Ketohexokinase-C regulates global protein acetylation to decrease carnitine palmitoyltransferase 1a-mediated fatty acid oxidation.

BACKGROUND & AIMS: The consumption of sugar and a high-fat diet (HFD) promotes the development of obesity and metabolic dysfunction. Despite their well-known synergy, the mechanisms by which sugar worsens the outcomes associated with a HFD are largely elusive. METHODS: Six-week-old, male, C57Bl/6 J mice were fed either chow or a HFD and were provided with regular, fructose- or glucose-sweetened water. Moreover, cultured AML12 hepatocytes were engineered to overexpress ketohexokinase-C (KHK-C) using a lentivirus vector, while CRISPR-Cas9 was used to knockdown CPT1α. The cell culture experiments were complemented with in vivo studies using mice with hepatic overexpression of KHK-C and in mice with liver-specific CPT1α knockout. We used comprehensive metabolomics, electron microscopy, mitochondrial substrate phenotyping, proteomics and acetylome analysis to investigate underlying mechanisms. RESULTS: Fructose supplementation in mice fed normal chow and fructose or glucose supplementation in mice fed a HFD increase KHK-C, an enzyme that catalyzes the first step of fructolysis. Elevated KHK-C is associated with an increase in lipogenic proteins, such as ACLY, without affecting their mRNA expression. An increase in KHK-C also correlates with acetylation of CPT1α at K508, and lower CPT1α protein in vivo. In vitro, KHK-C overexpression lowers CPT1α and increases triglyceride accumulation. The effects of KHK-C are, in part, replicated by a knockdown of CPT1α. An increase in KHK-C correlates negatively with CPT1α protein levels in mice fed sugar and a HFD, but also in genetically obese db/db and lipodystrophic FIRKO mice. Mechanistically, overexpression of KHK-C in vitro increases global protein acetylation and decreases levels of the major cytoplasmic deacetylase, SIRT2. CONCLUSIONS: KHK-C-induced acetylation is a novel mechanism by which dietary fructose augments lipogenesis and decreases fatty acid oxidation to promote the development of metabolic complications. IMPACT AND IMPLICATIONS: Fructose is a highly lipogenic nutrient whose negative consequences have been largely attributed to increased de novo lipogenesis. Herein, we show that fructose upregulates ketohexokinase, which in turn modifies global protein acetylation, including acetylation of CPT1a, to decrease fatty acid oxidation. Our findings broaden the impact of dietary sugar beyond its lipogenic role and have implications on drug development aimed at reducing the harmful effects attributed to sugar metabolism.

Adipocyte-Derived PXR Signaling Is Dispensable for Diet-Induced Obesity and Metabolic Disorders in Mice.

Pregnane X receptor (PXR) is a xenobiotic receptor that can be activated by numerous chemicals including endogenous hormones, dietary steroids, pharmaceutical agents, and environmental chemicals. PXR has been established to function as a xenobiotic sensor to coordinately regulate xenobiotic metabolism by regulating the expression of many enzymes and transporters required for xenobiotic metabolism. Recent studies have implicated a potentially important role for PXR in obesity and metabolic disease beyond xenobiotic metabolism, but how PXR action in different tissues or cell types contributes to obesity and metabolic disorders remains elusive. To investigate the role of adipocyte PXR in obesity, we generated a novel adipocyte-specific PXR deficient mouse model (PXRΔAd). Notably, we found that loss of adipocyte PXR did not affect food intake, energy expenditure, and obesity in high-fat diet-fed male mice. PXRΔAd mice also had similar obesity-associated metabolic disorders including insulin resistance and hepatic steatosis as control littermates. PXR deficiency in adipocytes did not affect expression of key adipose genes in PXRΔAd mice. Our findings suggest that adipocyte PXR signaling may be dispensable in diet-induced obesity and metabolic disorders in mice. Further studies are needed to understand the role of PXR signaling in obesity and metabolic disorders in the future. SIGNIFICANCE STATEMENT: The authors demonstrate that deficiency of adipocyte pregnane X receptor (PXR) does not affect diet-induced obesity or metabolic disorders in mice and infers that adipocyte PXR signaling may not play a key role in diet-induced obesity. More studies are needed to understand the tissue-specific role of PXR in obesity.

