Bioenergetic Differences Research Articles

An update on the role of sex hormones in the function of the cardiorenal mitochondria.

Multiple studies have highlighted the crucial role of mitochondrial bioenergetics in understanding the progression of cardiorenal diseases, revealing new potential treatment targets related to mitochondrial metabolism. There are well-established sexual dimorphisms in cardiac and renal physiology, with premenopausal females being generally protected from pathology compared with males. The mechanisms of this protection remain to be fully elucidated, however, they clearly depend, at least in part, on sex hormones. Sex hormones contribute to regulating mitochondrial function, and vice versa, highlighting the existence of a bidirectional relationship pivotal for cellular energy metabolism; however, there are still large gaps in knowledge when the sex differences in mitochondrial bioenergetics in health and disease are concerned. This manuscript provides an overview of the new evidence that has been accumulated regarding the role of sex hormones in renal and cardiac mitochondria-dependent cellular energetics, metabolism, and signaling, mainly focusing on the data obtained within the last 3-5 years. We briefly discuss mitochondrial function and different types of sex hormones for the reader and then focus on novel research underscoring the emerging mitochondrial pathways regulated by sex hormones, which might be of interest for the development of novel therapeutic strategies for cardiorenal conditions.

Age- and sex-associated alterations in hypothalamic mitochondrial bioenergetics and inflammatory-associated signaling in the 3xTg mouse model of Alzheimer’s disease

Mitochondrial dysfunction and associated inflammatory signaling are pivotal in both aging and in Alzheimer’s disease (AD). Studies have also shown that hypothalamic function is affected in AD. The hypothalamus may be a target for AD drugs given that mitochondrial alterations are observed in the hypothalamus. This study investigated how age and sex affect mitochondrial bioenergetics and inflammatory signaling in the hypothalamic mitochondria of 3xTg and control mice at 2, 6, and 13 months, aiming to enhance our understanding of these processes in aging and AD. Parameters included oxygen consumption rates, expression levels of subunits comprising mitochondrial complexes I-V, the enzymatic activity of cytochrome c oxidase (COX), transcription factors associated with inflammation such as NF-κB, pIκB-α, Nrf2, and other inflammatory biomarkers. Hypothalamic mitochondrial dysfunction was observed in 3xTg females as early as 2 months, but no changes were detected in 3xTg males until 6 months of age. In 3xTg mice, subunit expression levels for mitochondrial complexes I-II were significantly reduced in both sexes. Significant sex-based differences in COX activity were also observed at 13 months of age, with levels being lower in females compared to males. In addition, significant sex differences were indicated in NF-κB, pIκB-α, Nrf2, and other inflammatory biomarkers at different age groups during normal aging and AD progression. These findings highlight important sex differences in hypothalamic bioenergetics and inflammation, offering insights into potential new targets for preventing and/or treating AD.

Respiratory bioenergetics is enhanced in human, but not bovine macrophages after exposure to M. bovis PPD: Exploratory insights into overall similar Cellular Metabolic Profiles.

The role of macrophage (MØ) cellular metabolism and reprogramming during TB infection is of great interest due to the influence of Mycobacterium spp. on MØ bioenergetics. Recent studies have shown that M. tuberculosis induces a TLR2-dependent shift towards aerobic glycolysis, comparable to the established LPS induced pro-inflammatory M1 MØ polarisation. Distinct differences in the metabolic profile of murine and human MØ indicates species-specific differences in bioenergetics. So far, studies examining the metabolic potential of bovine MØ are lacking, thus the basic bioenergetics of bovine and human MØ were explored in response to a variety of innate immune stimuli. Cellular energy metabolism kinetics were measured concurrently for both species on a Seahorse XFe96 platform to generate bioenergetic profiles for the response to the bona-fide TLR2 and TLR4 ligands, FSL-1 and LPS respectively. Despite previous reports of species-specific differences in TLR signalling and cytokine production between human and bovine MØ, we observed similar respiratory profiles for both species. Basal respiration remained constant between stimulated MØ and controls, whereas addition of TLR ligands induced increased glycolysis, as measured by the surrogate parameter ECAR. In contrast to MØ stimulation with M. tuberculosis PPD, another TLR2 ligand, M. bovis PPD treatment significantly enhanced basal respiration rates and glycolysis only in human MØ. Respiratory profiling further revealed significant elevation of ATP-linked OCR and maximal respiration suggesting a strong OXPHOS activation upon M. bovis PPD stimulation in human MØ. Our results provide an exploratory set of data elucidating the basic respiratory profile of bovine vs. human MØ that will not only lay the foundation for future studies to investigate host-tropism of the M. tuberculosis complex but may explain inflammatory differences observed for other zoonotic diseases.

