Hepatic Mitochondrial Respiration Research Articles

Dysglycemia and liver lipid content determine the relationship of insulin resistance with hepatic OXPHOS capacity in obesity

Hepatic mitochondrial respiration is higher in steatosis, but lower in overt type 2 diabetes. We hypothesized that hepatic OXPHOS capacity increases with a greater degree of insulin resistance in obesity, independent of other metabolic diseases. We analysed 65 humans without diabetes (BMI 50±7 kg/m2, HbA1c 5.5±0.4%) undergoing bariatric surgery. MASLD stages were assessed by histology, whole-body insulin sensitivity (PREDIcted-M index) by oral glucose tolerance tests, and maximal ADP-stimulated mitochondrial OXPHOS capacity by high-resolution respirometry of liver samples. Prediabetes was present in 30 participants, and MASLD in 46 participants. Thereof, 25 had metabolic dysfunction-associated steatohepatitis (MASH), and seven had F2-F3 fibrosis. While simple regression did not detect an association of insulin sensitivity with hepatic OXPHOS capacity, interaction analyses revealed that the regression coefficient of OXPHOS capacity depended on fasting plasma glucose (FPG) and liver lipid content. Interestingly, the respective slopes were negative for FPG ≤100 mg/dl, but positive for FPG >100 mg/dl. Liver lipid content displayed similar behavior, with a threshold value of 24%. Post-challenge glycemia affected the association between insulin sensitivity and OXPHOS capacity normalized for citrate synthase activity. Presence of prediabetes affected hepatic insulin signaling, mitochondrial dynamics and fibrosis prevalence, while the presence of MASLD related to higher biomarkers of hepatic inflammation, cell damage and lipid peroxidation in people with normal glucose tolerance. Rising liver lipid contents and plasma glucose concentrations, even in the non-diabetic range, are associated with a progressive decline of hepatic mitochondrial adaptation in people with obesity and insulin resistance. CLINTRIALS. NCT01477957.

VCD-induced menopause mouse model reveals reprogramming of hepatic metabolism

ObjectiveMenopause adversely impacts systemic energy metabolism and increases the risk of metabolic disease(s) including hepatic steatosis, but the mechanisms are largely unknown. Dosing female mice with vinyl cyclohexene dioxide (VCD) selectively causes follicular atresia in ovaries, leading to a murine menopause-like phenotype. MethodsIn this study, we treated female C57BL6/J mice with VCD (160 mg/kg i.p. for 20 consecutive days followed by verification of the lack of estrous cycling) to investigate changes in body composition, energy expenditure (EE), hepatic mitochondrial function, and hepatic steatosis across different dietary conditions. ResultsVCD treatment induced ovarian follicular loss and increased follicle-stimulating hormone (FSH) levels in female mice, mimicking a menopause-like phenotype. VCD treatment did not affect body composition, or EE in mice on a low-fat diet (LFD) or in response to a short-term (1-week) high-fat, high sucrose diet (HFHS). However, the transition to a HFHS lowered cage activity in VCD mice. A chronic HFHS diet (16 weeks) significantly increased weight gain, fat mass, and hepatic steatosis in VCD-treated mice compared to HFHS-fed controls. In the liver, VCD mice showed suppressed hepatic mitochondrial respiration on LFD, while chronic HFHS resulted in compensatory increases in hepatic mitochondrial respiration. Also, liver RNA sequencing revealed that VCD promoted global upregulation of hepatic lipid/cholesterol synthesis pathways. ConclusionOur findings suggest that the VCD-induced menopause model compromises hepatic mitochondrial function and lipid/cholesterol homeostasis that sets the stage for HFHS diet-induced steatosis while also increasing susceptibility to obesity.

Consuming a Linoleate-Rich Diet Increases Concentrations of Tetralinoleoyl Cardiolipin in Mouse Liver and Alters Hepatic Mitochondrial Respiration