Non-hematopoietic IL-4Rα expression contributes to fructose-driven obesity and metabolic sequelae.

OBJECTIVE: The risks of excess sugar intake in addition to high-fat diet consumption on immunopathogenesis of obesity-associated metabolic diseases are poorly defined. Interleukin-4 (IL-4) and IL-13 signaling via IL-4Rα regulates adipose tissue lipolysis, insulin sensitivity, and liver fibrosis in obesity. However, the contribution of IL-4Rα to sugar rich diet-driven obesity and metabolic sequelae remains unknown. METHODS: WT, IL-4Rα-deficient (IL-4Rα-/-) and STAT6-deficient mice (STAT6-/-) male mice were fed low-fat chow, high fat (HF) or HF plus high carbohydrate (HC/fructose) diet (HF + HC). Analysis included quantification of: (i) body weight, adiposity, energy expenditure, fructose metabolism, fatty acid oxidation/synthesis, glucose dysmetabolism and hepatocellular damage; (ii) the contribution of the hematopoietic or non-hematopoietic IL-4Rα expression; and (iii) the relevance of IL-4Rα downstream canonical STAT6 signaling pathway in this setting. RESULTS: We show that IL-4Rα regulated HF + HC diet-driven weight gain, whole body adiposity, adipose tissue inflammatory gene expression, energy expenditure, locomotor activity, glucose metabolism, hepatic steatosis, hepatic inflammatory gene expression and hepatocellular damage. These effects were potentially, and in part, dependent on non-hematopoietic IL-4Rα expression but were independent of direct STAT6 activation. Mechanistically, hepatic ketohexokinase-A and C expression was dependent on IL-4Rα, as it was reduced in IL-4Rα-deficient mice. KHK activity was also affected by HF + HC dietary challenge. Further, reduced expression/activity of KHK in IL-4Rα mice had a significant effect on fatty acid oxidation and fatty acid synthesis pathways. CONCLUSION: Our findings highlight potential contribution of non-hematopoietic IL-4Rα activation of a non-canonical signaling pathway that regulates the HF + HC diet-driven induction of obesity and severity of obesity-associated sequelae.

Myeloid-specific deficiency of pregnane X receptor decreases atherosclerosis in LDL receptor-deficient mice.

The pregnane X receptor (PXR) is a nuclear receptor that can be activated by numerous drugs and xenobiotic chemicals. PXR thereby functions as a xenobiotic sensor to coordinately regulate host responses to xenobiotics by transcriptionally regulating many genes involved in xenobiotic metabolism. We have previously reported that PXR has pro-atherogenic effects in animal models, but how PXR contributes to atherosclerosis development in different tissues or cell types remains elusive. In this study, we generated an LDL receptor-deficient mouse model with myeloid-specific PXR deficiency (PXRΔMyeLDLR-/-) to elucidate the role of macrophage PXR signaling in atherogenesis. The myeloid PXR deficiency did not affect metabolic phenotypes and plasma lipid profiles, but PXRΔMyeLDLR-/- mice had significantly decreased atherosclerosis at both aortic root and brachiocephalic arteries compared with control littermates. Interestingly, the PXR deletion did not affect macrophage adhesion and migration properties, but reduced lipid accumulation and foam cell formation in the macrophages. PXR deficiency also led to decreased expression of the scavenger receptor CD36 and impaired lipid uptake in macrophages of the PXRΔMyeLDLR-/- mice. Further, RNA-Seq analysis indicated that treatment with a prototypical PXR ligand affects the expression of many atherosclerosis-related genes in macrophages in vitro. These findings reveal a pivotal role of myeloid PXR signaling in atherosclerosis development and suggest that PXR may be a potential therapeutic target in atherosclerosis management.

Non-nucleoside reverse transcriptase inhibitor efavirenz activates PXR to induce hypercholesterolemia and hepatic steatosis.

