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Increased aerobic glycolysis in keratinocytes has been reported as a hallmark of skin diseases while its pharmacological inhibition restores keratinocyte homeostasis. Pyruvate kinase muscle (PKM) isoforms are key enzymes in the glycolytic pathway and, therefore, an attractive therapeutic target. Simon Nold and colleagues used CRISPR/Cas9-mediated gene editing to investigate the outcomes of PKM splicing perturbations and specific PKM1 or PKM2 deficiency in human HaCaT keratinocytes. Collectively, the study demonstrated different effects of PKM1 or PKM2 depletion on the reciprocal PKM isoform and on keratinocyte gene expression, metabolism and proliferation. Findings from this study provide novel insights into the role of PKM in keratinocyte homeostasis, warranting additional investigations into the underlying molecular mechanisms and potential therapeutic applications.

Colorectal cancer (CRC) cells shift metabolism toward aerobic glycolysis and away from using oxidative substrates such as butyrate. Pyruvate kinase M1/2 (PKM) is an enzyme that catalyzes the last step in glycolysis, which converts phosphoenolpyruvate to pyruvate. M1 and M2 are alternatively spliced isoforms of the Pkm gene. The PKM1 isoform promotes oxidative metabolism, whereas PKM2 enhances aerobic glycolysis. We hypothesize that the PKM isoforms are involved in the shift away from butyrate oxidation towards glycolysis in CRC cells. Here, we find that PKM2 is increased and PKM1 is decreased in human colorectal carcinomas as compared to non-cancerous tissue. To test whether PKM1/2 alter colonocyte metabolism, we created a knockdown of PKM2 and PKM1 in CRC cells to analyze how butyrate oxidation and glycolysis would be impacted. We report that butyrate oxidation in CRC cells is regulated by PKM1 levels, not PKM2. Decreased butyrate oxidation observed through knockdown of PKM1 and PKM2 is rescued through re-addition of PKM1. Diminished PKM1 lowered mitochondrial basal respiration and decreased mitochondrial spare capacity. We demonstrate that PKM1 suppresses glycolysis and inhibits hypoxia-inducible factor-1 alpha. These data suggest that reduced PKM1 is, in part, responsible for increased glycolysis and diminished butyrate oxidation in CRC cells.

Background Acute kidney injury (AKI) is associated with a severe decline in kidney function caused by abnormalities within the podocytes' glomerular matrix. Recently, AKI has been linked to alterations in glycolysis and the activity of glycolytic enzymes, including pyruvate kinase M2 (PKM2). However, the contribution of this enzyme to AKI remains largely unexplored. Methods Cre-loxP technology was used to examine the effects of PKM2 specific deletion in podocytes on the activation status of key signaling pathways involved in the pathophysiology of AKI by lipopolysaccharides (LPS). In addition, we used lentiviral shRNA to generate murine podocytes deficient in PKM2 and investigated the molecular mechanisms mediating PKM2 actions in vitro. Results Specific PKM2 deletion in podocytes ameliorated LPS-induced protein excretion and alleviated LPS-induced alterations in blood urea nitrogen and serum albumin levels. In addition, PKM2 deletion in podocytes alleviated LPS-induced structural and morphological alterations to the tubules and to the brush borders. At the molecular level, PKM2 deficiency in podocytes suppressed LPS-induced inflammation and apoptosis. In vitro, PKM2 knockdown in murine podocytes diminished LPS-induced apoptosis. These effects were concomitant with a reduction in LPS-induced activation of β-catenin and the loss of Wilms’ Tumor 1 (WT1) and nephrin. Notably, the overexpression of a constitutively active mutant of β-catenin abolished the protective effect of PKM2 knockdown. Conversely, PKM2 knockdown cells reconstituted with the phosphotyrosine binding–deficient PKM2 mutant (K433E) recapitulated the effect of PKM2 depletion on LPS-induced apoptosis, β-catenin activation, and reduction in WT1 expression. Conclusions Taken together, our data demonstrates that PKM2 plays a key role in podocyte injury and suggests that targetting PKM2 in podocytes could serve as a promising therapeutic strategy for AKI. Trial registration Not applicable.

