Uncoupled Mitochondrial Respiration Research Articles

Insulin sensitivity and skeletal muscle mitochondrial respiration in Black and White women with obesity.

Non-Hispanic black women (BW) have a greater risk of type 2 diabetes (T2D) and insulin resistance (IR) compared to non-Hispanic white women (WW). The mechanisms leading to these differences are not understood, and it is unclear whether synergistic effects of race and obesity impact disease risk. To understand the interaction of race and weight, hepatic and peripheral IR were compared in WW and BW with and without obesity. Hepatic and peripheral IR was measured by a labeled, hyperinsulinemic-euglycemic clamp in BW (n=32) and WW (n=32) with and without obesity. Measurements of body composition, cardiorespiratory fitness, and skeletal muscle (SM) respiration were completed. Data were analyzed by mixed model ANOVA. Subjects with obesity had greater hepatic and peripheral IR and lower SM respiration (P<0.001). Despite 14% greater insulin (P=0.066), BW tended to have lower peripheral glucose disposal (Rd; P=0.062), which was driven by women without obesity (P=0.002). BW had significantly lower glucose production (P=0.005), hepatic IR (P=0.024), and maximal coupled and uncoupled respiration (P<0.001) than WW. Maximal coupled and uncoupled SM mitochondrial respiration was strongly correlated with peripheral and hepatic IR (P<0.01). While BW without obesity had lower Rd than WW, race and obesity did not synergistically impact peripheral IR. Paradoxically, WW with obesity had greater hepatic IR compared to BW. Relationships between SM respiration and IR persisted across a range of body weight. These data provide support for therapies in BW, like exercise, that improve SM mitochondrial respiration to reduce IR and T2D risk.

#489 UCP1 regulates cardiolipin metabolism and mediates mitochondrial homeostasis maintenance of ANXA1 in diabetic nephropathy

Abstract Background and Aims Diabetic nephropathy (DN) is accounted for a large proportion of chronic kidney disease (CKD) and is the leading cause of end-stage renal disease (ESRD) nowadays. The complex pathogenesis of DN has hindered the development of a single effective medicine, so it is of great significance to seek new therapeutic targets. Uncoupling of mitochondrial respiration by chemical uncouplers has proven effective in ameliorating obesity, insulin resistance, and hyperglycemia, which were risk factors for DN. Recently, we found that ANXA1 could improve mitochondrial function to mitigate DN progression. However, the underlying mechanism is not fully clear yet. Method We used kidney samples from patients in our hospital. ANXA1-KO mice with local renal overexpression of UCP1 were constructed, UCP1-KO mice were constructed for STZ/HFD modeling, and db/db mice were treated with CL316243. UCP1-KO mice with renal overexpression of CRLS1 were constructed. Mice with PTECs-specific deletion of ANXA1 were constructed. Cell lines with UCP1/ANXA1/GATA3 knockdown and UCP1/ANXA1/GATA3 overexpression were constructed, and cell reversal tests were performed using Ac2-26, CL316243 and siRNA targeting the above genes. Results Here, we identified uncoupling protein 1 (UCP1), an inner membrane protein of mitochondria, as a key to mitochondrial homeostasis improved by ANXA1. Specifically, ANXA1 attenuated mitochondrial dysfunction via appropriately upregulating UCP1 by stabilizing its transcription factor GATA binding protein 3 (GATA3) through combining with thioredoxin. Moreover, specific overexpression of UCP1 in renal cortex rescued renal injuries in diabetic Anxa1-KO mice. UCP1 deletion aggravated renal injuries in HFD/STZ-induced diabetic mice. Mechanistically, UCP1 reduced mitochondrial fission through the aristaless-related homeobox (ARX)/cardiolipin synthase 1 (CRLS1) pathway. Therapeutically, CL316243, a UCP1 agonist, could attenuate established DN in db/db mice. Conclusion Thus, our data unraveled specific mechanism by which UCP1 mediated mitochondrial homeostasis maintenance via the ANXA1-GATA3-UCP1-ARX-CRLS1 signaling pathway. This work established a novel principle to harness the power of uncouplers for the treatment of DN.