BackgroundHepatic mitochondrial dysfunction is a major cause of fat accumulation in the liver. Individuals with fatty liver conditions have hepatic mitochondrial structural abnormalities and a switch in the side chain composition of the mitochondrial phospholipid, cardiolipin, from poly- to monounsaturated fatty acids. Linoleic acid (LA), an essential dietary fatty acid, is required to remodel nascent cardiolipin (CL) to its tetralinoleoyl cardiolipin (L4CL, CL with 4 LA side chains) form, which is integral for mitochondrial membrane structure and function to promote fatty acid oxidation. It is unknown, however, whether increasing LA in the diet can increase hepatic L4CL concentrations and improve mitochondrial respiration in the liver compared with a diet rich in monounsaturated and saturated fatty acids. ObjectivesThe main aim of this study was to test the ability of a diet fortified with LA-rich safflower oil (SO), compared with the one fortified with lard (LD), to increase concentrations of L4CL and improve mitochondrial respiration in the livers of mice. MethodsTwenty-four (9-wk-old) C57 BL/J6 male mice were fed either the SO or LD diets for ∼100 d, whereas food intake and body weight, fasting glucose, and glucose tolerance tests were performed to determine any changes in glycemic control. ResultsLivers from mice fed SO diet had higher relative concentrations of hepatic L4CL species compared with LD diet-fed mice (P value = 0.004). Uncoupled mitochondria of mice fed the SO diet, compared with LD diet, had an increased baseline oxygen consumption rate (OCR) and succinate-driven respiration (P values = 0.03 and 0.01). SO diet-fed mice had increased LA content in all phospholipid classes compared with LD-fed mice (P < 0.05). ConclusionsOur findings reveal that maintaining or increasing hepatic L4CL may result in increased OCR in uncoupled hepatic mitochondria in healthy mice whereas higher oleate content of CL reduced mitochondrial function shown by lower OCR in uncoupled mitochondria.

OR15-03 Lower Hepatic Mitochondrial Respiration Relates To Dysmetabolism In Adolescents With Severe Obesity

Abstract Disclosure: E. Finn: None. K.N. Fuller: None. L.A. Knaub: None. Y. Garcia Reyes: None. S.C. Derderian: None. J. Schoen: None. T. Inge: None. J.E. Reusch: Advisory Board Member; Self; Medtronic Diabetes. K. Nadeau: None. M. Cree: None. Introduction: The severity of metabolic disease in youth with obesity is varied and determinants of disease progression are not known. Non-alcoholic fatty liver disease appears to be central to the development of cardiometabolic disease in youth with severe obesity. We tested the hypothesis that hepatic mitochondrial respiratory capacity would correlate with cardiometabolic measures in youth with severe obesity and a range of glycemia. Methods: Adolescents with severe obesity and a range of dysglycemia undergoing bariatric surgery were enrolled. Participants underwent anthropomorphic assessments, fasting labs, and a 4-hour mixed meal tolerance test (MMTT, 45 g carbs, 14 g protein, and 14 g fat) over 30 minutes followed by serial measures of glucose, insulin, C-peptide, glucagon-like peptide-1 (GLP-1), and free fatty acids. Liver biopsies obtained at time of vertical sleeve gastrectomy surgery were analyzed for mitochondrial respiration. Permeabilized liver tissue was assessed for mitochondrial oxygen consumption by respirometry (Oxygraph-2k, OROBOROS) under both pyruvate (carbohydrate) and palmitoyl-carnitine (lipids) substrate conditions. Results: Fifteen youth with severe obesity (female=60%, mean age 16.9 ± 1.5 years, mean BMI 47.4 ± 7.2 kg/m2) were enrolled. Lower hepatic carbohydrate-supported respiration relates to higher serum glucose concentrations throughout the 4-hr MMTT (P=&amp;lt; 0.001 to 0.05) as well as higher fasting serum triglycerides (p =&amp;lt;0.001-0.05). Lower liver lipid-supported respiration is associated with higher C-peptide levels (p =0.001-0.01) throughout MMTT, increased Matsuda Index (p =0.001-0.05), and higher fasting hepatic transaminases, alanine aminotransferase (p =0.006-0.04), aspartame aminotransferase (p =0.007-0.02), and gamma-glutamyl transferase (p =0.01-0.03). Insulin, GLP-1, and free fatty acids are not correlated with mitochondrial respiration. Conclusion: Consistent with findings in adults and animals, youth with severe obesity have lower hepatic mitochondrial respiration which correlates with markers of poorer cardiometabolic health including higher triglycerides, higher glucose concentraions, lower insulin sensitivity, higher insulin secretion (C-peptide), and higher liver transaminases. Based on these findings, dysregulated hepatic mitochondrial respiration should be further explored as a possible contributor to the development of early liver disease and glucose dysregulation. Presentation: Friday, June 16, 2023

Pre- and Post-Sexual Maturity Liver-specific ERα Knockout Does Not Impact Hepatic Mitochondrial Function.