Pancreatic β-cells are crucial regulators of systemic glucose homeostasis, and their dysfunction and loss are central features in type 2 diabetes. Interventions that rectify β-cell dysfunction and loss are essential to combat this deadly malady. In the current study, we sought to delineate the role of soluble epoxide hydrolase (sEH) in β-cells under diet-induced metabolic stress. The expression of sEH was upregulated in murine and macaque diabetes models and islets of diabetic human patients. We postulated that hyperglycemia-induced elevation in sEH leads to a reduction in its substrates, epoxyeicosatrienoic acids (EETs), and attenuates the function of β-cells. Genetic deficiency of sEH potentiated glucose-stimulated insulin secretion in mice, likely in a cell-autonomous manner, contributing to better systemic glucose control. Consistent with this observation, genetic and pharmacological inactivation of sEH and the treatment with EETs exhibited insulinotropic effects in isolated murine islets ex vivo. Additionally, sEH deficiency enhanced glucose sensing and metabolism with elevated ATP and cAMP concentrations. This phenotype was associated with attenuated oxidative stress and diminished β-cell death in sEH deficient islets. Moreover, pharmacological inhibition of sEH in vivo mitigated, albeit partly, high fat diet-induced β-cell loss and dedifferentiation. The current observations provide new insights into the role of sEH in β-cells and information that may be leveraged for the development of a mechanism-based intervention to rectify β-cell dysfunction and loss.

17,18-Epoxyeicosatetraenoic acid (17,18-EEQ) and 19,20-epoxydocosapentaenoic acid (19,20-EDP) are bioactive epoxides produced from n-3 polyunsaturated fatty acid eicosapentaenoic acid and docosahexaenoic acid, respectively. However, these epoxides are quickly metabolized into less active diols by soluble epoxide hydrolase (sEH). We have previously demonstrated that an sEH inhibitor, t-TUCB, decreased serum triglycerides (TG) and increased lipid metabolic protein expression in the brown adipose tissue (BAT) of diet-induced obese mice. This study investigates the preventive effects of t-TUCB (T) alone or combined with 19,20-EDP (T + EDP) or 17,18-EEQ (T + EEQ) on BAT activation in the development of diet-induced obesity and metabolic disorders via osmotic minipump delivery in mice. Both T + EDP and T + EEQ groups showed significant improvement in fasting glucose, serum triglycerides, and higher core body temperature, whereas heat production was only significantly increased in the T + EEQ group. Moreover, both the T + EDP and T + EEQ groups showed less lipid accumulation in the BAT. Although UCP1 expression was not changed, PGC1α expression was increased in all three treated groups. In contrast, the expression of CPT1A and CPT1B, which are responsible for the rate-limiting step for fatty acid oxidation, was only increased in the T + EDP and T + EEQ groups. Interestingly, as a fatty acid transporter, CD36 expression was only increased in the T + EEQ group. Furthermore, both the T + EDP and T + EEQ groups showed decreased inflammatory NFκB signaling in the BAT. Our results suggest that 17,18-EEQ or 19,20-EDP combined with t-TUCB may prevent high-fat diet-induced metabolic disorders, in part through increased thermogenesis, upregulating lipid metabolic protein expression, and decreasing inflammation in the BAT.

OBJECTIVES: Brown adipose tissue (BAT) is a promising target for obesity prevention. N-3 epoxides are fatty acid epoxides produced from n-3 polyunsaturated fatty acids and shown to be beneficial for health. However, these epoxides are unstable and quickly metabolized by the cytosolic soluble epoxide hydrolase (sEH). Here, we investigated the effects of sEH inhibitor (t-TUCB) alone or combined with two different n-3 epoxides on BAT activation in the development of diet-induced obesity and associated metabolic disorders. METHODS: Male C57BL6/J mice were fed a high-fat diet and received either of the following treatment: the vehicle control, t-TUCB alone (T), or t-TUCB combined with 19,20-EDP (T + EDP) or 17,18-EEQ (T + EEQ) via osmotic minipump delivery near the interscapular BAT for 6 weeks. Mice were examined for changes in body weight, food intake, glucose, insulin, and cold tolerance tests, and indirect calorimetry. Blood and tissue biochemical analyses were also performed to assess changes in metabolic homeostasis. RESULTS: Although no differences in food intake were observed, there were small but significant increases in body weight in both T and T + EDP groups. Mice in the T + EDP and T + EEQ groups showed significant decreases in fasting glucose and serum TG levels, higher core body temperature, and better cold tolerance compared to the controls. However, heat production was significantly increased only in the T + EEQ group. Thermogenic UCP1 protein expression showed a moderate, but not significant, increase in the T + EEQ group. On the other hand, PGC1 α protein expression was significantly increased in the T, T + EDP, and T + EEQ groups compared to the controls. Perilipin protein expression and phosphorylation were also significantly increased in the three treated groups. In contrast, protein expression of FABP4 and HSL was only increased in the T and T + EDP groups, and CD36 protein expression was only increased in the T + EEQ group. CONCLUSIONS: Our results suggest that sEH pharmacological inhibition by t-TUCB combined with n-3 epoxides may prevent high-fat diet-induced glucose and lipid disorders, in part through increased thermogenesis and upregulating of protein expression of thermogenic and lipid metabolic genes. FUNDING SOURCES: The work was supported by NIH grants to L.Z., A.B., and B.D.H.

