17α-Estradiol alleviates high-fat diet-induced inflammatory and metabolic dysfunction in skeletal muscle of male and female mice.

17α-estradiol (17α-E2) is a naturally occurring nonfeminizing diastereomer of 17ß-estradiol that has life span-extending effects in rodent models. To date, studies of the systemic and tissue-specific benefits of 17α-E2 have largely focused on the liver, brain, and white adipose tissue with far less focus on skeletal muscle. Skeletal muscle has an important role in metabolic and age-related disease. Therefore, this study aimed to determine whether 17α-E2 treatment has positive, tissue-specific effects on skeletal muscle during a high-fat feeding. We hypothesized that male, but not female, mice, would benefit from 17α-E2 treatment during a high-fat diet (HFD) with changes in the mitochondrial proteome to support lipid oxidation and subsequent reductions in diacylglycerol (DAG) and ceramide content. To test this hypothesis, we used a multiomics approach to determine changes in lipotoxic lipid intermediates, metabolites, and proteins related to metabolic homeostasis. Unexpectedly, we found that 17α-E2 had marked, but different, beneficial effects within each sex. In male mice, we show that 17α-E2 alleviates HFD-induced metabolic detriments of skeletal muscle by reducing the accumulation of diacylglycerol (DAG), and inflammatory cytokine levels, and altered the abundance of most of the proteins related to lipolysis and ß-oxidation. Similar to male mice, 17α-E2 treatment reduced fat mass while protecting muscle mass in female mice but had little muscle inflammatory cytokine levels. Although female mice were resistant to HFD-induced changes in DAGs, 17α-E2 treatment induced the upregulation of six DAG species. In female mice, 17α-E2 treatment changed the relative abundance of proteins involved in lipolysis, ß-oxidation, as well as structural and contractile proteins but to a smaller extent than male mice. These data demonstrate the metabolic benefits of 17α-E2 in skeletal muscle of male and female mice and contribute to the growing literature of the use of 17α-E2 for multi tissue health span benefits.NEW & NOTEWORTHY Using a multiomics approach, we show that 17α-E2 alleviates HFD-induced metabolic detriments in skeletal muscle by altering bioactive lipid intermediates, inflammatory cytokines, and the abundance of proteins related to lipolysis and muscle contraction. The positive effects of 17α-E2 in skeletal muscle occur in both sexes but differ in their outcome.

Chronic Ketosis Modulates HIF1α-Mediated Inflammatory Response in Rat Brain.

Hypoxia inducible factor alpha (HIF1α) is associated with neuroprotection conferred by diet-induced ketosis but the underlying mechanism remains unclear. In this study we use a ketogenic diet in rodents to induce a metabolic state of chronic ketosis, as measured by elevated blood ketone bodies. Chronic ketosis correlates with neuroprotection in both aged and following focal cerebral ischaemia and reperfusion (via middle cerebral artery occlusion, MCAO) in mouse and rat models. Ketone bodies are known to be used efficiently by the brain and metabolism of ketone bodies is associated with increased cytosolic succinate levels that inhibits prolyl hydroxylases allowing HIF1α to accumulate. Ketosis also regulates inflammatory pathways, and HIF1α is reported to be essential for gene expression of interleukin10 (IL10). Therefore we hypothesised that ketosis-stabilised HIF1α modulates the expression of inflammatory cytokines orchestrating neuroprotection. To test changes in cytokine levels in rodent brain, eight-week-old rats were fed either the standard chow diet (SD) or the ketogenic (KG) diet for 4 weeks before ischaemia experiments (MCAO) were performed and the brain tissues were collected. Consistent with our hypothesis, immunoblotting analysis shows IL10 levels were significantly higher in KG diet rat brain compared to SD, whereas the TNFα and IL6 levels were significantly lower in the brains of KG diet fed group.

Chronic Ketosis Modulates HIF1α-Mediated Inflammatory Response in Rat Brain.

