Attenuation of brown adipocyte whitening in high-fat diet-induced obese rats: Effects of melatonin and ß-hydroxybutyrate on Cidea, Fsp27 and MT1 expression.

AIM: To investigate the effects of ß-hydroxybutyrate (BHB) and melatonin on brown adipose tissue (BAT) plasticity in rats fed a high-fat diet (HFD). METHODS: We employed a 7-week experimental design for a study on 30 male Sprague-Dawley rats divided into five groups: (1) a control-diet fed group; (2) a high-fat diet (HFD)-fed group; (3) a group that received an HFD and a BHB solution in their drinking water; (4) a group that received an HFD with 10 mg/kg/day melatonin in their drinking water; and (5) a group that received an HFD and were also treated with the combination of BHB and melatonin. Following the treatment period, biochemical indices, gene expression levels of key thermogenic markers (including uncoupling protein 1 [UCP1], PR domain containing 16 [PRDM16], Cidea, fat-specific protein 27 [Fsp27], and metallothionein 1 [MT1]), and stereological assessments of BAT were evaluated. RESULTS: Treatment with BHB and melatonin significantly boosted blood ketone levels, improved lipid profiles, and reduced weight gain from an HFD. It also downregulated genes linked to WAT, namely, Cidea and Fsp27, and upregulated key BAT markers, including UCP1, PRDM16 and peroxisome proliferator-activated receptor-gamma coactivator-1-alpha. Additionally, the co-treatment increased MT1 receptor expression and enhanced the structural density of BAT. CONCLUSION: The combined oral administration of BHB and melatonin successfully prevented the whitening of BAT in obese rats fed an HFD, indicating its potential as a therapeutic strategy for obesity-related BAT dysfunction. The synergistic effects of this treatment underscore the potential of a combined approach to address BAT dysfunction in obesity.

Quercetin declines LPS induced inflammation and augments adiponectin expression in 3T3-L1 differentiated adipocytes SIRT-1 dependently.

BACKGROUND: Inflammation is an important factor contributing to obesity-induced metabolic disorders. Different investigations confirm that local inflammation in adipose issues is the primary reason for such disorder, resulting in low-grade systemic inflammation. Anti-inflammatory, antioxidant, and epigenetic modification are among the varied properties of Quercetin (QCT) as a natural flavonoid. OBJECTIVE: The precise molecular mechanism followed by QCT to alleviate inflammation has been unclear. This study explores whether the anti-inflammatory effects of QCT in 3T3-L1 differentiated adipocytes may rely on SIRT-1. METHODS: The authors isolated 3T3-L1 pre-adipocyte cells and exposed them to varying concentrations of QCT, lipopolysaccharide (LPS), and a selective inhibitor of silent mating type information regulation 2 homolog 1 (SIRT-1) called EX-527. After determining the optimal dosages of QCT, LPS, and EX-527, they assessed the mRNA expression levels of IL-18, IL-1, IL-6, TNF-α, SIRT-1, and adiponectin using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). RESULTS: The study showed considerable cytotoxic effects of LPS (200 ng/mL) + QCT (100 µM) + EX-527 (10 µM) on 3T3-L1 differentiated adipocytes after 48 h of incubation. QCT significantly upregulated the expression levels of adiponectin and SIRT-1 (p < 0.0001). However, introducing SIRT-1 inhibitor (p < 0.0001) reversed the impact of QCT on adiponectin expression. Additionally, QCT reduced SIRT-1-dependent pro-inflammatory cytokines in 3T3-L1 differentiated adipocytes (p < 0.0001). CONCLUSION: This study revealed that QCT treatment reduced crucial pro-inflammatory cytokines levels and increased adiponectin levels following LPS treatment. This finding implies that SIRT-1 may be a crucial factor for the anti-inflammatory activity of QCT.

ß-Hydroxybutyrate and melatonin suppress maladaptive UPR, excessive autophagy and pyroptosis in Aß 1-42 and LPS-Induced SH-SY5Y cells.

