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Beyond glycemic control, SGLT2 inhibitors (SGLT2is) have protective effects on cardiorenal function. Renoprotection has been suggested to involve inhibition of NHE3 leading to reduced ATP-dependent tubular workload and mitochondrial oxygen consumption. NHE3 activity is also important for regulation of endosomal pH, but the effects of SGLT2i on endocytosis are unknown. We used a highly differentiated cell culture model of proximal tubule (PT) cells to determine the direct effects of SGLT2i on Na+-dependent fluid transport and endocytic uptake in this nephron segment. Strikingly, canagliflozin but not empagliflozin reduced fluid transport across cell monolayers and dramatically inhibited endocytic uptake of albumin. These effects were independent of glucose and occurred at clinically relevant concentrations of drug. Canagliflozin acutely inhibited surface NHE3 activity, consistent with a direct effect, but did not affect endosomal pH or NHE3 phosphorylation. In addition, canagliflozin rapidly and selectively inhibited mitochondrial complex I activity. Inhibition of mitochondrial complex I by metformin recapitulated the effects of canagliflozin on endocytosis and fluid transport, whereas modulation of downstream effectors AMPK and mTOR did not. Mice given a single dose of canagliflozin excreted twice as much urine over 24 h compared with empagliflozin-treated mice despite similar water intake. We conclude that canagliflozin selectively suppresses Na+-dependent fluid transport and albumin uptake in PT cells via direct inhibition of NHE3 and of mitochondrial function upstream of the AMPK/mTOR axis. These additional targets of canagliflozin contribute significantly to reduced PT Na+-dependent fluid transport in vivo. NEW & NOTEWORTHY: Reduced NHE3-mediated Na+ transport has been suggested to underlie the cardiorenal protection provided by SGLT2 inhibitors. We found that canagliflozin, but not empagliflozin, reduced NHE3-dependent fluid transport and endocytic uptake in cultured proximal tubule cells. These effects were independent of SGLT2 activity and resulted from inhibition of mitochondrial complex I and NHE3. Studies in mice are consistent with greater effects of canagliflozin versus empagliflozin on fluid transport. Our data suggest that these selective effects of canagliflozin contribute to reduced Na+-dependent transport in proximal tubule cells. [ABSTRACT FROM AUTHOR]

Background: Thermogenic beige adipocytes, which dissipate energy as heat, are found in neonates and adults. Recent studies show that neonatal beige adipocytes are highly plastic and contribute to >50% of beige adipocytes in adults. Neonatal beige adipocytes are distinct from recruited beige adipocytes in that they develop independently of temperature and sympathetic innervation through poorly defined mechanisms. Methods: We characterized the neonatal beige adipocytes in the inguinal white adipose tissue (iWAT) of C57BL6 postnatal day 3 and 20 mice (P3 and P20) by imaging, genome-wide RNA-seq analysis, ChIP-seq analysis, qRT-PCR validation, and biochemical assays. Results: We found an increase in acetylated histone 3 lysine 27 (H3K27ac) on the promoter and enhancer regions of beige-specific gene UCP1 in iWAT of P20 mice. Furthermore, H3K27ac ChIP-seq analysis in the iWAT of P3 and P20 mice revealed strong H3K27ac signals at beige adipocyte-associated genes in the iWAT of P20 mice. The integration of H3K27ac ChIP-seq and RNA-seq analysis in the iWAT of P20 mice reveal epigenetically active signatures of beige adipocytes, including oxidative phosphorylation and mitochondrial metabolism. We identify the enrichment of GA-binding protein alpha (GABPα) binding regions in the epigenetically active chromatin regions of the P20 iWAT, particularly on beige genes, and demonstrate that GABPα is required for beige adipocyte differentiation. Moreover, transcriptomic analysis and glucose oxidation assays revealed increased glycolytic activity in the neonatal iWAT from P20. Conclusions: Our findings demonstrate that epigenetic mechanisms regulate the development of peri-weaning beige adipocytes via GABPα. Further studies to better understand the upstream mechanisms that regulate epigenetic activation of GABPα and characterization of the metabolic identity of neonatal beige adipocytes will help us harness their therapeutic potential in metabolic diseases. [ABSTRACT FROM AUTHOR]