Brown adipose tissue (BAT) is an important target for obesity treatment and prevention. Soluble epoxide hydrolase (sEH) converts bioactive epoxy fatty acids (EpFAs) into less active diols. sEH inhibitors (sEHI) are beneficial in many chronic diseases by stabilizing EpFAs. However, roles of sEH and sEHI in brown adipogenesis and BAT activity in treating diet-induced obesity (DIO) have not been reported. sEH expression was studied in in vitro models of brown adipogenesis and the fat tissues of DIO mice. The effects of the sEHI, trans-4-{4-[3-(4-trifluoromethoxy-phenyl)-ureido]-cyclohexyloxy-benzoic acid (t-TUCB), were studied in vitro and in the obese mice via mini osmotic pump delivery. sEH expression was increased in brown adipogenesis and the BAT of the DIO mice. t-TUCB promoted brown adipogenesis in vitro. Although t-TCUB did not change body weight, fat pad weight, or glucose and insulin tolerance in the obese mice, it decreased serum triglycerides and increased protein expression of genes important for lipid metabolism in the BAT. Our results suggest that sEH may play a critical role in brown adipogenesis, and sEHI may be beneficial in improving BAT protein expression involved in lipid metabolism. Further studies using the sEHI combined with EpFA generating diets for obesity treatment and prevention are warranted.

Brown adipose tissue (BAT) is an important target for obesity treatment and prevention. Soluble epoxide hydrolase (sEH) converts bioactive epoxy fatty acids (EpFAs) into less active diols. sEH inhibitors (sEHI) are beneficial in many chronic diseases by stabilizing EpFAs. However, roles of sEH and sEHI in brown adipogenesis and BAT activity in treating diet-induced obesity (DIO) have not been reported. sEH expression was studied in in vitro models of brown adipogenesis and the fat tissues of DIO mice. The effects of the sEHI, trans-4-{4-[3-(4-trifluoromethoxy-phenyl)-ureido]-cyclohexyloxy-benzoic acid (t-TUCB), were studied in vitro and in the obese mice via mini osmotic pump delivery. sEH expression was increased in brown adipogenesis and the BAT of the DIO mice. t-TUCB promoted brown adipogenesis in vitro. Although t-TCUB did not change body weight, fat pad weight, or glucose and insulin tolerance in the obese mice, it decreased serum triglycerides and increased protein expression of genes important for lipid metabolism in the BAT. Our results suggest that sEH may play a critical role in brown adipogenesis, and sEHI may be beneficial in improving BAT protein expression involved in lipid metabolism. Further studies using the sEHI combined with EpFA generating diets for obesity treatment and prevention are warranted.

OBJECTIVES: Brown adipocytes have emerged as novel targets for obesity treatment and prevention. Cytochrome P450 (CYP) epoxygenases, primarily CYP2J and CYP2C isoforms, produce epoxy fatty acids (EpFAs), which have been suggested to play important roles in the regulation of white adipogenesis and obesity. However, the roles of CYP epoxygenases in brown adipogenesis remain unexplored. In this study, we sought to characterize mRNA expression patterns of Cyp2j and 2c subfamily members during adipogenesis of human and murine brown adipocytes and in obese mice and investigate the impact of modulating the expression of Cyp2j13 on brown adipogenesis. METHODS: The mRNA expression of various Cyp2j and Cyp2c isoforms were examined throughout murine and human brown adipocyte differentiation and in the brown adipose tissue (BAT) of diet-induced obese and control mice. To induce epoxygenase overexpression, stable transfection of murine brown preadipocytes with either Cyp2j13 or a vector control was performed. Protein and mRNA expression of Cyp2j13 and brown marker genes were analyzed. RESULTS: Expression of murine Cyp2j isoforms Cyp2j6, Cyp2j8, Cyp2j9, and Cyp2j13, and the human isoform CYP2J2 consistently decreased throughout brown adipocyte differentiation, while expression of Cyp2c isoforms did not elicit consistent patterns. Moreover, Cyp2j expression in BAT was enhanced in diet-induced obese mice compared to the controls. Due to its high relative abundance and significance, Cyp2j13 was selected for further investigation. Overexpression of Cyp2j13 significantly suppressed murine brown adipocyte differentiation as evaluated by lipid accumulation and brown marker gene UCP1 expression. CONCLUSIONS: Our results suggest that CYP epoxygenases may play important roles in brown adipogenesis. Cyp2j13, in particular, may be a novel target for brown adipogenesis, and consequently, for obesity treatment and prevention. Further studies using CYP2J inhibitors and Cyp2j13 knockdown are warranted. FUNDING SOURCES: The work was supported by NIH 1R15DK114790–01A1 (to L.Z.), K99DK100736 and R00DK100736 (to A.B.).