Insights into sex differences in mitochondrial bioenergetics and metabolism in the kidney

Insights into sex differences in mitochondrial bioenergetics and metabolism in the kidney

Abstract P357: Renal Male and Female Cortical Mitochondria Display Bioenergetic Differences in Response to Varying Substrates

Introduction: In general, renal disease susceptibility is higher in males than in age-matched females until menopause. The damage to renal proximal tubules (PT) is key to the onset of kidney disease development due to their high capacity for reabsorption and intense mitochondrial metabolism. Our previous data identified higher oxygen consumption in the renal cortex of healthy male Sprague Dawley rats and lower H 2 O 2 production compared to females. We hypothesize that sex differences in renal mitochondrial substrate reliance trigger differential bioenergetic responses in PTs, which contributes to kidney disease development later in life. Methods: 11-week-old male and female Sprague Dawley rats were acquired from Charles River Labs. The kidneys were flushed, and the renal cortex was used for ex vivo experiments. Mitochondria were isolated with differential centrifugation, and rovided with different substrate combinations: 10mM pyruvate and 2mM malate (PM), 5mM of glutamate, malate, and succinate (GMS), or 5mM succinate only (S). Spectrofluorimetry was employed to measure membrane potential (TMRM) and H 2 O 2 production (Amplex Red), XF24e Seahorse was used for oxygen consumption rate (OCR). OriginPro was used for statistical analysis (one-way ANOVA with post hoc test) and data displayed as mean ± SEM. Results: Seahorse revealed that male renal cortical mitochondria exhibit higher basal and maximum OCR compared to females when provided with the GMS or PM substrate combinations (39.4 ± 1.18 vs 26.5± 0.88, respectively for basal GMS, and 52.0 ± 2.33 vs 32.6 ± 1.24 maximum respiration GMS, p<0.001). However, similar OCR was observed when mitochondria were provided succinate only (31.2 ± 1.67 vs 28.7 ± 1.30, respectively for basal and 50.5 ± 2.49 vs 51.1 ± 1.76 maximum respiration, p>0.05). Mitochondrial membrane potential was higher in females compared to males when succinate only was provided (p<0.001), whereas similar mitochondrial membrane potential was observed between males and females when GMS or PM were used. Regardless of the substrate combination used, H 2 O 2 production was higher in female renal mitochondria compared to males (p<0.001). Conclusions: We report that male and female renal cortical mitochondria display unique bioenergetic properties in response to varying substrates. These basal bioenergetic differences may contribute to the difference in susceptibility to kidney diseases later in life.

Bioenergetics and mitochondrial deregulation underlie self-sustained persistency of atrial fibrillation in an ovine model

Abstract Background Atrial fibrillation (AF) often transitions from paroxysmal to more sustained forms in a 4-15/100 person-year range(1). It is well-recognized from both clinical and animal studies that some individuals undergo this transition much more readily than others, but the underlying mechanisms remain unclear. Purpose To identify potential differences in atrial bioenergetics between animals developing stable AF (AF-S) versus those resistant to this transition (AF-R) in a sheep AF-model. Methods AF was induced through repeated bursts of atrial tachystimulation (2). Stability of AF was monitored with telemetry. In-vivo electrophysiological remodeling was assessed via contact mapping. Metabolic and bioenergetic remodeling were evaluated using frozen left atrial appendage (LAA) tissues and isolated LAA mitochondria. Results AF-S sheep developed stable (>24-hour self-sustained) AF after 13 (range 5-22) days on average, whereas AF-R sheep failed to develop stable AF despite 120-day tachystimulation. Contact mapping and histological analysis revealed similar electro-anatomical remodeling in AF-S and AF-R groups, with the only significant difference observed in the decrease of activation time and conduction velocity in the AF-S atria. Both AF-S and AF-R animals displayed a notable increase in mitochondrial mass. Proteomic analysis showed significant differences in metabolism-related proteins and pathways between AF-S and AF-R, in particular concerning the anaplerotic supplementation of the tricarboxylic acid cycle at the level of α-ketoglutarate. Furthermore, compared to AF-R, AF-S LAA exhibited a 45% increase in succinate content, and a 23% decrease in ATP and Creatine Phosphate (PCr) content, suggesting a pathological metabolic alteration linked to a bioenergetic default. AF-S mitochondria presented abnormal succinate-oxidation, associated with a significant 20% decrease in ATP-synthesis rate, 22% increase in ROS-production, mitochondrial-membrane hyperpolarization, and decreased Complex I/II specific activity ratio. AF-S mitochondria alterations were prevented by complex I inhibitor S1QEL1.1, hinting to a participation of mitochondrial ROS into the mechanism of AF stabilization. In vivo acute intravenous perfusion of succinate in rats made them more susceptible to more frequent (OR 35 CI=[1.63-449.80]) and longer AF episodes upon treansesophageal stimulation; this vulnerability to AF was prevented by intraperitoneal pretreatment with SQEL1.1. Conclusions Resistance to AF-stabilization is associated with specific bioenergetic remodeling. These findings provide new mechanistic insights into AF-resistance and might open avenues for novel therapeutic strategies for AF-management.

