Elamipretide treatment during pregnancy ameliorates the progression of polycystic kidney disease in maternal and neonatal mice with PKD1 mutations.

Pregnancy is proposed to aggravate cyst progression in autosomal dominant polycystic kidney disease (ADPKD) but Tolvaptan, the only FDA-approved drug for adult ADPKD, is not recommended for pregnant ADPKD patients because of potential fetal harm. Since pregnancy itself may increase the risk for ADPKD progression, we investigated the safety and efficacy of Elamipretide, a mitochondrial-protective tetrapeptide. Elamipretide was found to ameliorate the progression of kidney disease in pregnant Pkd1RC/RC mice, in parallel with attenuation of ERK1/2 phosphorylation and improvement of mitochondrial supercomplex formation. Furthermore, Elamipretide was found to pass through the placenta and breast milk and ameliorate aggressive infantile polycystic kidney disease without any observed teratogenic or harmful effect. Elamipretide has an excellent safety profile and is currently tested in multiple phase II and phase III clinical trials. These preclinical studies support a potential clinical trial of Elamipretide for the treatment of ADPKD, particularly for patients that cannot take Tolvaptan.

Loss of FoxOs in muscle increases strength and mitochondrial function during aging.

BACKGROUND: Muscle mitochondrial decline is associated with aging-related muscle weakness and insulin resistance. FoxO transcription factors are targets of insulin action and deletion of FoxOs improves mitochondrial function in diabetes. However, disruptions in proteostasis and autophagy are hallmarks of aging and the effect of chronic inhibition of FoxOs in aged muscle is unknown. This study investigated the role of FoxOs in regulating muscle strength and mitochondrial function with age. METHODS: We measured muscle strength, cross-sectional area, muscle fibre-type, markers of protein synthesis/degradation, central nuclei, glucose/insulin tolerance, and mitochondrial bioenergetics in 4.5-month (Young) and 22-24-month-old (Aged) muscle-specific FoxO1/3/4 triple KO (TKO) and littermate control (Ctrl) mice. RESULTS: Lean mass was increased in Aged TKO compared with both Aged Ctrl and younger groups by 26-33% (P < 0.01). Muscle strength, measured by max force of tibialis anterior (TA) contraction, was 20% lower in Aged Ctrl compared with Young Ctrls (P < 0.01) but was not decreased in Aged TKOs. Increased muscle strength in Young and Aged TKO was associated with 18-48% increased muscle weights compared with Ctrls (P < 0.01). Muscle cross-sectional analysis of TA, soleus, and plantaris revealed increases in fibre size distribution and a 2.5-10-fold increase in central nuclei in Young and Aged TKO mice, without histologic signs of muscle damage. Age-dependent increases in Gadd45a and Ube4a expression as well accumulation of K48 polyubiquitinated proteins were observed in quad and TA but were prevented by FoxO deletion. Young and Aged TKO muscle showed minimal changes in autophagy flux and no accumulation of autophagosomes compared with Ctrl groups. Increased strength in Young and Aged TKO was associated with a 10-20% increase in muscle mitochondrial respiration using glutamate/malate/succinate compared with controls (P < 0.05). OXPHOS subunit expression and complex I activity were decreased 16-34% in Aged Ctrl compared with Young Ctrl but were prevented in Aged TKO. Both Aged Ctrl and Aged TKO showed impaired glucose tolerance by 33% compared to young groups (P < 0.05) indicating improved strength and mitochondrial respiration are not due to improved glycemia. CONCLUSIONS: FoxO deletion increases muscle strength even during aging. Deletion of FoxOs maintains muscle strength in part by mild suppression of atrophic pathways, including inhibition of Gadd45a and Ube4a expression, without accumulation of autophagosomes in muscle. Deletion of FoxOs also improved mitochondrial function by maintenance of OXPHOS in both young and aged TKO.

Transcriptomic Regulation of Muscle Mitochondria and Calcium Signaling by Insulin/IGF-1 Receptors Depends on FoxO Transcription Factors.