Hypoxia inducible factor alpha (HIF1α) is associated with neuroprotection conferred by diet-induced ketosis, but the underlying mechanism remains unclear. In this study, we use a ketogenic diet in rodents to induce a metabolic state of chronic ketosis, as measured by elevated blood ketone bodies. Chronic ketosis correlates with neuroprotection in both aged and following focal cerebral ischemia and reperfusion (via middle cerebral artery occlusion, MCAO) in mouse and rat models. Ketone bodies are known to be used efficiently by the brain, and metabolism of ketone bodies is associated with increased cytosolic succinate levels that inhibits prolyl hydroxylases allowing HIF1α to accumulate. Ketosis also regulates inflammatory pathways, and HIF1α is reported to be essential for gene expression of interleukin 10 (IL10). Therefore, we hypothesized that ketosis-stabilized HIF1α modulates the expression of inflammatory cytokines orchestrating neuroprotection. To test changes in cytokine levels in rodent brain, 8-week-rats were fed either the standard chow diet (SD) or the KG diet for 4 weeks before ischemia experiments (MCAO) were performed and the brain tissues were collected. Consistent with our hypothesis, immunoblotting analysis shows IL10 levels were significantly higher in KG diet rat brain compared to SD, whereas the TNFα and IL6 levels were significantly lower in the brains of KG diet-fed group.

Loss of Cardiac PFKFB2 Drives Metabolic, Functional, and Electrophysiological Remodeling in the Heart.

BACKGROUND: Phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2) is a critical glycolytic regulator responsible for upregulation of glycolysis in response to insulin and adrenergic signaling. PFKFB2, the cardiac isoform of PFK-2, is degraded in the heart in the absence of insulin signaling, contributing to diabetes-induced cardiac metabolic inflexibility. However, previous studies have not examined how the loss of PFKFB2 affects global cardiac metabolism and function. METHODS AND RESULTS: To address this, we have generated a mouse model with a cardiomyocyte-specific knockout of PFKFB2 (cKO). Using 9-month-old cKO and control mice, we characterized the impacts of PFKFB2 on cardiac metabolism, function, and electrophysiology. cKO mice have a shortened life span of 9 months. Metabolically, cKO mice are characterized by increased glycolytic enzyme abundance and pyruvate dehydrogenase activity, as well as decreased mitochondrial abundance and beta oxidation, suggesting a shift toward glucose metabolism. This was supported by a decrease in the ratio of palmitoyl carnitine to pyruvate-dependent mitochondrial respiration in cKO relative to control animals. Metabolomic, proteomic, and Western blot data support the activation of ancillary glucose metabolism, including pentose phosphate and hexosamine biosynthesis pathways. Physiologically, cKO animals exhibited impaired systolic function and left ventricular dilation, represented by reduced fractional shortening and increased left ventricular internal diameter, respectively. This was accompanied by electrophysiological alterations including increased QT interval and other metrics of delayed ventricular conduction. CONCLUSIONS: Loss of PFKFB2 results in metabolic remodeling marked by cardiac ancillary pathway activation. This could delineate an underpinning of pathologic changes to mechanical and electrical function in the heart.

Skeletal and cardiac muscle have different protein turnover responses in a model of right heart failure.

Right heart failure (RHF) is a common and deadly disease in aged populations. Extra-cardiac outcomes of RHF such as skeletal muscle atrophy contribute to morbidity and mortality. Despite the significance of maintaining right ventricular (RV) and muscle function, the mechanisms of RHF and muscle atrophy are unclear. Metformin (MET) improves cardiac and muscle function through the regulation of metabolism and the cellular stress response. However, whether MET is a viable therapeutic for RHF and muscle atrophy is not yet known. We used deuterium oxide labeling to measure individual protein turnover in the RV as well as subcellular skeletal muscle proteostasis in aged male mice subjected to 4 weeks of hypobaric hypoxia (HH)-induced RHF. Mice exposed to HH had elevated RV mass and impaired RV systolic function, neither of which was prevented by MET. HH resulted in a higher content of glycolytic, cardiac, and antioxidant proteins in the RV, most of which were inhibited by MET. The synthesis of these key RV proteins was generally unchanged by MET, suggesting MET accelerated protein breakdown. HH resulted in a loss of skeletal muscle mass due to inhibited protein synthesis alongside myofibrillar protein breakdown. MET did not impact HH-induced muscle protein turnover and did not prevent muscle wasting. Together, we show tissue-dependent responses to HH-induced RHF where the RV undergoes hypertrophic remodeling with higher expression of metabolic and stress response proteins. Skeletal muscle undergoes loss of protein mass and atrophy, primarily due to myofibrillar protein breakdown. MET did not prevent HH-induced RV dysfunction or muscle wasting, suggesting that the identification of other therapies to attenuate RHF and concomitant muscle atrophy is warranted.

Loss of cardiac PFKFB2 drives Metabolic, Functional, and Electrophysiological Remodeling in the Heart.