Characterization of Sex-Dependent Differences in Brain Microvascular Bioenergetics

Background: Women experience increased incidence of stroke-related deaths and lifetime risk of Alzheimer's disease (AD). Microvascular dysfunction has been implicated in the pathogenesis of ischemic stroke and AD. Our objective is to investigate the sex-dependent differences in bioenergetics of young mouse brain microvessels (BMVs). Methods: BMVs were isolated from young male and female mice (3-4 months), using a combination of gradient centrifugation and filtration (300μm and 40μm filters) methods. Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of BMVs were measured utilizing the Agilent Seahorse XFe 24 analyzer. The levels of proteins and mRNAs of mitochondrial respiratory complexes were measured by western blot and RT-PCR, respectively. Citrate synthase (CS) enzyme activity and ATP levels were determined in lysates of BMVs. Results: Real-Time ATP Rate assay demonstrated a decreased total ATP production rate and ATP contribution from both glycolysis and oxidative phosphorylation (OxPhos) in BMVs from females compared to males. Mitochondrial stress test assay revealed significantly lower values of several mitochondrial respiratory parameters, including basal respiration, ATP production, maximal respiration, spare respiratory capacity, and proton leak in BMVs from females compared to males. Glycolytic Rate assay uncovered reduced basal glycolysis, proton efflux rate (PER) originating from basal glycolysis, and the mitoOCR/glycoPER ratio in BMVs from females compared to males. Interestingly, no sex-dependent differences were observed in basal PER and post-2-DG acidification rates in BMVs. Mito Fuel Flex Test assay showed no sex-dependent difference in the utilization of glucose, glutamine, and long-chain fatty acids as fuel substrates in BMVs. ATP measurements confirmed the reduced ATP levels in BMVs from females compared to males. Measurements of CS activity revealed no sex-dependent difference in the BMVs. Lastly, Western blot and RT-PCR experiments indicated no differences in either protein or mRNA levels of various mitochondrial complex protein levels.Conclusions: Female BMVs showed reduced mitochondrial respiratory and glycolytic parameters compared to male BMVs although relative contribution of various substrates for energy production were not different. These sex-dependent differences involving glycolysis and OxPhos make the microvasculature vulnerable to injury in females. Funding sources: NIH grants AG074489 and NS114286 (PVGK and RM). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A patient-based iPSC-derived hepatocyte model of alcohol-associated cirrhosis reveals bioenergetic insights into disease pathogenesis.

Only ~20% of heavy drinkers develop alcohol cirrhosis (AC). While differences in metabolism, inflammation, signaling, microbiome signatures and genetic variations have been tied to the pathogenesis of AC, the key underlying mechanisms for this interindividual variability, remain to be fully elucidated. Induced pluripotent stem cell-derived hepatocytes (iHLCs) from patients with AC and healthy controls differ transcriptomically, bioenergetically and histologically. They include a greater number of lipid droplets (LDs) and LD-associated mitochondria compared to control cells. These pre-pathologic indicators are effectively reversed by Aramchol, an inhibitor of stearoyl-CoA desaturase. Bioenergetically, AC iHLCs have lower spare capacity, slower ATP production and their mitochondrial fuel flexibility towards fatty acids and glutamate is weakened. MARC1 and PNPLA3, genes implicated by GWAS in alcohol cirrhosis, show to correlate with lipid droplet-associated and mitochondria-mediated oxidative damage in AC iHLCs. Knockdown of PNPLA3 expression exacerbates mitochondrial deficits and leads to lipid droplets alterations. These findings suggest that differences in mitochondrial bioenergetics and lipid droplet formation are intrinsic to AC hepatocytes and can play a role in its pathogenesis.

