PPARα Knockout Mice Research Articles

PPAR-α Insufficiency Enhances Doxorubicin-Induced Nephropathy in PPAR-α Knockout Mice and a Murine Podocyte Cell Line.

Peroxisome proliferator-activated receptor-alpha (PPAR-α) and its exogenous activators (fibrates) promote autophagy. However, whether the deleterious effects of PPAR-α deficiency on doxorubicin (DOX)-induced podocytopathy are associated with reduced autophagy remains to be clarified. We investigated the mechanisms of PPAR-α in DOX-induced podocytopathy and tubular injury in PPAR-α knockout (PAKO) mice and in a murine podocyte cell line. DOX-treated PAKO mice showed higher serum levels of triglycerides and non-esterified fatty acids and more severe podocytopathy than DOX-treated wild-type mice, as evidenced by higher urinary levels of proteins and podocalyxin at 3 days to 2 weeks and higher blood urea nitrogen and serum creatinine levels at 4 weeks. Additionally, there was an increased accumulation of p62, a negative autophagy marker, in the glomerular and tubular regions in DOX-treated PAKO mice at Day 9. Moreover, DOX-treated PAKO mice showed more severe glomerulosclerosis and tubular damage and lower podocalyxin expression in the kidneys than DOX-treated control mice at 4 weeks. Furthermore, DOX treatment increased p-p53, an apoptosis marker, and cleaved the caspase-3 levels and induced apoptosis, which was ameliorated by fenofibrate, a PPAR-α activator. Fenofibrate further enhanced AMPK activation and autophagy under fed and fasting conditions. Conclusively, PPAR-α deficiency enhances DOX-induced podocytopathy, glomerulosclerosis, and tubular injury, possibly by reducing autophagic activity in mouse kidneys.

Oleuropein mitigates non-alcoholic fatty liver disease (NAFLD) and modulates liver metabolites in high-fat diet-induced obese mice via activating PPARα.

This study aimed to elucidate the mechanism of oleuropein (OLE) ameliorates non-alcoholic fatty liver disease (NAFLD) and its underlying mechanisms. Male C57BL/6J mice were fed either a low-fat diet (LFD), a high-fat diet (HFD), or a HFD supplemented with 0.03% (w/w) OLE for 16 weeks. OLE supplementation decreased body weight and liver weight, improved serum lipid profiles, and ameliorated HFD-induced hepatic dysfunction. Liver metabolomics analysis revealed that OLE increased the levels of nicotinamide, tauroursodeoxycholic acid, taurine, and docosahexaenoic acid, which were beneficial for lipid homeostasis and inflammation regulation. OLE exerted its protective effects by activating peroxisome proliferator-activated receptor alpha (PPARα), a key transcription factor that regulates fibroblast growth factor 21 (FGF21) expression and modulates lipid oxidation, lipogenesis and inflammation pathways. Importantly, OLE supplementation did not significantly affect body weight or liver weight in PPARα knockout (PPARα KO) mice, indicating that PPARα is essential for OLE-mediated NAFLD prevention. Our results suggest that OLE alleviates NAFLD in mice by activating PPARα and modulating liver metabolites. © 2024 Society of Chemical Industry.

Chrysanthemum morifolium Ramat extract and probiotics combination ameliorates metabolic disorders through regulating gut microbiota and PPARα subcellular localization

BackgroundChrysanthemum morifolium Ramat, a traditional Chinese medicine, has the effects on liver clearing, vision improving, and anti-inflammation. C. morifolium and probiotics have been individually studied for their beneficial effects on metabolic diseases. However, the underlying molecular mechanisms were not completely elucidated. This study aims to elucidate the potential molecular mechanisms of C. morifolium and probiotics combination (CP) on alleviating nonalcoholic fatty liver disease (NAFLD) and the dysregulation of glucose metabolism in high-fat diet (HFD)-fed mice.MethodsThe therapeutic effect of CP on metabolism was evaluated by liver histology and serum biochemical analysis, as well as glucose tolerance test. The impact of CP on gut microbiota was analyzed by 16S rRNA sequencing and fecal microbiota transplantation. Hepatic transcriptomic analysis was performed with the key genes and proteins validated by RT-qPCR and western blotting. In addition, whole body Pparα knockout (Pparα−/−) mice were used to confirm the CP-mediated pathway.ResultsCP supplementation ameliorated metabolic disorders by reducing body weight and hepatic steatosis, and improving glucose intolerance and insulin resistance in HFD fed mice. CP intervention mitigated the HFD-induced gut microbiota dysbiosis, which contributed at least in part, to the beneficial effect of improving glucose metabolism. In addition, hepatic transcriptomic analysis showed that CP modulated the expression of genes associated with lipid metabolism. CP downregulated the mRNA level of lipid droplet-binding proteins, such as Cidea and Cidec in the liver, leading to more substrates for fatty acid oxidation (FAO). Meanwhile, the expression of CPT1α, the rate-limiting enzyme of FAO, was significantly increased upon CP treatment. Mechanistically, though CP didn’t affect the total PPARα level, it promoted the nuclear localization of PPARα, which contributed to the reduced expression of Cidea and Cidec, and increased expression of CPT1α, leading to activated FAO. Moreover, whole body PPARα deficiency abolished the anti-NAFLD effect of CP, suggesting the importance of PPARα in CP-mediated beneficial effect.ConclusionThis study revealed the hypoglycemic and hepatoprotective effect of CP by regulating gut microbiota composition and PPARα subcellular localization, highlighting its potential for therapeutic candidate for metabolic disorders.

