Epoxygenase Cyp2c44 regulates hepatic lipid metabolism and insulin signaling by controlling FATP2 localization and activation of the DAG/PKCδ axis.

Cytochrome P450 epoxygenase Cyp2c44, a murine epoxyeicosatrienoic acid (EET) producing enzyme, promotes insulin sensitivity and Cyp2c44(-/-) mice show hepatic insulin resistance. Because insulin resistance leads to hepatic lipid accumulation and hyperlipidemia, we hypothesized that Cyp2c44 regulates hepatic lipid metabolism. Standard chow diet (SD) fed male Cyp2c44(-/-) mice had significantly decreased EET levels and increased hepatic and plasma lipid levels compared to wild-type mice. We showed increased hepatic plasma membrane localization of the FA transporter 2 (FATP2) and total unsaturated fatty acids and diacylglycerol levels. Cyp2c44(-/-) mice had impaired glucose tolerance and increased hepatic plasma membraneassociated PKCδ and phosphorylated IRS-1, two negative regulators of insulin signaling. Surprisingly, SD and high fat diet fed (HFD) Cyp2c44(-/-) mice had similar glucose tolerance and hepatic plasma membrane PKCδ levels, suggesting that SD-fed Cyp2c44(-/-) mice have reached their maximal glucose intolerance. Inhibition of PKCδ resulted in decreased IRS-1 serine phosphorylation and improved insulin-mediated signaling in Cyp2c44(-/-) hepatocytes. Finally, Cyp2c44(-/-) HFD-fed mice treated with the analog EET-A showed decreased hepatic plasma membrane FATP2 and PCKDδ levels with improved glucose tolerance and insulin signaling. In conclusion, loss of Cyp2c44 with concomitant decreased EET levels leads to increased hepatic FATP2 plasma membrane localization, diacylglycerol accumulation, and PKCδ-mediated attenuation of insulin signaling. Thus, Cyp2c44 acts as a regulator of lipid metabolism by linking it to insulin signaling.

Pharmacological SERCA activation limits diet-induced steatohepatitis and restores liver metabolic function in mice.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most common form of liver disease and poses significant health risks to patients who progress to metabolic dysfunction-associated steatohepatitis (MASH). Fatty acid (FA) overload alters endoplasmic reticulum (ER) calcium stores and induces mitochondrial oxidative stress in hepatocytes, leading to hepatocellular inflammation and apoptosis. Obese mice have impaired liver sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) function, which normally maintains intracellular calcium homeostasis by transporting Ca2+ ions from the cytoplasm to ER. We hypothesized that restoration of SERCA activity would improve diet-induced steatohepatitis in mice by limiting ER stress and mitochondrial dysfunction. Wild-type and melanocortin-4 receptor knockout (Mc4r-/-) mice were placed on either chow or Western diet (WD) for 8 weeks. Half of the WD-fed mice were administered CDN1163 to activate SERCA, which reduced liver fibrosis and inflammation. SERCA activation also restored glucose tolerance and insulin sensitivity, improved histological markers of MASH, increased expression of antioxidant enzymes, and decreased expression of oxidative stress and ER stress genes. CDN1163 decreased hepatic citric acid cycle flux and liver pyruvate cycling, enhanced expression of mitochondrial respiratory genes, and shifted hepatocellular [NADH]/[NAD+] and [NADPH]/[NADP+] ratios to a less oxidized state, which was associated with elevated polyunsaturated fatty acid (PUFA) content of liver lipids. In sum, the data demonstrate that pharmacological SERCA activation limits MASLD progression and prevents metabolic dysfunction induced by WD feeding in mice.

Tubular CPT1A deletion minimally affects aging and chronic kidney injury.