Compared with males, premenopausal women and female rodents are protected against hepatic steatosis and present with higher functioning mitochondria (greater hepatic mitochondrial respiration and reduced H2O2 emission). Despite evidence that estrogen action mediates female protection against steatosis, mechanisms remain unknown. Here we validated a mouse model with inducible reduction of liver estrogen receptor alpha (ERα) (LERKO) via adeno-associated virus (AAV) Cre. We phenotyped the liver health and mitochondrial function of LERKO mice (n = 10-12 per group) on a short-term high-fat diet (HFD), and then tested whether timing of LERKO induction at 2 timepoints (sexually immature: 4 weeks old [n = 11 per group] vs sexually mature: 8-10 weeks old [n = 8 per group]) would impact HFD-induced outcomes. We opted for an inducible LERKO model due to known estrogen-mediated developmental programming, and we reported both receptor and tissue specificity with our model. Control mice were ERαfl/fl receiving AAV with green fluorescent protein (GFP) only. Results show that there were no differences in body weight/composition or hepatic steatosis in LERKO mice with either short-term (4-week) or chronic (8-week) high-fat feeding. Similarly, LERKO genotype nor timing of LERKO induction (pre vs post sexual maturity) did not alter hepatic mitochondrial O2 and H2O2 flux, coupling, or OXPHOS protein. Transcriptomic analysis showed that hepatic gene expression in LERKO was significantly influenced by developmental stage. Together, these studies suggest that hepatic ERα is not required in female protection against HFD-induced hepatic steatosis nor does it mediate sexual dimorphism in liver mitochondria function.

Impact of GLP-1 receptor agonist versus omega-3 fatty acids supplement on obesity-induced alterations of mitochondrial respiration.

To compare administration of the glucagon-like peptide-1 (GLP-1) analogue, exenatide, versus dietary supplementation with the omega-3 fatty acid-rich Calanus oil on obesity-induced alterations in mitochondrial respiration. Six-week-old female C57BL/6JOlaHSD mice were given high fat diet (HFD, 45% energy from fat) for 12 weeks to induce obesity. Thereafter, they were divided in three groups where one received exenatide (10 μg/kg/day) via subcutaneously implanted mini-osmotic pumps, a second group received 2% Calanus oil as dietary supplement, while the third group received HFD without any treatment. Animals were sacrificed after 8 weeks of treatment and tissues (skeletal muscle, liver, and white adipose tissue) were collected for measurement of mitochondrial respiratory activity by high-resolution respirometry, using an Oroboros Oxygraph-2k (Oroboros instruments, Innsbruck, Austria). It was found that high-fat feeding led to a marked reduction of mitochondrial respiration in adipose tissue during all three states investigated - LEAK, OXPHOS and ETS. This response was to some extent attenuated by exenatide treatment, but not with Calanus oil treatment. High-fat feeding had no major effect on hepatic mitochondrial respiration, but exenatide treatment resulted in a significant increase in the various respiratory states in liver. Mitochondrial respiration in skeletal muscle was not significantly influenced by high-fat diet or any of the treatments. The precise evaluation of mitochondrial respiration considering absolute oxygen flux and ratios to assess flux control efficiency avoided misinterpretation of the results. Exenatide increased hepatic mitochondrial respiration in high-fat fed mice, but no clear beneficial effect was observed in skeletal muscle or fat tissue. Calanus oil did not negatively affect respiratory activity in these tissues, which maintains its potential as a dietary supplement, due to its previously reported benefits on cardiac function.

Indole-3-acetic acid improves the hepatic mitochondrial respiration defects by PGC1a up-regulation

Recent evidences have linked indole-3-acetic acid (I3A), a gut microbiota-derived metabolite from dietary tryptophan, with the protection against non-alcoholic fatty liver disease (NAFLD). However, the values of I3A on mitochondrial homeostasis in NAFLD have yet to be analyzed. In this study, we verified that I3A alleviated dietary-induced metabolic impairments, particularly glucose dysmetabolism and liver steatosis. Importantly, we expanded the understanding of I3A further to enhance mitochondrial oxidative phosphorylation in the liver by RNA-seq. Consistently, I3A restored the deficiency of mitochondrial respiration complex (MRC) capacity in palmitic acid (PA)-induced HepG2 without initiating oxidative stress in vitro. These changes were dependent on peroxisome proliferator-activated receptor γ coactivator 1 (PGC1)-a, a key regulator of mitochondrial biogenesis. Silencing of PGC1a by siRNA and pharmacologic inhibitor SR-18292, blocked the restoration of I3A on mitochondrial oxidative phosphorylation. In addition, pre-treatment of I3A guarded against the deficiency of MRC capacity. In conclusion, our findings uncovered that I3A increased hepatic PGC1a expression, contributing to mitochondrial respiration improvement in NAFLD.