BACKGROUND: Alzheimer's disease is a neurological disease characterized by the build-up of amyloid beta peptide (Aß) and lipopolysaccharide (LPS), which causes synapse dysfunction, cell death, and neuro-inflammation. A maladaptive unfolded protein response (UPR), excessive autophagy, and pyroptosis aggravate the disease. Melatonin (MEL) and hydroxybutyrate (BHB) have both shown promise in terms of decreasing Aß pathology. The goal of this study was to see how BHB and MEL affected the UPR, autophagy, and pyroptosis pathways in Aß1-42 and LPS-induced SH-SY5Y cells. MATERIALS AND METHODS: Human neuroblastoma SH-SY5Y cells were treated with BHB, MEL, or a combination of the two after being exposed to A ß1-42 and LPS. Cell viability was determined using the MTT test, and gene expression levels of UPR (ATF6, PERK, and CHOP), autophagy (Beclin-1, LC3II, P62, and Atg5), and pyroptosis-related markers (NLRP3, TXNIP, IL-1ß, and NFκB1) were determined using quantitative Real-Time PCR (qRT-PCR). For statistical analysis, one-way ANOVA was employed, followed by Tukey's post hoc test. RESULTS: BHB and MEL significantly increased SH-SY5Y cell viability in the presence of A ß1-42 and LPS. Both compounds inhibited the expression of maladaptive UPR and autophagy-related genes, as well as inflammatory and pyroptotic markers caused by Aß1-42 and LPS-induced SH-SY5Y cells. CONCLUSION: BHB and MEL rescue neurons in A ß1-42 and LPS-induced SH-SY5Y cells by reducing maladaptive UPR, excessive autophagy, and pyroptosis. More research is needed to fully comprehend the processes behind their beneficial effects and to discover their practical applications in the treatment of neurodegenerative disorders.

Targeting apoptosis and unfolded protein response: the impact of ß-hydroxybutyrate in clear cell renal cell carcinoma under glucose-deprived conditions.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) plays a significant role in the mortality associated with kidney cancer. Targeting biological processes that inhibit cancer growth opens up new treatment possibilities. The unfolded protein response (UPR) and apoptosis have crucial roles in RCC progression. This study investigates the impact of ß-hydroxybutyrate (BHB) on ccRCC cells under glucose deprivation resembling as a ketogenic diet. METHOD: Caki-1 ccRCC cells were exposed to decreasing glucose concentrations alone or in combination with 10 or 25 mM BHB during 48 and 72 h. Cell viability was determined using MTT assay. The mRNA expression level of apoptosis-and UPR-related markers (Bcl-2, Bax, caspase 3, XBP1s, BIP, CHOP, ATF4, and ATF6) were assayed by qRT-PCR. RESULTS: Cell viability experiments demonstrated that combining different doses of BHB with decreasing glucose levels initially improved cell viability after 48 h. Nevertheless, this trend reversed after 72 h, with higher impacts disclosed at 25 mM BHB. Apoptosis was induced in BHB-treated cells as caspase-3 and Bax were increased and Bcl-2 was downregulated. BHB supplementation reduced UPR-related gene expression (XBP1s, BIP, CHOP, ATF4, and ATF6), revealing a possible mechanism by which BHB affects cell survival. CONCLUSION: This research emphasizes the dual effect of BHB, initially suppressing cell- survival under glucose deprivation but eventually triggering apoptosis and suppressing UPR signaling. These data highlight the intricate connection between metabolic reprogramming and cellular stress response in ccRCC. Further research is recommended to explore the potential of BHB as a therapeutic strategy for managing ccRCC.