Kidney tubules use fatty acid oxidation (FAO) to support their high energetic requirements. Carnitine palmitoyltransferase 1A (CPT1A) is the rate-limiting enzyme for FAO, and it is necessary to transport long-chain fatty acids into mitochondria. To define the role of tubular CPT1A in aging and injury, we generated mice with tubule-specific deletion of Cpt1a (Cpt1aCKO mice), and the mice were either aged for 2 years or injured by aristolochic acid or unilateral ureteral obstruction. Surprisingly, Cpt1aCKO mice had no significant differences in kidney function or fibrosis compared with wild-type mice after aging or chronic injury. Primary tubule cells from aged Cpt1aCKO mice had a modest decrease in palmitate oxidation but retained the ability to metabolize long-chain fatty acids. Very-long-chain fatty acids, exclusively oxidized by peroxisomes, were reduced in kidneys lacking tubular CPT1A, consistent with increased peroxisomal activity. Single-nuclear RNA-Seq showed significantly increased expression of peroxisomal FAO enzymes in proximal tubules of mice lacking tubular CPT1A. These data suggest that peroxisomal FAO may compensate in the absence of CPT1A, and future genetic studies are needed to confirm the role of peroxisomal ß-oxidation when mitochondrial FAO is impaired.

Microvascular insulin resistance associates with enhanced muscle glucose disposal in CD36 deficiency.

Dysfunction of endothelial insulin delivery to muscle associates with insulin resistance. CD36, a fatty acid transporter and modulator of insulin signaling is abundant in endothelial cells, especially in capillaries. Humans with inherited 50% reduction in CD36 expression have endothelial dysfunction but whether it is associated with insulin resistance is unclear. Using hyperinsulinemic/euglycemic clamps in Cd36-/- and wildtype mice, and in 50% CD36 deficient humans and matched controls we found that Cd36-/- mice have enhanced systemic glucose disposal despite unaltered transendothelial insulin transfer and reductions in microvascular perfusion and blood vessel compliance. Partially CD36 deficient humans also have better glucose disposal than controls with no capillary recruitment by insulin. CD36 knockdown in primary human-derived microvascular cells impairs insulin action on AKT, endothelial nitric oxide synthase, and nitric oxide release. Thus, insulin resistance of microvascular function in CD36 deficiency paradoxically associates with increased glucose utilization, likely through a remodeling of muscle gene expression.

Insulin at the intersection of thermoregulation and glucose homeostasis.

Mammals are protected from changes in environmental temperature by altering energetic processes that modify heat production. Insulin is the dominant stimulus of glucose uptake and metabolism, which are fundamental for thermogenic processes. The purpose of this work was to determine the interaction of ambient temperature induced changes in energy expenditure (EE) on the insulin sensitivity of glucose fluxes. Short-term and adaptive responses to thermoneutral temperature (TN, ∼28 °C) and room (laboratory) temperature (RT, ∼22 °C) were studied in mice. This range of temperature does not cause detectable changes in circulating catecholamines or shivering and postabsorptive glucose homeostasis is maintained. We tested the hypothesis that a decrease in EE that occurs with TN causes insulin resistance and that this reduction in insulin action and EE is reversed upon short term (<12h) transition to RT. Insulin-stimulated glucose disposal (Rd) and tissue-specific glucose metabolic index were assessed combining isotopic tracers with hyperinsulinemic-euglycemic clamps. EE and insulin-stimulated Rd are both decreased (∼50%) in TN-adapted vs RT-adapted mice. When RT-adapted mice are switched to TN, EE rapidly decreases and Rd is reduced by ∼50%. TN-adapted mice switched to RT exhibit a rapid increase in EE, but whole-body insulin-stimulated Rd remains at the low rates of TN-adapted mice. In contrast, whole body glycolytic flux rose with EE. This higher EE occurs without increasing glucose uptake from the blood, but rather by diverting glucose from glucose storage to glycolysis. In addition to adaptations in insulin action, 'insulin-independent' glucose uptake in brown fat is exquisitely sensitive to thermoregulation. These results show that insulin action adjusts to non-stressful changes in ambient temperature to contribute to the support of body temperature homeostasis without compromising glucose homeostasis.