Influence of NAFLD and bariatric surgery on hepatic and adipose tissue mitochondrial biogenesis and respiration

Impaired mitochondrial oxidative phosphorylation (OXPHOS) in liver tissue has been hypothesised to contribute to the development of nonalcoholic steatohepatitis in patients with nonalcoholic fatty liver disease (NAFLD). It is unknown whether OXPHOS capacities in human visceral (VAT) and subcutaneous adipose tissue (SAT) associate with NAFLD severity and how hepatic OXPHOS responds to improvement in NAFLD. In biopsies sampled from 62 patients with obesity undergoing bariatric surgery and nine control subjects without obesity we demonstrate that OXPHOS is reduced in VAT and SAT while increased in the liver in patients with obesity when compared with control subjects without obesity, but this was independent of NAFLD severity. In repeat liver biopsy sampling in 21 patients with obesity 12 months after bariatric surgery we found increased hepatic OXPHOS capacity and mitochondrial DNA/nuclear DNA content compared with baseline. In this work we show that obesity has an opposing association with mitochondrial respiration in adipose- and liver tissue with no overall association with NAFLD severity, however, bariatric surgery increases hepatic OXPHOS and mitochondrial biogenesis.

Critical Role for Hepatocyte-Specific eNOS in NAFLD and NASH.

Regulation of endothelial nitric oxide synthase (eNOS) in hepatocytes may be an important target in nonalcoholic fatty liver disease (NAFLD) development and progression to nonalcoholic steatohepatitis (NASH). In this study, we show genetic deletion and viral knockdown of hepatocyte-specific eNOS exacerbated hepatic steatosis and inflammation, decreased hepatic mitochondrial fatty acid oxidation and respiration, increased mitochondrial H2O2 emission, and impaired the hepatic mitophagic (BNIP3 and LC3II) response. Conversely, overexpressing eNOS in hepatocytes in vitro and in vivo increased hepatocyte mitochondrial respiration and attenuated Western diet-induced NASH. Moreover, patients with elevated NAFLD activity score (histology score of worsening steatosis, hepatocyte ballooning, and inflammation) exhibited reduced hepatic eNOS expression, which correlated with reduced hepatic mitochondrial fatty acid oxidation and lower hepatic protein expression of mitophagy protein BNIP3. The current study reveals an important molecular role for hepatocyte-specific eNOS as a key regulator of NAFLD/NASH susceptibility and mitochondrial quality control with direct clinical correlation to patients with NASH.

1165-P: Alterations of Hepatic Mitochondrial Respiration in Murine Models of Diabetes and Nonalcoholic Fatty Liver Disease (NAFLD)

Diabetes mellitus (DM) accelerates progression of fatty liver to nonalcoholic steatohepatitis (NASH) and fibrosis. Mitochondrial function is also frequently impaired in DM, but maybe differently regulated in NAFLD. Thus, we examined hepatic mitochondrial respiration during DM and NAFLD development. Two-days old male C57BL/6j mice received streptozotocin (STZ) or vehicle (placebo, PLC) and then high-fat diet (HFD) or continued regular chow diet (RCD) from week 4 to 12, yielding 4 models (n=8 each): obesity [PLC+HFD], DM [STZ+RCD], NASH [STZ+HFD] and control [PLC+RCD]. Histology and immunohistochemistry were performed to describe NAFLD, including the NAFLD activity score (NAS) derived from steatosis, lobular inflammation and hepatocellular ballooning. High-resolution respirometry and hyperinsulinemic-euglycemic clamps were used to measure mitochondrial respiration and insulin sensitivity. DM and NASH showed &amp;gt;200% higher blood glucose levels (p&amp;lt;0.0001 vs. control). The NAS (mean±SEM) was not different in obese (1.38±0.32; p&amp;gt;0.99), gradually higher in DM (2.50±0.42; p=0.063) and markedly increased in NASH (3.25±0.45; p&amp;lt;0.01) compared to control (1.00±0.27). Main contributors to NAS were steatosis and hepatocellular ballooning in NASH (p&amp;lt;0.001 and p&amp;lt;0.05 vs. control) and hepatocellular ballooning in DM (p&amp;lt;0.01 vs. control). Maximal uncoupled β-oxidation-associated respiration was 1.6-, 1.7-, and 1.9-fold higher in obesity, DM and NASH compared to control (all p&amp;lt;0.01). Mitochondrial density was decreased in both DM and NASH (p&amp;lt;0.05 vs. control), whereas β-oxidation-dependent mitochondrial leak control ratio was not different between all groups. Insulin-stimulated glucose disposal was 44% and 66% lower in obesity and NASH (both p&amp;lt;0.001 vs. control). In conclusion, hyperglycemia and high-fat diet feeding associate with higher respiration but lower content of hepatic mitochondria, which may both contribute to progression of NAFLD. Disclosure B. Dewidar: None. M. Reinadofundo: None. C. Englisch: None. L. Mastrototaro: None. D. Pesta: None. C. Ress: None. I. Esposito: None. M. Roden: Advisory Panel; Self; Allergan plc, Bristol-Myers Squibb Company, Novo Nordisk A/S, Research Support; Self; Boehringer Ingelheim International GmbH, Danone Nutricia, Sanofi-Aventis Deutschland GmbH.