BACKGROUND & AIMS: The most prescribed non-nucleoside reverse transcriptase inhibitor, efavirenz, has been associated with elevated risk of dyslipidemia and hepatic steatosis in HIV-infected patients but the underlying mechanisms remain elusive. Herein, we investigated the role of pregnane X receptor (PXR) in mediating the adverse effects of efavirenz on lipid homeostasis. METHODS: Cell-based reporter assays, primary cell culture, and multiple mouse models including conditional knockout and humanized mice were combined to study the impact of efavirenz on PXR activities and lipid homeostasis in vitro and in vivo. A novel liver-specific Pxr knockout mouse model was also generated to determine the contribution of hepatic PXR signaling to efavirenz-elicited dyslipidemia and hepatic steatosis. RESULTS: We found that efavirenz is a potent PXR-selective agonist that can efficiently activate PXR and induce its target gene expression in vitro and in vivo. Treatment with efavirenz-induced hypercholesterolemia and hepatic steatosis in mice but deficiency of hepatic PXR abolished these adverse effects. Interestingly, efavirenz-mediated PXR activation regulated the expression of several key hepatic lipogenic genes including fatty acid transporter CD36 and cholesterol biosynthesis enzyme squalene epoxidase (SQLE), leading to increased lipid uptake and cholesterol biosynthesis in hepatic cells. While CD36 is a known PXR target gene, we identified a DR-2-type of PXR-response element in the SQLE promoter and established SQLE as a direct transcriptional target of PXR. Since PXR exhibits considerable differences in its pharmacology across species, we also confirmed these findings in PXR-humanized mice and human primary hepatocytes. CONCLUSIONS: The widely prescribed antiretroviral drug efavirenz induces hypercholesterolemia and hepatic steatosis by activating PXR signaling. Activation of PXR should be taken into consideration for patients undergoing long-term treatment with PXR agonistic antiretroviral drugs. LAY SUMMARY: Efavirenz is widely prescribed for HIV-infected patients but has some side effects. It can increase lipid levels in patients' blood and liver. Here we show that efavirenz can activate a unique liver protein called PXR which mediates the adverse effects of efavirenz on lipid levels in mouse models.

Myeloid ß-Catenin Deficiency Exacerbates Atherosclerosis in Low-Density Lipoprotein Receptor-Deficient Mice.

OBJECTIVE: The Wnt/ß-catenin signaling is an ancient and evolutionarily conserved pathway that regulates essential aspects of cell differentiation, proliferation, migration and polarity. Canonical Wnt/ß-catenin signaling has also been implicated in the pathogenesis of atherosclerosis. Macrophage is one of the major cell types involved in the initiation and progression of atherosclerosis, but the role of macrophage ß-catenin in atherosclerosis remains elusive. This study aims to investigate the impact of ß-catenin expression on macrophage functions and atherosclerosis development. APPROACH AND RESULTS: To investigate the role of macrophage canonical Wnt/ß-catenin signaling in atherogenesis, we generated ß-cateninΔmyeLDLR-/- mice (low-density lipoprotein receptor-deficient mice with myeloid-specific ß-catenin deficiency). As expected, deletion of ß-catenin decreased macrophage adhesion and migration properties in vitro. However, deficiency of ß-catenin significantly increased atherosclerotic lesion areas in the aortic root of LDLR-/- (low-density lipoprotein receptor-deficient) mice without affecting the plasma lipid levels and atherosclerotic plaque composition. Mechanistic studies revealed that ß-catenin can regulate activation of STAT (signal transducer and activator of transcription) pathway in macrophages, and ablation of ß-catenin resulted in STAT3 downregulation and STAT1 activation, leading to elevated macrophage inflammatory responses and increased atherosclerosis. CONCLUSIONS: This study demonstrates a critical role of myeloid ß-catenin expression in atherosclerosis by modulating macrophage inflammatory responses.

Targeting IκB kinase ß in Adipocyte Lineage Cells for Treatment of Obesity and Metabolic Dysfunctions.

