Dietary fructose feeds hepatic lipogenesis via microbiota-derived acetate.

Consumption of fructose has risen markedly in recent decades owing to the use of sucrose and high-fructose corn syrup in beverages and processed foods1, and this has contributed to increasing rates of obesity and non-alcoholic fatty liver disease2-4. Fructose intake triggers de novo lipogenesis in the liver4-6, in which carbon precursors of acetyl-CoA are converted into fatty acids. The ATP citrate lyase (ACLY) enzyme cleaves cytosolic citrate to generate acetyl-CoA, and is upregulated after consumption of carbohydrates7. Clinical trials are currently pursuing the inhibition of ACLY as a treatment for metabolic diseases8. However, the route from dietary fructose to hepatic acetyl-CoA and lipids remains unknown. Here, using in vivo isotope tracing, we show that liver-specific deletion of Acly in mice is unable to suppress fructose-induced lipogenesis. Dietary fructose is converted to acetate by the gut microbiota9, and this supplies lipogenic acetyl-CoA independently of ACLY10. Depletion of the microbiota or silencing of hepatic ACSS2, which generates acetyl-CoA from acetate, potently suppresses the conversion of bolus fructose into hepatic acetyl-CoA and fatty acids. When fructose is consumed more gradually to facilitate its absorption in the small intestine, both citrate cleavage in hepatocytes and microorganism-derived acetate contribute to lipogenesis. By contrast, the lipogenic transcriptional program is activated in response to fructose in a manner that is independent of acetyl-CoA metabolism. These data reveal a two-pronged mechanism that regulates hepatic lipogenesis, in which fructolysis within hepatocytes provides a signal to promote the expression of lipogenic genes, and the generation of microbial acetate feeds lipogenic pools of acetyl-CoA.

Acetyl-CoA promotes glioblastoma cell adhesion and migration through Ca2+-NFAT signaling.

The metabolite acetyl-coenzyme A (acetyl-CoA) is the required acetyl donor for lysine acetylation and thereby links metabolism, signaling, and epigenetics. Nutrient availability alters acetyl-CoA levels in cancer cells, correlating with changes in global histone acetylation and gene expression. However, the specific molecular mechanisms through which acetyl-CoA production impacts gene expression and its functional roles in promoting malignant phenotypes are poorly understood. Here, using histone H3 Lys27 acetylation (H3K27ac) ChIP-seq (chromatin immunoprecipitation [ChIP] coupled with next-generation sequencing) with normalization to an exogenous reference genome (ChIP-Rx), we found that changes in acetyl-CoA abundance trigger site-specific regulation of H3K27ac, correlating with gene expression as opposed to uniformly modulating this mark at all genes. Genes involved in integrin signaling and cell adhesion were identified as acetyl-CoA-responsive in glioblastoma cells, and we demonstrate that ATP citrate lyase (ACLY)-dependent acetyl-CoA production promotes cell migration and adhesion to the extracellular matrix. Mechanistically, the transcription factor NFAT1 (nuclear factor of activated T cells 1) was found to mediate acetyl-CoA-dependent gene regulation and cell adhesion. This occurs through modulation of Ca2+ signals, triggering NFAT1 nuclear translocation when acetyl-CoA is abundant. The findings of this study thus establish that acetyl-CoA impacts H3K27ac at specific loci, correlating with gene expression, and that expression of cell adhesion genes are driven by acetyl-CoA in part through activation of Ca2+-NFAT signaling.

Differential effects of acute versus chronic dietary fructose consumption on metabolic responses in FVB/N mice.

Increased human consumption of high-fructose corn syrup has been linked to the marked increase in obesity and metabolic syndrome. Previous studies on the rapid effects of a high-fructose diet in mice have largely been confined to the C57BL/6 strains. In the current study, the FVB/N strain of mice that are resistant to diet-induced weight gain were used and fed a control or high-fructose diet for 48 h or for 12 wk. Many of the previously reported changes that occurred upon high-fructose feeding for 48 h in C57BL/6 mice were recapitulated in the FVB/N mice. However, the acute increases in fructolytic and lipogenic gene expression were completely lost during the 12-wk dietary intervention protocol. Furthermore, there was no significant weight gain in FVB/N mice fed a high-fructose diet for 12 wk, despite an overall increase in caloric consumption and an increase in average epididymal adipocyte cell size. These findings may be in part explained by a commensurate increase in energy expenditure and in carbohydrate utilization in high-fructose-fed animals. Overall, these findings demonstrate that FVB/N mice are a suitable model for the study of the effects of dietary intervention on metabolic and molecular parameters. Furthermore, the rapid changes in hepatic gene expression that have been widely reported were not sustained over a longer time course. Compensatory changes in energy expenditure and utilization may be in part responsible for the differences obtained between acute and chronic high-fructose feeding protocols.