Non-alcoholic fatty liver disease in type 2 diabetes - A specific entity?

Individuals with obesity or type 2 diabetes (T2D) have an increased risk of developing non-alcoholic fatty liver disease (NAFLD). In insulin-resistant states, altered adipose tissue function may be the initial abnormality underlying NAFLD. Hepatic lipid oversupply interferes with insulin signalling and mitochondrial function. In obese individuals, adaptation of hepatic mitochondrial respiration fails with the progression of NAFLD and can activate pro-inflammatory pathways. T2D as well as type 1 diabetes are associated with altered hepatic mitochondrial function. Screening for NAFLD remains challenging especially in those with diabetes because liver enzymes are often in the normal range and the performance of NAFLD scores is limited. Patients with T2D and severe insulin-resistant diabetes (SIRD) have the highest prevalence of NAFLD at diagnosis and the greatest risk of progression. In this subgroup, the single-nucleotide-polymorphism (SNP) rs738409(G) of the patatin-like phospholipase domain-containing protein 3 (PNPLA3) gene is associated with high liver fat content and adipose tissue insulin resistance. This frequent SNP is also known to be associated with lean NAFLD so that genetic testing for this and other SNPs could improve future screening strategies to identify high-risk individuals. Although lifestyle modifications are effective, this approach is limited owing to difficulties with compliance and several classes of drugs are being tested to treat NAFLD. Antihyperglycaemic drugs such as glucagon-like peptide 1 receptor agonists (GLP-1 RA), sodium-glucose cotransporter 2 inhibitors (SGLT2i) and pioglitazone are promising and halt the progression of NAFLD. In conclusion, although NAFLD in diabetes may not be a separate entity, there are specific features to its pathogenesis and clinical management.

Prebiotic Xylo-Oligosaccharides Ameliorate High-Fat-Diet-Induced Hepatic Steatosis in Rats.

Understanding the importance of the gut microbiota (GM) in non-alcoholic fatty liver disease (NAFLD) has raised the hope for therapeutic microbes. We have shown that high hepatic fat content associated with low abundance of Faecalibacterium prausnitzii in humans and, further, the administration of F. prausnitzii prevented NAFLD in mice. Here, we aimed at targeting F. prausnitzii by prebiotic xylo-oligosaccharides (XOS) to treat NAFLD. First, the effect of XOS on F. prausnitzii growth was assessed in vitro. Then, XOS was supplemented or not with high (HFD, 60% of energy from fat) or low (LFD) fat diet for 12 weeks in Wistar rats (n = 10/group). XOS increased F. prausnitzii growth, having only a minor impact on the GM composition. When supplemented with HFD, XOS ameliorated hepatic steatosis. The underlying mechanisms involved enhanced hepatic β-oxidation and mitochondrial respiration. Nuclear magnetic resonance (1H-NMR) analysis of cecal metabolites showed that, compared to the HFD, the LFD group had a healthier cecal short-chain fatty acid profile and on the HFD, XOS reduced cecal isovalerate and tyrosine, metabolites previously linked to NAFLD. Cecal branched-chain fatty acids associated positively and butyrate negatively with hepatic triglycerides. In conclusion, XOS supplementation can ameliorate NAFLD by improving hepatic oxidative metabolism and affecting GM.

Sex and BNIP3 genotype, rather than acute lipid injection, modulate hepatic mitochondrial function and steatosis risk in mice.

Both lipid oversupply and poor mitochondrial function (low respiration and elevated H2O2 emission) have been implicated in the development of hepatic steatosis and liver injury. Mitophagy, the targeted degradation of low-functioning mitochondria, is critical for maintaining mitochondrial quality control. Here, we used intralipid injection combined with acute (4 day) and chronic (4-7wk) high-fat diets (HFD) to examine whether hepatic mitochondrial respiration would decrease and H2O2 emission would increase with lipid overload. We tested these effects in male and female wild type (WT) mice and mice null for a critical mediator of mitophagy, BCL-2/adenovirus EIB 19-kDa interacting protein knockout (BNIP3 KO) housed at thermoneutral temperatures. Intralipid injection was successful in elevating serum triglycerides and nonesterified fatty acids but had no impact on hepatic mitochondrial respiratory function or H2O2 emission. However, female mice had greater mitochondrial respiration on the acute HFD and lower H2O2 emission across both HFD durations and were protected against hepatic steatosis. Unexpectedly, BNIP3 KO animals had greater hepatic mitochondrial respiration, better coupled respiration, and increased electron chain protein content after the 4-day HFD, compared with WT animals. Altogether, these data suggest that acute lipid overload delivered by a single intralipid bolus does not alter hepatic mitochondrial outcomes, but rather sex and genotype profoundly impact hepatic mitochondrial respiration and H2O2 emission.NEW & NOTEWORTHY This is the first study focusing on hepatic mitochondrial respiratory outcomes in response to lipid overload via a high-fat diet (HFD) combined with intralipid injection. Novel findings include no effect of intralipid injection on mitochondrial outcomes of interest despite increased circulating lipid concentrations. However, we report pronounced differences in hepatic mitochondrial respiration, complex protein expression, and H2O2 production by sex and BCL-2/adenovirus EIB 19-kDa interacting protein (BNIP3) genotype. Specifically, female mice had lower H2O2 emission globally and on an acute HFD, females had greater hepatic mitochondrial respiration than males while BNIP3 knockout (KO) animals had greater mitochondrial coupling and complex protein expression than wild-type (WT) animals.