Angiotensin II participates in mitochondrial thermogenic functions via the activation of glycolysis in chemically induced human brown adipocytes

Brown adipocytes are potential therapeutic targets for the prevention of obesity-associated metabolic diseases because they consume circulating glucose and fatty acids for heat production. Angiotensin II (Ang II) peptide is involved in the pathogenesis of obesity- and cold-induced hypertension; however, the mechanism underlying the direct effects of Ang II on human brown adipocytes remains unclear. Our transcriptome analysis of chemical compound-induced brown adipocytes (ciBAs) showed that the Ang II type 1 receptor (AGTR1), but not AGTR2 and MAS1 receptors, was expressed. The Ang II/AGTR1 axis downregulated the expression of mitochondrial uncoupling protein 1 (UCP1). The simultaneous treatment with β-adrenergic receptor agonists and Ang II attenuated UCP1 expression, triglyceride lipolysis, and cAMP levels, although cAMP response element-binding protein (CREB) phosphorylation was enhanced by Ang II mainly through the protein kinase C pathway. Despite reduced lipolysis, both coupled and uncoupled mitochondrial respiration was enhanced in Ang II-treated ciBAs. Instead, glycolysis and glucose uptake were robustly activated upon treatment with Ang II without a comprehensive transcriptional change in glucose metabolic genes. Elevated mitochondrial energy status induced by Ang II was likely associated with UCP1 repression. Our findings suggest that the Ang II/AGTR1 axis participates in mitochondrial thermogenic functions via glycolysis.

Mitochondrial flashes are interlinked with adaptive thermogenesis in brown adipocytes

Mitochondrial flash (mitoflash) is the electrochemical excitation of a single mitochondrion and plays diverse signaling roles in a variety of cells. From the viewpoint of bioenergetics, it represents the transient uncoupling of mitochondrial respiration from ATP synthesis. Brown adipose tissue (BAT) produces heat, primarily via mitochondrial uncoupled respiration. Here, we aim to illustrate the mitoflash activity in BAT thermogenesis. We show that BAT mitochondria undergo stochastic and intermittent mitoflashes at the levels of isolated mitochondria, cultured brown adipocytes, and even intact BAT. A BAT mitoflash contains multiple signals, including transient depolarization of mitochondrial membrane potential, transient matrix alkalization, and bursting production of reactive oxygen species. Importantly, thermogenic activation by β3-adrenergic stimulation dramatically augments mitoflash incidence, with mild effects on its unitary characteristics. Ablation of the thermogenic uncoupling protein 1 (UCP1) showed little effect on mitoflash biogenesis during heat production. Collectively, our results suggest that mitoflash might constitute an elemental signaling activity of mitochondria to regulate BAT thermogenesis.

Critical roles of tubular mitochondrial ATP synthase dysfunction in maleic acid-induced acute kidney injury.

Maleic acid (MA) induces renal tubular cell dysfunction directed to acute kidney injury (AKI). AKI is an increasing global health burden due to its association with mortality and morbidity. However, targeted therapy for AKI is lacking. Previously, we determined mitochondrial-associated proteins are MA-induced AKI affinity proteins. We hypothesized that mitochondrial dysfunction in tubular epithelial cells plays a critical role in AKI. In vivo and in vitro systems have been used to test this hypothesis. For the in vivo model, C57BL/6 mice were intraperitoneally injected with 400mg/kg body weight MA. For the in vitro model, HK-2 human proximal tubular epithelial cells were treated with 2mM or 5mM MA for 24h. AKI can be induced by administration of MA. In the mice injected with MA, the levels of blood urea nitrogen (BUN) and creatinine in the sera were significantly increased (p < 0.005). From the pathological analysis, MA-induced AKI aggravated renal tubular injuries, increased kidney injury molecule-1 (KIM-1) expression and caused renal tubular cell apoptosis. At the cellular level, mitochondrial dysfunction was found with increasing mitochondrial reactive oxygen species (ROS) (p < 0.001), uncoupled mitochondrial respiration with decreasing electron transfer system activity (p < 0.001), and decreasing ATP production (p < 0.05). Under transmission electron microscope (TEM) examination, the cristae formation of mitochondria was defective in MA-induced AKI. To unveil the potential target in mitochondria, gene expression analysis revealed a significantly lower level of ATPase6 (p < 0.001). Renal mitochondrial protein levels of ATP subunits 5A1 and 5C1 (p < 0.05) were significantly decreased, as confirmed by protein analysis. Our study demonstrated that dysfunction of mitochondria resulting from altered expression of ATP synthase in renal tubular cells is associated with MA-induced AKI. This finding provides a potential novel target to develop new strategies for better prevention and treatment of MA-induced AKI.

Western diet-induced obesity results in brain mitochondrial dysfunction in female Ossabaw swine.