Precarious hope: Ethical considerations for offering experimental fetal therapies outside of research after initial studies in humans.

OBJECTIVE: Risks and benefits of experimental fetal therapies can remain uncertain after initial clinical studies, especially long-term effects. Nevertheless, pregnant individuals may request them, hoping to benefit their future child. Guidance about offering experimental fetal therapies outside research (as "innovative therapy") is limited, despite their ethical complexity. We propose points for clinicians and reviewers to consider when deciding whether and how to offer experimental fetal therapies as innovative therapies after initial clinical studies. METHOD: We used conceptual analysis and a current case to develop points for consideration, grounded in broader debates on innovative therapy and the unique challenges associated with experimental fetal therapies. RESULTS: Clinicians should evaluate whether offering experimental fetal therapies as innovative therapy is appropriate for a pregnant individual and their fetus. The anticipated risk-benefit ratio for the fetus should be favorable. For the pregnant individual, risks may outweigh benefits, within reasonable limits. Medical resources should be sufficient to ensure appropriate care. Clinicians should support pregnant individuals in making informed choices. Clinicians offering innovative therapies with more than minimal risk should collect and report data on outcomes. Independent review should take place. CONCLUSION: Considering these points may advance the interests of fetuses, future children, and their families.

Exercise training adaptations in liver glycogen and glycerolipids require hepatic AMP-activated protein kinase in mice.

Regular exercise elicits adaptations in glucose and lipid metabolism that allow the body to meet energy demands of subsequent exercise bouts more effectively and mitigate metabolic diseases including fatty liver. Energy discharged during the acute exercise bouts that comprise exercise training may be a catalyst for liver adaptations. During acute exercise, liver glycogenolysis and gluconeogenesis are accelerated to supply glucose to working muscle. Lower liver energy state imposed by gluconeogenesis and related pathways activates AMP-activated protein kinase (AMPK), which conserves ATP partly by promoting lipid oxidation. This study tested the hypothesis that AMPK is necessary for liver glucose and lipid adaptations to training. Liver-specific AMPKα1α2 knockout (AMPKα1α2fl/fl+AlbCre) mice and littermate controls (AMPKα1α2fl/fl) completed sedentary and exercise training protocols. Liver nutrient fluxes were quantified at rest or during acute exercise following training. Liver metabolites and molecular regulators of metabolism were assessed. Training increased liver glycogen in AMPKα1α2fl/fl mice, but not in AMPKα1α2fl/fl+AlbCre mice. The inability to increase glycogen led to lower glycogenolysis, glucose production, and circulating glucose during acute exercise in trained AMPKα1α2fl/fl+AlbCre mice. Deletion of AMPKα1α2 attenuated training-induced declines in liver diacylglycerides. In particular, training lowered the concentration of unsaturated and elongated fatty acids comprising diacylglycerides in AMPKα1α2fl/fl mice, but not in AMPKα1α2fl/fl+AlbCre mice. Training increased liver triacylglycerides and the desaturation and elongation of fatty acids in triacylglycerides of AMPKα1α2fl/fl+AlbCre mice. These lipid responses were independent of differences in tricarboxylic acid cycle fluxes. In conclusion, AMPK is required for liver training adaptations that are critical to glucose and lipid metabolism.NEW & NOTEWORTHY This study shows that the energy sensor and transducer, AMP-activated protein kinase (AMPK), is necessary for an exercise training-induced: 1) increase in liver glycogen that is necessary for accelerated glycogenolysis during exercise, 2) decrease in liver glycerolipids independent of tricarboxylic acid (TCA) cycle flux, and 3) decline in the desaturation and elongation of fatty acids comprising liver diacylglycerides. The mechanisms defined in these studies have implications for use of regular exercise or AMPK-activators in patients with fatty liver.