Efficacy of Intravenous N-acetylcysteine As an Adjuvant Therapy in Acute Iron Toxicity in Rats

Iron is a one of the heavy metals that is necessary for cell function. Acute or chronic exposure to high dose lead to oxidative damage. In the liver, iron overload affect hepatic mitochondrial respiration. Deferoxamine is the established antidote of iron toxicity which act as a chelator. N-acetylcysteine is a safe over the counter mucolytic which is antioxidant and glutathione substitute properities. Its use as an antidote for some toxins is well established nowadays. The aim of this work is to study the efficacy of intravenous N-acetylcysteine as an adjuvant therapy with deferoxamine in acute iron intoxication in rats. This study was carried out on twenty male albino rats, weighted 200-250 gm and divided into group I: 10 rats received 400 mg/kg elemental iron orally followed by 25 mg/kg subcutaneous deferroxamine and group II: 10 rats received 400 mg/kg elemental iron followed by 150 mg/kg IV NAC and 25 mg/kg subcutaneous deferroxamine. The results revealed that N-acetylcysteine use lowered both oxidative stress markers malondialdehyde MDA and cyclic adenosine monophosphate cAMP. On the other hand, the reduction of serum and hepatic iron levels and the elevation of Alanine Transaminase (ALT) and Aspartate Aminotransferase (AST) were statistically insignificant. It was concluded that intravenous N-acetylcysteine helps in reduction of oxidative stress caused by acute iron toxicity.

Assessment of mitochondrial function in metabolic dysfunction-associated fatty liver disease using obese mouse models.

Metabolic dysfunction-associated fatty liver disease (MAFLD) is characterized by deregulated hepatic lipid metabolism; however, the association between MAFLD development and mitochondrial dysfunction has yet to be confirmed. Herein, we employed high-resolution respirometry, blue native polyacrylamide gel electrophoresis-based in-gel activity measurement and immunoblot analysis to assess mitochondrial function in obesity-induced mouse models with varying degrees of MAFLD. Results showed a slight but significant decrease in hepatic mitochondrial respiration in some MAFLD mice compared to mice fed a standard diet. However, the activities and levels of mitochondrial oxidative phosphorylation complexes remained unchanged during obesity-induced MAFLD progression. These results suggest that mitochondrial function, particularly oxidative phosphorylation, was mildly affected during obesity-induced MAFLD development. Moreover, transcriptome profiling of mouse and human liver tissues with varying degrees of MAFLD revealed that the decreased activation of mitochondria-related pathways was only associated with MAFLD of a high histological grade, whereas the major regulators of mitochondrial biogenesis were not altered in mice or humans during MAFLD development. Collectively, our results suggest that impaired hepatic mitochondrial function is not closely associated with obesity-induced MAFLD. Therefore, therapeutic strategies targeting mitochondria for the treatment of MAFLD should be reconsidered.

Acetaminophen toxicity induces mitochondrial complex I inhibition in human liver tissue.

Acetaminophen (APAP) is used worldwide and is regarded as safe in therapeutic concentrations but can cause acute liver failure in higher doses. High doses of APAP have been shown to inhibit complex I and II mitochondrial respiratory capacity in mouse hepatocytes, but human studies are lacking. Here, we studied mitochondrial respiratory capacity in human hepatic tissue ex vivo with increasing doses of APAP. Hepatic biopsies were obtained from 12 obese patients who underwent a Roux-en-Y gastric bypass (RYGB) or a sleeve gastrectomy surgery. Mitochondrial respiration was measured by high-resolution respirometry. Therapeutic concentrations (≤0.13mmol/L) of APAP did not inhibit state 3 complex I-linked respiration. APAP concentrations of ≥2.0mmol/L in the medium significantly reduced hepatic mitochondrial respiration in a dose-dependent manner. Complex II-linked mitochondrial respiration was not inhibited by APAP. We conclude that the mitochondrial respiratory capacity is affected by a hepato-toxic effect of APAP, which involved complex I, but not complex II.