Background: Phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2) is a critical glycolytic regulator responsible for upregulation of glycolysis in response to insulin and adrenergic signaling. PFKFB2, the cardiac isoform of PFK-2, is degraded in the heart in the absence of insulin signaling, contributing to diabetes-induced cardiac metabolic inflexibility. However, previous studies have not examined how the loss of PFKFB2 affects global cardiac metabolism and function. Methods: To address this, we have generated a mouse model with a cardiomyocyte-specific knockout of PFKFB2 (cKO). Using 9-month-old cKO and control (CON) mice, we characterized impacts of PFKFB2 on cardiac metabolism, function, and electrophysiology. Results: cKO mice have a shortened lifespan of 9 months. Metabolically, cKO mice are characterized by increased glycolytic enzyme abundance and pyruvate dehydrogenase (PDH) activity, as well as decreased mitochondrial abundance and beta oxidation, suggesting a shift toward glucose metabolism. This was supported by a decrease in the ratio of palmitoyl carnitine to pyruvate-dependent mitochondrial respiration in cKO relative to CON animals. Metabolomic, proteomic, and western blot data support the activation of ancillary glucose metabolism, including pentose phosphate and hexosamine biosynthesis pathways. Physiologically, cKO animals exhibited impaired systolic function and left ventricular (LV) dilation, represented by reduced fractional shortening and increased LV internal diameter, respectively. This was accompanied by electrophysiological alterations including increased QT interval and other metrics of delayed ventricular conduction. Conclusions: Loss of PFKFB2 results in metabolic remodeling marked by cardiac ancillary pathway activation. This could delineate an underpinning of pathologic changes to mechanical and electrical function in the heart. Clinical Perspective: What is New?: We have generated a novel cardiomyocyte-specific knockout model of PFKFB2, the cardiac isoform of the primary glycolytic regulator Phosphofructokinase-2 (cKO).The cKO model demonstrates that loss of cardiac PFKFB2 drives metabolic reprogramming and shunting of glucose metabolites to ancillary metabolic pathways.The loss of cardiac PFKFB2 promotes electrophysiological and functional remodeling in the cKO heart.What are the Clinical Implications?: PFKFB2 is degraded in the absence of insulin signaling, making its loss particularly relevant to diabetes and the pathophysiology of diabetic cardiomyopathy.Changes which we observe in the cKO model are consistent with those often observed in diabetes and heart failure of other etiologies.Defining PFKFB2 loss as a driver of cardiac pathogenesis identifies it as a target for future investigation and potential therapeutic intervention.

The loss of cardiac SIRT3 decreases metabolic flexibility and proteostasis in an age-dependent manner.

SIRT3 is a longevity factor that acts as the primary deacetylase in mitochondria. Although ubiquitously expressed, previous global SIRT3 knockout studies have shown primarily a cardiac-specific phenotype. Here, we sought to determine how specifically knocking out SIRT3 in cardiomyocytes (SIRTcKO mice) temporally affects cardiac function and metabolism. Mice displayed an age-dependent increase in cardiac pathology, with 10-month-old mice exhibiting significant loss of systolic function, hypertrophy, and fibrosis. While mitochondrial function was maintained at 10 months, proteomics and metabolic phenotyping indicated SIRT3 hearts had increased reliance on glucose as an energy substrate. Additionally, there was a significant increase in branched-chain amino acids in SIRT3cKO hearts without concurrent increases in mTOR activity. Heavy water labeling experiments demonstrated that, by 3 months of age, there was an increase in protein synthesis that promoted hypertrophic growth with a potential loss of proteostasis in SIRT3cKO hearts. Cumulatively, these data show that the cardiomyocyte-specific loss of SIRT3 results in severe pathology with an accelerated aging phenotype.

Kinetic proteomics identifies targeted changes in liver metabolism and the ribo-interactome by dietary sulfur amino acid restriction.

Dietary sulfur amino acid restriction (SAAR) protects against diet-induced obesity, extends healthspan, and coincides with an overall reduction in hepatic protein synthesis. To explore the underpinnings of SAAR-induced slowed growth and its impact on liver metabolism and proteostasis, we resolved changes in hepatic mRNA and protein abundances and compared synthesis rates of individual liver proteins. To achieve this, adult male mice were provided deuterium-labeled drinking water while freely consuming either a regular-fat or high-fat diet that was SAA restricted. Livers from these mice and their respective dietary controls were used to conduct transcriptomic, proteomic, and kinetic proteomic analyses. We found that remodeling of the transcriptome by SAAR was largely agnostic to dietary fat content. Shared signatures included activation of the integrated stress response alongside alterations in metabolic processes impacting lipids, fatty acids, and amino acids. Changes to the proteome correlated poorly with the transcriptome, and yet, functional clustering of kinetic proteomic changes in the liver during SAAR revealed that the management of fatty acids and amino acids were altered to support central metabolism and redox balance. Dietary SAAR also strongly influenced the synthesis rates of ribosomal proteins and ribosome-interacting proteins regardless of dietary fat. Taken together, dietary SAAR alters the transcriptome and proteome in the liver to safely manage increased fatty acid flux and energy use and couples this with targeted changes in the ribo-interactome to support proteostasis and slowed growth.