Diet-induced obesity is implicated in the development of a variety of neurodegenerative disorders. Concurrently, the loss of mitochondrial Complex I protein or function is emerging as a key phenotype across an array of neurodegenerative disorders. Therefore, the objective of this study was to determine if Western diet (WD) feeding in swine [carbohydrate = 40.8% kCal (17.8% of total calories from high fructose corn syrup), protein = 16.2% kcal, fat = 42.9% kCal, and 2% cholesterol] would result in Complex I syndrome pathology. To characterize the effects of WD-induced obesity on brain mitochondria in swine, high resolution respirometry measurements from isolated brain mitochondria, oxidative phosphorylation Complex expression, and indices of oxidative stress and mitochondrial biogenesis were assessed in female Ossabaw swine fed a WD for 6-months. In line with Complex I syndrome, WD feeding severely reduced State 3 Complex I, State 3 Complex I and II, and uncoupled mitochondrial respiration in the hippocampus and prefrontal cortex (PFC). State 3 Complex I mitochondrial respiration in the PFC inversely correlated with serum total cholesterol. WD feeding also significantly reduced protein expression of oxidative phosphorylation Complexes I-V in the PFC. WD feeding significantly increased markers of antioxidant defense and mitochondrial biogenesis in the hippocampi and PFC. These data suggest WD-induced obesity may contribute to Complex I syndrome pathology by increasing oxidative stress, decreasing oxidative phosphorylation Complex protein expression, and reducing brain mitochondrial respiration. Furthermore, these findings provide mechanistic insight into the clinical link between obesity and mitochondrial Complex I related neurodegenerative disorders.

OR14-06 Serum Thyroid Hormones Increase At The Onset Of Lactation And Promote Metabolic Pathways Required For Human Milk Synthesis

Abstract Disclosure: H. Rostom: None. X. Meng: None. R. Humphrey: None. A. Fry: None. T. Elajnaf: None. F. Hannan: None. An increase in mammary metabolism is critical for initiating lactation during postpartum days 1-4. We utilised clinical and cellular approaches to investigate whether thyroid hormones, which promote lactation in rodents, are involved in initiating human lactation and regulating mammary metabolism. We recruited n=35 healthy pregnant women intending to breastfeed following informed consent and measured serum thyroid hormones (TSH and free T4) at 36 weeks’ gestation and on postpartum days 1-4 (1). Serum TSH significantly increased at post-partum day 1 compared with 36 weeks’ gestation (2.5±0.3 vs. 1.6±0.1 mU/L, p=0.001). This early postpartum rise in TSH was followed by a significant increase in free T4 on post-partum days 3 and 4 (11.7±0.2 vs. 10.5±0.2pmol/L at 36 weeks’ gestation, p&amp;lt;0.01). We hypothesised that thyroid hormones increase mammary metabolism to promote breast milk production. Reverse transcription-quantitative PCR (RT-qPCR) of genes encoding deiodinase enzymes (DIO1-3), which regulate intracellular thyroid hormone activity, was undertaken in lactating human mammary epithelial cells (HMECs) isolated from breast milk. This showed that DIO1, which converts T4 to T3, was expressed in lactating HMECs, whereas DIO2 and DIO3 were not detected. Moreover, RT-qPCR of the thyroid hormone receptor alpha and beta (THRA and THRB) genes in lactating HMECs showed that THRB has ∼4-fold greater expression than THRA (p&amp;lt;0.05, n=4). THRB phosphorylates Akt, which is required for initiating lactation. Consistent with this, cultured HMECs stimulated with 10nM T3, the most potent thyroid hormone, for 15min showed an ∼2-fold increase in Akt phosphorylation (p&amp;lt;0.0001, n=4). Akt regulates oxidative phosphorylation, which we assessed by measuring oxygen consumption rate (OCR) and ATP synthesis. HMECs treated with 10nM T3 for ≤8hrs showed a 2-fold increase in OCR (p&amp;lt;0.05, n=4) without any change in cellular ATP. These findings suggested that T3 uncoupled mitochondrial respiration from ATP synthesis. In support of this, RT-qPCR of cultured HMECs stimulated with 10nM T3 showed increased expression of genes encoding uncoupling proteins 2 and 3 (≥2-fold, p&amp;lt;0.01, n=4), which also function to divert substrates away from mitochondrial catabolism; and &amp;gt;20-fold increased expression of PPARGC1A (p&amp;lt;0.0001, n=4), which promotes mitochondrial biogenesis. In summary, these findings demonstrate that serum TSH and free T4 are increased at the onset of lactation. Moreover, our cellular studies indicate that thyroid hormones promote mammary mitochondrial biogenesis and may divert substrates used for ATP generation towards the synthesis of breast milk. Reference: (1) Rostom, H. et al. Protocol for an observational study investigating hormones triggering the onset of sustained lactation: the INSIGHT study. BMJ Open 12, e062478. Presentation: Friday, June 16, 2023

Scalable Generation of Pre-Vascularized and Functional Human Beige Adipose Organoids.