Magnetic resonance angiography reveals increased arterial blood supply and tumorigenesis following high fat feeding in a mouse model of triple-negative breast cancer.

Breast cancer is the second most commonly diagnosed malignancy among women globally. Past MRI studies have linked a high animal fat diet (HAFD) to increased mammary cancer risk in the SV40Tag mouse model of triple-negative breast cancer. Here, serial MRI examines tumor progression and measures the arterial blood volume feeding mammary glands in low fat diet (LFD) or HAFD fed mice. Virgin female C3(1)SV40Tag mice (n = 8), weaned at 3 weeks old, were assigned to an LFD (n = 4, 3.7 kcal/g, 17.2% kcal from vegetable oil) or an HAFD (n = 4, 5.3 kcal/g, 60% kcal from lard) group. From ages 8 to 12 weeks, weekly fast spin echo MR images and time-of-flight (TOF) MR angiography of inguinal mammary glands were acquired at 9.4 T. Following in vivo MRI, mice were sacrificed. Inguinal mammary glands were excised and fixed for ex vivo MRI and histology. Tumor, blood, and mammary gland volumes for each time point were measured from manually traced regions of interest; tumors were classified as invasive by histopathology-blinded observers. Our analysis confirmed a strong correlation between total tumor volume and blood volume in the mammary gland. Tumor growth rates from weeks 8-12 were twice as high in HAFD-fed mice (0.42 ± 0.14/week) as in LFD-fed mice (0.21 ± 0.03/week), p < 0.004. Mammary gland blood volume growth rate was 2.2 times higher in HAFD mice (0.29 ± 0.11/week) compared with LFD mice (0.13 ± 0.06/week), p < 0.02. The mammary gland growth rate of HAFD-fed mice (0.071 ± 0.011/week) was 2.7 times larger than that of LFD-fed mice (0.026 ± 0.009/week), p < 0.01. This is the first non-invasive, in vivo MRI study to demonstrate a strong correlation between an HAFD and increased cancer burden and blood volume in mammary cancer without using contrast agents, strengthening the evidence supporting the adverse effects of an HAFD on mammary cancer. These results support the potential future use of TOF angiography to evaluate vasculature of suspicious lesions.

Psychosocial Stress Exposure Disrupts Mammary Gland Development.

Exposure to psychosocial stressors and ensuing stress physiology have been associated with spontaneous invasive mammary tumors in the Sprague-Dawley rat model of human breast cancer. Mammary gland (MG) development is a time when physiologic and environmental exposures influence breast cancer risk. However, the effect of psychosocial stress exposure on MG development remains unknown. Here, in the first comprehensive longitudinal study of MG development in nulliparous female rats (from puberty through young adulthood; 8-25 wks of age), we quantify the spatial gradient of differentiation within the MG of socially stressed (isolated) and control (grouped) rats. We then demonstrate that social isolation increased stress reactivity to everyday stressors, resulting in downregulation of glucocorticoid receptor (GR) expression in the MG epithelium. Surprisingly, given that chemical carcinogens increase MG cancer risk by preventing normal terminal end bud (TEB) differentiation, chronic isolation stress did not alter TEBs. Instead, isolation blunted MG growth and alveolobular differentiation and reduced epithelial cell proliferation in these structures. Social isolation also enhanced corpora luteal progesterone at all ages but reduced estrogenization only in early adulthood, a pattern that precludes modulated ovarian function as a sufficient mechanism for the effects of isolation on MG development. This longitudinal study of natural variation provides an integrated view of MG development and the importance of increased GR activation in nulliparous ductal growth and alveolobular differentiation. Thus, social isolation and its physiological sequelae disrupt MG growth and differentiation and suggest a contribution of stress exposure during puberty and young adulthood to the previously observed increase in invasive MG cancer observed in chronically socially-isolated adult Sprague-Dawley rats.