Assessing the ability of dihydrosterculic acid to activate PPARα

Peroxisome proliferator-activated receptor α (PPARα) is a ligand-activated transcription factor associated with the expression of specific target genes involved in fatty acid β-oxidation. Activation of PPARα could potentially be utilized to treat diseases associated with the accumulation of hepatic lipids such as non-alcoholic fatty liver disease (NAFLD), which currently impacts approximately 25% of the world’s population. At present, a class of synthetic ligands called fibrates can be prescribed to help manage NAFLD but are weak agonists for PPARα and therefore have limited effcacy. Consequently, alternative ligands for PPARα must be established and no natural endogenous ligands have been conclusively identified. This study expands on our previous work and aims to identify the direct effects of dihydrosterculic acid (DHSA), a cyclopropyl fatty acid found in cottonseed oil (CSO), on PPARα activity. To identify downstream effects of DHSA, male mice were placed on a CSO- (DHSA-containing) or isocaloric oil-enriched diet for 6 weeks and tissues were sent for genome-wide RNA sequencing analysis. CSO-fed mice demonstrated increased PPARα gene expression in the liver and concomitant decreased triglyceride in plasma (-75.1±11.5 milligrams/deciliter (mg/dL); p<0.01) and liver (-86.7±71.7 mg/gram (g) protein; p=0.1) and decreased free fatty acid in liver (-16.5±3.4 micromolar (μM)/g protein; p<0.01) relative to the oil-enriched control diet. Confirmational tissue analysis of liver samples show an increase in PPARα target gene expression in the DHSA containing groups compared to control diet groups, indicating that DHSA could potentially serve as an endogenous ligand for PPARα to increase its transcriptional activity to increase fat oxidation. To further test this hypothesis, female PPARα knockout mice were fed a DHSA containing diet or control diet for 4 weeks (n=8). Following the 4-week period, the chow-fed wild type females gained more weight (1.3±0.6 g; p<0.05) than the PPARα knockout mice. Otherwise, there were no significant differences in body weight or food intake between diets or genotypes. Additional in vitro studies show increased PPARα target gene expression following treatments with DHSA, specifically for CPT1a (2.7±0.2 fold increase; p<0.01), ACOX1 (1.3±0.1 fold increase; p<0.05), and ACADVL (1.6±0.1 fold increase, p<0.01), which encode key enzymes in lipid oxidation pathways. SCD1, the rate limiting enzyme in lipogenesis, was also downregulated (0.5±0.03 fold decrease, p<0.01) in response to a 24-hour treatment with DHSA indicating a reduction in endogenous lipogenesis. From these results, we believe that DHSA could function through a PPARα-dependent mechanism. Further analysis of the tissues collected from the PPARα knockout mouse model will aid in determining the mechanism of DHSA as well as identifying its potential for the treatment of excess lipid accumulation. This project is funded by Cotton Incorporated. This is the full abstract presented at the American Physiology Summit 2024 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.

Regulation of Myocardial Lipid Accumulation in the Development of Obesity

Energy metabolism is important in the development of heart disease, which is the leading cause of death worldwide. The importance of energy metabolism in disease is becoming even more clear with the rise in obesity, metabolic diseases, and the recognition that they all fit together as with the recent offcial report of a new syndrome named Cardiovascular-Kidney-Metabolic (CKM) Syndrome. A complete understanding of myocardial lipid storage and subsequent utilization of lipids is essential to understanding the impact of these metabolic diseases in the heart and on the development of cardiac dysfunction and hypertrophy. In this study we investigate how the heart controls fuel storage by taking advantage of the impact of the transcription factor peroxisome proliferator activated receptor α (PPARα) on 1) fatty acid metabolism and lipid accumulation, 2) its key role in sex differences in cardiac hypertrophy which we previously reported, and 3) the regulation of lipid droplets. It is controversial whether a decline in myocardial PPARα mediates cardiac hypertrophy and whether it increases or reduces lipid accumulation. Using our tamoxifen inducible cardiac-specific PPARα knockout mouse (cPPAR−/−) we see less cardiac lipid accumulation in response to a 5 week high fat diet. Our data indicate that this difference in triglyceride levels is present in both males and females. Importantly, this is not driven by a difference in fatty acid supply to these hearts. Serum fatty acid levels are not lower in cPPAR−/− vs Control mice fed a high fat diet. Further, we have not observed a difference in high fat diet induced body weight gain between cPPAR−/− and Control mice. We have begun to examine how these differences in high fat diet-induced lipid accumulation correlate with cardiac hypertrophy by looking at heart weight normalized to tibia length. While we did not expect 5 weeks of a high fat diet to induce cardiac hypertrophy, it is possible that the difference in cardiac lipid storage in the cPPAR−/− hearts could impact this. This measurement did not indicate a difference in cardiac hypertrophy between high fat diet fed cPPAR−/− and Control mice. We are utilizing this mouse as a model to investigate the regulation of lipid droplets and the lipid droplet proteome. So far, our results are indicating that the regulation of lipid droplet proteins is not affected in a uniform manner. For example, our data is suggesting that the expression of different members of the perilipin family are affected in distinct ways. Overall, these data indicate that a reduction in cardiac PPARα protects against obesity induced cardiac lipid accumulation. Going forward, it will be interesting to see how these changes occur in relation to the lipid droplet proteome and whether any changes that occur differentially in males vs female may be involved in sex differences in cardiac hypertrophy. Better understanding these factors will be essential for the development of safe and effective future therapies for CKM syndrome and other cardiovascular diseases. ND-EPSCoR. This is the full abstract presented at the American Physiology Summit 2024 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.

