Inducible hepatic expression of CREBH mitigates diet-induced obesity, insulin resistance, and hepatic steatosis in mice.

Cyclic AMP-responsive element-binding protein H (CREBH encoded by Creb3l3) is a transcription factor that regulates the expression of genes that control lipid and glucose metabolism as well as inflammation. CREBH is upregulated in the liver under conditions of overnutrition, and mice globally lacking the gene (CREBH-/-) are highly susceptible to diet-induced obesity, insulin resistance, and hepatic steatosis. The net protective effects of CREBH have been attributed in large part to the activities of fibroblast growth factor (Fgf)-21 (Fgf21), a target gene that promotes weight loss, improves glucose homeostasis, and reduces hepatic lipid accumulation. To explore the possibility that activation of the CREBH-Fgf21 axis could ameliorate established effects of high-fat feeding, we generated an inducible transgenic hepatocyte-specific CREBH overexpression mouse model (Tg-rtTA). Acute overexpression of CREBH in livers of Tg-rtTA mice effectively reversed diet-induced obesity, insulin resistance, and hepatic steatosis. These changes were associated with increased activities of thermogenic brown and beige adipose tissues in Tg-rtTA mice, leading to reductions in fat mass, along with enhanced insulin sensitivity and glucose tolerance. Genetically silencing Fgf21 in Tg-rtTA mice abrogated the CREBH-mediated reductions in body weight loss, but only partially reversed the observed improvements in glucose metabolism. These findings reveal that the protective effects of CREBH activation may be leveraged to mitigate diet-induced obesity and associated metabolic abnormalities in both Fgf21-dependent and Fgf21-independent pathways.

Preemptive Activation of the Integrated Stress Response Protects Mice From Diet-Induced Obesity and Insulin Resistance by Fibroblast Growth Factor 21 Induction.

Integrated stress response (ISR) is a signaling system in which phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) by stress-specific kinases and subsequent activation of activation transcription factor (ATF) 4 help restore cellular homeostasis following exposure to environmental stresses. ISR activation has been observed in metabolic diseases, including hepatic steatosis (HS), steatohepatitis (SH), and insulin resistance (IR), but it remains unclear whether ISR contributes to disease pathogenesis or represents an innate defense mechanism against metabolic stresses. Constitutive repressor of eIF2α phosphorylation (CReP) is a critical regulatory subunit of the eIF2α phosphatase complex. Here, we show that CReP ablation causes constitutive eIF2α phosphorylation in the liver, which leads to activation of the ATF4 transcriptional program including increased fibroblast growth factor 21 (FGF21) production. Liver-specific CReP knockout (CRePLKO ) mice exhibited marked browning of white adipose tissue (WAT) and increased energy expenditure and insulin sensitivity in an FGF21-dependent manner. Furthermore, CRePLKO mice were protected from high-fat diet (HFD)-induced obesity, HS, and IR. Acute CReP ablation in liver of HFD-induced obese mice also reduced adiposity and improved glucose homeostasis. Conclusion: These data suggest that CReP abundance is a critical determinant for eIF2α phosphorylation and ensuing ISR activation in the liver. Constitutive ISR activation in the liver induces FGF21 and confers protection from HFD-induced adiposity, IR, and HS in mice. Augmenting hepatic ISR may represent a therapeutic approach to treat metabolic disorders.

Effect of circulating glucagon and free fatty acids on hepatic FGF21 production in dairy cows.

Modern dairy cows meet the energy demand of early lactation by calling on hormonally driven mechanisms to increase the use of lipid reserves. In this context, we recently reported that fibroblast growth factor-21 (FGF21), a hormone required for efficient use of lipid reserves in rodents, is upregulated in periparturient dairy cows. Increased plasma FGF21 in early lactation coincides with elevated circulating concentrations of glucagon (GCG) and nonesterified fatty acids (NEFA). To assess the relative contribution of these factors in regulating FGF21, two experiments were performed in energy-sufficient, nonpregnant, nonlactating dairy cows. In the first study, cows were injected with saline or GCG every 8 h over a 72-h period. GCG increased hepatic FGF21 mRNA by an average of fivefold over matched controls but had no effect on plasma FGF21. In the second study, cows were infused and injected with saline, infused with Intralipid and injected with saline, or infused with Intralipid and injected with GCG. Infusions and injections were administered intravenously over 16 h and subcutaneously every 8 h, respectively. Intralipid infusion increased plasma NEFA from 92 to 550 µM within 3 h and increased plasma FGF21 from 1.3 to >11 ng/ml 6 h later; FGF21 mRNA increased by 34-fold in liver but remained invariant in adipose tissue. GCG injections during the Intralipid infusion had no additional effects on plasma NEFA, liver FGF21 mRNA, or plasma FGF21. These data implicate plasma NEFA as a key factor triggering hepatic production and increased circulating concentrations of FGF21 in early lactation.