Extracellular Matrix Abnormalities Contribute to Cardiac Insulin Resistance and Associated Dysfunction in Diet-induced Obese Mice.

Increased deposition of extracellular matrix (ECM) components such as collagens and hyaluronan contributes to the pathogenesis of obesity-associated insulin resistance in muscle, liver, and adipose tissue. Despite the significance of the heart in cardiovascular and metabolic diseases, maladaptive ECM remodelling in obesity-associated cardiac insulin resistance and cardiac dysfunction has not been studied. Using genetic and pharmacological approaches in mice fed a high fat (HF) diet, we demonstrated a tight association between increased ECM deposition with cardiac insulin resistance. Increased collagen deposition by genetic deletion of matrix metalloproteinase 9 (MMP9) exacerbated cardiac insulin resistance and decreased hyaluronan deposition by treatment with PEGylated human recombinant hyaluronidase PH20 (PEGPH20) improved cardiac insulin resistance in obese mice. These relationships corresponded to functional changes in the heart. PEGPH20 treatment in obese mice ameliorated HF diet-induced abnormal myocardial remodelling. In addition to hyaluronan, increased collagen deposition is a characteristic of the obese mouse heart. We further demonstrated that pirfenidone, a clinically available anti-fibrotic medication which inhibits collagen expression, improved cardiac insulin resistance and cardiac function in obese mice. Our results provide important new insights into the role of ECM remodelling in the pathogenesis of cardiac insulin resistance and associated dysfunction in obesity of distinct mouse models. These findings support the novel therapeutic potential of targeting early cardiac ECM abnormalities in the prevention and treatment of obesity-related cardiovascular complications.

Insulin at the Intersection of Thermoregulation and Glucose Homeostasis.

Mammals are protected from changes in environmental temperature by altering energetic processes that modify heat production. Insulin is the dominant stimulus of glucose uptake and metabolism, which are fundamental for thermogenic processes. The purpose of this work was to determine the interaction of ambient temperature induced changes in energy expenditure (EE) on the insulin sensitivity of glucose fluxes. Short-term and adaptive responses to thermoneutral temperature (TN, ~28°C) and room (laboratory) temperature (RT, ~22°C) were studied in mice. This range of temperature does not cause detectable changes in circulating catecholamines or shivering and postabsorptive glucose homeostasis is maintained. We tested the hypothesis that a decrease in EE that occurs with TN causes insulin resistance and that this reduction in insulin action and EE is reversed upon short term (<12h) transition to RT. Insulin-stimulated glucose disposal (Rd) and tissue specific glucose uptake were assessed combining isotopic tracers with hyperinsulinemic-euglycemic clamps. EE and insulin-stimulated Rd are both decreased (~50%) in TN-adapted vs RT-adapted mice. When RT-adapted mice are switched to TN, EE rapidly decreases and Rd is reduced by ~50%. TN-adapted mice switched to RT exhibit a rapid increase in EE, but whole body insulin-stimulated Rd remains at the low rates of TN-adapted mice. In contrast, whole body glycolytic flux rose with EE. This higher EE occurs without increasing glucose uptake from the blood, but rather by diverting glucose from glucose storage to glycolysis. In addition to adaptations in insulin action, 'insulin-independent' glucose uptake in brown fat is exquisitely sensitive to thermoregulation. These results show that insulin action adjusts to non-stressful changes in ambient temperature to contribute to the support of body temperature homeostasis without compromising glucose homeostasis.

Disability training for genetic counseling students: The advocates' perspective.