Background: Mitochondrial energetics are an important property of aging muscle, as generation of energy is pivotal to the execution of muscle contraction. However, its association with functional outcomes, including leg power and cardiorespiratory fitness is largely understudied.Methods: In the Study of Muscle, Mobility, and Aging (SOMMA), we collected vastus lateralis biopsies from older adults (n=879,70-94 years,59.2% women). Maximal state 3 respiration (Max OXPHOS) was assessed in permeabilized fiber bundles by high-resolution respirometry. Capacity for maximal adenosine triphosphate production (ATPmax) was measured in vivo by 31P magnetic resonance spectroscopy. Leg extension power was measured with a Keiser press system, and VO2 peak was determined using a standardized cardiopulmonary exercise test. Gender-stratified multivariate linear regression models were adjusted for age, race, technician/site, adiposity, and physical activity with beta-coefficients expressed per 1 SD increment in the independent variable.Results: Max OXPHOS was associated with leg power for both women (β=0.12Watts/kg,p<0.001) and men (β=0.11Watts/kg,p<0.050). ATPmax was associated with leg power for men (β=0.09Watts/kg p<0.05) but was not significant for women (β=0.03Watts/kg,p=0.11). Max OXPHOS and ATPmax were associated with VO2 peak in women and men (Max OXPHOS, βwomen=1.03mL/kg/min, βmen=1.32 mL/kg/min; ATPmax βwomen=0.87mL/kg/min, βmen=1.50mL/kg/min;all p<0.001).Conclusions: Higher muscle mitochondrial energetics measures were associated with both better cardiorespiratory fitness and greater leg power in older adults. Muscle mitochondrial energetics explained a greater degree of variance in VO2 peak compared to leg power. [ABSTRACT FROM AUTHOR]

The prevalence of cardiometabolic heart failure with preserved ejection fraction (HFpEF) continues to grow, representing over half of heart failure cases in the United States. As no specific medication for HFpEF exists, treatment guidelines focus on the management of comorbidities related to metabolic syndrome (e.g. obesity, diabetes, hypertension) that promote the disease. These same comorbidities also drive pathology in non-cardiac tissues, and the links between cardiometabolic disease presentations in different organs are increasingly being recognized. Preclinical studies on the potential crosstalk between HFpEF and metabolic disease in the liver (e.g. metabolic-associated fatty liver disease; MAFLD) have focused on how liver dysfunction may affect the heart, particularly through the release of secreted liver proteins. This may reflect the situation in the clinic, where incident MAFLD is a risk factor for future HFpEF development. Here, in contrast to this developing paradigm of liver-initiated cardiac disease, we report for the first time a defect in cardiac metabolism related to the mitochondrial metabolic protein GCN5L1 that drives hepatic steatosis and MAFLD in HFpEF., Competing Interests: The authors have declared that no conflict of interest exists.

Proteostasis is vital for cellular health, with disruptions leading to pathologies including aging, neurodegeneration and metabolic disorders. Traditionally, proteotoxic stress responses were studied as acute reactions to various noxious factors; however, recent evidence reveals that many proteostasis stress-response genes exhibit ~12-hour ultradian rhythms under physiological conditions in mammals. These rhythms, driven by an XBP1s-dependent 12h oscillator, are crucial for managing proteostasis. By exploring the chromatin landscape of the murine 12h hepatic oscillator, we identified RBBP5, a key subunit of the COMPASS complex writing H3K4me3, as an essential epigenetic regulator of proteostasis. RBBP5 is indispensable for regulating both the hepatic 12h oscillator and transcriptional response to acute proteotoxic stress, acting as a co-activator for proteostasis transcription factor XBP1s. RBBP5 ablation leads to increased sensitivity to proteotoxic stress, chronic inflammation, and hepatic steatosis in mice, along with impaired autophagy and reduced cell survival in vitro . In humans, lower RBBP5 expression is associated with reduced adaptive stress-response gene expression and hepatic steatosis. Our findings establish RBBP5 as a central regulator of proteostasis, essential for maintaining mammalian organismal health., Competing Interests: Disclosure and competing interests: The authors have no competing interests to declare.