Pravastatin and Gemfibrozil Modulate Differently Hepatic and Colonic Mitochondrial Respiration in Tissue Homogenates from Healthy Rats.

Statins and fibrates are widely used for the management of hypertriglyceridemia but they also have limitations, mostly due to pharmacokinetic interactions or side effects. It is conceivable that some adverse events like liver dysfunction or gastrointestinal discomfort are caused by mitochondrial dysfunction. Data about the effects of statins and fibrates on mitochondrial function in different organs are inconsistent and partially contradictory. The aim of this study was to investigate the effect of pravastatin (statin) and gemfibrozil (fibrate) on hepatic and colonic mitochondrial respiration in tissue homogenates. Mitochondrial oxygen consumption was determined in colon and liver homogenates from 48 healthy rats after incubation with pravastatin or gemfibrozil (100, 300, 1000 μM). State 2 (substrate dependent respiration) and state 3 (adenosine diphosphate: ADP-dependent respiration) were assessed. RCI (respiratory control index)—an indicator for coupling between electron transport chain system (ETS) and oxidative phosphorylation (OXPHOS) and ADP/O ratio—a parameter for the efficacy of OXPHOS, was calculated. Data were presented as a percentage of control (Kruskal–Wallis + Dunn’s correction). In the liver both drugs reduced state 3 and RCI, gemfibrozil-reduced ADP/O (complex I). In the colon both drugs reduced state 3 but enhanced ADP/O. Pravastatin at high concentration (1000 µM) decreased RCI (complex II). Pravastatin and gemfibrozil decrease hepatic but increase colonic mitochondrial respiration in tissue homogenates from healthy rats.

Sex‐Dependent Murine Hepatic Mitochondrial and Mitophagy Responses to High‐Fat Diet and Exercise

Excessive accumulation of hepatic fat (steatosis) is closely associated with obesity and hepatic insulin resistance, a critical factor for the development of type 2 diabetes. Exercise is a therapeutic that mitigates hepatic steatosis putatively through enhanced mitochondrial function and quality, but sex specific differences are unknown. Here we examined if a high fat diet (HFD) elicited sex‐specific reductions in hepatic mitochondrial respiration and mitochondrial recycling (autophagy/mitophagy) pathways and whether exercise (voluntary wheel running [VWR]) restored these impingements. Male and female wild type (WT, C57BL/6J) mice (n=6–7 per sex, per treatment) housed at thermoneutrality (~80°F) were fed low fat diet (LFD) or HFD (45%kcal fat) for 16 weeks with or without access to VWR for the final 8 weeks. Female mice had significantly reduced hepatic triacylglycerol (TAG) compared to males (p&lt;0.0001) and both sexes showed resistance to increases in TAG with HFD, potentially due to thermoneutral housing inducing higher TAG on a control diet. There was a dose‐dependent increase in maximal state 3 (ADP stimulated) respiratory capacity with HFD and HFD+VWR that was potentiated in females. HFD elicited an increase in H2O2 in males that was reduced with exercise while females had reduced mitochondrial H2O2 production in both conditions. Interestingly, neither glutathione (GSH), glutathione disulfide (GSSG) or the GSH:GSSG ratio, markers of oxidative stress, were different between the groups. Males had a consistent reduction in mitochondrial complex I‐V expression with HFD+VWR. Females had reduced expression of complex I with HFD and HFD+VWR but otherwise retained expression of complexes II‐V. Citrate synthase, a marker of hepatic mitochondrial content, was reduced by HFD in both males and females. However, HFD+VWR further reduced citrate synthase in males but restored activity in females to the level of the LFD cohort. Since mitochondrial content is regulated by mitophagy proteins, we examined the mitophagy regulating BCL2/adenovirus E1B 19 kDa protein‐interacting protein 3 (BNIP3) and Parkin proteins. Males had reduced BNIP3 expression on the LFD that was increased with HFD. HFD elicited no change in females, however, HFD+VWR drastically reduced BNIP3 levels in both sexes. Parkin was unchanged with HFD in both sexes yet had divergent responses with HFD+VWR as males had reduced expression and female expression increased. Overall, these data show sex‐specific hepatic triglyceride, mitochondrial, and mitophagy responses to HFD and exercise and provide cautionary evidence of studying hepatic steatosis in single sex models.Support or Funding InformationFunded by: Veterans Affairs Merit Review; 1I01BX002567‐01 (JPT), KUMC Biomedical Research Training Program (CSM), K‐INBRE grant number P20 GM103418 (CSM), CTSA grant from NCATS awarded to the University of Kansas for Frontiers: University of Kansas Clinical and Translational Science Institute (# TL1TR002368) (CSM)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hepatocyte‐Specific Deletion of eNOS Impairs Mitochondrial Function and Exacerbates Hepatic Steatosis