When insulin binds insulin receptor, IRS1 signaling is stimulated to trigger the maximal insulin response. p52Shc protein competes directly with IRS1, thus damping and diverting maximal insulin response. Genetic reduction of p52Shc minimizes competition with IRS1, and improves insulin signaling and glucose control in mice, and improves pathophysiological consequences of hyperglycemia. Given the multiple benefits of Shc reduction in vivo, we investigated whether any of 1680 drugs used in humans may function as Shc inhibitors, and thus potentially serve as novel anti-diabetics. Of the 1680, 30 insulin sensitizers were identified by screening in vitro, and of these 30 we demonstrated that 7 bound Shc protein. Of the 7 drugs, idebenone dose-dependently bound Shc protein in the 50-100 nM range, and induced insulin sensitivity and cytoprotection in this same 100 nM range that clinically dosed idebenone reaches in human plasma. By contrast we observe mitochondrial effects of idebenone in the 5,000 nM range that are not reached in human dosing. Multiple assays of target engagement demonstrate that idebenone physically interacts with Shc protein. Idebenone sensitizes mice to insulin in two different mouse models of prediabetes. Genetic depletion of idebenone's target eliminates idebenone's ability to insulin-sensitize in vivo. Thus, idebenone is the first-in-class member of a novel category of insulin-sensitizing and cytoprotective agents, the Shc inhibitors. Idebenone is an approved drug and could be considered for other indications such as type 2 diabetes and fatty liver disease, in which insulin resistance occurs.

Background and objective Soluble epoxide hydrolase (sEH) is an enzyme in the arachidonate cascade which converts epoxy fatty acids (EpFAs), such as epoxyeicosatrienoic acids (EETs) produced by cytochrome P450 enzymes, to dihydroxy-eicosatrienoic acids. In the last 20 years with the development of inhibitors to sEH it has been possible to increase the levels of EETs and other EpFAs in in vivo models. Recently, studies have shown that EETs play a key role in blocking inflammation in a bone resorption process, but the mechanism is not clear. In the current study we used the sEH inhibitor (1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea [TPPU]) to investigate the immunomodulatory effects in a mouse periodontitis model. Material and methods Mice were infected on days 0, 2, and 4 with Aggregatibacter actinomycetemcomitans and divided into groups (n = 6) that were treated orally, daily for 15 days, with 1 mg/kg of TPPU. Then, the mice were killed and their jaws were analyzed for bone resorption using morphometry. Immunoinflammatory markers in the gingival tissue were analyzed by microarray PCR or western blotting. Results Infected mice treated with TPPU showed lower bone resorption than infected mice without treatment. Interestingly, infected mice showed increased expression of sEH; however, mice treated with TPPU had a reduction in expression of sEH. Besides, several proinflammatory cytokines and molecular markers were downregulated in the gingival tissue in the group treated with 1 mg/kg of TPPU. Conclusion The sEH inhibitor, TPPU, showed immunomodulatory effects, decreasing bone resorption and inflammatory responses in a bone resorption mouse model.