Objective: Examine the association of ectopic adipose tissue (AT) with skeletal muscle (SM) mitochondrial bioenergetics in older adults., Methods: Cross-sectional data from 829 older adults ≥70 years was used. Total abdominal, subcutaneous, and visceral AT; and thigh muscle fat infiltration (MFI) was quantified by MRI. SM mitochondrial energetics were characterized using in vivo 31 P-MRS (ATP max ) and ex vivo high-resolution respirometry (maximal oxidative phosphorylation (OXPHOS)). ActivPal was used to measure PA (step count). Linear regression models adjusted for covariates were applied, with sequential adjustment for BMI and PA., Results: Independent of BMI, total abdominal (standardized (Std.) β=-0.21; R 2 =0.09) and visceral AT (Std. β=-0.16; R 2 =0.09) were associated with ATP max ( p <0.01), but not after further adjustment for PA (p≥0.05). Visceral AT (Std. β=-0.16; R 2 =0.25) and thigh MFI (Std. β=-0.11; R 2 =0.24) were negatively associated with carbohydrate-supported maximal OXPHOS independent of BMI and PA ( p <0.05). Total abdominal AT (Std. β=-0.19; R 2 =0.24) and visceral AT (Std. β=-0.17; R 2 =0.24) were associated with fatty acid-supported maximal OXPHOS independent of BMI and PA (p<0.05)., Conclusions: Skeletal MFI and abdominal visceral, but not subcutaneous AT, are inversely associated with SM mitochondrial bioenergetics in older adults independent of BMI. Associations between ectopic AT and in vivo mitochondrial bioenergetics are attenuated by PA.

Rationale: Cardiorespiratory fitness and mitochondrial energetics are associated with reduced walking speed in older adults. The impact of cardiorespiratory fitness and mitochondrial energetics on walking speed in older adults with diabetes has not been clearly defined., Objective: To examine differences in cardiorespiratory fitness and skeletal muscle mitochondrial energetics between older adults with and without diabetes. We also assessed the contribution of cardiorespiratory fitness and skeletal muscle mitochondrial energetics to slower walking speed in older adults with diabetes., Findings: Participants with diabetes had lower cardiorespiratory fitness and mitochondrial energetics when compared to those without diabetes, following adjustments for covariates including BMI, chronic comorbid health conditions, and physical activity. 4-m and 400-m walking speeds were slower in those with diabetes. Mitochondrial oxidative capacity alone or combined with cardiorespiratory fitness mediated ∼20-70% of the difference in walk speed between older adults with and without diabetes. Further adjustments of BMI and co-morbidities further explained the group differences in walk speed., Conclusions: Skeletal muscle mitochondrial energetics and cardiorespiratory fitness contribute to slower walking speeds in older adults with diabetes. Cardiorespiratory fitness and mitochondrial energetics may be therapeutic targets to maintain or improve mobility in older adults with diabetes., Article Highlights: Why did we undertake this study? To determine if mitochondrial energetics and cardiorespiratory fitness contribute to slower walking speed in older adults with diabetes. What is the specific question(s) we wanted to answer? Are mitochondrial energetics and cardiorespiratory fitness in older adults with diabetes lower than those without diabetes? How does mitochondrial energetics and cardiorespiratory fitness impact walking speed in older adults with diabetes? What did we find? Mitochondrial energetics and cardiorespiratory fitness were lower in older adults with diabetes compared to those without diabetes, and energetics, and cardiorespiratory fitness, contributed to slower walking speed in those with diabetes. What are the implications of our findings? Cardiorespiratory fitness and mitochondrial energetics may be key therapeutic targets to maintain or improve mobility in older adults with diabetes.

Background: Muscle mass loss may be associated with liver fat accumulation, yet scientific consensus is lacking and evidence in older adults is scant. It is unclear which muscle characteristics might contribute to this association in older adults., Methods: We associated comprehensive muscle-related phenotypes including muscle mass normalized to body weight (D 3 -creatine dilution), muscle fat infiltration (MRI), carbohydrate-supported muscle mitochondrial maximal oxidative phosphorylation (respirometry), and cardiorespiratory fitness (VO 2 peak) with liver fat among older adults. Linear regression models adjusted for age, gender, technician (respirometry only), daily minutes of moderate to vigorous physical activity, and prediabetes/diabetes status tested main effects and interactions of each independent variable with waist circumference (high: women-≥88 cm, men-≥102 cm) and gender., Results: Among older adults aged 75 (IQR 73, 79 years; 59.8% women), muscle mass and liver fat were not associated overall but were positively associated among participants with a high waist circumference (β: 25.2; 95%CI 11.7, 40.4; p =.0002; N=362). Muscle fat infiltration and liver fat were positively associated (β: 15.2; 95%CI 6.8, 24.3; p =.0003; N=378). Carbohydrate-supported maximum oxidative phosphorylation and VO 2 peak (adjusted β: -12.9; 95%CI -20.3, -4.8; p =0.003; N=361) were inversely associated with liver fat; adjustment attenuated the estimate for maximum oxidative phosphorylation although the point estimate remained negative (β: -4.0; 95%CI -11.6, 4.2; p =0.32; N=321)., Conclusions: Skeletal muscle-related characteristics are metabolically relevant factors linked to liver fat in older adults. Future research should confirm our results to determine whether trials targeting mechanisms common to liver and muscle fat accumulation are warranted.