IκB kinase ß (IKKß), a central coordinator of inflammation through activation of nuclear factor-κB, has been identified as a potential therapeutic target for the treatment of obesity-associated metabolic dysfunctions. In this study, we evaluated an antisense oligonucleotide (ASO) inhibitor of IKKß and found that IKKß ASO ameliorated diet-induced metabolic dysfunctions in mice. Interestingly, IKKß ASO also inhibited adipocyte differentiation and reduced adiposity in high-fat (HF)-fed mice, indicating an important role of IKKß signaling in the regulation of adipocyte differentiation. Indeed, CRISPR/Cas9-mediated genomic deletion of IKKß in 3T3-L1 preadipocytes blocked these cells differentiating into adipocytes. To further elucidate the role of adipose progenitor IKKß signaling in diet-induced obesity, we generated mice that selectively lack IKKß in the white adipose lineage and confirmed the essential role of IKKß in mediating adipocyte differentiation in vivo. Deficiency of IKKß decreased HF-elicited adipogenesis in addition to reducing inflammation and protected mice from diet-induced obesity and insulin resistance. Further, pharmacological inhibition of IKKß also blocked human adipose stem cell differentiation. Our findings establish IKKß as a pivotal regulator of adipogenesis and suggest that overnutrition-mediated IKKß activation serves as an initial signal that triggers adipose progenitor cell differentiation in response to HF feeding. Inhibition of IKKß with antisense therapy may represent as a novel therapeutic approach to combat obesity and metabolic dysfunctions. Stem Cells 2016;34:1883-1895.

Pregnane X receptor mediates dyslipidemia induced by the HIV protease inhibitor amprenavir in mice.

Human immunodeficiency virus (HIV) protease inhibitors (PIs) have been used successfully in extending the life span of people infected with HIV. The use of PIs has also been associated with dyslipidemia and an increased risk of cardiovascular disease, but the underlying mechanisms remain elusive. Several PIs have been implicated in activating the nuclear receptor pregnane X receptor (PXR), which acts as a xenobiotic sensor to regulate xenobiotic metabolism in the liver and intestine. Recent studies indicate that PXR may also play an important role in the regulation of lipid homeostasis. In the present study, we identified amprenavir, a widely used HIV PI, as a potent PXR-selective agonist. Computational docking studies combined with site-direct mutagenesis identified several key residues within the ligand-binding pocket of PXR that constitute points of interaction with amprenavir. Amprenavir efficiently activated PXR and induced PXR target gene expression in vitro and in vivo. Short-term exposure to amprenavir significantly increased plasma total cholesterol and atherogenic low-density lipoprotein cholesterol levels in wild-type mice, but not in PXR-deficient mice. Amprenavir-mediated PXR activation stimulated the expression of several key intestinal genes involved in lipid homeostasis. These findings provide critical mechanistic insight for understanding the impact of PIs on cardiovascular disease and demonstrate a potential role of PXR in mediating the adverse effects of HIV PIs in humans.

Monoclonal antibody against α-actinin 4 from human umbilical vein endothelial cells inhibits endothelium-dependent vasorelaxation.

BACKGROUND: This study was attempted to identify new molecules expressed on the plasma membrane of human umbilical vein endothelial cells (HUVECs) using monoclonal antibody-based proteomics technology and to determine the effect of the identified antibody on vascular reactivity. METHODS: Twenty-two antibodies were developed from rats inoculated with HUVECs, and their effects were determined by observing vascular reactivity. RESULTS: Among the 22 antibodies, the C-7 antibody significantly inhibited endothelium-dependent vasorelaxation in response to acetylcholine (ACh) but not to histamine. Moreover, the C-7 antibody did not affect norepinephrine-induced contraction in either the endothelium-intact or -denuded aorta. A proteomics study involving immunoprecipitation of the C-7 antibody with biotinylated HUVECs showed that this antibody binds to plasma membrane proteins corresponding to immunoglobulin heavy chain (VHDJ region), chaperonin-containing T-complex polypeptide 1 and α-actinin 4. The muscarinic M3 ACh receptor and α-actinin 4 were colocalized on the plasma membrane of HUVECs, and the colocalization was found to increase in response to ACh and was inhibited by pretreatment with the C-7 antibody. CONCLUSIONS: These results demonstrate that monoclonal C-7 antibody exerts an inhibitory effect on endothelium-dependent vasorelaxation induced by ACh and that this response may at least partially result from the inhibition of α-actinin 4.

