP). Thus not only do mitochondrial inhibitors mimic the effects of hypoxia, but they also abolish oxygen sensitivity. These observations suggest that there is a close link between oxygen sensing and mitochondrial function in type I cells. Mechanisms that could account for this link and the actions of mitochondrial inhibitors are discussed.

Two CD95 (APO-1/Fas) signaling pathways

We have identified two cell types, each using almost exclusively one of two different CD95 (APO-1/Fas) signaling pathways. In type I cells, caspase-8 was activated within seconds and caspase-3 within 30 min of receptor engagement, whereas in type II cells cleavage of both caspases was delayed for approximately 60 min. However, both type I and type II cells showed similar kinetics of CD95-mediated apoptosis and loss of mitochondrial transmembrane potential (DeltaPsim). Upon CD95 triggering, all mitochondrial apoptogenic activities were blocked by Bcl-2 or Bcl-xL overexpression in both cell types. However, in type II but not type I cells, overexpression of Bcl-2 or Bcl-xL blocked caspase-8 and caspase-3 activation as well as apoptosis. In type I cells, induction of apoptosis was accompanied by activation of large amounts of caspase-8 by the death-inducing signaling complex (DISC), whereas in type II cells DISC formation was strongly reduced and activation of caspase-8 and caspase-3 occurred following the loss of DeltaPsim. Overexpression of caspase-3 in the caspase-3-negative cell line MCF7-Fas, normally resistant to CD95-mediated apoptosis by overexpression of Bcl-xL, converted these cells into true type I cells in which apoptosis was no longer inhibited by Bcl-xL. In summary, in the presence of caspase-3 the amount of active caspase-8 generated at the DISC determines whether a mitochondria-independent apoptosis pathway is used (type I cells) or not (type II cells).

Mitochondrial function in type I cells isolated from rabbit arterial chemoreceptors.

1. In this, and the accompanying paper (Duchen & Biscoe, 1992), we test the hypothesis that the oxygen sensitivity of mitochondrial electron transport forms a basis for transduction in the carotid body, the primary peripheral arterial oxygen sensor. We here describe for isolated type I cells the changes in autofluorescence of mitochondrial NAD(P)H that accompany changes in PO2. 2. NAD(P)H autofluorescence (excitation, 340-360 nm; emission peak, 450 nm) increased with anoxia, reflecting a rise in the NAD(P)H/NAD(P) ratio. Graded increases in autofluorescence were seen in response to graded decreases in PO2, suggesting that mitochondrial function is progressively altered below a PO2 of about 60 mmHg. 3. A mitochondrial origin for the NAD(P)H autofluorescence was suggested by the mutual exclusion of the responses to anoxia and cyanide. 4. Oxidized flavoproteins fluoresce when excited at 450 nm with an emission peak at 550 nm. The small signals obtained under these conditions increased with uncoupler and showed a graded decrease with falling PO2 reflecting a rise in the FADH/FAD ratio. 5. Hypoxia raises [Ca2+]i. The hypoxia-induced changes in mitochondrial function were not secondary to this rise. A brief K(+)-induced depolarization leads to a transient increase in [Ca2+]i. At the same time there is a rapid decrease in NAD(P)H autofluorescence followed by an increase that far outlasts the rise in [Ca2+]i. This delayed increase in autofluorescence was smaller than was the increase with anoxia, even though K(+)-induced depolarization raised [Ca2+]i more than does anoxia. In Ca(2+)-free solutions the depolarization-induced changes were abolished, while those associated with hypoxia were maintained. 6. The changes of autofluorescence with K(+)-induced depolarization appear to reflect (i) oxidation of NAD(P)H by stimulation of respiration following mitochondrial Ca2+ uptake and (ii) reduction of NAD(P) by the Ca(2+)-dependent activation of mitochondrial dehydrogenases. This activation could last several minutes following only 100 ms depolarization, while the changes accompanying hypoxia closely followed the time course of the change in PO2. 7. In similarly isolated rat or mouse chromaffin cells and mouse dorsal root ganglion neurons under identical conditions, no measurable change in autofluorescence or in [Ca2+]i was seen until the PO2 fell below about 5 mmHg. 8. Carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP) increases O2 consumption, oxidizing mitochondrial NADH and hence decreasing autofluorescence, (delta FFCCP). Blockade of electron transport by anoxia or CN- decreases O2 consumption, increasing mitochondrial NADH/NAD and autofluorescence (delta FCN). The fractional change in autofluorescence with FCCP, delta FFCCP/delta FFCCP+FCN), is thus a measure of resting O2 consumption.(ABSTRACT TRUNCATED AT 400 WORDS)