A High-Fat and High-Fructose Diet Exacerbates Liver Dysfunction by Regulating Sirtuins in a Murine Model.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is rapidly emerging as the most prevalent chronic liver disease, closely linked to the escalating rates of diabesity. The Western diet's abundance of fat and fructose significantly contributes to MASLD, disrupting hepatic glucose metabolism. We previously demonstrated that a high-fat and high-fructose diet (HFHFD) led to increased body and liver weight compared to the low-fat diet (LFD) group, accompanied by glucose intolerance and liver abnormalities, indicating an intermediate state between fatty liver and liver fibrosis in the HFHFD group. Sirtuins are crucial epigenetic regulators associated with energy homeostasis and play a pivotal role in these hepatic dysregulations. Our investigation revealed that HFHFD significantly decreased Sirt1 and Sirt7 gene and protein expression levels, while other sirtuins remained unchanged. Additionally, glucose 6-phosphatase (G6Pase) gene expression was reduced in the HFHFD group, suggesting a potential pathway contributing to fibrosis progression. Chromatin immunoprecipitation analysis demonstrated a significant increase in histone H3 lysine 18 acetylation within the G6Pase promoter in HFHFD livers, potentially inhibiting G6Pase transcription. In summary, HFHFD may inhibit liver gluconeogenesis, potentially promoting liver fibrosis by regulating Sirt7 expression. This study offers an epigenetic perspective on the detrimental impact of fructose on MASLD progression.

The mitochondrial link: Phthalate exposure and cardiovascular disease.

Phthalates' pervasive presence in everyday life poses concern as they have been revealed to induce perturbing health defects. Utilized as a plasticizer, phthalates are riddled throughout many common consumer products including personal care products, food packaging, home furnishings, and medical supplies. Phthalates permeate into the environment by leaching out of these products which can subsequently be taken up by the human body. It is previously established that a connection exists between phthalate exposure and cardiovascular disease (CVD) development; however, the specific mitochondrial link in this scenario has not yet been described. Prior studies have indicated that one possible mechanism for how phthalates exert their effects is through mitochondrial dysfunction. By disturbing mitochondrial structure, function, and signaling, phthalates can contribute to the development of the foremost cause of death worldwide, CVD. This review will examine the potential link among phthalates and their effects on the mitochondria, permissive of CVD development.

Shikonin Causes an Apoptotic Effect on Human Kidney Cancer Cells through Ras/MAPK and PI3K/AKT Pathways.

(1) Background: Shikonin, the main ingredient in Chinese herbal medicine, is described as a novel angiogenesis inhibitor, and its anticancer effects have already been studied. Shikonin and its derivatives induce apoptosis and suppress metastasis, which further enhance the effectiveness of chemotherapy. However, their mechanism of function has not been completely elucidated on human renal cancer cells. (2) Methods: In our study, CAKI-2 and A-498 cells were treated with increasing concentrations (2.5-40 µM) of shikonin, when colony formation ability and cytotoxic activity were tested. The changes in the expression of the main targets of apoptotic pathways were measured by RT-qPCR and Western blot. The intracellular levels of miR-21 and miR-155 were quantified by RT-qPCR. (3) Results: Shikonin exerted a dose-dependent effect on the proliferation of the cell lines examined. In 5 µM concentration of shikonin in vitro elevated caspase-3 and -7 levels, the proteins of the Ras/MAPK and PI3K/AKT pathways were activated. However, no significant changes were detected in the miR-21 and miR-155 expressions. (4) Conclusions: Our findings indicated that shikonin causes apoptosis of renal cancer cells by activating the Ras/MAPK and PI3K/AKT pathways. These effects of shikonin on renal cancer cells may bear important potential therapeutic implications for the treatment of renal cancer.

Synthesis of Mixed-Phase TiO2-ZrO2 Nanocomposite for Photocatalytic Wastewater Treatment.

