Oral combined contraceptives induce liver mitochondrial reactive oxygen species and whole-body metabolic adaptations in female mice.

Compared to age-matched men, pre-menopausal women show greater resilience against cardiovascular disease (CVD), hepatic steatosis, diabetes and obesity - findings that are widely attributed to oestrogen. However, meta-analysis data suggest that current use of oral combined contraceptives (OC) is a risk factor for myocardial infarction, and OC use further compounds with metabolic disease risk factors to increase CVD susceptibility. While mitochondrial function in tissues such as the liver and skeletal muscle is an emerging mechanism by which oestrogen may confer its protection, effects of OC use on mitochondria and metabolism in the context of disease risk remain unexplored. To answer this question, female C57Bl/6J mice were fed a high fat diet and treated with vehicle or OCs for 3, 12 or 20 weeks (n = 6 to 12 per group) at a dose and ratio that mimic the human condition of cycle cessation in the low oestrogen, high progesterone stage. Liver and skeletal muscle mitochondrial function (respiratory capacity, H2 O2 , coupling) was measured along with clinical outcomes of cardiometabolic disease such as obesity, glucose tolerance, hepatic steatosis and aortic atherosclerosis. The main findings indicate that regardless of treatment duration, OCs robustly increase hepatic mitochondrial H2 O2 levels, likely due to diminished antioxidant capacity, but have no impact on muscle mitochondrial H2 O2 . Furthermore, OC-treated mice had lower adiposity and hepatic triglyceride content compared to control mice despite reduced wheel running, spontaneous physical activity and total energy expenditure. Together, these studies describe tissue-specific effects of OC use on mitochondria as well as variable impacts on markers of metabolic disease susceptibility. KEY POINTS: Oestrogen loss in women increases risk for cardiometabolic diseases, a link that has been partially attributed to negative impacts on mitochondria and energy metabolism. To study the effect of oral combined contraceptives (OCs) on hepatic and skeletal muscle mitochondria and whole-body energy metabolism, we used an animal model of OCs which mimics the human condition of cessation of hormonal cycling in the low oestrogen, high progesterone state. OC-treated mice have increased hepatic mitochondrial oxidative stress and decreased physical activity and energy expenditure, despite displaying lower adiposity and liver fat at this time point. These pre-clinical data reveal tissue-specific effects of OCs that likely underlie the clinical findings of increased cardiometabolic disease in women who use OCs compared to non-users, when matched for obesity.

Estradiol treatment or modest exercise improves hepatic health and mitochondrial outcomes in female mice following ovariectomy.

We recently reported that compared with males, female mice have increased hepatic mitochondrial respiratory capacity and are protected against high-fat diet-induced steatosis. Here, we sought to determine the role of estrogen in hepatic mitochondrial function, steatosis, and bile acid metabolism in female mice and investigate potential benefits of exercise in the absence or presence of estrogen via ovariectomy (OVX). Female C57BL mice (n = 6 per group) were randomly assigned to sham surgery (sham), ovariectomy (OVX), or OVX plus estradiol replacement therapy (OVX + Est). Half of the mice in each treatment group were sedentary (SED) or had access to voluntary wheel running (VWR). All mice were fed a high-fat diet (HFD) and were housed at thermoneutral temperatures. We assessed isolated hepatic mitochondrial respiratory capacity using the Oroboros O2k with both pyruvate and palmitoylcarnitine as substrates. As expected, OVX mice presented with greater hepatic steatosis, weight gain, and fat mass gain compared with sham and OVX + Est animals. Hepatic mitochondrial coupling (basal/state 3 respiration) with pyruvate was impaired following OVX, but both VWR and estradiol treatment rescued coupling to levels greater than or equal to sham animals. Estradiol and exercise also had different effects on liver electron transport chain protein expression depending on OVX status. Markers of bile acid metabolism and excretion were also impaired by ovariectomy but rescued with estradiol add-back. Together our data suggest that estrogen depletion impairs hepatic mitochondrial function and liver health, and that estradiol replacement and modest exercise can aid in rescuing this phenotype.NEW & NOTEWORTHY OVX induces hepatic steatosis in sedentary mice which can be prevented by modest physical activity (VWR) and/or estradiol treatment. Estrogen impacts hepatic mitochondrial coupling in a substrate-specific manner. OVX mice have impaired fecal bile acid excretion, which was rescued with estradiol treatment.