Sodium and fluid retention in liver disease is classically thought to result from reduced effective circulating volume and stimulation of the renin-angiotensin-aldosterone system (RAAS). Aldosterone dives Na + retention by activating the mineralocorticoid receptor and promoting the maturation and apical surface expression of the epithelial Na + channel (ENaC), found in the aldosterone-sensitive distal nephron. However, evidence of fluid retention without RAAS activation suggests the involvement of additional mechanisms. Liver disease can greatly increase plasma and urinary bile acid concentrations and have been shown to activate ENaC in vitro . We hypothesize that elevated bile acids in liver disease activate ENaC and drive fluid retention independent of RAAS. We therefore increased circulating bile acids in mice through bile duct ligation (BDL) and measured effects on urine and body composition, while using spironolactone to antagonize the mineralocorticoid receptor. We found BDL lowered blood [K + ] and hematocrit, and increased benzamil-sensitive natriuresis compared to sham, consistent with ENaC activation. BDL mice also gained significantly more body water. Blocking ENaC reversed fluid gains in BDL mice but had no effect in shams. In isolated collecting ducts from rabbits, taurocholic acid stimulated net Na + absorption but had no effect on K + secretion or flow-dependent ion fluxes. Our results provide experimental evidence for a novel aldosterone-independent mechanism for sodium and fluid retention in liver disease which may provide additional therapeutic options for liver disease patients., Competing Interests: Conflict of Interest: Authors declare no conflict of interest.

Nonalcoholic fatty liver disease (NAFLD) encompasses a spectrum of pathologies that includes steatosis, steatohepatitis (NASH) and fibrosis and is strongly associated with insulin resistance and type 2 diabetes. Changes in mitochondrial function are implicated in the pathogenesis of NAFLD, particularly in the transition from steatosis to NASH. Mitophagy is a mitochondrial quality control mechanism that allows for the selective removal of damaged mitochondria from the cell via the autophagy pathway. While past work demonstrated a negative association between liver fat content and rates of mitophagy, when changes in mitophagy occur during the pathogenesis of NAFLD and whether such changes contribute to the primary endpoints associated with the disease are currently poorly defined. We therefore undertook the studies described here to establish when alterations in mitophagy occur during the pathogenesis of NAFLD, as well as to determine the effects of genetic inhibition of mitophagy via conditional deletion of a key mitophagy regulator, PARKIN, on the development of steatosis, insulin resistance, inflammation and fibrosis. We find that loss of mitophagy occurs early in the pathogenesis of NAFLD and that loss of PARKIN hastens the onset but not severity of key NAFLD disease features. These observations suggest that loss of mitochondrial quality control in response to nutritional stress may contribute to mitochondrial dysfunction and the pathogenesis of NAFLD.

Sodium-glucose co-transporter type 2 (SGLT2) inhibitor therapy to treat type 2 diabetes unexpectedly reduced all-cause mortality and hospitalization due to heart failure in several large-scale clinical trials, and has since been shown to produce similar cardiovascular disease-protective effects in patients without diabetes. How SGLT2 inhibitor therapy improves cardiovascular disease outcomes remains incompletely understood. Metabolic flexibility refers to the ability of a cell or organ to adjust its use of metabolic substrates, such as glucose or fatty acids, in response to physiological or pathophysiological conditions, and is a feature of a healthy heart that may be lost during diabetic cardiomyopathy and in the failing heart. We therefore undertook studies to determine the effects of SGLT2 inhibitor therapy on cardiac metabolic flexibility in vivo in obese, insulin resistant mice using a [U 13 C]-glucose infusion during fasting and hyperinsulinemic euglycemic clamp. Relative rates of cardiac glucose versus fatty acid use during fasting were unaffected by EMPA, whereas insulin-stimulated rates of glucose use were significantly increased by EMPA, alongside significant improvements in cardiac insulin signaling. These metabolic effects of EMPA were associated with reduced cardiac hypertrophy and protection from ischemia. These observations suggest that the cardiovascular disease-protective effects of SGLT2 inhibitors may in part be explained by beneficial effects on cardiac metabolic substrate selection., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Author(s).)