Insulin and IGF-1, acting through the insulin receptor (IR) and IGF-1 receptor (IGF1R), maintain muscle mass and mitochondrial function, at least part of which occurs via their action to regulate gene expression. Here, we show that while muscle-specific deletion of IR or IGF1R individually results in only modest changes in the muscle transcriptome, combined deletion of IR/IGF1R (MIGIRKO) altered > 3000 genes, including genes involved in mitochondrial dysfunction, fibrosis, cardiac hypertrophy, and pathways related to estrogen receptor, protein kinase A (PKA), and calcium signaling. Functionally, this was associated with decreased mitochondrial respiration and increased ROS production in MIGIRKO muscle. To determine the role of FoxOs in these changes, we performed RNA-Seq on mice with muscle-specific deletion of FoxO1/3/4 (M-FoxO TKO) or combined deletion of IR, IGF1R, and FoxO1/3/4 in a muscle quintuple knockout (M-QKO). This revealed that among IR/IGF1R regulated genes, >97% were FoxO-dependent, and their expression was normalized in M-FoxO TKO and M-QKO muscle. FoxO-dependent genes were related to oxidative phosphorylation, inflammatory signaling, and TCA cycle. Metabolomic analysis showed accumulation of TCA cycle metabolites in MIGIRKO, which was reversed in M-QKO muscle. Likewise, calcium signaling genes involved in PKA signaling and sarcoplasmic reticulum calcium homeostasis were markedly altered in MIGIRKO muscle but normalized in M-QKO. Thus, combined loss of insulin and IGF-1 action in muscle transcriptionally alters mitochondrial function and multiple regulatory and signaling pathways, and these changes are mediated by FoxO transcription factors.

Insulin and IGF-1 receptors regulate complex I-dependent mitochondrial bioenergetics and supercomplexes via FoxOs in muscle.

Decreased skeletal muscle strength and mitochondrial dysfunction are characteristic of diabetes. The actions of insulin and IGF-1 through the insulin receptor (IR) and IGF-1 receptor (IGF1R) maintain muscle mass via suppression of forkhead box O (FoxO) transcription factors, but whether FoxO activation coordinates atrophy in concert with mitochondrial dysfunction is unknown. We show that mitochondrial respiration and complex I activity were decreased in streptozotocin (STZ) diabetic muscle, but these defects were reversed in muscle-specific FoxO1, -3, and -4 triple-KO (M-FoxO TKO) mice rendered diabetic with STZ. In the absence of systemic glucose or lipid abnormalities, muscle-specific IR KO (M-IR-/-) or combined IR/IGF1R KO (MIGIRKO) impaired mitochondrial respiration, decreased ATP production, and increased ROS. These mitochondrial abnormalities were not present in muscle-specific IR, IGF1R, and FoxO1, -3, and -4 quintuple-KO mice (M-QKO). Acute tamoxifen-inducible deletion of IR and IGF1R also decreased muscle pyruvate respiration, complex I activity, and supercomplex assembly. Although autophagy was increased when IR and IGF1R were deleted in muscle, mitophagy was not increased. Mechanistically, RNA-Seq revealed that complex I core subunits were decreased in STZ-diabetic and MIGIRKO muscle, and these changes were not present with FoxO KO in STZ-FoxO TKO and M-QKO mice. Thus, insulin-deficient diabetes or loss of insulin/IGF-1 action in muscle decreases complex I-driven mitochondrial respiration and supercomplex assembly in part by FoxO-mediated repression of complex I subunit expression.

Loss of iRhom2 accelerates fat gain and insulin resistance in diet-induced obesity despite reduced adipose tissue inflammation.