Differences in Brain Region Mitochondrial Bioenergetics Detected Using CK Clamp

The brain is a non-uniform organ comprised of discrete structures of varying function and cell composition. The differing function of brain regions ostensibly would necessitate disparate energetic demands, thereby suggesting brain bioenergetics would be different among brain regions. This study aimed to address whether brain regions indeed have different bioenergetics by evaluating substrate preference, fuel-specific mitochondrial function at physiologically relevant energy demands as well as associated membrane potential and electron conductance using high-resolution respirometry (Oroboros O2k) in the frontal cortex, hypothalamus, and hippocampus. We hypothesize there will be different bioenergetics among brain regions. Substrate preference was established using a substrate-uncoupler-inhibitor-titration (SUIT) with additions, in order: ADP, Pyruvate/Malate, UK5099, Octanoylcarnitine, Rotenone, Succinate, and Antimycin A. There was no statistically significant interaction between region and substrate (p=0.15) nor a main effect of region (p=0.91), however, there was a main effect of substrate in which pyruvate/malate (+275%) and succinate/rotenone (+170%) had significantly greater respiration than octanoylcarnitine, independent of region (p<0.001). Given the results of the SUIT test, pyruvate/malate was used for subsequent tests. The SUIT method relies upon saturating ADP levels and non-physiological ATP:ADP concentration ratios, the extent to which this test obscures the interpretation of brain region respiration is unclear. Instead, an alternative approach that leverages the enzymatic reaction of creatine kinase and phosphocreatine to assess mitochondrial respiration and membrane potential at clamped physiological ATP:ADP ratios, “CK Clamp,” was used. Function of mitochondria with a pyruvate fuel was then evaluated in all three brain regions in which energetic demand was clamped at ΔGATP = -12.87 (high demand), -14.08, -14.5, and -14.85 (low demand). There was a statistically significant interaction between brain region and clamped ΔGATP values (p = 0.02). Frontal cortex displayed higher respiration than hippocampus at -12.87 ΔGATP (33%). There was no statistically significant interaction between region and clamp for mitochondrial membrane potential assessed during the CK clamp (p=0.16), however there was a significant main effect of clamped ΔGATP (p<0.001) in which membrane potential increased (i.e., hyperpolarized) as energetic demand decreased. This agrees with the bioenergetic model that a decrease in energetic demand increases the proton motive force (aka, hyperpolarization), thereby applying a backpressure on the electron transport chain, slowing respiration. Electron conductance was then assessed by calculating the slope of respiration versus clamped ΔGATP. Electron conductance was greater in the frontal cortex than hypothalamus (+70%, p = 0.015). The conclusion of this study is there are differences in mitochondrial bioenergetics among brain regions and the CK clamp was more sensitive at detecting these differences than the SUIT method. None. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Peripheral Blood Cells From Older Adults Exhibit Sex-Associated Differences in Mitochondrial Function.

Blood-based mitochondrial bioenergetic profiling is a feasible, economical, and minimally invasive approach that can be used to examine mitochondrial function and energy metabolism in human subjects. In this study, we use 2 complementary respirometric techniques to evaluate mitochondrial bioenergetics in both intact and permeabilized peripheral blood mononuclear cells (PBMCs) and platelets to examine sex dimorphism in mitochondrial function among older adults. Employing equal numbers of PBMCs and platelets to assess mitochondrial bioenergetics, we observe significantly higher respiration rates in female compared to male participants. Mitochondrial bioenergetic differences remain significant after controlling for independent parameters including demographic parameters (age, years of education), and cognitive parameters (mPACC5, COGDX). Our study illustrates that circulating blood cells, immune cells in particular, have distinctly different mitochondrial bioenergetic profiles between females and males. These differences should be taken into account as blood-based bioenergetic profiling is now commonly used to understand the role of mitochondrial bioenergetics in human health and aging.

Characterization of bioenergetic and kinematic plasticity of bovine sperm reveals novel and dynamic temporal traits.

Sperm transport through the female reproductive tract requires energy for motility and fertilization. Sperm kinematic assessment is conducted as an industry standard to estimate semen quality prior to bovine insemination. However, individual samples with similar post-thaw motility result in different pregnancy outcomes, suggesting that differences in bioenergetics may be important for sperm function. Thus, characterization of bioenergetic and kinematic parameters of sperm over time may reveal novel metabolic requirements for sperm function. Post-thawed sperm from five samples of individual (A, B, C) and pooled bulls (AB, AC) were assessed at 0 and 24 hr after thawing. Sperm were evaluated for kinematics via Computer-Assisted Sperm Analyses and bioenergetic profiles using a Seahorse Analyzer for basal respiration [BR], mitochondrial stress test [MST] and energy map [EM]. Motility was nearly identical among samples after thawing and no differences in bioenergetics were detected. However, after 24 hr of sperm storage, pooled sperm samples (AC) presented with higher BR and proton leakage compared to other samples. Sperm kinematic variability among samples was higher after 24 hr, suggesting difference in sperm quality may manifest over time. Despite a reduction in motility and mitochondrial membrane potential, BR was higher at 24 hr compared to 0 hr for nearly all samples. A metabolic divergence between samples was detected by EM, indicating a shift in bioenergetic profiles over time that was undetected after thawing. These new bioenergetic profiles elucidate a novel dynamic plasticity of sperm metabolism over time while suggesting an influence of heterospermic interactions for further investigation.