Obesity and type 2 diabetes are becoming a global sociobiomedical burden. Beige adipocytes are emerging as key inducible actors and putative relevant therapeutic targets for improving metabolic health. However, in vitro models of human beige adipose tissue are currently lacking and hinder research into this cell type and biotherapy development. Unlike traditional bottom-up engineering approaches that aim to generate building blocks, here a scalable system is proposed to generate pre-vascularized and functional human beige adipose tissue organoids using the human stromal vascular fraction of white adipose tissue as a source of adipose and endothelial progenitors. This engineered method uses a defined biomechanical and chemical environment using tumor growth factor β (TGFβ) pathway inhibition and specific gelatin methacryloyl (GelMA) embedding parameters to promote the self-organization of spheroids in GelMA hydrogel, facilitating beige adipogenesis and vascularization. The resulting vascularized organoids display key features of native beige adipose tissue including inducible Uncoupling Protein-1 (UCP1) expression, increased uncoupled mitochondrial respiration, and batokines secretion. The controlled assembly of spheroids allows to translate organoid morphogenesis to a macroscopic scale, generating vascularized centimeter-scale beige adipose micro-tissues. This approach represents a significant advancement in developing in vitro human beige adipose tissue models and facilitates broad applications ranging from basic research to biotherapies.

Adverse bioenergetic effects of N-acyl amino acids in human adipocytes overshadow beneficial mitochondrial uncoupling

ObjectiveEnhancing energy turnover via uncoupled mitochondrial respiration in adipose tissue has great potential to improve human obesity and other metabolic complications. However, the amount of human brown adipose tissue and its uncoupling protein 1 (UCP1) is low in obese patients. Recently, a class of endogenous molecules, N-acyl amino acids (NAAs), was identified as mitochondrial uncouplers in murine adipocytes, presumably acting via the adenine nucleotide translocator (ANT). Given the translational potential, we investigated the bioenergetic effects of NAAs in human adipocytes, characterizing beneficial and adverse effects, dose ranges, amino acid derivatives and underlying mechanisms. MethodNAAs with neutral (phenylalanine, leucine, isoleucine) and polar (lysine) residues were synthetized and assessed in intact and permeabilized human adipocytes using plate-based respirometry. The Seahorse technology was applied to measure bioenergetic parameters, dose-dependency, interference with UCP1 and adenine nucleotide translocase (ANT) activity, as well as differences to the established chemical uncouplers niclosamide ethanolamine (NEN) and 2,4-dinitrophenol (DNP). ResultNAAs with neutral amino acid residues potently induce uncoupled respiration in human adipocytes in a dose-dependent manner, even in the presence of the UCP1-inhibitor guanosine diphosphate (GDP) and the ANT-inhibitor carboxyatractylate (CAT). However, neutral NAAs significantly reduce maximal oxidation rates, mitochondrial ATP-production, coupling efficiency and reduce adipocyte viability at concentrations above 25 μM. The in vitro therapeutic index (using induced proton leak and viability as determinants) of NAAs is lower than that of NEN and DNP. ConclusionNAAs are potent mitochondrial uncouplers in human adipocytes, independent of UCP1 and ANT. However, previously unnoticed adverse effects harm adipocyte functionality, reduce the therapeutic index of NAAs in vitro and therefore question their suitability as anti-obesity agents without further chemical modifications.

Apical and mechanistic effects of 6PPD-quinone on different life-stages of the fathead minnow (Pimephales promelas)

N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine-quinone (6PPD-quinone) is an emerging contaminant of concern that is generated through the environmental oxidation of the rubber tire anti-degradant 6PPD. Since the initial report of 6PPD-quinone being the cause of urban runoff mortality syndrome of Coho salmon, numerous species have been identified as either sensitive or insensitive to acute lethality caused by 6PPD-quinone. In sensitive species, acute lethality might be caused by uncoupling of mitochondrial respiration in gills. However, little is known about effects of 6PPD-quinone on insensitive species. Here we demonstrate that embryos of fathead minnows (Pimephales promelas) are insensitive to exposure to concentrations as great as 39.97 μg/L for 168 h, and adult fathead minnows are insensitive to exposure to concentrations as great as 9.4 μg/L for 96 h. A multi-omics approach using a targeted transcriptomics array, (EcoToxChips), and proton nuclear magnetic resonance (1H NMR) was used to assess responses of the transcriptomes and metabolomes of gills and livers from adult fathead minnows exposed to 6PPD-quinone for 96 h to begin to identify sublethal effects of 6PPD-quinone. There was little agreement between results of the EcoToxChip and metabolomics analyses, likely because genes present on the EcoToxChip were not representative of pathways suggested to be perturbed by metabolomic analysis. Changes in abundances of transcripts and metabolites in livers and gills suggest that disruption of one‑carbon metabolism and induction of oxidative stress might be occurring in gills and livers, but that tissues differ in their sensitivity or responsiveness to 6PPD-quinone. Overall, several pathways impacted by 6PPD-quinone were identified as candidates for future studies of potential sublethal effects of this chemical.