IGF binding protein-3 mediates stress-induced apoptosis in non-transformed mammary epithelial cells.

Mammary epithelial cell (MEC) number is an important determinant of milk production in lactating dairy cows. IGF-I increases IGF binding protein-3 (IGFBP-3) production in these cells, which plays a role in its ability to enhance proliferation. In the present study, we show that the apoptotic factor anisomycin (ANS) also increases IGFBP-3 mRNA and protein in a dose- and concentration-dependent manner that mirrors activation of caspase-3 and -7, with significant increases in both IGFBP-3 protein and caspase activation observed by 3 h. Knock-down of IGFBP-3 with small interfering (si) RNA attenuated the ability of ANS to induce apoptosis, while knock-down of IGFBP-2, the other major IGFBP made by bovine MEC, had no effect. Reducing IGFBP-3 also decreased the ability of ANS to induce mitochondrial cytochrome c release, indicating its involvement in the intrinsic apoptotic pathway. In contrast, transfection with IGFBP-3 in the absence of ANS failed to induce apoptosis. Since both the mitogen IGF-I and the apoptotic inducer ANS increase IGFBP-3 production in MEC, we proposed that cellular localization might determine IGFBP-3 action. While both IGF-I and ANS stimulated the release of IGFBP-3 into conditioned media, only ANS induced nuclear localization of IGFBP-3. A pan-caspase inhibitor had no effect on ANS-induced nuclear localization of IGFBP-3, indicating that nuclear entry of IGFBP-3 precedes caspase activation. Treatment with IGF-I had no effect on ANS-induced nuclear localization, but did block ANS-induced apoptosis. In summary, our data indicate that IGFBP-3 plays a role in stress-induced apoptosis that may require nuclear localization in non-transformed MEC.

High-throughput screening identifies small molecule inhibitors of thioesterase superfamily member 1: Implications for the management of non-alcoholic fatty liver disease.

OBJECTIVE: Thioesterase superfamily member 1 (Them1) is a long chain acyl-CoA thioesterase comprising two N-terminal HotDog fold enzymatic domains linked to a C-terminal lipid-sensing steroidogenic acute regulatory transfer-related (START) domain, which allosterically modulates enzymatic activity. Them1 is highly expressed in thermogenic adipose tissue, where it functions to suppress energy expenditure by limiting rates of fatty acid oxidation, and is induced markedly in liver in response to high fat feeding, where it suppresses fatty acid oxidation and promotes glucose production. Them1-/- mice are protected against non-alcoholic fatty liver disease (NAFLD), suggesting Them1 as a therapeutic target. METHODS: A high-throughput small molecule screen was performed to identify promising inhibitors targeting the fatty acyl-CoA thioesterase activity of purified recombinant Them1.Counter screening was used to determine specificity for Them1 relative to other acyl-CoA thioesterase isoforms. Inhibitor binding and enzyme inhibition were quantified by biophysical and biochemical approaches, respectively. Following structure-based optimization, lead compounds were tested in cell culture. RESULTS: Two lead allosteric inhibitors were identified that selectively inhibited Them1 by binding the START domain. In mouse brown adipocytes, these inhibitors promoted fatty acid oxidation, as evidenced by increased oxygen consumption rates. In mouse hepatocytes, they promoted fatty acid oxidation, but also reduced glucose production. CONCLUSION: Them1 inhibitors could prove attractive for the pharmacologic management of NAFLD.

Aflatoxin B1 metabolism: Regulation by phase I and II metabolizing enzymes and chemoprotective agents.