Bilirubin, an endogenous antioxidant that affects p53 protein and its downstream apoptosis/autophagy-related genes in LS180 and SW480 cell culture models of colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is one of the commonest neoplasms worldwide, which its pathogenesis is strongly correlated with p53 mutations. Antioxidants are believed to decelerate the CRC progression, possibly through interfering with p53 and its downstream target genes and mechanisms. Regarding the potential antioxidant effects of bilirubin, as an incredible endogenous antioxidant, we sought to investigate how bilirubin affected the expression levels of p53 protein and its downstream target genes, including Mdm2, Bcl-2, BECN1 and LC3, in LS180 and SW480 cell culture models of CRC. METHODS AND RESULTS: Using the MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazolium bromide) assay, 50 and 100 µM concentrations of bilirubin were determined to be non-toxic for both LS180 and SW480 cell lines. Western blot analysis was employed to evaluate the protein expression levels of p53. The results revealed that p53 protein levels were higher in LS180 cells treated with bilirubin compared to the control group. Notwithstanding, in SW480 cells, no considerable changes were observed in p53 protein levels of treated cells compared to the control ones. The quantitative reverse transcriptase-polymerase chain reaction (q RT-PCR) method was used to measure the mRNA expression levels of the apoptosis/autophagy-related genes, Mdm2, Bcl-2, BECN1, and LC3 , as the p53's downstream target genes. Consequently, the expression of Bcl-2 and Mdm2 genes were affected by p53, while BECN1 and LC3 expression levels were decreased in both cell lines. CONCLUSION: Bilirubin is an endogenous antioxidant with significant anti-tumor effects in the studied CRC cell lines, probably through the regulation of p53 protein expression levels and subsequent control of apoptosis and autophagy, as two key processes involved in cell survival and progression of tumor cells.

Bilirubin, once a toxin but now an antioxidant alleviating non-alcoholic fatty liver disease in an autophagy-dependent manner in high-fat diet-induced rats: a molecular and histopathological analysis.

Background and purpose: As an endogenous antioxidant, bilirubin has surprisingly been inversely correlated with the risk of non-alcoholic fatty liver disease (NAFLD). Thereupon, the current evaluation was designed to assess the positive effects of bilirubin on the autophagy flux, as well as the other pathogenic processes and parameters involved in the expansion of NAFLD. Experimental approach: Thirty adult male rats weighing 150-200 g with free access to sucrose solution (18%) were randomly subdivided into 5 groups (n = 6). Subsequently, the animals were euthanized, and their blood specimens and liver tissue samples were collected to measure serum biochemical indices, liver histopathological changes, intrahepatic triglycerides content, and tissue stereological alterations. Furthermore, the expression levels of autophagy-related genes (Atgs) were measured to assess the state of the autophagy flux. Findings/Results: Fasting blood glucose, body weight, as well as liver weight, liver-specific enzyme activity, and serum lipid profile indices markedly decreased in rats that underwent a six-week bilirubin treatment compared to the control group. In addition, histopathological studies showed that hepatic steatosis, fibrosis, inflammation, and necrosis significantly decreased in the groups that received bilirubin compared to the control animals. Bilirubin also caused significant alterations in the expression levels of the Atgs, as well as the Beclin- 1 protein. Conclusion and implication: Bilirubin may have potential ameliorative effects on NAFLD-associated liver damage. Moreover, the beneficial effects of bilirubin on intrahepatic lipid accumulation and steatosis were comparable with the group that did not ever receive bilirubin.

The effect of quercetin on adipogenesis, lipolysis, and apoptosis in 3T3-L1 adipocytes: The role of SIRT1 pathways.

Background: Lipotoxicity, caused by adipocyte triglyceride over-accumulation, contributes to obesity-related comorbidities such as hypertension, type 2 diabetes, coronary heart disease, respiratory dysfunction, and osteoarthritis. This study focuses on determining how sirtuin-1 (SIRT-1) mediates quercetin's (QCT) effect on 3T3-L1 adipocytes. Key aspects of this study include preventing adipogenesis, inducing lipolysis, and stimulating adipocyte apoptosis. Methods: 3T3-L1 adipocytes underwent treatment with varying QCT doses, lipopolysaccharide (LPS), and the SIRT-1 inhibitor EX-527, followed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide [MTT] assay for cell viability assessment. Furthermore, quantitative real-time polymerase chain reaction measured mRNA expression levels of adipogenesis markers (fatty acid synthase [FASN] and peroxisome proliferator-activated receptor gamma [PPARγ]), lipolysis markers (adipose triglyceride lipase [ATGL] and hormone-sensitive lipase [HSL]), and apoptosis markers (B-cell lymphoma2 [Bcl-2], Bcl-2 Associated -X-protein [BAX] and Caspase-3). Results: The data showed that LPS + QCT significantly reduced cell viability in a dose- and time-dependent manner, unaffected by LPS + QCT + EX-527. Treatment with LPS + QCT did not affect FASN and PPARγ expression but significantly increased ATGL and HSL mRNA expression compared with LPS alone. Interestingly, EX-527 reversed the effects of LPS + QCT on lipogenesis and lipolysis markers completely. QCT enhanced apoptosis in a SIRT-1 independent pattern. Conclusion: The data suggest that QCT suppresses adipogenesis while increasing lipolysis via SIRT-1. However, QCT's effects on apoptosis appear to be independent of SIRT-1. These findings provide further evidence for QCT's effects on adipocytes, particularly its interaction with SIRT-1.