UNCOUPLING PROTEIN UCP1 EXPRESSION DYNAMICS IN ADIPOSE TISSUES OF THE OUTBRED ICR MICE IN POSTNATAL ONTOGENESIS

Uncoupling protein (UCP1) uncouples mitochondrial respiration from ATP synthesis, resulting in heat production in brown and beige adipocytes. The presence of adipocytes with UCP1 expression in fat depots has been shown to promote metabolic health and provide protection against metabolic disorders. It stimulates interest in studying the age dynamics of UCP1 expression. There are few data available, mainly obtained on the C57Bl/6J mouse line predisposed to obesity and cover either early or late ontogenesis. In our study, for the first time, the expression of the UCP1 protein in the adipose tissues of male ICR mice was studied from the weaning to old age. Interscapular brown adipose tissue (BAT), inguinal and perigonadal white adipose tissue (IWAT and GWAT) of 20-day, 1.5, 6, 18 months mice were collected. UCP1 levels were detected by western-blotting. IWAT UCP1 expression decreased by 2 times between 20 days and 1.5 months. No UCP1 bands on blots from mice older than 1.5 months were observed. In gonadal depot UCP1 was detected only in 30% of the samples from 1.5- and 6‑months old mice, and UCP1 expression level was ten times lower in compare to inguinal depot. No statistically significant changes in UCP1 protein expression were detected in brown adipose tissue. The physiological role of UCP1-expressing cells in GWAT is discussed, as well as a possible relationship between the timing and rate of UCP1 expression decrease during the growth and maturation of reproductive function with the activation of lipogenesis in inguinal adipose tissue.

Invasive and noninvasive markers of human skeletal muscle mitochondrial function.

Mitochondria are organelles that fuel cellular energy requirements by ATP formation via aerobic metabolism. Given the wide variety of methods to assess skeletal muscle mitochondrial capacity, we tested how well different invasive and noninvasive markers of skeletal muscle mitochondrial capacity reflect mitochondrial respiration in permeabilized muscle fibers. Nineteen young men (mean age: 24 ± 4 years) were recruited, and a muscle biopsy was collected to determine mitochondrial respiration from permeabilized muscle fibers and to quantify markers of mitochondrial capacity, content such as citrate synthase (CS) activity, mitochondrial DNA copy number, TOMM20, VDAC, and protein content for complex I-V of the oxidative phosphorylation (OXPHOS) system. Additionally, all participants underwent noninvasive assessments of mitochondrial capacity: PCr recovery postexercise (by 31 P-MRS), maximal aerobic capacity, and gross exercise efficiency by cycling exercise. From the invasive markers, Complex V protein content and CS activity showed the strongest concordance (Rc = 0.50 to 0.72) with ADP-stimulated coupled mitochondrial respiration, fueled by various substrates. Complex V protein content showed the strongest concordance (Rc = 0.72) with maximally uncoupled mitochondrial respiration. From the noninvasive markers, gross exercise efficiency, VO2max , and PCr recovery exhibited concordance values between 0.50 and 0.77 with ADP-stimulated coupled mitochondrial respiration. Gross exercise efficiency showed the strongest concordance with maximally uncoupled mitochondrial respiration (Rc = 0.67). From the invasive markers, Complex V protein content and CS activity are surrogates that best reflect skeletal muscle mitochondrial respiratory capacity. From the noninvasive markers, exercise efficiency and PCr recovery postexercise most closely reflect skeletal muscle mitochondrial respiratory capacity.

Can exercise prevent the age‐related decline in adaptive homeostasis? Evidence across organisms and tissues

Can exercise prevent the age‐related decline in adaptive homeostasis? Evidence across organisms and tissues

High fat diet feeding induces brain mitochondrial dysfunction in female Ossabaw swine