Cardioprotective Role of Beta Hydroxybutyrate in Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD)

Background: Cardiovascular disease (CVD) is the leading cause of mortality in metabolic dysfunction-associated steatotic liver disease (MASLD). Beta hydroxybutyrate (BHOB), a liver metabolite, is the major ketone that serves as an alternative fuel source in the body. Previously, we observed cardiovascular dysfunction that was associated with reduced circulating BHOB in hepatocyte-specific PPARα knockout mice (PparaHEPKO), a mouse model that exhibits hepatic steatosis independent of obesity and insulin resistance. Hypothesis: We hypothesize that restoring plasma BHOB levels will attenuate the mechanisms underlying hepatic steatosis-induced cardiovascular dysfunction and improve cardiovascular function in PparaHEPKO mice.Aims: To determine the cardioprotective role of increased plasma levels of BHOB in CVDs induced by MASLD. Methods: 30 week old male PparaHEPKO mice were given 1,3 butanediol (20% in drinking water) (PparaHEPKO+ 1,3 butanediol) or vehicle (PparaHEPKO) (n=6) for 6 weeks. Plasma BHOB was measured at baseline and after treatment. Cardiac structure and function were measured by high resolution ultrasound echocardiography (VEVO 3100). Mean arterial blood pressure was measured by radio telemetry. Cardiac lipid accumulation was determined by Oil Red O (ORO) and cardiac triglyceride levels. Cardiac apoptosis and fibrosis were determined by TUNEL and picrosirius red staining, further confirmed by western blot. Cardiac natriuretic peptides were determined by real-time PCR. Liver fat was determined by EchoMRI, ORO staining and hepatic triglyceride levels.Results: After 6 weeks of 1,3 butanediol treatment, PparaHEPKO exhibit increased plasma BHOB compared to baseline (0.5 ± 0.01 vs. 0.2 ± 0.02mmol/L), attenuated arterial blood pressure compared to control (109 ± 3 vs. 121 ± 4mmHg), improved cardiac output (13.8 ± 0.8 vs. 11.1 ± 0.7mL/min), stroke volume (31.1 ± 2.1 vs. 23.4 ± 1.3μL), and isovolumic relaxation time (18.7 ± 0.8 vs. 20.6 ± 0.9ms). 1,3 butanediol treatment also attenuated vascular stiffness, cardiac lipid (0.7 ± 0.27 vs. 1.5 ± 0.17), ANP (1.1 ± 0.03 vs. 1.3 ± 0.03), COL1A1 (0.9 ± 0.1 vs. 9.0 ± 0.5), and cleaved caspase-3 (1.8 ± 0.3 vs. 3.7 ± 0.7). Interestingly, 1,3 butanediol did not alleviate hepatic fat compared to control as demonstrated by EchoMRI (0.8 ± 0.3 vs. 0.7 ± 0.3%), hepatic triglyceride (1.4 ± 0.3 vs. 1.3 ± 0.2mM) and Oil Red O staining.Conclusion: Our findings indicate that increasing plasma BHOB level improves arterial blood pressure, exercise tolerance, systolic, diastolic, and vascular functions in MASLD-induced CVD. Furthermore, BHOB attenuates cardiac lipid, apoptosis and fibrosis. However, BHOB did not alleviate hepatic steatosis suggesting that BHOB improves cardiovascular functions in PparaHEPKO mice independent of hepatic fat content. This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Grant 1R01DK121748-01A1 (D.E. Stec), the National Institute of General Medical Sciences Grant P20GM104357-02, P30GM149404 and P20GM144041 (D.E. Stec), and by American Heart Association Postdoctoral Award (23POST1020493, O.O. Badmus). This is the full abstract presented at the American Physiology Summit 2024 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.