The SLC39A12 gene encodes the zinc transporter protein ZIP12, which is expressed across many tissues and is highly abundant in the vertebrate nervous system. As a zinc transporter, ZIP12 functions to transport zinc across cellular membranes, including cellular zinc influx across the plasma membrane. Genome-wide association and exome sequencing studies have shown that brain susceptibility-weighted magnetic resonance imaging (MRI) intensity is associated with ZIP12 polymorphisms and rare mutations. ZIP12 is required for neural tube closure and embryonic development in Xenopus tropicalis. Frog embryos depleted of ZIP12 by antisense morpholinos develop an anterior neural tube defect and lack viability. ZIP12 is also necessary for neurite outgrowth and mitochondrial function in mouse neural cells. ZIP12 mRNA is increased in brain regions of schizophrenic patients. Outside of the nervous system, hypoxia induces ZIP12 expression in multiple mammalian species, including humans, which leads to endothelial and smooth muscle thickening in the lung and contributes towards pulmonary hypertension. Other studies have associated ZIP12 with other diseases such as cancer. Given that ZIP12 is highly expressed in the brain and that susceptibility-weighted MRI is associated with brain metal content, ZIP12 may affect neurological diseases and psychiatric illnesses such as Parkinson's disease, Alzheimer's disease, and schizophrenia. Furthermore, the induction of ZIP12 and resultant zinc uptake under pathophysiological conditions may be a critical component of disease pathology, such as in pulmonary hypertension. Drug compounds that bind metals like zinc may be able to treat diseases associated with impaired zinc homeostasis and altered ZIP12 function.

The arachidonic acid cascade is arguably the most widely known biologic regulatory pathway. Decades after the seminal discoveries involving its cyclooxygenase and lipoxygenase branches, studies of this cascade remain an active area of research. The third and less widely known branch, the cytochrome P450 pathway leads to highly active oxygenated lipid mediators, epoxy fatty acids (EpFAs) and hydroxyeicosatetraenoic acids (HETEs), which are of similar potency to prostanoids and leukotrienes. Unlike the COX and LOX branches, no pharmaceuticals currently are marketed targeting the P450 branch. However, data support therapeutic benefits from modulating these regulatory lipid mediators. This is being approached by stabilizing or mimicking the EpFAs or even by altering the diet. These approaches lead to predominantly beneficial effects on a wide range of apparently unrelated states resulting in an enigma of how this small group of natural chemical mediators can have such diverse effects. EpFAs are degraded by soluble epoxide hydrolase (sEH) and stabilized by inhibiting this enzyme. In this review, we focus on interconnected aspects of reported mechanisms of action of EpFAs and inhibitors of soluble epoxide hydrolase (sEHI). The sEHI and EpFAs are commonly reported to maintain homeostasis under pathological conditions while remaining neutral under normal physiological conditions. Here we provide a conceptual framework for the unique and broad range of biological activities ascribed to epoxy fatty acids. We argue that their mechanism of action pivots on their ability to prevent mitochondrial dysfunction, to reduce subsequent ROS formation and to block resulting cellular signaling cascades, primarily the endoplasmic reticulum stress. By stabilizing the mitochondrial – ROS – ER stress axis, the range of activity of EpFAs and sEHI display an overlap with the disease conditions including diabetes, fibrosis, chronic pain, cardiovascular and neurodegenerative diseases, for which the above outlined mechanisms play key roles.

Objective Diabetic nephropathy is one of the most devastating complications of diabetes, and growing evidence implicates podocyte dysfunction in disease pathogenesis. The objective of this study was to investigate the contribution of protein tyrosine phosphatase 1B (PTP1B) in podocytes to hyperglycemia-induced renal injury. Methods To determine the in vivo function of PTP1B in podocytes we generated mice with podocyte-specific PTP1B disruption (hereafter termed pod-PTP1B KO). Kidney functions were determined in control and pod-PTP1B KO mice under normoglycemia and high-fat diet (HFD)- and streptozotocin (STZ)-induced hyperglycemia. Results PTP1B expression increased in murine kidneys following HFD and STZ challenges. Under normoglycemia control and pod-PTP1B KO mice exhibited comparable renal functions. However, podocyte PTP1B disruption attenuated hyperglycemia-induced albuminuria and renal injury and preserved glucose control. Also, podocyte PTP1B disruption was accompanied with improved renal insulin signaling and enhanced autophagy with decreased inflammation and fibrosis. Moreover, the beneficial effects of podocyte PTP1B disruption in vivo were recapitulated in E11 murine podocytes with lentiviral-mediated PTP1B knockdown. Reconstitution of PTP1B in knockdown podocytes reversed the enhanced insulin signaling and autophagy suggesting that they were likely a consequence of PTP1B deficiency. Further, pharmacological attenuation of autophagy in PTP1B knockdown podocytes mitigated the protective effects of PTP1B deficiency. Conclusions These findings demonstrate that podocyte PTP1B deficiency attenuates hyperglycemia-induced renal damage and suggest that PTP1B may present a therapeutic target in renal injury.