Study objective: Emerging evidence now suggest reduced mitochondrial function as a key initiator in Alzheimer’s disease (AD)-like pathology increasing the need for models of AD that display mitochondrial dysfunction. Dyslipidemia is a common aliment from overconsumption of a high fat diet (HFD) that increases the risk of AD and may contribute to mitochondrial dysfunction. Rodent studies utilize HFDs to induce brain pathology resembling that of AD. Unsurprisingly, rodent studies are rarely recapitulated in humans, which leads to a very low clinical success rate and highlights the need for more translatable models to study AD-like pathology. Swine seem to share many characteristics of humans and might offer a more translatable model to study HFD induced pathology. Thus, the objective of this study was to determine if HFD feeding in swine could offer a more translatable model to study mitochondrial dysfunction during neurodegeneration. Hypothesis: 6-months of HFD feeding will decrease indices of mitochondrial function and biogenesis in female Ossabaw swine. Methods: To characterize the effects of a HFD on brain mitochondrial health in a highly translatable swine model, high resolution respirometry measurements were taken from isolated brain mitochondria from in female Ossabaw swine fed a HFD for 6-months. Additionally, indices of mitochondrial biogenesis in whole tissue was measured (transcriptional markers of biogenesis and citrate synthase activity). Results: HFD feeding significantly increased body weight (p&lt;0.05) with trending increases in blood glucose (p&lt;0.10). Maximal uncoupled mitochondrial respiration was significantly decreased (p&lt;0.05) with state 3-complex I and state 3 complex I &amp; II trending down in the dorsal hippocampus (DH) after HFD diet feeding (p&lt;0.10). Additionally, state 2 respiration was significantly decreased in the prefrontal cortex (PFC) after HFD feeding (p&lt;0.05). HFD feeding increased transcriptional markers of mitochondrial biogenesis in the DH and PFC (p&lt;0.05), without altering whole homogenate citrate synthase activity in either brain region. Conclusions: These data indicate HFD feeding in Ossabaw pigs could offer a potential model to study pathologies relating to mitochondrial dysfunction during neurodegenerative process. Additionally, the DH was found to be more vulnerable to HFD-induced perturbations, evident by the exacerbated deficits in mitochondrial respiration in this brain region. Finally, these data suggest HFD-induced increase in transcriptional markers of mitochondrial biogenesis may be a compensatory response to HFD-induced mitochondrial respiration deficits. However, citrate synthase activity was not different between groups indicating this upregulation of transcriptional markers was unable to translate to an increase in mitochondrial content in the DH and PFC. The project was supported by the MU Office of Postdoctoral Education Research Grant Program. Additional funding was also generously provided Scott Rector. Images in figure 2 adapted and modified from “Krebs cycle template”, by BioRender.com (2022). Retrieved from https://app.biorender.com/biorender-templates . This work was also supported with resources and the use of facilities at the University of Missouri and Harry S. Truman Memorial Veterans Hospital in Columbia, Missouri. This is the full abstract presented at the American Physiology Summit 2023 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.

Phytochemical compound PB125 attenuates skeletal muscle mitochondrial dysfunction and impaired proteostasis in a model of musculoskeletal decline.

Impaired mitochondrial function and disrupted proteostasis contribute to musculoskeletal dysfunction. However, few interventions simultaneously target these two drivers to prevent musculoskeletal decline. Nuclear factor erythroid 2-related factor 2 (Nrf2) activates a transcriptional programme promoting cytoprotection, metabolism, and proteostasis. We hypothesized daily treatment with a purported Nrf2 activator, PB125, in Hartley guinea pigs, a model of musculoskeletal decline, would attenuate the progression of skeletal muscle mitochondrial dysfunction and impaired proteostasis and preserve musculoskeletal function. We treated 2- and 5-month-old male and female Hartley guinea pigs for 3 and 10months, respectively, with the phytochemical compound PB125. Longitudinal assessments of voluntary mobility were measured using Any-MazeTM open-field enclosure monitoring. Cumulative skeletal muscle protein synthesis rates were measured using deuterium oxide over the final 30days of treatment. Mitochondrial oxygen consumption in soleus muscles was measured using high resolution respirometry. In both sexes, PB125 (1) increased electron transfer system capacity; (2) attenuated the disease/age-related decline in coupled and uncoupled mitochondrial respiration; and (3) attenuated declines in protein synthesis in the myofibrillar, mitochondrial and cytosolic subfractions of the soleus. These effects were not associated with statistically significant prolonged maintenance of voluntary mobility in guinea pigs. Collectively, treatment with PB125 contributed to maintenance of skeletal muscle mitochondrial respiration and proteostasis in a pre-clinical model of musculoskeletal decline. Further investigation is necessary to determine if these documented effects of PB125 are also accompanied by slowed progression of other aspects of musculoskeletal dysfunction. KEY POINTS: Aside from exercise, there are no effective interventions for musculoskeletal decline, which begins in the fifth decade of life and contributes to disability and cardiometabolic diseases. Targeting both mitochondrial dysfunction and impaired protein homeostasis (proteostasis), which contribute to the age and disease process, may mitigate the progressive decline in overall musculoskeletal function (e.g. gait, strength). A potential intervention to target disease drivers is to stimulate nuclear factor erythroid 2-related factor 2 (Nrf2) activation, which leads to the transcription of genes responsible for redox homeostasis, proteome maintenance and mitochondrial energetics. Here, we tested a purported phytochemical Nrf2 activator, PB125, to improve mitochondrial function and proteostasis in male and female Hartley guinea pigs, which are a model for musculoskeletal ageing. PB125 improved mitochondrial respiration and attenuated disease- and age-related declines in skeletal muscle protein synthesis, a component of proteostasis, in both male and female Hartley guinea pigs.