Myeloid-specific IκB kinase ß deficiency decreases atherosclerosis in low-density lipoprotein receptor-deficient mice.

OBJECTIVE: Inflammatory responses are the driving force of atherosclerosis development. IκB kinase ß (IKKß), a central coordinator in inflammation through regulation of nuclear factor-κB, has been implicated in the pathogenesis of atherosclerosis. Macrophages play an essential role in the initiation and progression of atherosclerosis, yet the role of macrophage IKKß in atherosclerosis remains elusive and controversial. This study aims to investigate the impact of IKKß expression on macrophage functions and to assess the effect of myeloid-specific IKKß deletion on atherosclerosis development. METHODS AND RESULTS: To explore the issue of macrophage IKKß involvement of atherogenesis, we generated myeloid-specific IKKß-deficient low-density lipoprotein receptor-deficient mice (IKKß(ΔMye)LDLR(-/-)). Deficiency of IKKß in myeloid cells did not affect plasma lipid levels but significantly decreased diet-induced atherosclerotic lesion areas in the aortic root, brachiocephalic artery, and aortic arch of low-density lipoprotein receptor-deficient mice. Ablation of myeloid IKKß attenuated macrophage inflammatory responses and decreased atherosclerotic lesional inflammation. Furthermore, deficiency of IKKß decreased adhesion, migration, and lipid uptake in macrophages. CONCLUSIONS: The present study demonstrates a pivotal role for myeloid IKKß expression in atherosclerosis by modulating macrophage functions involved in atherogenesis. These results suggest that inhibiting nuclear factor-κB activation in macrophages may represent a feasible approach to combat atherosclerosis.

Fructose impairs fat oxidation: Implications for the mechanism of western diet-induced NAFLD.

Increased fructose intake from sugar-sweetened beverages and highly processed sweets is a well-recognized risk factor for the development of obesity and its complications. Fructose strongly supports lipogenesis on a normal chow diet by providing both, a substrate for lipid synthesis and activation of lipogenic transcription factors. However, the negative health consequences of dietary sugar are best observed with the concomitant intake of a HFD. Indeed, the most commonly used obesogenic research diets, such as "Western diet", contain both fructose and a high amount of fat. In spite of its common use, how the combined intake of fructose and fat synergistically supports development of metabolic complications is not fully elucidated. Here we present the preponderance of evidence that fructose consumption decreases oxidation of dietary fat in human and animal studies. We provide a detailed review of the mitochondrial ß-oxidation pathway. Fructose affects hepatic activation of fatty acyl-CoAs, decreases acylcarnitine production and impairs the carnitine shuttle. Mechanistically, fructose suppresses transcriptional activity of PPARα and its target CPT1α, the rate limiting enzyme of acylcarnitine production. These effects of fructose may be, in part, mediated by protein acetylation. Acetylation of PGC1α, a co-activator of PPARα and acetylation of CPT1α, in part, account for fructose-impaired acylcarnitine production. Interestingly, metabolic effects of fructose in the liver can be largely overcome by carnitine supplementation. In summary, fructose decreases oxidation of dietary fat in the liver, in part, by impairing acylcarnitine production, offering one explanation for the synergistic effects of these nutrients on the development of metabolic complications, such as NAFLD.

Fructose Induced KHK-C Increases ER Stress and Modulates Hepatic Transcriptome to Drive Liver Disease in Diet-Induced and Genetic Models of NAFLD.