The use of TiO2 nanoparticles for photocatalysis for the degradation of organic dyes under UV light for wastewater treatment has been widely studied. However, the photocatalytic characteristics of TiO2 nanoparticles are inadequate due to their UV light response and higher band gap. In this work, three nanoparticles were synthesized: (i) TiO2 nanoparticle was synthesized by a sol-gel process. (ii) ZrO2 was prepared using a solution combustion process and (iii) mixed-phase TiO2-ZrO2 nanoparticles were synthesized by a sol-gel process to remove Eosin Yellow (EY) from aqueous solutions in the wastewater. XRD, FTIR, UV-VIS, TEM, and XPS analysis methods were used to examine the properties of the synthesized products. The XRD investigation supported the tetragonal and monoclinic crystal structures of the TiO2 and ZrO2 nanoparticles. TEM studies identified that mixed-phase TiO2-ZrO2 nanoparticles have the same tetragonal structure as pure mixed-phase. The degradation of Eosin Yellow (EY) was examined using TiO2, ZrO2, and mixed-phase TiO2-ZrO2 nanoparticles under visible light. The results confirmed that the mixed-phase TiO2-ZrO2nanoparticles show a higher level of photocatalytic activity, and the process is accomplished at a high degradation rate in lesser time and at a lower power intensity.

Traditional Therapeutics and Potential Epidrugs for CVD: Why Not Both?

Cardiovascular disease (CVD) is the leading cause of death worldwide. In addition to the high mortality rate, people suffering from CVD often endure difficulties with physical activities and productivity that significantly affect their quality of life. The high prevalence of debilitating risk factors such as obesity, type 2 diabetes mellitus, smoking, hypertension, and hyperlipidemia only predicts a bleak future. Current traditional CVD interventions offer temporary respite; however, they compound the severe economic strain of health-related expenditures. Furthermore, these therapeutics can be prescribed indefinitely. Recent advances in the field of epigenetics have generated new treatment options by confronting CVD at an epigenetic level. This involves modulating gene expression by altering the organization of our genome rather than altering the DNA sequence itself. Epigenetic changes are heritable, reversible, and influenced by environmental factors such as medications. As CVD is physiologically and pathologically diverse in nature, epigenetic interventions can offer a ray of hope to replace or be combined with traditional therapeutics to provide the prospect of addressing more than just the symptoms of CVD. This review discusses various risk factors contributing to CVD, perspectives of current traditional medications in practice, and a focus on potential epigenetic therapeutics to be used as alternatives.

Role of microRNA in Endocrine Disruptor-Induced Immunomodulation of Metabolic Health.

The prevalence of poor metabolic health is growing exponentially worldwide. This condition is associated with complex comorbidities that lead to a compromised quality of life. One of the contributing factors recently gaining attention is exposure to environmental chemicals, such as endocrine-disrupting chemicals (EDCs). Considerable evidence suggests that EDCs can alter the endocrine system through immunomodulation. More concerning, EDC exposure during the fetal development stage has prominent adverse effects later in life, which may pass on to subsequent generations. Although the mechanism of action for this phenomenon is mostly unexplored, recent reports implicate that non-coding RNAs, such as microRNAs (miRs), may play a vital role in this scenario. MiRs are significant contributors in post-transcriptional regulation of gene expression. Studies demonstrating the immunomodulation of EDCs via miRs in metabolic health or towards the Developmental Origins of Health and Disease (DOHaD) Hypothesis are still deficient. The aim of the current review was to focus on studies that demonstrate the impact of EDCs primarily on innate immunity and the potential role of miRs in metabolic health.

Obesity II: Establishing causal links between chemical exposures and obesity.

Obesity is a multifactorial disease with both genetic and environmental components. The prevailing view is that obesity results from an imbalance between energy intake and expenditure caused by overeating and insufficient exercise. We describe another environmental element that can alter the balance between energy intake and energy expenditure: obesogens. Obesogens are a subset of environmental chemicals that act as endocrine disruptors affecting metabolic endpoints. The obesogen hypothesis posits that exposure to endocrine disruptors and other chemicals can alter the development and function of the adipose tissue, liver, pancreas, gastrointestinal tract, and brain, thus changing the set point for control of metabolism. Obesogens can determine how much food is needed to maintain homeostasis and thereby increase the susceptibility to obesity. The most sensitive time for obesogen action is in utero and early childhood, in part via epigenetic programming that can be transmitted to future generations. This review explores the evidence supporting the obesogen hypothesis and highlights knowledge gaps that have prevented widespread acceptance as a contributor to the obesity pandemic. Critically, the obesogen hypothesis changes the narrative from curing obesity to preventing obesity.