Hepatic ketone body regulation of renal gluconeogenesis

ObjectivesDuring fasting, liver pivotally regulates blood glucose levels through glycogenolysis and gluconeogenesis. Kidney also produces glucose through gluconeogenesis. Gluconeogenic genes are transactivated by fasting, but their expression patterns are chronologically different between the two organs. We find that renal gluconeogenic gene expressions are positively correlated with the blood β-hydroxybutyrate concentration. Thus, we herein aim to investigate the regulatory mechanism and its physiological implications. MethodsGluconeogenic gene expressions in liver and kidney were examined in hyperketogenic mice such as high-fat diet (HFD)-fed and ketogenic diet-fed mice, and in hypoketogenic PPARα knockout (PPARα−/−) mice. Renal gluconeogenesis was evaluated by rise in glycemia after glutamine loading in vivo. Functional roles of β-hydroxybutyrate in the regulation of renal gluconeogenesis were investigated by metabolome analysis and RNA-seq analysis of proximal tubule cells. ResultsRenal gluconeogenic genes were transactivated concurrently with blood β-hydroxybutyrate uprise under ketogenic states, but the increase was blunted in hypoketogenic PPARα−/− mice. Administration of 1,3-butandiol, a ketone diester, transactivated renal gluconeogenic gene expression in fasted PPARα−/− mice. In addition, HFD-fed mice showed fasting hyperglycemia along with upregulated renal gluconeogenic gene expression, which was blunted in HFD-fed PPARα−/− mice. In vitro experiments and metabolome analysis in renal tubular cells showed that β-hydroxybutyrate directly promotes glucose and NH3 production through transactivating gluconeogenic genes. In addition, RNA-seq analysis revealed that β-hydroxybutyrate-induced transactivation of Pck1 was mediated by C/EBPβ. ConclusionsOur findings demonstrate that β-hydroxybutyrate mediates hepato–renal interaction to maintain homeostatic regulation of blood glucose and systemic acid-base balance through renal gluconeogenesis regulation.

The lack of PPARα exacerbated the progression of non-alcoholic steatohepatitis in mice with spleen deficiency syndrome by triggering an inflammatory response.

In addition to abnormal liver inflammation, the main symptoms of non-alcoholic steatohepatitis (NASH) are often accompanied by gastrointestinal digestive dysfunction, consistent with the concept of spleen deficiency (SD) in traditional Chinese medicine. As an important metabolic sensor, whether peroxisome proliferator-activated receptor alpha (PPARα) participates in regulating the occurrence and development of NASH with SD (NASH-SD) remains to be explored. Clinical liver samples were collected for RNA-seq analysis. C57BL/6J mice induced by folium sennae (SE) were used as an SD model. qPCR analysis was conducted to evaluate the inflammation and metabolic levels of mice. PPARα knockout mice (PPARαko) were subjected to SE and methionine-choline-deficient (MCD) diet to establish the NASH-SD model. The phenotype of NASH and the inflammatory indicators were measured using histopathologic analysis and qPCR as well. The abnormal expression of PPARα signaling, coupled with metabolism and inflammation, was found in the results of RNA-seq analysis from clinical samples. SD mice showed a more severe inflammatory response in the liver evidenced by the increases in macrophage biomarkers, inflammatory factors, and fibrotic indicators in the liver. qPCR results also showed differences in PPARα between SD mice and control mice. In PPARαko mice, further evidence was found that the lack of PPARα exacerbated the inflammatory response phenotype as well as the lipid metabolism disorder in NASH-SD mice. The abnormal NR signaling accelerated the vicious cycle between lipotoxicity and inflammatory response in NAFLD with SD. Our results provide new evidence for nuclear receptors as potential therapeutic targets for NAFLD with spleen deficiency.

Bezafibrate alleviates diabetes-induced spermatogenesis dysfunction by inhibiting inflammation and oxidative stress

The metabolic disorders caused by diabetes can lead to various complications, including male spermatogenesis dysfunction. Exploring effective therapeutics that attenuate diabetes mellitus (DM)-induced male subfertility is of great importance. Pharmaceuticals targeting PPARα activation such as bezafibrate have been regarded as an important strategy for patients with diabetes. In this study, we use streptozocin (STZ) injection to establish a type 1 DM mice model and use bezafibrate to treat DM mice and evaluate the effects of bezafibrate on the spermatogenic function of the DM male mice. Bezafibrate treatment exhibited protective effects on DM-induced spermatogenesis deficiency, as reflected by increased testis weight, improved histological morphology of testis, elevated sperm parameters, increased serum testosterone concentration as well as increased mRNA levels of steroidogenesis enzymes. Meanwhile, testicular cell apoptosis, inflammation accumulation and oxidative stress status were also shown to be alleviated by bezafibrate compared with the DM group. In vivo and in vitro studies, PPARα specific inhibitor and PPARα knockout mice were further used to investigate the role of PPARα in the protective effects of bezafibrate on DM-induced spermatogenesis dysfunction. Our results indicated that the protection of bezafibrate on DM-induced spermatogenesis deficiency was abrogated by PPARα inhibition or deletion. Our study suggested that bezafibrate administration could ameliorate DM-induced spermatogenesis dysfunction and may represent a novel practical strategy for male infertility.

Emerging contaminants at trace levels of pesticides perturbs biomolecules in different organs in mice: Role of peroxisome proliferator-activated receptor-alpha