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.

Sex modulates hepatic mitochondrial adaptations to high-fat diet and physical activity.

The impact of sexual dimorphism and mitophagy on hepatic mitochondrial adaptations during the treatment of steatosis with physical activity are largely unknown. Here, we tested if deficiencies in liver-specific peroxisome proliferative activated-receptor-γ coactivator-1α (PGC-1α), a transcriptional coactivator of biogenesis, and BCL-2/ADENOVIRUS EIB 19-kDa interacting protein (BNIP3), a mitophagy regulator, would impact hepatic mitochondrial adaptations (respiratory capacity, H2O2 production, mitophagy) to a high-fat diet (HFD) and HFD plus physical activity via voluntary wheel running (VWR) in both sexes. Male and female wild-type (WT), liver-specific PGC-1α heterozygote (LPGC-1α), and BNIP3 null mice were thermoneutral housed (29-31°C) and divided into three groups: sedentary-low-fat diet (LFD), 16 wk of (HFD), or 16 wk of HFD with VWR for the final 8 wk (HFD + VWR) (n = 5-7/sex/group). HFD did not impair mitochondrial respiratory capacity or coupling in any group; however, HFD + VWR significantly increased maximal respiratory capacity only in WT and PGC-1α females. Males required VWR to elicit mitochondrial adaptations that were inherently present in sedentary females including greater mitochondrial coupling control and reduced H2O2 production. Females had overall reduced markers of mitophagy, steatosis, and liver damage. Steatosis and markers of liver injury were present in sedentary male mice on the HFD and were effectively reduced with VWR despite no resolution of steatosis. Overall, reductions in PGC-1α and loss of BNIP3 only modestly impacted mitochondrial adaptations to HFD and HFD + VWR with the biggest effect seen in BNIP3 females. In conclusion, hepatic mitochondrial adaptations to HFD and treatment of HFD-induced steatosis with VWR are more dependent on sex than PGC-1α or BNIP3.

Hepatic mitochondrial adaptations to physical activity: impact of sexual dimorphism, PGC1α and BNIP3-mediated mitophagy.

KEY POINTS: Hepatic mitochondrial adaptations to physical activity may be regulated by mitochondrial biogenesis (PGC1α) and mitophagy (BNIP3). Additionally, these adaptations may be sex-dependent. Chronic increase in physical activity lowers basal mitochondrial respiratory capacity in mice. Female mice have higher hepatic electron transport system protein content, elevated respiratory capacity, lowered mitophagic flux, and emit less mitochondrial H2 O2 independent of physical activity. Males require chronic daily physical activity to attain a similar mitochondrial phenotype compared to females. In contrast, females have limited hepatic adaptations to chronic physical activity. Livers deficient in PGC1α and BNIP3 display similar mitochondrial adaptations to physical activity to those found in wild-type mice. ABSTRACT: Hepatic mitochondrial adaptations to physical activity may be regulated by biogenesis- and mitophagy-associated pathways in a sex-dependent manner. Here, we tested if mice with targeted deficiencies in liver-specific peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α; LPGC1α+/- ) and BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3)-mediated mitophagy (BNIP3-/- ) would have reduced physical activity-induced adaptations in respiratory capacity, H2 O2 emission and mitophagy compared to wild-type (WT) controls and if these effects were impacted by sex. Male and female WT, LPGC1α+/- and BNIP3-/- C57BL6/J mice were divided into groups that remained sedentary or had access to daily physical activity via voluntary wheel running (VWR) (n = 6-10/group) for 4 weeks. Mice had ad libitum access to low-fat diet and water. VWR reduced basal mitochondrial respiration, increased mitochondrial coupling and altered ubiquitin-mediated mitophagy in a sex-specific manner in WT mice. Female mice of all genotypes displayed higher electron transport system content, displayed increased ADP-stimulated respiration, produced less mitochondrially derived reactive oxygen species, exhibited reduced mitophagic flux, and were less responsive to VWR compared to males. Males responded more robustly to VWR-induced changes in hepatic mitochondrial function resulting in a match to adaptations found in females. Deficiencies in PGC1α and BNIP3 alone did not largely alter mitochondrial adaptations to VWR. However, VWR restored sex-dependent abnormalities in mitophagic flux in LPGC1α+/- . Finally, BNIP3-/- mice had elevated mitochondrial content and increased mitochondrial respiration putatively through repressed mitophagic flux. In conclusion, hepatic mitochondrial adaptations to physical activity are more dependent on sex than PGC1α and BNIP3.