Non-alcoholic fatty liver disease (NAFLD) is a liver manifestation of metabolic syndrome, and is estimated to affect one billion individuals worldwide. An increased intake of a high-fat diet (HFD) and sugar-sweetened beverages are risk-factors for NAFLD development, but how their combined intake promotes progression to a more severe form of liver injury is unknown. Here we show that fructose metabolism via ketohexokinase (KHK) C isoform increases endoplasmic reticulum (ER) stress in a dose dependent fashion, so when fructose is coupled with a HFD intake it leads to unresolved ER stress. Conversely, a liver-specific knockdown of KHK in C57BL/6J male mice consuming fructose on a HFD is adequate to improve the NAFLD activity score and exert a profound effect on the hepatic transcriptome. Overexpression of KHK-C in cultured hepatocytes is sufficient to induce ER stress in fructose free media. Upregulation of KHK-C is also observed in genetically obesity ob/ob, db/db and lipodystrophic FIRKO male mice, whereas KHK knockdown in these mice improves metabolic function. Additionally, in over 100 inbred strains of male or female mice hepatic KHK expression correlates positively with adiposity, insulin resistance, and liver triglycerides. Similarly, in 241 human subjects and their controls, hepatic Khk expression is upregulated in early, but not late stages of NAFLD. In summary, we describe a novel role of KHK-C in triggering ER stress, which offers a mechanistic understanding of how the combined intake of fructose and a HFD propagates the development of metabolic complications.

Loss of Carnitine Palmitoyltransferase 1a Reduces Docosahexaenoic Acid-Containing Phospholipids and Drives Sexually Dimorphic Liver Disease in Mice.

Background and Aims: Genome and epigenome wide association studies identified variants in carnitine palmitoyltransferase 1a (CPT1a) that associate with lipid traits. The goal of this study was to determine the impact by which liver-specific CPT1a deletion impacts hepatic lipid metabolism. Approach and Results: Six-to-eight-week old male and female liver-specific knockout (LKO) and littermate controls were placed on a low-fat or high-fat diet (HFD; 60% kcal fat) for 15 weeks. Mice were necropsied after a 16 hour fast, and tissues were collected for lipidomics, matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI), kinome analysis, RNA-sequencing, and protein expression by immunoblotting. Female LKO mice had increased serum alanine aminotransferase (ALT) levels which were associated with greater deposition of hepatic lipids, while male mice were not affected by CPT1a deletion relative to male control mice. Mice with CPT1a deletion had reductions in DHA-containing phospholipids at the expense of monounsaturated fatty acids (MUFA)-containing phospholipids in both whole liver and at the level of the lipid droplet (LD). Male and female LKO mice increased RNA levels of genes involved in LD lipolysis ( Plin2 , Cidec , G0S2 ) and in polyunsaturated fatty acid (PUFA) metabolism ( Elovl5, Fads1, Elovl2 ), while only female LKO mice increased genes involved in inflammation ( Ly6d, Mmp12, Cxcl2 ). Kinase profiling showed decreased protein kinase A (PKA) activity, which coincided with increased PLIN2, PLIN5, and G0S2 protein levels and decreased triglyceride hydrolysis in LKO mice. Conclusions: Liver-specific deletion of CPT1a promotes sexually dimorphic steatotic liver disease (SLD) in mice, and here we have identified new mechanisms by which females are protected from HFD-induced liver injury.

Loss of carnitine palmitoyltransferase 1a reduces docosahexaenoic acid-containing phospholipids and drives sexually dimorphic liver disease in mice.