Aflatoxin B1 (AFB1) widely contaminates staple food and feed crops and is well-known as the most potent natural hepatocarcinogen in humans and domesticated animals. This review highlights significant advances in our understanding of the pivotal role of phase I and II metabolizing enzymes in the bioactivation and detoxification of AFB1 and its metabolites across species. In humans, cytochrome P450 (CYP) 1A2, CYP3A4, CYP3A5, and CYP3A7 in liver and CYP2A13 in lung are essential for the bioactivation of AFB1 to the extremely toxic exo-AFB1-8,9-epoxide (AFBO), whereas CYP1A1, CYP1A2, CYP2A6, and CYP3A4 are important in the turkey and duck, CYP1A1 and CYP2A6 are important in the chicken and quail, CYP3A11 and CYP3A13 are important in mice, and CYP2A5 are important in the hamster. In contrast, glutathione-S-transferase (GST) M1 and GSTT1 are primary responsible for detoxification of the AFB1 by catalyzing the conjugation of GSH to AFBO in humans, whereas GSTM2 in a nonhuman primate, GSTA3 in mice, GSTA5 in rats, and GSTA1, GSTA2, GSTA3 and GSTA4 in the turkey are important. Additionally, microsomal epoxide hydrolase (mEH) and aflatoxin-aldehyde reductase (AFAR) have also been shown to play key roles in AFB1 detoxification in the human, rat, and pig. Moreover, an overview of the chemoprotective agents, including synthetic compounds and naturally occurring plant compounds, which can be used to reduce aflatoxicosis is provided based on their ability to regulate these key enzymes. Collectively, this review summarizes the pivotal enzymes in the metabolism of AFB1 among humans, experimental and farm animals, as well as the chemoprotective agents that can be used to minimize risk of aflatoxicosis.

XBP1 Regulates the Biosynthetic Capacity of the Mammary Gland During Lactation by Controlling Epithelial Expansion and Endoplasmic Reticulum Formation.

Cells composing the mammary secretory compartment have evolved a high capacity to secrete not only proteins but also triglycerides and carbohydrates. This feature is illustrated by the mouse, which can secrete nearly twice its own weight in milk proteins, triglycerides and lactose over a short 20-day lactation. The coordination of synthesis and export of products in other secretory cells is orchestrated in part by the transcription factor X-box binding protein 1 (XBP1). To assess the role of XBP1 in mammary epithelial cells (MEC), we studied floxed XBP1 female mice lacking (wild type; WT) or expressing the Cre recombinase under the control of the ovine ß-lactoglobulin promoter (ΔXBP1(MEC)). Pregnant ΔXBP1(MEC) females had morphologically normal mammary development and gave birth to the same number of pups as WT mice. Their litters, however, suffered a weight gain deficit by lactation day 3 (L3)3 that grew to 80% by L14. ΔXBP1(MEC) dams had only modest changes in milk composition (-21% protein, +24% triglyceride) and in the expression of associated genes in isolated MEC. By L5, WT glands were fully occupied by dilated alveoli, whereas ΔXBP1(MEC) glands contained fewer, mostly unfilled alveoli and retained a prominent adipocyte population. The smaller epithelial compartment in ΔXBP1(MEC) glands was explained by lower MEC proliferation and increased apoptosis. Finally, endoplasmic reticulum ribbons were less abundant in ΔXBP1(MEC) at pregnancy day 18 and failed to increase in abundance by L5. Collectively, these results show that XBP1 is required for MEC population expansion during lactation and its ability to develop an elaborate endoplasmic reticulum compartment.

Increased plasma leptin attenuates adaptive metabolism in early lactating dairy cows.

Mammals meet the increased nutritional demands of lactation through a combination of increased feed intake and a collection of adaptations known as adaptive metabolism (e.g., glucose sparing via insulin resistance, mobilization of endogenous reserves, and increased metabolic efficiency via reduced thyroid hormones). In the modern dairy cow, adaptive metabolism predominates over increased feed intake at the onset of lactation and develops concurrently with a reduction in plasma leptin. To address the role of leptin in the adaptive metabolism of early lactation, we asked which adaptations could be countered by a constant 96-h intravenous infusion of human leptin (hLeptin) starting on day 8 of lactation. Compared to saline infusion (Control), hLeptin did not alter energy intake or milk energy output but caused a modest increase in body weight loss. hLeptin reduced plasma glucose by 9% and hepatic glycogen content by 73%, and these effects were associated with a 17% increase in glucose disposal during an insulin tolerance test. hLeptin attenuated the accumulation of triglyceride in the liver by 28% in the absence of effects on plasma levels of the anti-lipolytic hormone insulin or plasma levels of free fatty acids, a marker of lipid mobilization from adipose tissue. Finally, hLeptin increased the plasma concentrations of T4 and T3 by nearly 50% without affecting other neurally regulated hormones (i.e., cortisol and luteinizing hormone (LH)). Overall these data implicate the periparturient reduction in plasma leptin as one of the signals promoting conservation of glucose and energy at the onset of lactation in the energy-deficient dairy cow.

Response of the hepatic transcriptome to aflatoxin B1 in ducklings.