Objectives: Information is lacking on the consequences of chronic exposure to emerging contaminants at environmentally relevant (trace concentrations) on biomolecules. Environmental exposure to these chemical mixtures happens at trace concentrations and at multiple molecular interactions. The consequences of trace concentrations of multiple pesticides (MPs) on the regulation of selected biomolecules nitric oxide (NO), thiols, superoxide dismutase (SOD), and glutathione S-transferase (GST) in the tissues from wild type (WT) and genetically deficient- peroxisome proliferator-activated receptor-alpha (PPARα) knockout (Null) mice were investigated. Material and Methods: Mice were exposed to trace concentrations of MPs: Atrazine, dieldrin, endrin, endosulfan, and anthracene (1–100 ng/L) in drinking water for 6 weeks. Organs were collected and homogenized; NO, protein and non-protein thiol levels, as well as SOD and GST activities were determined. Results: Differential and organ selective effects of the treatments were observed in the WT and PPARα knockout. Increased NO levels were observed in the organs from WT with limited increase in the kidney (Null). SOD activity was decreased in the organs from the WT and was increased in the PPARα knockout when compared to the control. Thiol level was significantly increased in the heart and spleen in the WT and in the heart of the PPARα knockout mice when compared to the control. Non-protein thiol concentration was reduced in the heart and kidney (WT) and reduced in the liver of the PPARα knockout when compared to the control. GST activity was significantly decreased in the liver and spleen (WT) and was significantly elevated in all organs in the PPARα knockout mice when compared to the WT. Conclusion: The low concentrations of MPs may have caused selective dysregulation of biomolecules in different organs of the body. These effects observed may be influenced by genetic status such as in PPARα deficiency. These results present a scenario that implicates nanoconcentrations of series of organic contaminants that can cause cellular and molecular dysregulations of biomolecules precipitating toxicity and pathology that can be a threat to human health. Further, investigation into the molecular mechanism(s) and signaling pathway(s) implicated in these dysregulations is warranted.

Hyodeoxycholic acid ameliorates nonalcoholic fatty liver disease by inhibiting RAN-mediated PPARα nucleus-cytoplasm shuttling

Nonalcoholic fatty liver disease (NAFLD) is usually characterized with disrupted bile acid (BA) homeostasis. However, the exact role of certain BA in NAFLD is poorly understood. Here we show levels of serum hyodeoxycholic acid (HDCA) decrease in both NAFLD patients and mice, as well as in liver and intestinal contents of NAFLD mice compared to their healthy counterparts. Serum HDCA is also inversely correlated with NAFLD severity. Dietary HDCA supplementation ameliorates diet-induced NAFLD in male wild type mice by activating fatty acid oxidation in hepatic peroxisome proliferator-activated receptor α (PPARα)-dependent way because the anti-NAFLD effect of HDCA is abolished in hepatocyte-specific Pparα knockout mice. Mechanistically, HDCA facilitates nuclear localization of PPARα by directly interacting with RAN protein. This interaction disrupts the formation of RAN/CRM1/PPARα nucleus-cytoplasm shuttling heterotrimer. Our results demonstrate the therapeutic potential of HDCA for NAFLD and provide new insights of BAs on regulating fatty acid metabolism.

Regulation of renal function by the peroxisome proliferator-activated receptor-alpha: A novel target for treating hypertension

Approximately 37 million people in the U.S. have chronic kidney disease, which is a major risk factor for cardiovascular and end stage renal diseases. We have identified a nuclear receptor, peroxisome proliferator-activated receptor – alpha (PPAR-α), that plays a major role in regulating renal glomerular filtration rate in an experimental model of hypertension. Targeting PPAR-α signaling provides novel therapeutic strategies for reducing hypertension associated chronic kidney disease. PPAR-α knockout (KO) mice exhibit increased renal inflammation and blood pressure response to Angiotensin II (Ang II). In this study, we investigated the role of PPAR-α in renal function in a mouse model of hypertension. Male 4-month-old wild type (WT) and PPAR-α KO mice were instrumented with radio transmitters by artery canulation (Data Science International). This method minimizes stress and artifacts by avoiding the use of tethering, restraining, or anesthetizing the mice during data sampling. After recovery from surgery, we continuously measured mean arterial pressure (MAP) via radio telemetry in conscious ambulatory mice. After baseline MAP was established, vehicle (Veh; saline) or Ang II were infused using an osmotic minipump at a slow pressor dose (400 ng/kg/min) for 12 days. On day 12, we injected an intravenous bolus of fluorescin-sinistrin (3.74 ml/g body weight) and collected 8 blood samples (20 μl/sample) during a 75-minute period to enable calculation of the glomerular filtration rate (GFR) using [GFR = I/(A/a + B/b]. Similar to our prior observations, no significant (ns) differences in baseline MAP were observed between WT and PPAR-α KO mice [(mmHg): WT (n=6), 111±20 vs. PPAR-α KO (n=6), 113 ± 10; ns] whereas after 12 days of the slow pressor effect of Ang II, MAP was increased in both strains [(mmHg): WT (n=8), 138 ± 11# vs. PPAR-α KO (n=8), 156 ± 16#; #p<0.05 vs. basal, same strain]; however, the increase in MAP was greater in the PPAR-α KO (43 mmHg) compared to WT (27 mmHg) mice. The absence of PPAR-α reduced GFR by 24% under basal conditions [(μl/min): WT (n=3), 346 ± 20 vs. PPAR-α KO (n=3), 275 ± 27*, *p<0.05 vs WT]. After 12 days of Ang II infusion, GFR was reduced in WT mice by 27% and did not further reduce GFR in PPAR-α KO mice [(μl/min): WT (n=6), 251 ± 11# vs. PPAR-α KO (n=6), 277 ± 61; #p<0.05 vs. basal, same strain]. PPAR-α protects mice from worsening hypertension and is critical to preserving GFR during normotensive conditions. Ongoing studies are further investigating how PPAR-α regulates renal function. These finding suggest therapeutics designed to increase PPAR-α activity could have clinical benefit in chronic kidney disease. Howard University Research Centers at Minority Institutions, Georgetown-Howard Universities Center for Clinical and Translational Science, Howard University Aging Grant, K01HL092593 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.