β-Adrenergic Receptors and Adipose Tissue Metabolism: Evolution of an Old Story.

The role of β-adrenergic receptors (βARs) in adipose tissue to promote lipolysis and the release of fatty acids and nonshivering thermogenesis in brown fat has been studied for so many decades that one would think there is nothing left to discover. With the rediscovery of brown fat in humans and renewed interest in UCP1 and uncoupled mitochondrial respiration, it seems that a review of adipose tissue as an organ, pivotal observations, and the investigators who made them would be instructive to understanding where the field stands now. The discovery of the β3-adrenergic receptor was important for accurately defining the pharmacology of the adipocyte, while the clinical targeting of this receptor for obesity and metabolic disease has had its highs and lows. Many questions still remain about how βARs regulate adipocyte metabolism and the signaling molecules through which they do it.

Co-Evolution of Breast Milk Lipid Signaling and Thermogenic Adipose Tissue.

Breastfeeding is a unique and defining behavior of mammals and has a fundamental role in nourishing offspring by supplying a lipid-rich product that is utilized to generate heat and metabolic fuel. Heat generation from lipids is a feature of newborn mammals and is mediated by the uncoupling of mitochondrial respiration in specific fat depots. Breastfeeding and thermogenic adipose tissue have a shared evolutionary history: both have evolved in the course of homeothermy evolution; breastfeeding mammals are termed “thermolipials”, meaning “animals with warm fat”. Beyond its heat-producing capacity, thermogenic adipose tissue is also necessary for proper lipid metabolism and determines adiposity in offspring. Recent advances have demonstrated that lipid metabolism in infants is orchestrated by breast milk lipid signals, which establish mother-to-child signaling and control metabolic development in the infant. Breastfeeding rates are declining worldwide, and are paralleled by an alarming increase in childhood obesity, which at least in part may have its roots in the impaired metabolic control by breast milk lipid signals.

Combining Arsenic Trioxide and Mitocans Selectively Disrupts Cellular Energetics in Acute Myeloid Leukemia

In our earlier work with arsenic trioxide (ATO) resistance in acute promyelocytic leukemia (APL), we observed that ATO resistant cells reprogrammed their metabolism from glycolysis to oxidative phosphorylation (OXPHOS) as a mechanism of resistance. We further demonstrated that it could be overcome by targeting this metabolic switch using FCCP (mitocan) in combination with ATO (Balasundaram N et al. Biorxiv 2020). There is increasing evidence that acute myeloid leukemia (AML) cells have a greater metabolic plasticity unlike ATO resistant APL cells and most cancers that rely on glycolysis. AML leukemic stem cells preferentially utilize OXPHOS for their survival (Lagadinou ED et al. Cell stem cell 2013).Mitocans like venetoclax used in combination with hypo-methylating agents are already well established in the management of AML (Pollyea D, et al. Nat Med 2021). ATO is also an effective glycolytic inhibitor (Zhang H, et al. PNAS 2015) hence we hypothesized that a combination of ATO and mitocans could potentially target the metabolic plasticity of AML cells. As the combination of ATO and FCCP was found to be non-specific we performed a small-scale screening on an AML cell line (U937) using FDA-approved compounds that are reported to target mitochondria (Gohil V et al. Nature Biotechnology, 2010). Though most of the mitocans showed predicted synergy with ATO. We focused on artesunate (ART) as a candidate for further evaluation due to its specificity for malignant cells, high therapeutic index, bioavailability, route of administration, cost-effectiveness, and global usage as an antimalarial.The combination of ATO+ART significantly reduced the viability of different subtypes of AML cell lines (THP-1, MV4:11, and Kasumi-1) and acute lymphoblastic leukemia cell lines (Jurkat E6.1, SUP B15, and MOLT-4) with minimal effect on the normal cells (CD34 and peripheral blood mononuclear cells; n=10; 48hours) (figure 1a). We noted that the selective specificity of the combination was primarily due to the iron metabolism of the leukemic cells and a requirement of iron for the activity of ART. When an iron chelator deferoxamine (DFO) was used in combination with ATO+ART there was a significant reduction in the activity of the combination on the AML cells (Figure 1b, U937; n=10; 48hours). Seahorse extracellular flux analysis validated that ART (5uM) as a single agent promoted uncoupled mitochondrial respiration and the addition of ATO resulted in a metabolic catastrophe (figure 1c and d).Chemical drug proteomic analysis using biotinylated artesunate and pull down from the leukemic cells revealed that the top interacting partners were localized in the mitochondria. We also noted that ART treatment significantly affected the mitochondrial dynamics of leukemic cells, where ART and ATO+ART treated cells had fragmented mitochondria in comparison to the control and ATO alone treated cells where the mitochondria were more elongated (figure 1e, U937 cells; n=3). We evaluated the effect of ATO+ART and their combination with azacytidine (triplet) in-vitro. Dual and triple combinations showed greater toxicity on AML cell lines and primary AML cells (Figure 1f, n=50) in comparison to the normal peripheral blood mononuclear cells (PBMNCs) and normal CD34+ cells. Taken together, these findings highlight the selective specificity of these combinations and its clinical potential in AML. [Display omitted] DisclosuresAugustin: Christian Medical College: Patents & Royalties: US 2020/0345770 A1 - Pub.Date Nov.5, 2020; AML: Other: Co-Inventor. Krishna: Christian Medical College: Patents & Royalties: US 2020/0345770 A1 - Pub.Date Nov.5, 2020; KCM Vellore: Patents & Royalties; SGUL: Patents & Royalties; AML: Other: Co-Inventor. Mathews: Christian Medical College: Patents & Royalties: US 2020/0345770 A1 - Pub.Date Nov.5, 2020; AML: Other: Co-Inventor.