Brain zinc dysregulation is linked to many neurological disorders. However, the mechanisms regulating brain zinc homeostasis are poorly understood. We performed secondary analyses of brain MRI GWAS and exome sequencing data from adults in the UK Biobank. Coding ZIP12 polymorphisms in zinc transporter ZIP12 (SLC39A12) were associated with altered brain susceptibility weighted MRI (swMRI). Conditional and joint association analyses revealed independent GWAS signals in linkage disequilibrium with 2 missense ZIP12 polymorphisms, rs10764176 and rs72778328, with reduced zinc transport activity. ZIP12 rare coding variants predicted to be deleterious were associated with similar impacts on brain swMRI. In Neuro-2a cells, ZIP12 deficiency by short hairpin RNA (shRNA) depletion or CRISPR/Cas9 genome editing resulted in impaired mitochondrial function, increased superoxide presence, and detectable protein carbonylation. Inhibition of Complexes I and IV of the electron transport chain reduced neurite outgrowth in ZIP12 deficient cells. Transcriptional coactivator PGC-1α, mitochondrial superoxide dismutase (SOD2), and chemical antioxidants α-tocopherol, MitoTEMPO, and MitoQ restored neurite extension impaired by ZIP12 deficiency. Mutant forms of α-synuclein and tau linked to familial Parkinson's disease and frontotemporal dementia, respectively, reduced neurite outgrowth in cells deficient in ZIP12. Zinc and ZIP12 may confer resilience against neurological diseases or premature aging of the brain.

Pyruvate kinase M2 is a critical enzyme that regulates cell metabolism and growth under different physiological conditions. In its metabolic role, pyruvate kinase M2 catalyzes the last glycolytic step which converts phosphoenolpyruvate to pyruvate with the generation of ATP. Beyond this metabolic role in glycolysis, PKM2 regulates gene expression in the nucleus, phosphorylates several essential proteins that regulate major cell signaling pathways, and contribute to the redox homeostasis of cancer cells. The expression of PKM2 has been demonstrated to be significantly elevated in several types of cancer, and the overall inflammatory response. The unusual pattern of PKM2 expression inspired scientists to investigate the unrevealed functions of PKM2 and the therapeutic potential of targeting PKM2 in cancer and other disorders. Therefore, the purpose of this review is to discuss the mechanistic and therapeutic potential of targeting PKM2 with the focus on cancer metabolism, redox homeostasis, inflammation, and metabolic disorders. This review highlights and provides insight into the metabolic and non-metabolic functions of PKM2 and its relevant association with health and disease.

OBJECTIVES: Brown adipose tissue has recently emerged as a novel target for obesity treatment and prevention. In contrast to the lipid storing function of white adipocytes, brown adipocytes are responsible for dissipating energy as heat, a process involving uncoupling protein 1 (UCP1). Soluble epoxide hydrolase (sEH) is a cytosolic enzyme that converts epoxy fatty acids (EpFAs) into less active diols. By stabilizing endogenous EpFAs, potent small molecule sEH inhibitors have been shown to be beneficial for many chronic diseases. Several recent papers have reported that sEH inhibitors are able to reduce diet-induced obesity, possibly by upregulating UCP1 expression. In the current study, we sought to study the mechanisms by which sEH inhibitor acts on brown preadipocytes. METHODS: The effects of a potent sEH inhibitor, trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB), on murine brown adipocyte differentiation were evaluated by lipid accumulation and expression of brown adipocyte marker genes. PPAR alpha and PPAR gamma activation by t-AUCB was measured by their respective transactivation assays. The roles of PPARs were further studied by pharmacological antagonism and knockdown experiments by small RNA interference. RESULTS: We report that sEH expression was increased during murine brown adipocyte differentiation. t-AUCB dose-dependently promoted brown adipocyte differentiation. Moreover, we demonstrate that t-AUCB activated PPAR alpha, but not PPAR gamma. t-AUCB-induced upregulation of thermogenic gene Ucp1 and Pgc1 alpha and the general differentiation marker Fabp4 were significantly attenuated by the antagonist of PPAR alpha, GW6471. In contrast, they were only partially attenuated by the antagonist of PPAR gamma, GW9662, and specific knockdown of PPAR gamma. CONCLUSIONS: Our findings suggest that sEH may regulate brown adipogenesis and sEH pharmacological inhibition by t-AUCB promotes brown adipogenesis, possibly through activation of PPAR alpha. FUNDING SOURCES: The work is supported by NIH 1R15DK114790-01A1 (to LZ), R00DK100736 (to AB) and R01ES002710 (to BDH).