Obesity III: Obesogen assays: Limitations, strengths, and new directions.

There is increasing evidence of a role for environmental contaminants in disrupting metabolic health in both humans and animals. Despite a growing need for well-understood models for evaluating adipogenic and potential obesogenic contaminants, there has been a reliance on decades-old in vitro models that have not been appropriately managed by cell line providers. There has been a quick rise in available in vitro models in the last ten years, including commercial availability of human mesenchymal stem cell and preadipocyte models; these models require more comprehensive validation but demonstrate real promise in improved translation to human metabolic health. There is also progress in developing three-dimensional and co-culture techniques that allow for the interrogation of a more physiologically relevant state. While diverse rodent models exist for evaluating putative obesogenic and/or adipogenic chemicals in a physiologically relevant context, increasing capabilities have been identified for alternative model organisms such as Drosophila, C. elegans, zebrafish, and medaka in metabolic health testing. These models have several appreciable advantages, including most notably their size, rapid development, large brood sizes, and ease of high-resolution lipid accumulation imaging throughout the organisms. They are anticipated to expand the capabilities of metabolic health research, particularly when coupled with emerging obesogen evaluation techniques as described herein.

Obesity I: Overview and molecular and biochemical mechanisms.

Obesity is a chronic, relapsing condition characterized by excess body fat. Its prevalence has increased globally since the 1970s, and the number of obese and overweight people is now greater than those underweight. Obesity is a multifactorial condition, and as such, many components contribute to its development and pathogenesis. This is the first of three companion reviews that consider obesity. This review focuses on the genetics, viruses, insulin resistance, inflammation, gut microbiome, and circadian rhythms that promote obesity, along with hormones, growth factors, and organs and tissues that control its development. It shows that the regulation of energy balance (intake vs. expenditure) relies on the interplay of a variety of hormones from adipose tissue, gastrointestinal tract, pancreas, liver, and brain. It details how integrating central neurotransmitters and peripheral metabolic signals (e.g., leptin, insulin, ghrelin, peptide YY3-36) is essential for controlling energy homeostasis and feeding behavior. It describes the distinct types of adipocytes and how fat cell development is controlled by hormones and growth factors acting via a variety of receptors, including peroxisome proliferator-activated receptor-gamma, retinoid X, insulin, estrogen, androgen, glucocorticoid, thyroid hormone, liver X, constitutive androstane, pregnane X, farnesoid, and aryl hydrocarbon receptors. Finally, it demonstrates that obesity likely has origins in utero. Understanding these biochemical drivers of adiposity and metabolic dysfunction throughout the life cycle lends plausibility and credence to the "obesogen hypothesis" (i.e., the importance of environmental chemicals that disrupt these receptors to promote adiposity or alter metabolism), elucidated more fully in the two companion reviews.

LncRNA DLEU2 regulates sirtuins and mitochondrial respiratory chain complex IV: a novel pathway in obesity and offspring's health.

BACKGROUND: Long non-coding RNAs (lncRNAs) have emerged as a rapidly expanding area of interest in chronic diseases. They are mostly unknown for roles in metabolic regulation. Sirtuins, an epigenetic modulator class, regulate metabolic pathways. However, how sirtuins are regulated via lncRNA is unknown. We hypothesized that a high-fat high-fructose diet (HFD-HF) during pregnancy would increase the risk for obesity via lncRNA-Sirtuin pathways. METHODS: Female C57Bl/6 mice (F0) were fed either chow diet (CD) or HFD-HF for 6 weeks till birth. The pups (F1) were fed either CD or HFD-HF for 20 weeks. Expression of Dleu2, sirtuins, mitochondrial respiratory complexes, and oxidative stress were investigated in the F1 livers. Fasting blood glucose, insulin sensitivity, glucose tolerance, body and tissues weight were measured. A mechanistic interaction was then carried out using a DLEU2 knockdown experiment in the HepG2 cell. RESULTS: Dleu2 and sirtuins were both significantly decreased in the livers of HFD-HF fed male F1 whose mothers were either fed CD or HFD-HF during reproductive and pregnancy windows. Confirming this connection, upon silencing DLEU2, transcription levels of SIRT1 through 6 and translational levels of SIRT1, 3, 5, and 6 were significantly downregulated. Knockdown of DLEU2 significantly decreased the protein level of cytochrome-c oxidase (complex IV, MTCO1) without altering other mitochondrial complexes, decreased mitochondrial membrane potential, decreased ATP, and increased reactive oxygen species. Interestingly, in F1 livers, the protein level of MTCO1 was also significantly decreased under an HFD-HF diet or even under chow diet if the mother was exposed to HFD-HF. CONCLUSION: Our findings reveal for the first time that one lncRNA can regulate sirtuins and a specific mitochondrial complex. Furthermore, diet or maternal diet can modulate Dleu2 and its downstream regulators in offspring, suggesting a potential role of DLEU2 in metabolic disorders over one or more generations.