Cardiac specific PPAR alpha knock-down protects against cardiac lipid accumulation during metabolic stress

A dysregulated lipid metabolism contributes to the pathological development of a wide array of diseases including Type 2 Diabetes and associated cardiovascular diseases. One main regulator of cardiac lipid metabolism is the transcription factor Peroxisome Proliferator Activated Receptor α (PPARα). PPARα has been shown to have regulatory effects on many proteins that are integral for the proper regulation of lipid metabolism including those involved in fatty acid oxidation, mitochondria, and lipid droplets. After triglycerides (TG) accumulate and are stored in lipid droplets, the fat is then transferred from lipid droplets to mitochondria where the fatty acids undergo fatty acid oxidation and are used for ATP synthesis. In this research we aimed to determine how cardiac PPARα regulates lipid metabolism and the organelles responsible for its homeostasis in the heart during metabolic stress in the context of obesity. To study this, we used our tamoxifen-inducible cardiomyocyte specific PPARα knockout (cPPARα -/- ) mouse. To induce obesity, we fed Control and cPPARα -/- mice a high fat diet (HFD) for 5 weeks. Our results show that the TG content is lower in hearts from cPPARα -/- vs Control mice fed the HFD. cPPARα -/- vs Control hearts also had a lower expression of Plin2 (a key lipid droplet protein) when fed a HFD. also agrees with our previous findings in fasted/fed hearts. Interestingly, this agrees with our previous findings that the hearts in cPPARα -/- mice were protected against the elevation in TG that normally occurs in response to short term fasting (16 hr fast). Therefore, a reduction in cardiac PPARα protects against lipid accumulation in the heart. As mitochondrial dysfunction has been linked to the cardiac dysfunction present in diabetic hearts and may be involved in regulating lipid accumulation, we next wanted to test whether cPPARα -/- affected mitochondrial function. Measurements of mitochondrial stress proteins have not suggested any elevation in mitochondrial dysfunction in the cPPARα -/- hearts either at baseline or in response to the HFD or 16 hr fast. Interestingly, cPPARα -/- hearts had a higher expression of the uncut ATF6 protein than the control hearts when fasted which suggests that the cPPARα -/- hearts may have less mitochondrial stress due to less excess ROS in the heart. In agreement with this, echocardiography measurements have so far not indicated any reduction in the cardiac function of cPPARα -/- vs control mice. In the future it will be important to look into mitochondrial dysfunction in greater depth including the regulation of mitochondrial fission and fusion pathways. Overall, this suggests that cardiac PPARα may protect against cardiac lipid accumulation in obesity and that this may be through the regulation of Plin2 and mitochondrial stress pathways. ND EPSCoR 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.

Bile acid metabolism disorder mediates hepatotoxicity of Nafion by-product 2 and perfluorooctane sulfonate in male PPARα-KO mice

Perfluorooctane sulfonate (PFOS) and Nafion by-product 2 (H-PFMO2OSA) induce hepatotoxicity in male mice via activation of the peroxisome proliferator-activated receptor α (PPARα) pathway; however, accumulating evidence suggests that PPARα-independent pathways also play a vital role in hepatotoxicity after exposure to per- and polyfluoroalkyl substances (PFASs). Thus, to assess the hepatotoxicity of PFOS and H-PFMO2OSA more comprehensively, adult male wild-type (WT) and PPARα knockout (PPARα-KO) mice were exposed to PFOS and H-PFMO2OSA (1 or 5 mg/kg/d) for 28 d via oral gavage. Results showed that although elevations in alanine transaminase (ALT) and aspartate aminotransferase (AST) were alleviated in PPARα-KO mice, liver injury, including liver enlargement and necrosis, was still observed after PFOS and H-PFMO2OSA exposure. Liver transcriptome analysis identified fewer differentially expressed genes (DEGs) in the PPARα-KO mice than in the WT mice, but more DEGs associated with the bile acid secretion pathway after PFOS and H-PFMO2OSA treatment. Total bile acid content in the liver was increased in the 1 and 5 mg/kg/d PFOS-exposed and 5 mg/kg/d H-PFMO2OSA-exposed PPARα-KO mice. Furthermore, in PPARα-KO mice, proteins showing changes in transcription and translation levels after PFOS and H-PFMO2OSA exposure were involved in the synthesis, transportation, reabsorption, and excretion of bile acids. Thus, exposure to PFOS and H-PFMO2OSA in male PPARα-KO mice may disturb bile acid metabolism, which is not under the control of PPARα.

PGC-1α and MEF2 Regulate the Transcription of the Carnitine Transporter OCTN2 Gene in C2C12 Cells and in Mouse Skeletal Muscle.