This study was conducted to determine the effects of aflatoxin B1 (AFB1) on the hepatic transcriptome in ducklings through RNA-sequencing (RNA-Seq). Twenty four, 1-day-old ducklings were divided into 4 treatment groups. Each group received an oral dose of AFB1 at 0, 10, 20, 40 µg/kg BW per day for 2 weeks. Administration of 20 and 40 µg/kg BW of AFB1 significantly decreased body weight, feed intake, serum total protein and albumin, while increasing serum aspartate aminotransferase and alanine aminotransferase activities, and hepatic histopathological lesions. Furthermore, RNA was extracted from the liver of ducklings administrated 0 and 40 µg/kg BW of AFB1. Two RNA-Seq libraries were created from pooled samples and produced over 149 M reads, totaling 14.9 Gb of sequence. Approximately 96,953 predicted transcripts were assembled, 749 of which had significant differential expressions (≥ 2-fold) between the control and AFB1 treatment. GO and KEGG pathway analysis results showed that many genes involved in phase I metabolism, phase II detoxification, oxidation-reduction process, carcinogenesis, apoptosis and cell cycle, and fatty acid metabolism were affected by AFB1 exposure. Conclusion, this study determined the hepatic transcriptome responded to AFB1 exposure, and provide candidate genes can be targeted to prevent and/or reduce aflatoxicosis in ducklings.

Curcumin Prevents Aflatoxin B1 Hepatoxicity by Inhibition of Cytochrome P450 Isozymes in Chick Liver.

This study was designed to establish if Curcumin (CM) alleviates Aflatoxin B1 (AFB1)-induced hepatotoxic effects and to determine whether alteration of the expression of cytochrome P450 (CYP450) isozymes is involved in the regulation of these effects in chick liver. One-day-old male broilers (n = 120) were divided into four groups and used in a two by two factorial trial in which the main factors included supplementing AFB1 (< 5 vs. 100 µg/kg) and CM (0 vs. 150 mg/kg) in a corn/soybean-based diet. Administration of AFB1 induced liver injury, significantly decreasing albumin and total protein concentrations and increasing alanine aminotransferase and aspartate aminotransferase activities in serum, and induced hepatic histological lesions at week 2. AFB1 also significantly decreased hepatic glutathione peroxidase, catalase, and glutathione levels, while increasing malondialdehyde, 8-hydroxydeoxyguanosine, and exo-AFB1-8,9-epoxide (AFBO)-DNA concentrations. In addition, the mRNA and/or activity of enzymes responsible for the bioactivation of AFB1 into AFBO-including CYP1A1, CYP1A2, CYP2A6, and CYP3A4-were significantly induced in liver microsomes after 2-week exposure to AFB1. These alterations induced by AFB1 were prevented by CM supplementation. Conclusively, dietary CM protected chicks from AFB1-induced liver injury, potentially through the synergistic actions of increased antioxidant capacities and inhibition of the pivotal CYP450 isozyme-mediated activation of AFB1 to toxic AFBO.

Individual and combined cytotoxic effects of aflatoxin B1, zearalenone, deoxynivalenol and fumonisin B1 on BRL 3A rat liver cells.

This study was performed to determine the individual and combined cytotoxic effects of Aflatoxin B1 (AFB1), zearalenone (ZEA), deoxynivalenol (DON) and fumonisin B1 (FB1) on BRL 3A rat liver cells. After the mycotoxins treated the BRL 3A cells for 12, 24 and 48 h, cell viability was determined using the MTT assay. The cytotoxicity of individual mycotoxins on BRL 3A cell viability in decreasing order were DON > AFB1 > ZEA > FB1. The central composite design (CCD) was used to assess the toxicity of binary and ternary mixtures of these mycotoxins. The mixtures of AFB1+ZEA and AFB1+DON showed the synergetic toxic effects on BRL 3A cells. These toxins decreased the viability of cells by inducing intracellular reactive oxygen species (ROS) production and promoting apoptosis in the BRL 3A cells. This effect was mediated by an upregulation of the stress and apoptotic genes Hsp70, p53, Bax, Caspase-3 and Caspase-8, along with a downregulation of the antiapoptotic gene Bcl-2. In conclusion, our results suggested that the coexistence of AFB1 and ZEA or DON in agricultural products could be more hepatotoxic than individually, suggests that the toxicological interactions of these toxins need to be better understood to assess health risks.

A novel strain of Cellulosimicrobium funkei can biologically detoxify aflatoxin B1 in ducklings.