SEX DIFFERENCES IN MITOCHONDRIAL RESILIENCE: EVIDENCE FROM BABOON HEPATOCYTES

Abstract Events that occur in utero set the trajectory for later-life diseases and longevity. Compelling data exist for interactions between developmental programming and aging, but the underlying mechanisms are not clearly defined. Fetal exposure to glucocorticoids (GC) is associated with alteration in hepatic enzymes and metabolic function in later life. We previously reported increased hepatic lipid accumulation and obese phenotype in middle-age male baboons exposed to GC as fetuses. The mitochondria play significant roles in cellular processes including stress responses and possibly a nexus between developmental programming and aging. The present study investigated the long-term effects of in utero GC exposure on mitochondrial bioenergetics using hepatocytes derived from aging baboons (16–18 years, average lifespan 21 years). Mitochondrial bioenergetics of both left and right lobe liver hepatocytes were examined as well as potential sex differences in mitochondrial function. Cell viability following isolation was similar among sexes and liver lobes but hepatocytes from males were highly energetic compared to females. Significant bioenergetic differences were observed in hepatocytes isolated from female baboons’ left and right liver lobes, with higher basal, maximal, and ATP-linked respiration in left lobe hepatocytes compared to the right lobe. These lobe-specific bioenergetic differences were absent in males. Interestingly, H2O2-induced oxidative stress significantly modified male baboon hepatocyte bioenergetics but females were unaffected, suggesting mitochondrial resilience in females compared to males. These data demonstrate that early life exposure to GC elicits a sex-specific effect on mitochondrial function. These mitochondrial differences might drive differences in cell senescence between males and females.

REGULATION OF NEURONAL MITOCHONDRIAL BIOENERGETICS BY LIPID METABOLITES UPREGULATED IN DEMENTIA

Abstract Mitochondrial dysfunction occurs early in Alzheimer’s disease (AD) progression and is evident in the Central Nervous System (CNS) and peripheral circulating cells. While there is evidence indicating that bioenergetic decline can drive the early pathogenesis of AD, little is known about the extracellular factors that alter neuronal bioenergetic capacity. We hypothesized that circulating metabolites contribute to neuronal bioenergetic decline associated with AD. Using a cohort comprised of participants with normal cognition, mild cognitive impairment, and dementia, we show that human serum harbors circulating, non-cellular factors capable of mediating neuronal bioenergetic differences according to the cognitive status of the serum donor. We developed a novel screening-based approach to identify candidate “mito-active” lipid metabolites in human serum that could mediate differences in bioenergetic capacity. Among these, Nervonic Acid and 15-epi-PGA1 were predicted to be mitochondrial inhibitors upregulated in participants with dementia. Neurons exposed to physiologically-relevant ranges of these molecules in-vitro exhibited a dose-dependent reduction in maximal mitochondrial respiration. We found that 500ug/mL of Nervonic Acid and 9ug/mL of 15-epi-PGA1 reduced maximal mitochondrial respiration by 62.3% and 63.3%, respectively, thereby validating our screening and prediction approach. Future experiments may be directed towards investigating if and how these and other mito-active metabolites of interest cross the blood-brain barrier to meaningfully affect AD. Furthermore, identified mito-active molecules can be investigated in other clinical cohorts to examine their role/s in multiple age-related or neurological conditions. This work expands our mechanistic understanding of how extrinsic factors associated with age and cognitive status contribute to neuronal bioenergetic decline.