OCTN2 (SLC22A5) is a carnitine transporter whose main function is the active transport of carnitine into cells. In skeletal muscle and other organs, the regulation of the SLC22A5 gene transcription has been shown to depend on the nuclear transcription factor PPAR-α. Due to the observation that the muscle OCTN2 mRNA level is maintained in PPAR-α knock-out mice and that PGC-1α overexpression in C2C12 myoblasts increases OCTN2 mRNA expression, we suspected additional regulatory pathways for SLC22A5 gene transcription. Indeed, we detected several binding sites of the myocyte-enhancing factor MEF2 in the upstream region of the SLC22A5 gene, and MEF2C/MEF2D stimulated the activity of the OCTN2 promoter in gene reporter assays. This stimulation was increased by PGC-1α and was blunted for a SLC22A5 promoter fragment with a mutated MEF2 binding site. Further, we demonstrated the specific binding of MEF2 to the SLC22A5 gene promoter, and a supershift of the MEF2/DNA complex in electrophoretic mobility shift assays. In immunoprecipitation experiments, we could demonstrate the interaction between PGC-1α and MEF2. In addition, SB203580, a specific inhibitor of p38 MAPK, blocked and interferon-γ stimulated the transcriptional activity of the SLC22A5 gene promoter. Finally, mice with muscle-specific overexpression of OCTN2 showed an increase in OCTN2 mRNA and protein expression in skeletal muscle. In conclusion, we detected and characterized a second stimulatory pathway of SLC22A5 gene transcription in skeletal muscle, which involves the nuclear transcription factor MEF2 and co-stimulation by PGC-1α and which is controlled by the p38 MAPK signaling cascade.

The Loss of PPARα in Adipocytes Induces Lipogenesis via the PASK‐SREBP1 Signaling Axis

PPARα is a nuclear receptor and a key player in regulating adiposity by activating b‐oxidation while inhibiting de novo lipogenesis within the liver. Its role in extrahepatic tissues is currently unknown. Therefore, we established the first adipose‐specific PPARα knockout (PparaFatKO) mice to determine the signaling position of PPARα in adipose tissue expansion and adipocyte hypertrophy that occurs during the development of obesity. To assess the function of PPARα in adiposity, female and male mice were placed on a high‐fat diet (HFD) or normal chow for 30 weeks. Only the male PparaFatKO animals had significantly more adiposity in the inguinal white adipose tissue (iWAT) (p=0.0091) and brown adipose tissue (BAT) (p=0.0031) with HFD compared to littermates. No changes in adiposity were observed in female mice compared to littermate controls. Lipidomics analysis of iWAT via liquid chromatography–mass spectrometry (LC‐MS) showed that PparaFatKO males had an increase in cholesterol esters. Interrogation of the signaling mechanisms with the loss of PPARα in adipocytes showed that the transcription factor sterol regulatory element‐binding protein‐1 (SREBP‐1) had more of the mature form present in the KO males and its target genes were higher, which might be due to the significantly (p=0.009) elevated expression of the Per‐Arnt‐Sim Kinase (PASK), a kinase that activates SREBP‐1. The hyperactivity of the PASK‐SREBP‐1 axis significantly (p<0.05) increased the lipogenesis proteins fatty acid synthase (FAS) and stearoyl‐Coenzyme A desaturase 1 (SCD1) and raised the expression of genes for cholesterol metabolism (Scarb1, Abcg1, and Abca1). Hence, we have uncovered a new signaling paradigm, the PASK‐SREBP‐1 axis in adipocytes, that drives lipogenesis, which PPARα inhibits. Our results demonstrate the first adipose‐specific actions for PPARα in protecting against lipogenesis and cholesterol ester accumulation that leads to adipocyte hypertrophy in obesity.

Cardiac specific knock-down of peroxisome proliferator activated receptor α prevents fasting-induced cardiac lipid accumulation and reduces perilipin 2

While fatty acid metabolism is altered under physiological conditions, alterations can also be maladaptive in diseases such as diabetes and heart failure. Peroxisome Proliferator Activated Receptor α (PPARα) is a transcription factor that regulates fat metabolism but its role in regulating lipid storage in the heart is unclear. The aim of this study is to improve our understanding of how cardiac PPARα regulates cardiac health and lipid accumulation. To study the role of cardiac PPARα, tamoxifen inducible cardiac-specific PPARα knockout mouse (cPPAR-/-) were treated for 5 days with tamoxifen and then studied after 1–2 months. Under baseline conditions, cPPAR-/- mice appear healthy with normal body weight and mortality is not altered. Importantly, cardiac hypertrophy or reduced cardiac function was also not observed at baseline. Mice were fasted to elevate circulating fatty acids and induce cardiac lipid accumulation. After fasting, cPPAR-/- mice had dramatically lower cardiac triglyceride levels than control mice. Interestingly, cPPAR-/- hearts also had reduced Plin2, a key protein involved in lipid accumulation and lipid droplet regulation, which may contribute to the reduction in cardiac lipid accumulation. Overall, this suggests that a decline in cardiac PPARα may blunt cardiac lipid accumulation by decreasing Plin2 and that independent of differences in systemic metabolism a decline in cardiac PPARα does not seem to drive pathological changes in the heart.