Two experiments were conducted to screen microorganisms with aflatoxin B1 (AFB1 ) removal potential from soils and to evaluate their ability in reducing the toxic effects of AFB1 in ducklings. In experiment 1, we screened 11 isolates that showed the AFB1 biodegradation ability, and the one exhibited the highest AFB1 removal ability (97%) was characterized and identified as Cellulosimicrobium funkei (C. funkei). In experiment 2, 80 day-old Cherry Valley ducklings were divided into four groups with four replicates of five birds each and were used in a 2 by 2 factorial trial design, in which the main factors included administration of AFB1 versus solvent and C. funkei versus solvent for 2 weeks. The AFB1 treatment significantly decreased the body weight gain, feed intake and impaired feed conversion ratio. AFB1 also decreased serum albumin and total protein concentration, while it increased activities of alanine aminotransferase and aspartate aminotransferase and liver damage in the ducklings. Supplementation of C. funkei alleviated the adverse effects of AFB1 on growth performance, and provided protective effects on the serum biochemical indicators, and decreased hepatic injury in the ducklings. Conclusively, our results suggest that the novel isolated C. funkei strain could be used to mitigate the negative effects of aflatoxicosis in ducklings.

Effects of dietary tin on growth performance, hematology, serum biochemistry, antioxidant status, and tin retention in broilers.

Tin (Sn) is widely used in daily life and distributed in many tissues and nutrients. Although over-ingestion of Sn can cause health problems, relatively little attention has been given to the toxic effects of Sn in livestock health and productivity. This study was performed to investigate the toxic effects of prolonged high intake of dietary Sn on broilers. 150 one-day-old Avian broilers were randomly divided into five treatment groups, with five replicates of six birds. For 6 weeks, each group was fed a corn-soybean basal diet (BD) or BD + Sn (as SnCl2) at 120, 240, 480, or 720 mg/kg, respectively. Compared with the control, hepatic glutathione peroxidase (GPX) and superoxide dismutase (SOD) activities were significantly decreased when supplemented with Sn up to 480 mg/kg, while malondialdehyde (MDA) was increased until Sn supplementation at 720 mg/kg. Moreover, dietary Sn supplementation at 720 mg/kg decreased BW gain, feed intake, and impaired feed conversion ratio. The 720 mg Sn/kg group also increased activities of alkaline phosphatase (AKP), while decreased hemoglobin (HGB), red blood cell (RBC), and hematocrit (HCT) in the blood. Furthermore, the accumulation of Sn in various tissues was dose dependent on Sn ingestion. It was found that the tibia and feather are the two main tissues for Sn accumulation, followed by the liver, kidney, and other tissues in broilers. In conclusion, the adverse effects on broilers were induced when diets supplemented with Sn up to 480 mg/kg. Sn levels also managed to accumulate in the tibia and feather of broilers.

Hepatotoxic effects of mycotoxin combinations in mice.

This study was performed to assess the individual and combined toxic effects of aflatoxin B1 (AFB1), zearalenone (ZEA) and deoxynivalenol (DON) within the liver of mice. A total of 56 4-week-old weanling female mice were divided into seven groups (n = 8). For 2 weeks, each group received an oral administration of either solvent (control), AFB1, ZEA, DON, AFB1 + ZEA, AFB1 + DON or ZEA + DON per day. The results showed that AFB1, ZEA and DON induced liver injury, indicated by elevated relative liver weight, activities of alanine aminotransferase (ALT) and/or aspartate aminotransferase (AST), as well as decreased albumin (ALB) and/or total protein (TP) concentration in the serum. These mycotoxins also decreased hepatic total antioxidant capacity (T-AOC), and/or increased the concentration of malondialdehyde (MDA). Moreover, AFB1 + DON displayed synergistic effects, while AFB1 + ZEA displayed antagonistic effects on those parameters previously described. Furthermore, the apoptotic potential was demonstrated associated with an upregulation of the apoptotic genes Caspase-3 and Bax, along with a downregulation of the antiapoptotic gene Bcl-2 in liver. In conclusion, this study provides a better understanding of the toxic effects of AFB1, ZEA, DON, alone or in combinations on the liver of mice, which could contribute to the risk assessment of these mycotoxins in food and feed.

Effects of the regulation of follistatin mRNA expression by IGF-1 in duck (Anas platyrhynchos) skeletal muscle.

The IGF-1 and TGF-ß pathways have been shown to be involved in regulating muscle development. Many mediators that are associated with the regulation of muscle development have been found to participate in the cross-talk between these two pathways. To research the relationships between IGF-1 and the follistatin-mediated TGF-ß pathways in duck skeletal muscle development, a series of studies were conducted. The results showed that follistatin had similar expression patterns to IGF-1 during duck embryonic muscle development. The in ovo feeding of IGF-1 to duck eggs was shown to increase follistatin expression in the duck skeletal muscle. Thus, IGF-1 may induce the mRNA expression of follistatin. These results suggest that follistatin may be a key regulator of multiple signaling cascades responding to the cross-talk between the IGF-1 and TGF-ß pathways.