MRI reveals increased tumorigenesis following high fat feeding in a mouse model of triple-negative breast cancer.

High animal fat consumption is associated with an increase in triple-negative breast cancer (TNBC) risk. Based on previous MRI studies demonstrating the feasibility of detecting very early non-palpable mammary cancers in simian virus 40 large T antigen (SV40TAg) mice, we examined the effect of dietary fat fed from weaning to young adulthood in this model of TNBC. Virgin female C3(1)SV40TAg mice (n = 16) were weaned at 3-4 weeks of age and then fed either a low fat diet (LFD) (n = 8, 3.7 kcal/g; 17.2% kcal from vegetable oil) or a high animal fat diet (HAFD) (n = 8, 5.3 kcal/g; 60% kcal from lard). After 8 weeks on the diet (12 weeks of age), fast spin echo MR images of inguinal mammary glands were acquired at 9.4 T. Following in vivo MRI, mice were sacrificed and inguinal mammary glands were excised and formalin fixed for ex vivo MRI. 3D volume-rendered MR images were then correlated with mammary gland histology to assess the glandular parenchyma and tumor burden. Using in vivo MRI, an average of 3.88 ± 1.03 tumors were detected per HAFD-fed mouse compared with an average of 1.25 ± 1.16 per LFD-fed mouse (p < 0.007). Additionally, the average tumor volume was significantly higher following HAFD feeding (0.53 ± 0.45 mm3 ) compared with LFD feeding (0.20 ± 0.08 mm3 , p < 0.02). Analysis of ex vivo MR and histology images demonstrated that HAFD mouse mammary glands had denser parenchyma, irregular and enlarged ducts, dilated blood vessels, increased white adipose tissue, and increased tumor invasion. MRI and histological studies of the SV40TAg mice demonstrated that HAFD feeding also resulted in higher cancer incidence and larger mammary tumors. Unlike other imaging methods for assessing environmental effects on mammary cancer growth, MRI allows routine serial measurements and reliable detection of small cancers as well as accurate tumor volume measurements and assessment of the three-dimensional distribution of tumors over time.

Nuclear deadenylation/polyadenylation factors regulate 3' processing in response to DNA damage.

We previously showed that mRNA 3' end cleavage reaction in cell extracts is strongly but transiently inhibited under DNA-damaging conditions. The cleavage stimulation factor-50 (CstF-50) has a role in this response, providing a link between transcription-coupled RNA processing and DNA repair. In this study, we show that CstF-50 interacts with nuclear poly(A)-specific ribonuclease (PARN) using in vitro and in extracts of UV-exposed cells. The CstF-50/PARN complex formation has a role in the inhibition of 3' cleavage and activation of deadenylation upon DNA damage. Extending these results, we found that the tumour suppressor BARD1, which is involved in the UV-induced inhibition of 3' cleavage, strongly activates deadenylation by PARN in the presence of CstF-50, and that CstF-50/BARD1 can revert the cap-binding protein-80 (CBP80)-mediated inhibition of PARN activity. We also provide evidence that PARN along with the CstF/BARD1 complex participates in the regulation of endogenous transcripts under DNA-damaging conditions. We speculate that the interplay between polyadenylation, deadenylation and tumour-suppressor factors might prevent the expression of prematurely terminated messengers, contributing to control of gene expression under different cellular conditions.

Adipocyte ACLY Facilitates Dietary Carbohydrate Handling to Maintain Metabolic Homeostasis in Females.

Sugars and refined carbohydrates are major components of the modern diet. ATP-citrate lyase (ACLY) is upregulated in adipocytes in response to carbohydrate consumption and generates acetyl-coenzyme A (CoA) for both lipid synthesis and acetylation reactions. Here, we investigate the role of ACLY in the metabolic and transcriptional responses to carbohydrates in adipocytes and unexpectedly uncover a sexually dimorphic function in maintaining systemic metabolic homeostasis. When fed a high-sucrose diet, AclyFAT-/- females exhibit a lipodystrophy-like phenotype, with minimal fat accumulation, insulin resistance, and hepatic lipid accumulation, whereas AclyFAT-/- males have only mild metabolic phenotypes. We find that ACLY is crucial for nutrient-dependent carbohydrate response element-binding protein (ChREBP) activation in adipocytes and plays a key role, particularly in females, in the storage of newly synthesized fatty acids in adipose tissue. The data indicate that adipocyte ACLY is important in females for the systemic handling of dietary carbohydrates and for the preservation of metabolic homeostasis.