OBJECTIVES: Dietary fiber has been proposed to protect against colorectal cancer. Butyrate, a fiber metabolite that is produced by bacteria in the colon, is known to inhibit cell proliferation and promote cell differentiation, while also inducing apoptotic cell death in colorectal cancer cells at physiologically relevant concentrations. Unlike the majority of cells in the human body that prefer utilizing glucose, non-cancerous colonocytes use butyrate as their primary energy source. However, colorectal cancer cells shift away from utilizing butyrate towards glucose (the Warburg effect). A decrease in butyrate utilization by the colonocyte has been reported in ulcerative colitis (UC) and colorectal cancer (CRC). In both of these diseases, the protein called Pyruvate Kinase Isoform M2 (PKM2) is a factor that has been found to be elevated in colonocytes and is known to catalyze a key step in glycolysis. We hypothesize that upregulation of PKM2 in ulcerative colitis and colorectal cancer results in diminished butyrate oxidation, and increased glucose utilization in colonocytes. METHODS: Mitochondrial function, substrate utilization will be analyzed in several colorectal cell lines, isolated colonocytes, or colonocytes grown in 3-D culture where PKM2 is knocked down, knocked-out, or overexpressed. An in vivo mouse model of colitis will be used to study the impact of PKM2 in the injury and repair process. RESULTS: Knockdown of PKM2 in cancerous colonocytes was associated with reduced proliferation and increased apoptosis. Butyrate oxidation was also increased in PKM2 knockdown cells. PKM2 regulated mitochondrial function and impacted the expression of uncoupling proteins (UCPs). Elevated PKM2 in primary colonocytes was associated with diminished butyrate utilization. Finally, conditional knockout of PKM2 in the colon resulted inhibited DSS-induced colitis. CONCLUSIONS: These results show an important role for PKM2 in promoting ulcerative colitis and colorectal cancer through shifting colonocyte metabolism away from butyrate utilization. FUNDING SOURCES: University of Tennessee - Start-up Funds.

17,18-Epoxyeicosatetraenoic acid (17,18-EEQ) and 19,20-epoxydocosapentaenoic acid (19,20-EDP) are bioactive epoxides produced from n-3 polyunsaturated fatty acid eicosapentaenoic acid and docosahexaenoic acid, respectively. However, these epoxides are quickly metabolized into less active diols by soluble epoxide hydrolase (sEH). We have previously demonstrated that an sEH inhibitor, t-TUCB, decreased serum triglycerides (TG) and increased lipid metabolic protein expression in the brown adipose tissue (BAT) of diet-induced obese mice. This study investigates the preventive effects of t-TUCB (T) alone or combined with 19,20-EDP (T + EDP) or 17,18-EEQ (T + EEQ) on BAT activation in the development of diet-induced obesity and metabolic disorders via osmotic minipump delivery in mice. Both T + EDP and T + EEQ groups showed significant improvement in fasting glucose, serum triglycerides, and higher core body temperature, whereas heat production was only significantly increased in the T + EEQ group. Moreover, both the T + EDP and T + EEQ groups showed less lipid accumulation in the BAT. Although UCP1 expression was not changed, PGC1α expression was increased in all three treated groups. In contrast, the expression of CPT1A and CPT1B, which are responsible for the rate-limiting step for fatty acid oxidation, was only increased in the T + EDP and T + EEQ groups. Interestingly, as a fatty acid transporter, CD36 expression was only increased in the T + EEQ group. Furthermore, both the T + EDP and T + EEQ groups showed decreased inflammatory NFκB signaling in the BAT. Our results suggest that 17,18-EEQ or 19,20-EDP combined with t-TUCB may prevent high-fat diet-induced metabolic disorders, in part through increased thermogenesis, upregulating lipid metabolic protein expression, and decreasing inflammation in the BAT.