Autophagy and the unfolded protein response shape the non-alcoholic fatty liver landscape: decoding the labyrinth.

The incidence of nonalcoholic fatty liver disease (NAFLD) is on the rise, mirroring a global surge in diabetes and metabolic syndrome, as its major leading causes. NAFLD represents a spectrum of liver disorders, ranging from nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH), which can potentially progress to cirrhosis and hepatocellular carcinoma (HCC). Mechanistically, we know the unfolded protein response (UPR) as a protective cellular mechanism, being triggered under circumstances of endoplasmic reticulum (ER) stress. The hepatic UPR is turned on in a broad spectrum of liver diseases, including NAFLD. Recent data also defines molecular mechanisms that may underlie the existing correlation between UPR activation and NAFLD. More interestingly, subsequent studies have demonstrated an additional mechanism, i.e. autophagy, to be involved in hepatic steatosis, and thus NAFLD pathogenesis, principally by regulating the insulin sensitivity, hepatocellular injury, innate immunity, fibrosis, and carcinogenesis. All these findings suggest possible mechanistic roles for autophagy in the progression of NAFLD and its complications. Both UPR and autophagy are dynamic and interconnected fluxes that act as protective responses to minimize the harmful effects of hepatic lipid accumulation, as well as the ER stress during NAFLD. The functions of UPR and autophagy in the liver, together with findings of decreased hepatic autophagy in correlation with conditions that predispose to NAFLD, such as obesity and aging, suggest that autophagy and UPR, alone or combined, may be novel therapeutic targets against the disease. In this review, we discuss the current evidence on the interplay between autophagy and the UPR in connection to the NAFLD pathogenesis.

Protective and curative effects of unconjugated bilirubin on gene expression of LOX-1 and iNOS in the heart of rats receiving high-fat diet and low dose streptozotocin: a histomorphometric approach.

BACKGROUND: Atherosclerosis is a chronic inflammatory condition affecting the large arteries and is a major cause of cardiovascular diseases (CVDs) globally. Increased levels of adhesion molecules in cardiac tissue serve as prognostic markers for coronary artery occlusion risk. Given the antioxidant properties of bilirubin and its inverse correlation with atherosclerosis, this study aimed to assess the beneficial effects of bilirubin on atherosclerotic indices and heart structure in high-fat diet-fed diabetic rats with atherosclerosis. METHODS: Atherosclerosis was induced in three out of five groups of adult male Sprague Dawley rats through a 14-week period of high-fat diet (HFD) consumption and a single low dose of streptozotocin (STZ) (35 mg/kg). The atherosclerotic rats were then treated with intraperitoneal administration of 10 mg/kg/day bilirubin for either 6 or 14 weeks (treated and protected groups, respectively), or the vehicle. Two additional groups served as the control and bilirubin-treated rats. Subsequently, the mRNA expression levels of vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), lectin-like LDL receptor 1 (LOX-1), and the inducible nitric oxide synthase (iNOS) were analyzed using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Histopathological and stereological analyses were performed to assess changes in the heart structure. RESULTS: Bilirubin significantly decreased the expression of VCAM-1, ICAM-1, LOX-1, and iNOS genes in the treated group. Moreover, bilirubin mitigated pathological damage in the left ventricle of the heart. Stereological analysis revealed a decrease in the left ventricle and myocardium volume, accompanied by an increase in vessel volume in rats treated with bilirubin. CONCLUSION: These findings demonstrate that mild hyperbilirubinemia can protect against the progression of atherosclerosis and heart failure by improving lipid profile, modulating adhesion molecules, LOX-1, and iNOS gene expression levels.