Pyrroloquinoline quinone attenuated benzyl butyl phthalate induced metabolic aberration and a hepatic metabolomic analysis.

Benzyl butyl phthalate (BBP) has recently been implicated as an obesogen. Our recent study demonstrated that BBP can exacerbate high fat diet (HFD) induced diabesity in male mice. Here, we explored if pyrroloquinoline quinone (PQQ), a natural antioxidant andphytochemical, can attenuate metabolic aberrations induced by HFD or HFD-BBPcombination. C57Bl/6 male and female mice were fed either a chow diet (CD) or HFD with or without BBP (3 mg/kg body weight/day)and/or PQQ (20 mg/kg/day)for 16 weeks. The mice's body and tissue weight, fasting blood glucose, glucose and insulin tolerance test, and liver metabolites level weremeasured. In HFD-fed male mice, PQQ significantly attenuated the increased body weight, liver weight, fasting blood glucose, and insulin intolerance under BBP exposure.Even though female mice did show some reversal of metabolic characteristics by PQQ, the response was not similar nor consistent with the male population. Amongthe 14 hepatic metabolites that were significantly altered by HFD compared to CD, only three major metabolites (acetyl-L-carnitine, DL-stachytine, and propionylcarnitine) were decreased. These three were shown to have more reduction under BBP exposure in the presence of HFD whereas with addition of PQQ, these metabolites were restored. Pathway analysis and literature search revealed that these metabolites were negatively associated with obesity and were involved in several pathways including beta-oxidation, oxidative stress, and mitochondrial function. Overall,this finding indicated the potential use of PQQ to restore thewide range of aberrant metabolic effectinduced by an obesogen in the presence of a western diet.

Butyl Benzyl Phthalate Promotes Adipogenesis in 3T3-L1 Cells via the miRNA-34a-5p Signaling Pathway in the Absence of Exogenous Adipogenic Stimuli.

Phthalates, a plasticizer group, are used extensively in many of the products we use every day. Public health concerns are growing as recent studies have implicated butyl benzyl phthalate (BBP) as an obesogen. However, BBP-induced epigenetic regulation during adipogenesis is still unknown. We investigated if BBP altered miR-34a-5p, a key miRNA involved in obesity, and regulated its downstream pathway. Differentiating 3T3-L1 cells were exposed to various doses of BBP without exogenous adipogenic stimuli, tested for adipogenesis markers (PPARγ and aP2), and stained for lipid accumulation with Oil Red O staining. We then measured the expression of miR-34a-5p and its target genes, Nampt and Sirt1, along with another significant epigenetic modulator, Sirt3. Furthermore, using antagomiR, we examined whether miR-34a-5p knockdown decreased adipogenesis. BBP exposure resulted in augmented expression levels of miR-34a-5p with an associated increase in adipogenesis. BBP significantly decreased the Nampt, Sirt1, and Sirt3 gene expression levels. However, a decrease in the protein expression was observed only for Nampt, indicating that miR-34a-5p under BBP exposure may regulate Sirt1/Sirt3 only at the transcriptional level. Interestingly, in the presence of BBP, knockdown of miR-34a-5p decreased adipogenesis in the differentiating 3T3-L1 cells. Furthermore, miR-34a-5p knockdown increased the Nampt protein expression levels as well as NAD+ levels, indicating that miR-34a-5p regulates Nampt during BBP exposure. Additionally, the NAD+-dependent sirtuin activity decreased in BBP-treated cells and increased in miR-34a-5p knockdown cells with BBP treatment. BBP exposure demonstrated the involvement of epigenetic regulation by altering the expression patterns of miR-34a-5p and its target Nampt, which may perturb the energy homeostasis of the differentiating adipocytes by altering NAD+ levels and sirtuin activity, resulting in increased adipogenesis.