BACKGROUND AND AIMS: Genome and epigenome wide association studies identified variants in carnitine palmitoyltransferase 1a (CPT1a) that associate with lipid traits. The goal of this study was to determine the role of liver-specific CPT1a on hepatic lipid metabolism. APPROACH AND RESULTS: Male and female liver-specific knockout (LKO) and littermate controls were placed on a low-fat or high-fat diet (60% kcal fat) for 15 weeks. Mice were necropsied after a 16 h fast, and tissues were collected for lipidomics, matrix-assisted laser desorption ionization mass spectrometry imaging, kinome analysis, RNA-sequencing, and protein expression by immunoblotting. Female LKO mice had increased serum alanine aminotransferase levels which were associated with greater deposition of hepatic lipids, while male mice were not affected by CPT1a deletion relative to male control mice. Mice with CPT1a deletion had reductions in DHA-containing phospholipids at the expense of monounsaturated fatty acids (MUFA)-containing phospholipids in whole liver and at the level of the lipid droplet (LD). Male and female LKO mice increased RNA levels of genes involved in LD lipolysis (Plin2, Cidec, G0S2) and in polyunsaturated fatty acid metabolism (Elovl5, Fads1, Elovl2), while only female LKO mice increased genes involved in inflammation (Ly6d, Mmp12, Cxcl2). Kinase profiling showed decreased protein kinase A activity, which coincided with increased PLIN2, PLIN5, and G0S2 protein levels and decreased triglyceride hydrolysis in LKO mice. CONCLUSIONS: Liver-specific deletion of CPT1a promotes sexually dimorphic steatotic liver disease (SLD) in mice, and here we have identified new mechanisms by which females are protected from HFD-induced liver injury.

Fructose induced KHK-C can increase ER stress independent of its effect on lipogenesis to drive liver disease in diet-induced and genetic models of NAFLD.

Non-alcoholic fatty liver disease (NAFLD) is a liver manifestation of metabolic syndrome, and is estimated to affect one billion individuals worldwide. An increased intake of a high-fat diet (HFD) and sugar-sweetened beverages are risk-factors for NAFLD development, but how their combined intake promotes progression to a more severe form of liver injury is unknown. Here we show that fructose metabolism via ketohexokinase (KHK) C isoform leads to unresolved endoplasmic reticulum (ER) stress when coupled with a HFD intake. Conversely, a liver-specific knockdown of KHK in mice consuming fructose on a HFD is adequate to improve the NAFLD activity score and exert a profound effect on the hepatic transcriptome. Overexpression of KHK-C in cultured hepatocytes is sufficient to induce ER stress in fructose free media. Upregulation of KHK-C is also observed in mice with genetically induced obesity or metabolic dysfunction, whereas KHK knockdown in these mice improves metabolic function. Additionally, in over 100 inbred strains of male or female mice hepatic KHK expression correlates positively with adiposity, insulin resistance, and liver triglycerides. Similarly, in 241 human subjects and their controls, hepatic Khk expression is upregulated in early, but not late stages of NAFLD. In summary, we describe a novel role of KHK-C in triggering ER stress, which offers a mechanistic understanding of how the combined intake of fructose and a HFD propagates the development of metabolic complications.

A luminescence-based protocol for assessing fructose metabolism via quantification of ketohexokinase enzymatic activity in mouse or human hepatocytes.

Ketohexokinase (KHK) catalyzes the first step of fructose metabolism. Inhibitors of KHK enzymatic activity are being evaluated in clinical trials for the treatment of non-alcoholic fatty liver disease (NAFLD) and diabetes. Here, we present a luminescence-based protocol to quantify KHK activity. The accuracy of this technique has been validated using knockdown and overexpression of KHK in vivo and in vitro. The specificity of the assay has been verified using 3-O-methyl-D-fructose, a non-metabolizable analog of fructose, heat inactivation of hexokinases, and depletion of potassium. For complete details on the use of this protocol, please refer to Damen et al. (2021).

Estrogen regulates Snail and Slug in the down-regulation of E-cadherin and induces metastatic potential of ovarian cancer cells through estrogen receptor alpha.