The genetics and disability communities have had a complicated relationship that is rooted in the history of the eugenics movement. Disability scholars claim that in order for healthcare professionals to provide optimal services to disabled patients, disability education must be implemented into healthcare training programs. No studies have explored the perspectives of disability advocates regarding the implementation and use of disability training in genetic counseling. This exploratory study recruited 13 advocates with lived experience of disability and genetic counseling exposure to participate in a semi-structured interview to share their recommendations for disability education opportunities in genetic counseling training programs and their perceived benefits of increased student exposure to disability. All advocates received genetic counseling themselves and four advocates reported working with genetic counselors in the advocacy setting. Advocates recounted their experiences working with genetic counselors, identifying qualities they deemed critical for effective counseling. All the advocates expressed interest in participating in experiential opportunities, with few concerns noted. The most frequently discussed recommendations for disability training included inviting advocates to speak in classrooms, and having students shadow disabled individuals. Advocates noted barriers to consider when implementing such educational opportunities, such as accessibility issues. Potential benefits of implementing disability education for students included providing students with a broader scope of knowledge and a deeper understanding of disability and resources available to the disability community. This novel study found that advocates are interested in participating in genetic counseling education, with recommendations on preferred experiential learning. By increasing a genetic counseling student's exposure to a disability, they may develop a comprehensive understanding of life with a disability, which may improve genetic counseling services to those with newly diagnosed disabilities.

"It's Pretty Sad If You Get Used to It": A Qualitative Study of First Responder Experiences with Opioid Overdose Emergencies.

BACKGROUND: First responders play a vital role in the United States opioid drug overdose crisis, a public health emergency that has claimed many lives. OBJECTIVE: We sought to investigate first responders' experiences and attitudes toward opioid overdose emergencies and the ongoing crisis, as well as emotional effects, coping strategies, and support systems. METHODS: A convenience sample of first responders (n = 18) at the Columbus Fire Division, with experience responding to opioid emergencies, participated in semi-structured telephone interviews between September 2018 and February 2019. Interviews were recorded, transcribed verbatim, and analyzed using content analysis for themes. RESULTS: While almost all participants described overdose emergencies as routine, they recalled some as memorable and emotionally impactful. Almost all respondents were frustrated by the high rates of overdose among their patients and the lack of sustainable improvements in outcomes, yet expressed a strong moral commitment to caring for patients and saving lives. Themes of burnout, compassion fatigue, and hopelessness emerged, as did themes of increased compassion and empathy. Support for personnel experiencing emotional difficulty was either lacking or underutilized. Further, many felt public policies should prioritize more permanent resources and improve access to care, and believed that people who use drugs should face greater accountability. CONCLUSION: First responders perceive a moral and professional duty to treat patients who overdose, despite their frustrations. They may benefit from additional occupational support to cope with the resultant emotional effects of their role in the crisis. Addressing macro-level factors contributing to the overdose crisis and improving patient outcomes could also positively affect first responder wellbeing.

Home Care in America: The Urgent Challenge of Putting Ethical Care into Practice.

Home care is one of the fastest-growing industries in the United States, providing valuable opportunities for millions of older adults and people with disabilities to live at home rather than in institutional settings. Home care workers assist clients with essential activities of daily living, but their wages and working conditions generally fail to reflect the importance of their work. Drawing on the work of Eva Feder Kittay and other care ethicists, we argue that good care involves attending to the needs of another out of a concern for their well-being. Such care should be standard in the home care system. Yet, because of the pervasive racial, gender, and economic inequalities that the home care industry perpetuates, home care workers and their clients cannot reasonably be expected to care about each other. We endorse reforms aimed at enabling home care workers and their clients to form and maintain professional relationships that cultivate care..

Extracellular matrix remodelling in obesity and metabolic disorders.