IntroductionWhole body loss of endothelial nitric oxide synthase (eNOS) worsens hepatic mitochondrial function and exacerbates nonalcoholic fatty liver disease (NAFLD) development and progression. However, the precise role of eNOS in hepatocytes in the contribution to NAFLD has not been established. Here, we examine the effects of in vivo hepatocyte‐specific ablation of eNOS on hepatic steatosis and NAFLD susceptibility.MethodsHepatocyte‐specific eNOS knockout (eNOShep−/−) mice were generated by crossing homozygous eNOS floxed (eNOSfl/fl) mice on a C57/BL6J background with albumin‐Cre recombinase transgenic mice. At 10 weeks of age, male eNOSfl/fl and eNOShep−/− mice (n = 12–14) were fed a control diet (10% fat) for 16 weeks.ResultsBody weight, body fat, and food intake did not differ between eNOSfl/fl and eNOShep−/− mice. eNOShep−/− mice developed elevated histological hepatic steatosis and increased fasting serum insulin (~30%) compared to eNOSfl/fl mice (p&lt;0.05), along with trends for elevations in serum liver enzymes AST and ALT (p&lt;0.17). Elevated hepatic steatosis in eNOShep−/− was related to significant reductions (−50%) in hepatic mitochondrial fatty acid oxidation and ADP‐stimulated and maximal uncoupled hepatic mitochondrial respiration (p&lt;0.05). However, hepatic citrate synthase activity, a marker of mitochondrial mass, was significantly elevated in eNOShep−/− mice compared to eNOSfl/fl mice (p&lt;0.05), while protein content of oxidative phosphorylation complexes and peroxisome proliferator‐activated receptor‐γ coactivator‐1α (PGC‐1α) did not differ between groups. Furthermore, eNOShep−/− mice presented with significant reductions (p&lt;0.05) in key proteins involved in mitophagy and autophagy including PTEN‐induced kinase 1 (PINK1) and phosphorylated Unc‐51 Like Autophagy Activating Kinase 1 (pUlk1/Ulk1) and tended to have significant reductions in BCL2/adenovirus E1B 19 kDa protein‐interacting protein 3 (BNIP3) compared to eNOSfl/fl mice.ConclusionHere we demonstrate for the first time that hepatocyte‐specific deletion of eNOS impairs hepatic mitochondrial function, reduces markers of autophagy/mitophagy, and exacerbates hepatic steatosis development. Further studies are necessary to determine the mechanisms by which deletion of eNOS regulates mitochondrial homeostasis.Support or Funding InformationVA Merit Grant I01BX003271‐01 (R.S.R.)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Time-related changes in hepatic and colonic mitochondrial oxygen consumption after abdominal infection in rats

BackgroundEvidence suggests that early adaptive responses of hepatic mitochondria occur in experimentally induced sepsis. Little is known about both colonic mitochondrial function during abdominal infection and long-term changes in mitochondrial function under inflammatory conditions. We hypothesize that hepatic and colonic mitochondrial oxygen consumption changes time-dependently after sterile laparotomy and in the course of abdominal infection. The aim of the present study was to investigate the hepatic and colonic mitochondrial respiration after sterile laparotomy and abdominal infection over up to 96 h.MethodsAfter approval of the local Animal Care and Use Committee, 95 Wistar rats were randomized into 8 groups (n = 11–12): 1–4 sham (laparotomy only) and 5–8 colon ascendens stent peritonitis (CASP). Healthy, unoperated animals served as controls (n = 9). The mitochondrial respiration in colon and liver homogenates was assessed 24, 48, 72, and 96 h after surgery. Mitochondrial oxygen consumption was determined using a Clark-type electrode. State 2 (oxygen consumption in the presence of the substrates for complexes I and II) and state 3 respiration (ADP dependent) were assessed. The respiratory control ratio (RCR state 3/state 2) and ADP/O ratio (ADP added/oxygen consumed) were calculated for both complexes. Data are presented as means ± SD, two-way ANOVA followed by Tukey’s post hoc test.ResultsHepatic RCR was initially (after 24 h) elevated in both operated groups; after 48 h only, the septic group was elevated compared to controls. In CASP groups, the hepatic ADP/O ratio for complex I was elevated after 24 h (vs. controls) and after 48 h (vs. sham) but declined after 72 h (vs. controls). The ADP/O ratio for complex II stayed unchanged over the time period until 96 h.The colonic RCR and ADP/O did not change over time after sham or CASP operation.ConclusionHepatic, but not colonic, mitochondrial respiration is increased in the initial phase (until 48 h) and normalizes in the longer course of time (until 96 h) of abdominal infection.