BACKGROUND: Low-grade inflammation and metabolic dysregulation are common comorbidities of obesity, both of which are associated with alterations in iRhom2-regulated pro-inflammatory cytokine and epidermal growth factor receptor (EGFR) ligand signaling. OBJECTIVE: Our objective was to determine the role of iRhom2 in the regulation of low-grade inflammation and metabolic dysregulation in a murine model of diet-induced obesity. METHODS: Wild type (WT) and iRhom2-deficient mice were fed normal chow (NC) or a high-fat diet (HFD) starting at 5 weeks of age for up to 33 weeks. Body composition, glucose and insulin tolerance, feeding behavior, and indirect calorimetry were measured at defined time points. Adipose tissue cytokine expression and inflammatory lesions known as crown-like structures (CLS) were analyzed at the end-point of the study. RESULTS: iRhom2-deficient mice show accelerated fat gain on a HFD, accompanied by insulin resistance. Indirect calorimetry did not demonstrate changes in energy expenditure or food intake, but locomotor activity was significantly reduced in HFD iRhom2-deficient mice. Interestingly, CLS, macrophage infiltration, and tumor necrosis factor (TNF) production were decreased in adipose tissue from HFD iRhom2-deficient mice, but circulating cytokines were unchanged. In inguinal and perigonadal fat, the EGFR ligand amphiregulin was markedly induced in HFD controls but completely prevented in iRhom2-deficient mice, suggesting a potentially dominant role of EGFR-dependent mechanisms over TNF in the modulation of insulin sensitivity. CONCLUSIONS: This study elucidates a novel role for iRhom2 as an immuno-metabolic regulator that affects adipose tissue inflammation independent of insulin resistance.

Loss of FoxOs in muscle reveals sex-based differences in insulin sensitivity but mitigates diet-induced obesity.

OBJECTIVE: Gender influences obesity-related complications, including diabetes. Females are more protected from insulin resistance after diet-induced obesity, which may be related to fat accumulation and muscle insulin sensitivity. FoxOs regulate muscle atrophy and are targets of insulin action, but their role in muscle insulin sensitivity and mitochondrial metabolism is unknown. METHODS: We measured muscle insulin signaling, mitochondrial energetics, and metabolic responses to a high-fat diet (HFD) in male and female muscle-specific FoxO1/3/4 triple knock-out (TKO) mice. RESULTS: In male TKO muscle, insulin-stimulated AKT activation was decreased. AKT2 protein and mRNA levels were reduced and insulin receptor protein and IRS-2 mRNA decreased. These changes contributed to decreased insulin-stimulated glucose uptake in glycolytic muscle in males. In contrast, female TKOs maintain normal insulin-mediated AKT phosphorylation, normal AKT2 levels, and normal glucose uptake in glycolytic muscle. When challenged with a HFD, fat gain was attenuated in both male and female TKO mice, and associated with decreased glucose levels, improved glucose homeostasis, and reduced muscle triglyceride accumulation. Furthermore, female TKO mice showed increased energy expenditure, relative to controls, due to increased lean mass and maintenance of mitochondrial function in muscle. CONCLUSIONS: FoxO deletion in muscle uncovers sexually dimorphic regulation of AKT2, which impairs insulin signaling in male mice, but not females. However, loss of FoxOs in muscle from both males and females also leads to muscle hypertrophy and increases in metabolic rate. These factors mitigate fat gain and attenuate metabolic abnormalities in response to a HFD.

FoxO Transcription Factors Are Critical Regulators of Diabetes-Related Muscle Atrophy.

Insulin deficiency and uncontrolled diabetes lead to a catabolic state with decreased muscle strength, contributing to disease-related morbidity. FoxO transcription factors are suppressed by insulin and thus are key mediators of insulin action. To study their role in diabetic muscle wasting, we created mice with muscle-specific triple knockout of FoxO1/3/4 and induced diabetes in these M-FoxO-TKO mice with streptozotocin (STZ). Muscle mass and myofiber area were decreased 20-30% in STZ-Diabetes mice due to increased ubiquitin-proteasome degradation and autophagy alterations, characterized by increased LC3-containing vesicles, and elevated levels of phosphorylated ULK1 and LC3-II. Both the muscle loss and markers of increased degradation/autophagy were completely prevented in STZ FoxO-TKO mice. Transcriptomic analyses revealed FoxO-dependent increases in ubiquitin-mediated proteolysis pathways in STZ-Diabetes, including regulation of Fbxo32 (Atrogin1), Trim63 (MuRF1), Bnip3L, and Gabarapl. These same genes were increased 1.4- to 3.3-fold in muscle from humans with type 1 diabetes after short-term insulin deprivation. Thus, FoxO-regulated genes play a rate-limiting role in increased protein degradation and muscle atrophy in insulin-deficient diabetes.