"Protective effects of artichoke extract and Bifidobacterium longum on male infertility in diabetic rats".

Background: Diabetes is a major global health concern and plays a significant role in male infertility and hormonal abnormalities by altering the tissue structure of spermatogenic tubes and decreasing the number of spermatogonia. This study investigated the effect of artichoke (Cynara scolymus L) hydroalcoholic extract and Bifidobacterium longum probiotic on sexual hormones, oxidative stress, apoptosis pathway, and histopathological changes in testicular tissues of diabetic rats to find an adjuvant therapy to manage the infertility complications of diabetes. Methods: In this experiment, 96 male-rats were randomly selected from eight groups. Control, Sham (normal saline), DM group (IP injected with 60 mg/kg STZ), Cynara (400 mg/kg hydroalcoholic extract of Cynara scolymus L), BBL (received 1 × 109 CFU/ml/day Bifidobacterium longum), DM + Cynara, DM + BBL, and DM + Cynara + BBL groups. After 48 days of orally gavage, serum level of FBS (fasting blood sugar), Malondi-aldehyde (MDA), Total-Anti-Oxidant Capacity (TAC), FSH (Follicle-stimulating hormone), LH (Luteinizing hormone), Testosterone, Testis mRNA-expressions of Protamin (prm1), BCL2, and Caspase-9 genes, as well as stereological changes were measured. Results: In comparison to the diabetic group, the hydroalcoholic extract of Cynara scolymus L combined with the probiotic Bifidobacterium longum resulted in a substantial decrease in FBS (p < 0.001) and MDA(p < 0.05) concentrations, and the expression of the Caspase-9 gene (1.33-fold change). In addition, serum levels of TAC, LH, FSH, Testosterone were significantly increased (p < 0.05). mRNA expression of protamine (p = 0.016) and BCL2 (0.72-fold change) were detected. Furthermore, in comparison with diabetic rats, the Cynara scolymus L-and Bifidobacterium longum-treated groups showed a significant increase in the number of sexual lineage cells, total weight, sperm count, motility, normal morphology, volume of the testis, and volume and length of seminiferous tubules (p < 0.05). Conclusion: The findings demonstrated that Cynara scolymus L extract and Bifidobacterium longum supplement had great therapeutic potential, including antioxidant, anti-apoptotic, anti-diabetic, fertility index improvement, and sex hormone modulators.

Adipose triglyceride lipase gene expression in peripheral blood mononuclear cells of subjects with obesity and its association with insulin resistance, inflammation and lipid accumulation in liver.

Introduction: Adipose triglyceride lipase (ATGL) is a crucial enzyme responsible for the release of fatty acids from various tissues. The expression of ATGL is regulated by insulin and this enzyme is linked to Insulin resistance (IR). On the other hand, ATGL-mediated lipolysis is connected to macrophage function and thus, ATGL is involved in inflammation and the pathogenesis of lipid-related disorders. This study aimed to investigate the correlation between ATGL, obesity, Metabolic Syndrome (MetS), and inflammation. Methods: A total of 100 participants, including 50 individuals with obesity and 50 healthy participants, were recruited for this study and underwent comprehensive clinical evaluations. Blood samples were collected to measure plasma lipid profiles, glycemic indices, and liver function tests. Additionally, peripheral blood mononuclear cells (PBMCs) were isolated and used for the assessment of the gene expression of ATGL, using real-time PCR. Furthermore, PBMCs were cultured and exposed to lipopolysaccharides (LPS) with simultaneous ATGL inhibition, and the gene expression of inflammatory cytokines, along with the secretion of prostaglandin E2 (PGE2), were measured. Results: The gene expression of ATGL was significantly elevated in PBMCs obtained from participants with obesity and was particularly higher in those diagnosed with MetS. It exhibited a correlation with insulin levels and Homeostatic Model Assessment for IR (HOMA-IR), and it was associated with lipid accumulation in the liver. Stimulation with LPS increased ATGL expression in PBMCs, while inhibition of ATGL attenuated the inflammatory responses induced by LPS. Conclusions: Obesity and MetS were associated with dysregulation of ATGL. ATGL might play a role in the upregulation of inflammatory cytokines and act as a significant contributor to the development of metabolic abnormalities related to obesity.