PPAR-α knockout leads to elevated blood pressure response to angiotensin II infusion associated with an increase in renal α-1 Na+/K+ ATPase protein expression and activity

Peroxisome proliferator activated receptor alpha (PPAR-α) deletion has been shown to increase blood pressure (BP). We hypothesized that the BP increase in PPAR-α KO mice was mediated by increased expression and activity of basolateral Na+/K+ ATPase (NKA) pump. To address this hypothesis, we treated wild-type (WT) and PPAR-α knockout (KO) mice with a slow-pressor dose of angiotensin II (400 ng/kg·min) for 12 days by osmotic minipump. Radiotelemetry showed no significant differences in baseline mean arterial pressure (MAP) between WT and PPAR-α KO mice; however, by day 12 of infusion, MAP was significantly higher in PPAR-α KO mice (156 ± 16) compared to WT mice (138 ± 11 mmHg). NKA activity and protein expression (α1 subunit) were significantly higher in PPAR-α KO mice compared to WT mice. There was no significant difference in NKA mRNA levels. Angiotensin II further increased the expression and activity of the NKA in both genotypes along with the water channel, aquaporin 1 (Aqp1). In contrast, angiotensin II decreased the expression (64–97% reduction in band density) of sodium‑hydrogen exchanger-3 (NHE3), NHE regulatory factor-1 (NHERF1, Slc9a3r1), sodium‑potassium-2-chloride cotransporter (NKCC2), and epithelial sodium channel (ENaC) β- and γ- subunits in the renal cortex of both WT and PPAR-α KO mice, with no difference between genotypes. The sodium-chloride cotransporter (NCC) was also decreased by angiotensin II, but significantly more in PPAR-α KO (59% WT versus 77% KO reduction from their respective vehicle-treated mice). Our results suggest that PPAR-α attenuates angiotensin II-mediated increased blood pressure potentially via reducing expression and activity of the NKA.

In vitro and in vivo study of the pathogenic role of PPARα in experimental periodontitis.

Objective The purpose of this study is to investigate the pathogenic role of PPARα in periodontal antigen treated gingival cells in vitro and in experimental periodontitis in vivo .Methodology Gingival fibroblasts, gingival epithelial cells and splenocytes were isolated from C57BL/6J wild type (WT) mice and treated with fixed P. gingivalis at for 48 hours. The mRNA levels of PPARs, TNFα, IL-1β and IL-10 were detected by Real-time quantitative PCR. Silk ligatures after being soaked in the P.gingivalis suspension were tied around both maxillary second molars of WT mice or PPARα knock-out (KO) mice for two weeks. PPARα agonist fenofibrate and vehicle control were injected into the different side of the palatal gingiva on days 3, 6, and 9. At day 14, bone resorption and gingival mRNA expression levels of PPARs, TNFα, IL-1β and IL-10 were measured by micro-computed tomography and RT-qPCR respectively.ResultsP. gingivalis treatment downregulated the expression of PPARα, but not PPARβ or PPARγ, and increased the expression of TNF-α and IL-1β in Gingival fibroblasts, gingival epithelial cells and splenocytes from WT mice. Gingival mRNA levels of PPARα were significantly decreased in experimental periodontitis in WT mice. The bone loss of PPARα KO mice in experimental periodontitis was significantly higher than WT mice and was not reduced by fenofibrate treatment. Gingival TNFα protein expressions were significantly increased by P. gingivalis associated ligation and decreased by fenofibrate treatment in WT mice but not in PPARα KO mice.Conclusion This study suggests that PPARα plays an essential role in periodontitis.

Lgr5+ cell fate regulation by coordination of metabolic nuclear receptors during liver repair

Background: Leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) is a target gene of Wnt/β-Catenin which plays a vital role in hepatic development and regeneration. However, the regulation of Lgr5 gene and the fate of Lgr5+ cells in hepatic physiology and pathology are little known. This study aims to clarify the effect of metabolic nuclear receptors on Lgr5+ cell fate in liver.Methods: We performed cell experiments with primary hepatocytes, Hep 1-6, Hep G2, and Huh 7 cells, and animal studies with wild-type (WT), farnesoid X receptor (FXR) knockout mice, peroxisome proliferator-activated receptor α (PPARα) knockout mice and Lgr5-CreERT2; Rosa26-mTmG mice. GW4064 and CDCA were used to activate FXR. And GW7647 or Wy14643 was used for PPARα activation. Regulation of Lgr5 by FXR and PPARα was determined by QRT-PCR, western blot (WB) and RNAscope® in situ hybridization (ISH) and immunofluorescence (IF), luciferase reporter assay, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). Diethyl 1,4-dihydro-2,4,6-trimethyl-3,5-pyridinedicarboxylate (DDC) diet was used to induce liver injury.Results: Pharmacologic activation of FXR induced Lgr5 expression, whereas activation of PPARα suppressed Lgr5 expression. Furthermore, FXR and PPARα competed for binding to shared site on Lgr5 promoter with opposite transcriptional outputs. DDC diet triggered the transition of Lgr5+ cells from resting state to proliferation. FXR activation enhanced Lgr5+ cell expansion mainly by symmetric cell division, but PPARα activation prevented Lgr5+ cell proliferation along with asymmetric cell division.Conclusion: Our findings unravel the opposite regulatory effects of FXR and PPARα on Lgr5+ cell fate in liver under physiological and pathological conditions, which will greatly assist novel therapeutic development targeting nuclear receptors.