Cold-induced Conversion of Connective Tissue Skeleton in Brown Adipose Tissues.

Thermogenesis via fatty acid-induced uncoupled mitochondrial respiration is the primary function of brown adipose tissue (BAT). In response to changes in ambient temperatures, the weight and specific gravity of BAT change, depending on the quantity of lipid droplets stored in brown adipocytes (BA). Such conditions should result in the reconstruction of connective tissue skeletons, especially of collagen fiber networks, although the mechanisms have not been clarified. This study showed that, within 4 hr of exposing mice to a cold environment, collagen fibers in the extracellular matrix (ECM) of BAT became discontinuous, twisted, emancipated, and curtailed. Surprisingly, the structure of collagen fibers returned to normal after the mice were kept at room temperature for 19 hr, indicating that the alterations in collagen fiber structures are physiological processes association with adaptation to cold environments. These dynamic changes in connective tissue skeletons were not observed in white adipose tissues, suggesting that they are unique to BAT. Interestingly, the vascular permeability of BAT was also augmented by exposure to cold. Collectively, these findings indicate that dynamic changes in ECM collagen fibers provide high flexibility to BAT, enabling the adjustment of tissue structures and the regulation of vascular permeability, resulting in adaptation to changes in ambient temperatures.

NAD+-Precursor Supplementation With L-Tryptophan, Nicotinic Acid, and Nicotinamide Does Not Affect Mitochondrial Function or Skeletal Muscle Function in Physically Compromised Older Adults

BackgroundBoosting NAD+ via supplementation with niacin equivalents has been proposed as a potential modality capable of promoting healthy aging and negating age-dependent declines of skeletal muscle mass and function. ObjectivesWe investigated the efficacy of NAD+-precursor supplementation (tryptophan, nicotinic acid, and nicotinamide) on skeletal muscle mitochondrial function in physically compromised older adults. MethodsA randomized, double-blind, controlled trial was conducted in 14 (female/male: 4/10) community-dwelling, older adults with impaired physical function [age, 72.9 ± 4.0 years; BMI, 25.2 ± 2.3 kg/m2]. Participants were supplemented with 207.5 mg niacin equivalents/day [intervention (INT)] and a control product (CON) that did not contain niacin equivalents, each for 32 days. The primary outcomes tested were mitochondrial oxidative capacity and exercise efficiency, analyzed by means of paired Student's t-tests. Secondary outcomes, such as NAD+ concentrations, were analyzed accordingly. ResultsFollowing supplementation, skeletal muscle NAD+ concentrations [7.5 ± 1.9 compared with 7.9 ± 1.6 AU, respectively] in INT compared with CON conditions were not significantly different compared to the control condition, whereas skeletal muscle methyl-nicotinamide levels were significantly higher under NAD+-precursor supplementation [INT, 0.098 ± 0.063 compared with CON, 0.025 ± 0.014; P = 0.001], suggesting an increased NAD+ metabolism. Conversely, neither ADP-stimulated [INT, 82.1 ± 19.0 compared with CON, 84.0 ± 19.2; P = 0.716] nor maximally uncoupled mitochondrial respiration [INT, 103.4 ± 30.7 compared with CON, 108.7 ± 33.4; P = 0.495] improved under NAD+-precursor supplementation, nor did net exercise efficiency during the submaximal cycling test [INT, 20.2 ± 2.77 compared with CON, 20.8 ± 2.88; P = 0.342]. ConclusionsOur findings are consistent with previous findings on NAD+ efficacy in humans, and we show in community-dwelling, older adults with impaired physical function that NAD+-precursor supplementation through L-tryptophan, nicotinic acid, and nicotinamide does not improve mitochondrial or skeletal muscle function. This study was registered at clinicaltrials.gov as NCT03310034.