Tumorigenesis is a multistep process involving dysregulated cell growth and metastasis. Considerable evidence implicates a mitogenic action of estrogen in early ovarian carcinogenesis. In contrast, its influence in the metastatic cascade of ovarian tumor cells remains obscure. In the present study, we showed that 17beta-estradiol (E2) increased the metastatic potential of human epithelial ovarian cancer cell lines. E2 treatment led to clear morphological changes characteristic of epithelial-mesenchymal transition (EMT) and an enhanced cell migratory propensity. These morphological and functional alterations were associated with changes in the abundance of EMT-related genes. Upon E2 stimulation, expression and promoter activity of the epithelial marker E-cadherin were strikingly suppressed, whereas EMT-associated transcription factors, Snail and Slug, were significantly up-regulated. This up-regulation was attributed to the increase in gene transcription activated by E2. Depletion of endogenous Snail or Slug using small interfering RNA (siRNA) attenuated E2-mediated decrease in E-cadherin. In addition, E2-induced cell migration was also neutralized by the siRNAs, suggesting that both transcription factors are indispensable for the prometastatic actions of E2. More importantly, by using selective estrogen receptor (ER) agonists, forced expression, and siRNA approaches, we identified that E2 triggered the metastatic behaviors exclusively through an ERalpha-dependent pathway. We also showed that ERbeta had an opposing action on ERalpha because the presence of ERbeta completely inhibited the EMT and down-regulation of E-cadherin induced by ERalpha. Collectively, this study provides a compelling argument that estrogen can potentiate tumor progression by EMT induction and highlights the crucial role of ERalpha in ovarian tumorigenesis.

The atypical antipsychotic quetiapine induces hyperlipidemia by activating intestinal PXR signaling.

Quetiapine, one of the most prescribed atypical antipsychotics, has been associated with hyperlipidemia and an increased risk for cardiovascular disease in patients, but the underlying mechanisms remain unknown. Here, we identified quetiapine as a potent and selective agonist for pregnane X receptor (PXR), a key nuclear receptor that regulates xenobiotic metabolism in the liver and intestine. Recent studies have indicated that PXR also plays an important role in lipid homeostasis. We generated potentially novel tissue-specific PXR-KO mice and demonstrated that quetiapine induced hyperlipidemia by activating intestinal PXR signaling. Quetiapine-mediated PXR activation stimulated the intestinal expression of cholesterol transporter Niemann-Pick C1-Like 1 (NPC1L1) and microsomal triglyceride transfer protein (MTP), leading to increased intestinal lipid absorption. While NPC1L1 is a known PXR target gene, we identified a DR-1-type PXR-response element in the MTP promoter and established MTP as a potentially novel transcriptional target of PXR. Quetiapine's effects on PXR-mediated gene expression and cholesterol uptake were also confirmed in cultured murine enteroids and human intestinal cells. Our findings suggest a potential role of PXR in mediating adverse effects of quetiapine in humans and provide mechanistic insights for certain atypical antipsychotic-associated dyslipidemia.

Deficiency of Adipocyte IKKß Affects Atherosclerotic Plaque Vulnerability in Obese LDLR Deficient Mice.

Background Obesity-associated chronic inflammation has been known to contribute to atherosclerosis development, but the underlying mechanisms remain elusive. Recent studies have revealed novel functions of IKK ß (inhibitor of NF -κB [nuclear factor κB] kinase ß), a key coordinator of inflammation through activation of NF -κB, in atherosclerosis and adipose tissue development. However, it is not clear whether IKK ß signaling in adipocytes can also affect atherogenesis. This study aims to investigate the impact of adipocyte IKK ß expression on atherosclerosis development in lean and obese LDLR (low-density lipoprotein receptor)-deficient ( LDLR -/-) mice. Methods and Results To define the role of adipocyte IKK ß in atherogenesis, we generated adipocyte-specific IKK ß-deficient LDLR -/- ( IKK ßΔAd LDLR -/-) mice. Targeted deletion of IKK ß in adipocytes did not affect adiposity and atherosclerosis in lean LDLR -/- mice when fed a low-fat diet. In response to high-fat feeding, however, IKK ßΔAd LDLR -/- mice had defective adipose remodeling and increased adipose tissue and systemic inflammation. Deficiency of adipocyte IKK ß did not affect atherosclerotic lesion sizes but resulted in enhanced lesional inflammation and increased plaque vulnerability in obese IKK ßΔAd LDLR -/- mice. Conclusions These data demonstrate that adipocyte IKK ß signaling affects the evolution of atherosclerosis plaque vulnerability in obese LDLR -/- mice. This study suggests that the functions of IKK ß signaling in atherogenesis are complex, and IKK ß in different cell types or tissues may have different effects on atherosclerosis development.