17α-estradiol Alleviates High-Fat Diet-Induced Inflammatory and Metabolic Dysfunction in Skeletal Muscle of Male and Female Mice.

Skeletal muscle has a central role in maintaining metabolic homeostasis. 17α-estradiol (17α-E2), a naturally-occurring non-feminizing diastereomer of 17ß-estradiol that demonstrates efficacy for improving metabolic outcomes in male, but not female, mice. Despite several lines of evidence showing that 17α-E2 treatment improves metabolic parameters in middle-aged obese and old male mice through effects in brain, liver, and white adipose tissue little is known about how 17α-E2 alters skeletal muscle metabolism, and what role this may play in mitigating metabolic declines. Therefore, this study aimed to determine if 17α-E2 treatment improves metabolic outcomes in skeletal muscle from obese male and female mice following chronic high fat diet (HFD) administration. We hypothesized that male, but not female, mice, would benefit from 17α-E2 treatment during HFD. To test this hypothesis, we used a multi-omics approach to determine changes in lipotoxic lipid intermediates, metabolites, and proteins related to metabolic homeostasis. In male mice, we show that 17α-E2 alleviates HFD-induced metabolic detriments of skeletal muscle by reducing the accumulation of diacylglycerol (DAGs) and ceramides, inflammatory cytokine levels, and reduced the abundance of most of the proteins related to lipolysis and beta-oxidation. In contrast to males, 17α-E2 treatment in female mice had little effect on the DAGs and ceramides content, muscle inflammatory cytokine levels, or changes to the relative abundance of proteins involved in beta-oxidation. These data support to the growing evidence that 17α-E2 treatment could be beneficial for overall metabolic health in male mammals.

Prognostic utility of autoantibodies to α-enolase and Hsp70 for cancer of the gingivo-buccal complex using immunoproteomics.

PURPOSE: Studies from our laboratory have reported 14 tumor antigens that elicit an autoantibody response in patients with cancer of the gingivobuccal complex (GBC) In this study, utility of the autoantibody response has been evaluated for prognosis of cancer of the GBC. EXPERIMENTAL DESIGN: Autoantibody response was evaluated using immunoproteomics and the prognostic significance was assessed by Kaplan-Meier survival and multivariate analysis. RESULTS: Autoantibody response against α-enolase isoforms a, b, and c and Hsp70 was detected in 27, 53, 64, and 26% of the 78 patients, respectively. Patients positive for autoantibody response to α-ENO and Hsp70 individually and in combination, showed significantly reduced disease-free survival (DFS) compared to those who do not show autoantibody response to either of them. Further the patients, who exhibit autoantibody response to α-ENO and Hsp70 in combination with nodal involvement and/or differentiation status, have significantly lowered DFS. The relative risk of recurrence is 3.41 for patients who exhibit autoantibody response to both the antigens. CONCLUSIONS AND CLINICAL RELEVANCE: Autoantibody response against α-ENO and Hsp70 provides an additional parameter and may be utilized along with nodal involvement and differentiation status for better prognosis of cancer of GBC.

Immunoproteomics reveals that cancer of the tongue and the gingivobuccal complex exhibit differential autoantibody response.

Autoantibody response to tumor antigens has been widely used to identify novel tumor markers for different cancers, including that of the head and neck. The oral cavity, which is in the head and neck region, comprises of many sub sites with distinct biologies and incidence of cancer of each sub site of the oral cavity is different. It is anticipated therefore that each sub site of the oral cavity may elicit a differential autoantibody response. This report evaluates the autoantibody response in 15 patients with cancer of gingivo-buccal complex and in 15 patients with cancer of tongue using Immunoproteomics, and shows that the autoantibody response to alpha-enolase, HSP 70, peroxiredoxin-VI, annexin II, pyruvate kinase, alpha-tubulin, beta-tubulin, ATP synthase, triose phosphate isomerase and aldose reductase seen in patients with cancer of gingivo-buccal complex is absent in patients with cancer of tongue. This suggests that cancer of these sub sites should be studied separately because of their different biology and emerging site specific molecular signatures including autoantibody responses to ensure unambiguous clinical interpretations.