Obesity causes extracellular matrix (ECM) remodelling which can develop into serious pathology and fibrosis, having metabolic effects in insulin-sensitive tissues. The ECM components may be increased in response to overnutrition. This review will focus on specific obesity-associated molecular and pathophysiological mechanisms of ECM remodelling and the impact of specific interactions on tissue metabolism. In obesity, complex network of signalling molecules such as cytokines and growth factors have been implicated in fibrosis. Increased ECM deposition contributes to the pathogenesis of insulin resistance at least in part through activation of cell surface integrin receptors and CD44 signalling cascades. These cell surface receptors transmit signals to the cell adhesome which orchestrates an intracellular response that adapts to the extracellular environment. Matrix proteins, glycoproteins, and polysaccharides interact through ligand-specific cell surface receptors that interact with the cytosolic adhesion proteins to elicit specific actions. Cell adhesion proteins may have catalytic activity or serve as scaffolds. The vast number of cell surface receptors and the complexity of the cell adhesome have made study of their roles challenging in health and disease. Further complicating the role of ECM-cell receptor interactions is the variation between cell types. This review will focus on recent insights gained from studies of two highly conserved, ubiquitously axes and how they contribute to insulin resistance and metabolic dysfunction in obesity. These are the collagen-integrin receptor-IPP (ILK-PINCH-Parvin) axis and the hyaluronan-CD44 interaction. We speculate that targeting ECM components or their receptor-mediated cell signalling may provide novel insights into the treatment of obesity-associated cardiometabolic complications.

Response to commentaries: 'autonomy-based criticisms of the patient preference predictor'.

The authors respond to four JME commentaries on their Feature Article, 'Autonomy-based criticisms of the patient preference predictor'.

How hummingbirds hover: Natural selection for energetics of hovering flight.

Osipova et al.1 recently identified an inactivating gene mutation that contributed to the evolution of the hummingbird species by increasing flux of pathways for energy production that are necessary for the unique ability for hovering flight. Lessons from the natural selection for this mutation are applied to physiology and medicine.

Improvement in insulin sensitivity and prevention of high fat diet-induced liver pathology using a CXCR2 antagonist.

BACKGROUND: Liver pathology (LP) characteristic of non-alcoholic fatty acid disease (NAFLD)/non-alcoholic steatohepatitis (NASH) is a prevalent co-morbidity of type 2 diabetes (T2D). Accumulating evidence indicates that neutrophils driving insulin resistance (IR), including hepatic IR, precipitate T2D-associated NAFLD/NASH. We hypothesized that targeting neutrophil accumulation into insulin-sensitive tissues in mice using a CXCR2 antagonist under T2D-precipitating high fat diet (HFD) could improve insulin sensitivity and prevent the progression towards liver pathology reminiscent of NAFLD/NASH. METHODS: Mice were age-matched and on standard rodent chow prior to 1:1 randomization into control and HFD formulated with the CXCR2 antagonist AZD5069 or with biologically inactive substitute. They were monitored for metabolic changes including insulin sensitivity using the hyperinsulinemic-euglycemic clamp and hepatic histopathologic evaluation in H&E-stained sections as well as via immunofluorescence microscopy of liver sections for leukocyte markers, collagen 1A1 formation, α-smooth muscle actin (SMA), and galectin-3 expression, for 16 weeks. Statistical tests used to determine significant differences among study groups and outcomes include Student's t-test, one-way ANOVA, repeated measures two-way ANOVA, and Fisher's exact test, depending on the analytical question. RESULTS: Compared to mice on HFD, mice in the AZD5069-formulated HFD exhibited improved insulin sensitivity, a modest reduction in weight gain, and a significant improvement in LP and markers related to NAFLD/NASH. Mice in the AZD5069-formulated HFD also exhibited reduced neutrophil accumulation into the liver at the end of the 16 week study period. CONCLUSIONS: These results show, for the first time, the effectiveness of a selective CXCR2 antagonist to improve insulin sensitivity, concomitantly preventing the progression towards LP characteristic of NAFLD/NASH. This represents a novel approach to target IR and developing LP under T2D-susceptible conditions using a single agent. Furthermore, our data extend the growing evidence in support of neutrophils as a leukocyte population that imprints and maintains a chronic inflammatory state in the progression of dysregulated metabolism in liver-specific co-morbid conditions.