REGULATION OF NEURONAL MITOCHONDRIAL BIOENERGETICS BY LIPID METABOLITES UPREGULATED IN DEMENTIA

Abstract Mitochondrial dysfunction occurs early in Alzheimer’s disease (AD) progression and is evident in the Central Nervous System (CNS) and peripheral circulating cells. While there is evidence indicating that bioenergetic decline can drive the early pathogenesis of AD, little is known about the extracellular factors that alter neuronal bioenergetic capacity. We hypothesized that circulating metabolites contribute to neuronal bioenergetic decline associated with AD. Using a cohort comprised of participants with normal cognition, mild cognitive impairment, and dementia, we show that human serum harbors circulating, non-cellular factors capable of mediating neuronal bioenergetic differences according to the cognitive status of the serum donor. We developed a novel screening-based approach to identify candidate “mito-active” lipid metabolites in human serum that could mediate differences in bioenergetic capacity. Among these, Nervonic Acid and 15-epi-PGA1 were predicted to be mitochondrial inhibitors upregulated in participants with dementia. Neurons exposed to physiologically-relevant ranges of these molecules in-vitro exhibited a dose-dependent reduction in maximal mitochondrial respiration. We found that 500ug/mL of Nervonic Acid and 9ug/mL of 15-epi-PGA1 reduced maximal mitochondrial respiration by 62.3% and 63.3%, respectively, thereby validating our screening and prediction approach. Future experiments may be directed towards investigating if and how these and other mito-active metabolites of interest cross the blood-brain barrier to meaningfully affect AD. Furthermore, identified mito-active molecules can be investigated in other clinical cohorts to examine their role/s in multiple age-related or neurological conditions. This work expands our mechanistic understanding of how extrinsic factors associated with age and cognitive status contribute to neuronal bioenergetic decline.

Plasma glycocholic acid and linoleic acid identified as potential mediators of mitochondrial bioenergetics in Alzheimer's dementia.

Mitochondrial bioenergetic alterations occur in the brain and peripheral cells of patients with Alzheimer’s disease (AD). This study focuses on plasma circulating factors, namely lipids, as mediators of systemic bioenergetic differences in participants with normal cognition (NC), mild cognitive impairment (MCI), and dementia due to probable AD (DEM). We examined bioenergetic differences across cognitive groups by measuring the mitochondrial respiration of peripheral blood mononuclear cells (PBMCs) from 37 participants (12 NC, 12 MCI, 13 DEM). PBMC bioenergetics were lower in the DEM group compared to the NC group. To determine whether circulating factors can mediate bioenergetic differences according to cognitive status, we exposed naïve neuronal Neuro-2a (N2a) cells to plasma from each participant in vitro. N2a bioenergetics were lower following plasma exposure from DEM compared to NC group participants. Notably, PBMC Max and N2a Max positively correlated, suggesting that circulating factors modulate the bioenergetics of naïve N2a cells according to the bioenergetic capacity of donor primary PBMCs. To identify lipid metabolites that may contribute to bioenergetic differences between cognitive groups, we performed liquid chromatography-mass spectrometry to assess the abundance of individual lipid species and correlated PBMC and N2a bioenergetics. Glycocholic acid (GCA) positively correlated with PBMC and N2a bioenergetics, while linoleic acid (LA) was negatively correlated. These data suggest that GCA and LA may contribute to the stimulatory and inhibitory bioenergetics effects related to cognitive status. Post hoc analyses revealed that GCA abundance was lower by 52.9% in the DEM group compared to the NC group and that LA abundance was higher by 55.7% in the DEM group compared to the NC group. To validate these findings, we examined the abundance of GCA and LA in the larger, more diverse, parent cohort (n = 378) and found similar results; GCA abundance was lower by 29.7% in the DEM group compared to the NC group and LA abundance was higher by 17.8% in the DEM group compared to the NC group. These data demonstrate that circulating factors have a direct effect on mitochondrial bioenergetics and that individual circulating factors identified to be associated with mitochondrial function are differentially expressed in patients with dementia.

Blood-based bioenergetic profiling reveals differences in mitochondrial function associated with cognitive performance and Alzheimer's disease.

Despite evidence for systemic mitochondrial dysfunction early in Alzheimer's disease (AD) pathogenesis, reliable approaches monitoring these key bioenergetic alterations are lacking. We used peripheral blood mononuclear cells (PBMCs) and platelets as reporters of mitochondrial function in the context of cognitive impairment and AD. Mitochondrial function was analyzed using complementary respirometric approaches in intact and permeabilized cells from older adults with normal cognition, mild cognitive impairment (MCI), and dementia due to probable AD. Clinical outcomes included measures of cognitive function and brain morphology. PBMC and platelet bioenergetic parameters were lowest in dementia participants. MCI platelets exhibited higher maximal respiration than normocognitives. PBMC and platelet respiration positively associated with cognitive ability and hippocampal volume, and negatively associated with white matter hyperintensities. Our findings indicate blood-based bioenergetic profiling can be used as a minimally invasive approach for measuring systemic bioenergetic differences associated with dementia, and may be used to monitor bioenergetic changes associated with AD risk and progression. Peripheral cell bioenergetic alterations accompanied cognitive decline in older adults with mild cognitive impairment (MCI) and Alzheimer's disease (AD) and related dementia (DEM). Peripheral blood mononuclear cells (PBMC) and platelet glucose-mediated respiration decreased in participants with dementia compared to normocognitive controls (NC). PBMC fatty-acid oxidation (FAO)-mediated respiration progressively declined in MCI and AD compared to NC participants, while platelet FAO-mediated respiration exhibited an inverse-Warburg effect in MCI compared to NC participants. Positive associations were observed between bioenergetics and Modified Preclinical Alzheimer's Cognitive Composite, and bioenergetics and hippocampal volume %, while a negative association was observed between bioenergetics and white matter hyperintensities. Systemic mitochondrial dysfunction is associated with cognitive decline.