Tumor-Associated Macrophages as Multifaceted Regulators of Breast Tumor Growth.

Breast cancer is the most commonly occurring cancer in women of Western countries and is the leading cause of cancer-related mortality. The breast tumor microenvironment contains immune cells, fibroblasts, adipocytes, mesenchymal stem cells, and extracellular matrix. Among these cells, macrophages or tumor-associated macrophages (TAMs) are the major components of the breast cancer microenvironment. TAMs facilitate metastasis of the breast tumor and are responsible for poor clinical outcomes. High TAM density was also found liable for the poor prognosis of breast cancer. These observations make altering TAM function a potential therapeutic target to treat breast cancer. The present review summarizes the origin of TAMs, mechanisms of macrophage recruitment and polarization in the tumor, and the contributions of TAMs in tumor progression. We have also discussed our current knowledge about TAM-targeted therapies and the roles of miRNAs and exosomes in re-educating TAM function.

Screening for alternative splicing of lncRNA Dleu2 in the mouse liver cell line AML-12.

The long non coding RNA deleted in leukemia 2 gene (Dleu2) has recently been demonstrated to be an active player in the progression of several types of cancer, including hepatocellular carcinoma. Dleu2 may serve a role in modulating the downstream effects-mediated by alternative splicing of its multiple exons. However, the proportional expression of these alternative splicing populations of the Dleu2 exons is currently unknown. To determine how Dleu2 could be affected by alternative splicing, a series of alternative splicing primer sets were designed to investigate which transcripts were preferentially activated when Dleu2 was targeted for downregulation or upregulation. A specific Dleu2 small interfering RNA that targeted an exon upstream of the tumor suppressor microRNA site significantly knocked down Dleu2 expression across all the primer sets used, which targeted 13 different alternative splicing transcripts over 5 different promoter sites in the mouse liver cell line, AML-12. Similarly, 50 µM Resveratrol led to significant upregulation of Dleu2 in 11 alternative splicing transcripts. These results show that Dleu2 is capable of successful modulation across alternative splicing transcripts that can be screened, and also that Resveratrol can be a potential nutraceutical, which may potentially lead to novel approaches in the use of lncRNA Dleu2 for diagnostics and regulation.

Role of macrophage autophagy in atherosclerosis: modulation by bioactive compounds.

Atherosclerosis is a chronic inflammatory disease associated with lipid metabolism disorder. Autophagy is a catabolic process and contributes to maintaining cellular homeostasis. Substantial evidence suggests that defective autophagy is implicated in several diseases, including atherosclerosis, while increased autophagy mitigates atherosclerosis development. Thus, understanding the mechanisms of autophagy regulation and its association with atherosclerosis is vital to develop new therapies against atherosclerosis. Dietary bioactive compounds are non-nutrient natural compounds that include phenolics, flavonoids, and carotenoids. Importantly, these bioactive compounds possess anti-inflammatory, antioxidant, and antibacterial properties that may alleviate various chronic diseases. Recently, examining the effects of bioactive compounds on autophagy activity in atherogenesis has drawn considerable attention. The current review discusses the role of macrophage autophagy in the development and progression of atherosclerosis. We also summarize our current knowledge of the therapeutic potential of bioactive compounds on atherosclerosis and autophagy.

Effect of Chronic Western Diets on Non-Alcoholic Fatty Liver of Male Mice Modifying the PPAR-γ Pathway via miR-27b-5p Regulation.