Long-chain acylcarnitines activate cell stress and myokine release in C2C12 myotubes: calcium-dependent and -independent effects.

Acylcarnitines, important lipid biomarkers reflective of acyl-CoA status, are metabolites that possess bioactive and inflammatory properties. This study examined the potential for long-chain acylcarnitines to activate cellular inflammatory, stress, and death pathways in a skeletal muscle model. Differentiated C2C12 myotubes treated with l-C14, C16, C18, and C18:1 carnitine displayed dose-dependent increases in IL-6 production with a concomitant rise in markers of cell permeability and death, which was not observed for shorter chain lengths. l-C16 carnitine, used as a representative long-chain acylcarnitine at initial extracellular concentrations ≥25 µM, increased IL-6 production 4.1-, 14.9-, and 31.4-fold over vehicle at 25, 50, and 100 µM. Additionally, l-C16 carnitine activated c-Jun NH2-terminal kinase, extracellular signal-regulated kinase, and p38 mitogen-activated protein kinase between 2.5- and 11-fold and induced cell injury and death within 6 h with modest activation of the apoptotic caspase-3 protein. l-C16 carnitine rapidly increased intracellular calcium, most clearly by 10 µM, implicating calcium as a potential mechanism for some activities of long-chain acylcarnitines. The intracellular calcium chelator BAPTA-AM blunted l-C16 carnitine-mediated IL-6 production by >65%. However, BAPTA-AM did not attenuate cell permeability and death responses, indicating that these outcomes are calcium independent. The 16-carbon zwitterionic compound amidosulfobetaine-16 qualitatively mimicked the l-C16 carnitine-associated cell stress outcomes, suggesting that the effects of high experimental concentrations of long-chain acylcarnitines are through membrane disruption. Herein, a model is proposed in which acylcarnitine cell membrane interactions take place along a spectrum of cellular concentrations encountered in physiological-to-pathophysiological conditions, thus regulating function of membrane-based systems and impacting cell biology.

Acylcarnitines activate proinflammatory signaling pathways.