Assessment of global histone acetylation in pediatric and adolescent obesity: Correlations with SIRT1 expression and metabolic-inflammatory profiles.

BACKGROUND: Epigenetic modifications, particularly histone acetylation-deacetylation and its related enzymes, such as sirtuin 1 (SIRT1) deacetylase, may have substantial roles in the pathogenesis of obesity and its associated health issues. This study aimed to evaluate global histone acetylation status and SIRT1 gene expression in children and adolescents with obesity and their association with metabolic and anthropometric parameters. METHODS: This study included 60 children and adolescents, 30 with obesity and 30 normal-weight. The evaluation consisted of the analysis of global histone acetylation levels and the expression of the SIRT1 gene in peripheral blood mononuclear cells, by specific antibody and real-time PCR, respectively. Additionally, insulin, fasting plasma glucose, lipid profile and tumor necrosis factor α (TNF-α) levels were measured. Insulin resistance was assessed using the homeostasis model assessment of insulin resistance (HOMA-IR). Metabolic syndrome was determined based on the diagnostic criteria established by IDF. RESULTS: Individuals with obesity, particularly those with insulin resistance, had significantly higher histone acetylation levels compared to control group. Histone acetylation was positively correlated with obesity indices, TNF-α, insulin, and HOMA-IR. Additionally, a significant decrease in SIRT1 gene expression was found among obese individuals, which was negatively correlated with the histone acetylation level. Furthermore, SIRT1 expression levels showed a negative correlation with various anthropometric and metabolic parameters. CONCLUSION: Histone acetylation was enhanced in children and adolescents with obesity, potentially resulting from down-regulation of SIRT1, and could play a role in the obesity-associated metabolic abnormalities and insulin resistance. Targeting global histone acetylation modulation might be considered as an epigenetic approach for early obesity management.

The protective effect of L-carnitine on testosterone synthesis pathway, and spermatogenesis in monosodium glutamate-induced rats.

BACKGROUND: Monosodium glutamate (MSG) is a food ingredient that is increasingly used commercially. MSG leads to oxidative stress, consequently suppressing steroid hormone production that causes defects in male reproductive system. This study aimed to evaluate the effect of L-carnitine as an antioxidant on testicular damage in MSG-induced male rats. METHODS: Sixty adult male Spargue-Dawley rats were randomly divided into six groups of ten as follows: control (water), sham (normal saline), L-carnitine (200 mg/kg b.w), MSG (3 g/kg b.w), MSG + L-carnitine 100 (3 g/kg b.w of MSG and 100 mg/kg b.w of L-carnitine), and MSG + L-carnitine 200 (3 g/kg b.w of MSG and 200 mg/kg b.w of L-carnitine). The treatment was administered by oral gavage for six months. Serum levels of Malondialdehyde (MDA), Total Anti-oxidant Capacity (TAC), LH, FSH, testosterone, and mRNA expressions of Star, Cyp11a1, and Hsd17b3 genes, and histological and stereological changes were assessed. RESULTS: L-carnitine led to a significant decrease in the level of MDA and a significant rise in the serum levels of TAC, LH, FSH, and mRNA expression of Star and Cyp11a1 compared to the MSG group (p < 0.05). Furthermore, stereological results indicated a significant increment in the number of sexual lineage cells, the total volume of the testis, length, diameter, and volume of seminiferous tubules, the height of the germinal epithelium, sperm count, and sperm motility (p < 0.05) in MSG + L-carnitine 200 compare to MSG group. CONCLUSION: The study's findings demonstrated that L-carnitine due to its anti-oxidant properties, ameliorated the reproductive abnormalities in the male rats exposed to MSG.