Western diets contribute to metabolic diseases. However, the effects of various diets and epigenetic mechanisms are mostly unknown. Here, six week-old C57BL/6J male and female mice were fed with a low-fat diet (LFD), high-fat diet (HFD), and high-fat high-fructose diet (HFD-HF) for 20 weeks. We determined that HFD-HF or HFD mice experienced significant metabolic dysregulation compared to the LFD. HFD-HF and HFD-fed male mice showed significantly increased body weight, liver size, and fasting glucose levels with downregulated PPARγ, SCD1, and FAS protein expression. In contrast, female mice were less affected by HFD and HFD-HF. As miR-27b contains a seed sequence in PPARγ, it was discovered that these changes are accompanied by male-specific upregulation of miR-27b-5p, which is even more pronounced in the HFD-HF group (p < 0.01 vs. LFD) compared to the HFD group (p < 0.05 vs. LFD). Other miR-27 subtypes were increased but not significantly. HFD-HF showed insignificant changes in fibrosis markers when compared to LFD. Interestingly, fat ballooning in hepatocytes was increased in HFD-fed mice compared to HFD-HF fed mice, however, the HFD-HF liver showed an increase in the number of small cells. Here, we concluded that chronic Western diet-composition administered for 20 weeks may surpass the non-alcoholic fatty liver (NAFL) stage but may be at an intermediate stage between fatty liver and fibrosis via miR-27b-5p-induced PPARγ downregulation.

Benzyl Butyl Phthalate Induced Early lncRNA H19 Regulation in C3H10T1/2 Stem Cell Line.

Exposure to endocrine-disrupting chemicals used in plastic manufacturing may contribute to the current obesity and diabetes epidemic. Our previous study demonstrated that benzyl butyl phthalate (BBP) induced adipogenesis in the C3H10T1/2 stem cell line. Here we investigated if BBP deregulated long noncoding RNA H19 and its downstream pathway and whether BBP plays a role in the insulin signaling pathway during adipocyte diiferentiation. Cells treated with an 8 day BBP regimen showed that H19 expression was decreased at day 2 with 50 µM BBP exposure (p < 0.05). However, no significant changes were observed from day 4 to day 8. Expression of miRNA-103/107, H19 regulated miRNAs, was upregulated at day 2 (p < 0.05) but not from day 4 to day 8. Similarly, expression of the let-7 family members (a, b, c, d, f, and g) was also significantly increased at day 2 (p < 0.05 or p < 0.01), except for let-7e. Both let-7 and miRNA-103/107 are targets of H19 and play roles in insulin signaling. Insulin receptor substrate (IRS)-1, one of the key insulin signal transduction regulators, was significantly downregulated from day 2 to day 8 (p < 0.05). Gene expression of insulin receptor (IR) and IRS-2 were not altered by BBP exposure. The ratio of IRS1/IRS2 was significantly decreased from day 2 to day 8. On day 4, phospho-Akt protein expression was significantly decreased (p < 0.05). In conclusion, BBP exposure may lead to metabolic dysregulation by altering vital epigenetic regulators such as lncRNA H19 and its target microRNAs at an earlier stage, which further regulates insulin signaling.

A moderate physiological dose of benzyl butyl phthalate exacerbates the high fat diet-induced diabesity in male mice.

Exposure to endocrine disrupting chemicals (EDCs) used in plastic manufacturing processes may be contributing to the current increase in metabolic disorders. Here, we determined that benzyl butyl phthalate (BBP), a common EDC and food packaging plasticizer, mixed into chow diet (CD) and high fat diets (HFD) at varying concentrations (4 µg/kg body weight (bw)/day, 169 µg/kg bw/day, 3 mg/kg bw/day, 50 mg/kg bw/day) produced a number of detrimental and sex-specific metabolic effects in C57BL/6 male and female mice after 16 weeks. Male mice exposed to moderate (3 mg/kg bw/day) concentrations of BBP in an HFD were especially affected, with significant increases in body weight due to significant increases in weight of liver and adipose tissue. Other doses did not show any significant changes when compared to only CD or HFD alone. HFD in the presence of 3 mg/kg bw/day BBP showed significant increases in fasting blood glucose, glucose intolerance, and insulin intolerance when compared to HFD alone. Furthermore, this group significantly alters transcriptional regulators involved in hepatic lipid synthesis and its downstream pathway. Interestingly, most of the BBP doses had no phenotypic effect when mixed with CD and compared to CD alone. The female mice did not show a similar response as the male population even though they consumed a similar amount of food. Overall, these data establish a dose which can be used for a BBP-induced metabolic research model and suggest that a moderate dosage level of EDC exposure can contribute to widely ranging metabolic effects.