Incomplete ß-oxidation of fatty acids in mitochondria is a feature of insulin resistance and type 2 diabetes mellitus (T2DM). Previous studies revealed that plasma concentrations of medium- and long-chain acylcarnitines (by-products of incomplete ß-oxidation) are elevated in T2DM and insulin resistance. In a previous study, we reported that mixed D,L isomers of C12- or C14-carnitine induced an NF-κB-luciferase reporter gene in RAW 264.7 cells, suggesting potential activation of proinflammatory pathways. Here, we determined whether the physiologically relevant L-acylcarnitines activate classical proinflammatory signaling pathways and if these outcomes involve pattern recognition receptor (PRR)-associated pathways. Acylcarnitines induced the expression of cyclooxygenase-2 in a chain length-dependent manner in RAW 264.7 cells. L-C14 carnitine (5-25 µM), used as a representative acylcarnitine, stimulated the expression and secretion of proinflammatory cytokines in a dose-dependent manner. Furthermore, L-C14 carnitine induced phosphorylation of JNK and ERK, common downstream components of many proinflammatory signaling pathways including PRRs. Knockdown of MyD88, a key cofactor in PRR signaling and inflammation, blunted the proinflammatory effects of acylcarnitine. While these results point to potential involvement of PRRs, L-C14 carnitine promoted IL-8 secretion from human epithelial cells (HCT-116) lacking Toll-like receptors (TLR)2 and -4, and did not activate reporter constructs in TLR overexpression cell models. Thus, acylcarnitines have the potential to activate inflammation, but the specific molecular and tissue target(s) involved remain to be identified.

Contributions of adipose tissue architectural and tensile properties toward defining healthy and unhealthy obesity.

The extracellular matrix (ECM) plays an important role in the maintenance of white adipose tissue (WAT) architecture and function, and proper ECM remodeling is critical to support WAT malleability to accommodate changes in energy storage needs. Obesity and adipocyte hypertrophy place a strain on the ECM remodeling machinery, which may promote disordered ECM and altered tissue integrity and could promote proinflammatory and cell stress signals. To explore these questions, new methods were developed to quantify omental and subcutaneous WAT tensile strength and WAT collagen content by three-dimensional confocal imaging, using collagen VI knockout mice as a methods validation tool. These methods, combined with comprehensive measurement of WAT ECM proteolytic enzymes, transcript, and blood analyte analyses, were used to identify unique pathophenotypes of metabolic syndrome and type 2 diabetes mellitus in obese women, using multivariate statistical modeling and univariate comparisons with weight-matched healthy obese individuals. In addition to the expected differences in inflammation and glycemic control, approximately 20 ECM-related factors, including omental tensile strength, collagen, and enzyme transcripts, helped discriminate metabolically compromised obesity. This is consistent with the hypothesis that WAT ECM physiology is intimately linked to metabolic health in obese humans, and the studies provide new tools to explore this relationship.

Roles for miRNA-378/378* in adipocyte gene expression and lipogenesis.

In this study, we explored the roles of microRNAs in adipocyte differentiation and metabolism. We first knocked down Argonaute2 (Ago2), a key enzyme in the processing of micro-RNAs (miRNAs), to investigate a potential role for miRNAs in adipocyte differentiation and/or metabolism. Although we did not observe dramatic differences in adipogenesis between Ago2 knock-down and control 3T3-L1 cells, incorporation of [(14)C]glucose or acetate into triacylglycerol, and steady-state levels of triacyglycerol were all reduced, suggesting a role for miRNAs in adipocyte metabolism. To study roles of specific miRNAs in adipocyte biology, we screened for miRNAs that are differentially expressed between preadipocytes and adipocytes for the 3T3-L1 and ST2 cell lines. Distinct subsets of miRNAs decline or increase during adipocyte conversion, whereas most miRNAs are not regulated. One locus encoding two miRNAs, 378/378*, contained within the intron of PGC-1beta is highly induced during adipogenesis. When overexpressed in ST2 mesenchymal precursor cells, miRNA378/378* increases the size of lipid droplets and incorporation of [(14)C]acetate into triacylglycerol. Although protein and mRNA expression levels of C/EBPalpha, C/EBPbeta, C/EBPdelta, and PPARgamma1 are unchanged, microarray and quantitative RT-PCR analyses indicate that a set of lipogenic genes are upregulated, perhaps due to increased expression of PPARgamma2. Knock-down of miRNA378 and/or miRNA378* decreases accumulation of triacylglycerol. Interestingly, we made the unexpected finding that miRNA378/378* specifically increases transcriptional activity of C/EBPalpha and C/EBPbeta on adipocyte gene promoters.