Mitochondrial Phenotype as a Driver of the Racial Dichotomy in Obesity and Insulin Resistance.

African Americans (AA) are disproportionately burdened by metabolic diseases. While largely unexplored between Caucasian (C) and AA, differences in mitochondrial bioenergetics may provide crucial insight to mechanisms for increased susceptibility to metabolic diseases. AA display lower total energy expenditure and resting metabolic rate compared to C, but paradoxically have a higher amount of skeletal muscle mass, suggestive of inherent energetic efficiency differences between these races. Such adaptations would increase the chances of overnutrition in AA; however, these disparities would not explain the racial difference in insulin resistance (IR) in healthy subjects. Hallmarks associated with insulin resistance (IR), such as reduced mitochondrial oxidative capacity and metabolic inflexibility are present even in healthy AA without a metabolic disease. These adaptations might be influential of mitochondrial “substrate preference” and could play a role in disproportionate IR rates among races. A higher glycolytic flux and provision of shuttles transferring electrons from cytosol to mitochondrial matrix could be a contributing factor in development of IR via heightened reactive oxygen species (ROS) production. This review highlights the above concepts and provides suggestions for future studies that could help delineate molecular premises behind potential impairments in insulin signaling and metabolic disease susceptibility in AA.

Diurnal Differences in Mitochondrial Bioenergetics is Lost in Bmal1 Knockout Rats

In the kidney, 95% of O2 consumed through mitochondrial respiration is used for Na+ transport. The circadian clock regulates Na+ transport with a necessary diurnal variation for optimal health. The circadian clock transcription factor BMAL1 is proposed to regulate mitochondrial bioenergetic function; however, this has not been extensively investigated in the kidney. Therefore, we tested the hypothesis that renal mitochondrial respiration may be impaired or altered in a novel Bmal1 clock gene knock‐out rat model and is associated with loss of diurnal control of Na+ excretion. Male and female global Bmal1+/+ and Bmal1‐/‐ rats at 12‐14 weeks of age maintained in a regular 12‐hour light/dark cycle was used to measure renal mitochondrial O2consumption. Outer renal medullary tissue was dissected and prepared for respiration measurements at either day (ZT2‐4) or night (ZT14‐16) periods to correspond with minimum and maximum whole‐body energy consumption as well as peak and trough BMAL1 protein expression, respectively (n = 10, Bmal1+/+; n = 22, Bmal1‐/‐). Respiration was assessed using permeabilized tissue in the Oroboros Oxygraph and analyzed using Data Lab 2 software (O2K, Oroboros Instruments GmbH). Mitochondrial gene expression analysis was assessed using digital drop PCR. Data from both sexes were combined for each genotype at the two time points and analyzed by two‐way ANOVA. Mitochondrial state 3 O2 consumption was significantly higher (main effect of genotype p = 0.0310) in Bmal1‐/‐ (118 ± 156 pmol/s*mg) compared to BMAL1+/+ rats (75 ± 15 pmol/s*mg; Tukey post‐hoc p = 0.0303). We also observed a significant increase in complex IV activity in Bmal1‐/‐ rats during the dark vs. light period (164 ± 40 vs. 306 ± 40 pmol/s*mg; p=0.0031, t‐test). Mitochondrial‐associated genes, optic atrophy 1 (Opal1) and mitofusion 1 (Mfn1) show as significant day‐night difference in mRNA expression in kidneys from BMAL1+/+ control rats that is lost in Bmal1‐/‐ rats consistent with disrupted mitochondrial fusion and fission processes. Our lab previously reported that male Bmal1‐/‐ rats do not have the typical night‐day difference in Na+ excretion, which is consistent with our new data showing an alteration in mitochondrial function in kidneys from Bmal1‐/‐ rats. Therefore, our findings demonstrate that BMAL1 may play a role in maintaining the coupling of mitochondrial respiration and ATP generation in the kidney and support the hypothesis that diurnal Na+handling is regulated in part by BMAL1‐dependent mitochondrial function.