Cancer, in part, is driven, by alterations in cellular metabolism that promote cell survival and cell proliferation. Identifying factors that influence this shift in cellular metabolism in cancer cells is important. Interleukin-1β (IL-1β) is a pro-inflammatory cytokine that has been reported to be elevated in colorectal cancer patients. While much is known toward the effect of dietary nutrients on regulating inflammation and the inflammatory response, which includes cytokines such as IL-1β, far less is understood how cytokines impact nutrient fate to alter cancer cell metabolism. Butyrate, a nutrient derived from the fermentation of dietary fiber in the colon, is the preferential exogenous energetic substrate used by non-cancerous colonocytes, but is used less efficiently by colorectal cancer cells. To test whether IL-1β alters colonocyte energy metabolism, we measured butyrate oxidation in HCT116 colorectal cancer cells with and without IL-1β. We hypothesize that IL-1β will push cancerous colonocytes away from the utilization and oxidation of butyrate. In this study, we demonstrate that pretreatment of colorectal cancer cells with IL-1β diminished butyrate oxidation and NADH levels. This effect was blocked with the interleukin receptor antagonist A (IL-1RA). Moreover, IL-1β suppressed basal mitochondrial respiration and lowered the mitochondrial spare capacity. By using inhibitors to block downstream targets of the interleukin-1 receptor pathway, we show that p38 is required for the IL-1β-mediated decrease in butyrate oxidation. These data provide insight into the metabolic effects induced by IL-1β in colorectal cancer, and identify relevant targets that may be exploited to block the effects of this cytokine. J. Cell. Biochem. 118: 1614-1621, 2017. © 2016 Wiley Periodicals, Inc.

Acute pancreatitis (AP) is a common and devastating gastrointestinal disorder that causes significant morbidity. The disease starts as local inflammation in the pancreas that may progress to systemic inflammation and complications. Protein tyrosine phosphatase 1B (PTP1B) is implicated in inflammatory signaling, but its significance in AP remains unclear. To investigate whether PTP1B may have a role in AP, we used pancreas PTP1B knockout (panc-PTP1B KO) mice and determined the effects of pancreatic PTP1B deficiency on cerulein- and arginine-induced acute pancreatitis. We report that PTP1B protein expression was increased in the early phase of AP in mice and rats. In addition, histological analyses of pancreas samples revealed enhanced features of AP in cerulein-treated panc-PTP1B KO mice compared with controls. Moreover, cerulein- and arginine-induced serum amylase and lipase were significantly higher in panc-PTP1B KO mice compared with controls. Similarly, pancreatic mRNA and serum concentrations of the inflammatory cytokines IL-1B, IL-6, and tumor necrosis factor-α were increased in panc-PTP1B KO mice compared with controls. Furthermore, panc-PTP1B KO mice exhibited enhanced cerulein- and arginine-induced NF-κB inflammatory response accompanied with increased mitogen-activated protein kinases activation and elevated endoplasmic reticulum stress. Notably, these effects were recapitulated in acinar cells treated with a pharmacological inhibitor of PTP1B. These findings reveal a novel role for pancreatic PTP1B in cerulein- and arginine-induced acute pancreatitis.

Obesity constitutes a major public health concern, being frequently associated with type 2 diabetes (T2D). Evidence from studies in humans and experimental animals suggest that consumption of the flavan-3-ol (-)-epicatechin (EC) and of EC-rich foods may improve insulin sensitivity. To further understand the potential benefits of dietary EC consumption on insulin resistance, this study investigated the capacity of EC supplementation to prevent high fat diet (HFD)-induced insulin resistance in mice. To assess the underlying mechanisms, the effects of HFD and EC consumption on the activation of the insulin cascade and of its negative modulators were evaluated. HFD consumption for 15 w caused obesity and insulin resistance in C57BL/6J mice as evidenced by high fasted and fed plasma glucose and insulin levels, and impaired ITT and GTT tests. This was associated with alterations in the activation of components of the insulin-triggered signaling cascade (insulin receptor, IRS1, ERK1/2, Akt) in adipose and liver tissues. EC supplementation prevented/ameliorated all these parameters. EC acted improving insulin sensitivity in the HFD-fed mice in part through a downregulation of the inhibitory molecules JNK, IKK, PKC and protein tyrosine phosphatase 1B (PTP1B). Thus, the above results suggest that consumption of EC-rich foods could constitute a dietary strategy to mitigate obesity-associated insulin resistance. Fil: Cremonini, Eleonora. University of California at Davis; Estados Unidos Fil: Bettaieb, Ahmed. University of California at Davis; Estados Unidos. University of Tennessee; Estados Unidos Fil: Haj, Fawaz G.. University of California at Davis; Estados Unidos Fil: Fraga, César Guillermo. University of California at Davis; Estados Unidos. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Analitica y Fisicoquímica. Cátedra de Fisicoquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Bioquímica y Medicina Molecular; Argentina Fil: Oteiza, Patricia Isabel. University of California at Davis; Estados Unidos