Key renal metabolic differences in healthy male and female Sprague Dawley rats

IntroductionUnderstanding and identifying differences in biological processes between males and females are critical for developing the sex‐based treatments for diseases. Mitochondrial dysfunction has been implemented in various kidney diseases yet baseline mitochondrial function and related sex differences within the kidney are not well established. Our previous data obtained in isolated renal mitochondria showed higher oxygen consumption and overall antioxidant capacity, and attenuated mitochondrial permeability transition in males. There is a pressing need for a more complete understanding of normal kidney function; to close this gap in knowledge, we hypothesized that divergent metabolic processes may influence the differences in mitochondrial bioenergetics and antioxidant capacity that we have previously identified.MethodsTissues were isolated from the kidneys collected from male and female Sprague Dawley (SD) rats at 11 weeks of age (Charles River labs, USA). The kidneys were perfused via the abdominal aorta with PBS/heparin solution to remove excess blood. Immediately following kidney collection, the cortex and medulla were isolated and snap frozen for metabolomic analysis. Metabolic profiles of renal cortices and medullae were generated using UHPLC‐HRMS, and metabolites were identified by retention time exact mass using MAVEN and MetaboAnalyst software. Ingenuity Pathway Analysis (IPA) was used to visualize and identify pathways of interests.Results. A total of 174 mitochondria‐focused metabolites were measured; to be considered significant, cutoffs of a 1.2 fold change (FC) with a corresponding p‐value of 0.05 were used. In the renal cortex and medulla, respectively, 22 and 38 metabolites were identified as increased or decreased in males compared to the females. Initial analysis demonstrated clear stratification of metabolite groups between male and female samples, as well as cortical and medullary tissues. Females exhibited upregulated metabolites implicated in protein and nucleic acid synthesis pathways such as UDP‐glucuronic acid (cortex/medulla, FC=1.796/1.615, p=0.013/<0.001), UDP‐D‐glucose (cortex/medulla, FC=1.415/1.594, p=<0.001/<0.001), guanosine(cortex/medulla, FC=1.290, p=0.018), and thymidine (cortex, FC=1.227, p=<0.001). Males, on the other hand, showed an upregulation of metabolites involved in the Kynurenine pathway (such as xanthurenic acid (cortex, FC=2.023, p=0.02), which is involved in NAD+ synthesis. Most interestingly, male kidneys also exhibited a major upregulation 5’‐methylthioadenosine (MTA) compared to females in both the cortex and the medulla (cortex/medulla, FC=41.88/62.88, p=0.002/0.004). MTA is involved in the polyamine metabolism and spermine/spermidine signaling, known to have antioxidant properties and attenuate diabetic kidney disease and AKI.ConclusionsWe report differences in metabolites in the cortex and medulla of young healthy male and female rats, primarily, related to their ability to utilize glucose and metabolize protein, and regulate spermine/spermidine pathway, which could explain differences in antioxidant capacity. The observed sex‐related dissimilarities indicate that male and female kidneys at least partially rely on different metabolic pathways, which opens up opportunities to develop new renal disease treatments targeting males and females separately.

Fish Specialize Their Metabolic Performance to Maximize Bioenergetic Efficiency in Their Local Environment: Conspecific Comparison Between Two Stocks of Pacific Chub Mackerel (Scomber japonicus)

Species-specific ecological traits in fishes are likely to vary between populations or stocks due to differences in regional oceanic conditions, such as latitudinal temperature. We examined potential intraspecific differences in the swimming performance and metabolism of Pacific chub mackerel (Scomber japonicus) from the Northwest and Northeast Pacific stocks, which are distributed on opposite sides of the North Pacific at similar latitudes, but where the temperature contrast is large. Swimming bioenergetics and metabolic data of Northwest stock mackerel were measured at 14, 18, and 24°C using variable-speed swim-tunnel respirometers, and then the resulting bioenergetic parameters were compared with previous findings from the Northeast stock. At a given size, the maximum sustainable swimming speed (Umax) of the Northwest stock showed no significant difference compared to the Northeast stock at 18 and 24°C, but was lower at 14°C. In addition, the oxygen consumption rate (MO2) of the Northwest stock showed lower mass dependence and different temperature dependence at a given swimming speed than in the Northeast stock. Combined with stock-specific data on growth and experienced temperatures in the wild, these bioenergetic differences indicate that the swimming performance and metabolism of the two stocks are specific to their local environment to maximize bioenergetic efficiency.