Magnetic resonance spectroscopy detects differential lipid composition in mammary glands on low fat, high animal fat versus high fructose diets.

The effects of consumption of different diets on the fatty acid composition in the mammary glands of SV40 T-antigen (Tag) transgenic mice, a well-established model of human triple-negative breast cancer, were investigated with magnetic resonance spectroscopy and spectroscopic imaging. Female C3(1) SV40 Tag transgenic mice (n = 12) were divided into three groups at 4 weeks of age: low fat diet (LFD), high animal fat diet (HAFD), and high fructose diet (HFruD). MRI scans of mammary glands were acquired with a 9.4 T scanner after 8 weeks on the diet. 1H spectra were acquired using point resolved spectroscopy (PRESS) from two 1 mm3 boxes on each side of inguinal mammary gland with no cancers, lymph nodes, or lymph ducts. High spectral and spatial resolution (HiSS) images were also acquired from nine 1-mm slices. A combination of Gaussian and Lorentzian functions was used to fit the spectra. The percentages of poly-unsaturated fatty acids (PUFA), mono-unsaturated fatty acids (MUFA), and saturated fatty acids (SFA) were calculated from each fitted spectrum. Water and fat peak height images (maps) were generated from HiSS data. The results showed that HAFD mice had significantly lower PUFA than both LFD (p < 0.001) and HFruD (p < 0.01) mice. The mammary lipid quantity calculated from 1H spectra was much larger in HAFD mice than in LFD (p = 0.03) but similar to HFruD mice (p = 0.10). The average fat signal intensity over the mammary glands calculated from HiSS fat maps was ~60% higher in HAFD mice than in LFD (p = 0.04) mice. The mean or median of calculated parameters for the HFruD mice were between those for LFD and HAFD mice. Therefore, PRESS spectroscopy and HiSS MRI demonstrated water and fat composition changes in mammary glands due to a Western diet, which was low in potassium, high in sodium, animal fat, and simple carbohydrates. Measurements of PUFA with MRI could be used to evaluate cancer risk, improve cancer detection and diagnosis, and guide preventative therapy.

Metabolically Activated Adipose Tissue Macrophages Perform Detrimental and Beneficial Functions during Diet-Induced Obesity.

During obesity, adipose tissue macrophages (ATMs) adopt a metabolically activated (MMe) phenotype. However, the functions of MMe macrophages are poorly understood. Here, we combine proteomic and functional methods to demonstrate that, in addition to potentiating inflammation, MMe macrophages promote dead adipocyte clearance through lysosomal exocytosis. We identify NADPH oxidase 2 (NOX2) as a driver of the inflammatory and adipocyte-clearing properties of MMe macrophages and show that, compared to wild-type, Nox2-/- mice exhibit a time-dependent metabolic phenotype during diet-induced obesity. After 8 weeks of high-fat feeding, Nox2-/- mice exhibit attenuated ATM inflammation and mildly improved glucose tolerance. After 16 weeks of high-fat feeding, Nox2-/- mice develop severe insulin resistance, hepatosteatosis, and visceral lipoatrophy characterized by dead adipocyte accumulation and defective ATM lysosomal exocytosis, a phenotype reproduced in myeloid cell-specific Nox2-/- mice. Collectively, our findings suggest that MMe macrophages perform detrimental and beneficial functions whose contribution to metabolic phenotypes during obesity is determined by disease progression.

Insulin regulates liver metabolism in vivo in the absence of hepatic Akt and Foxo1.

Considerable data support the idea that forkhead box O1 (Foxo1) drives the liver transcriptional program during fasting and is then inhibited by thymoma viral proto-oncogene 1 (Akt) after feeding. Here we show that mice with hepatic deletion of Akt1 and Akt2 were glucose intolerant, insulin resistant and defective in their transcriptional response to feeding in the liver. These defects were normalized with concomitant liver-specific deletion of Foxo1. Notably, in the absence of both Akt and Foxo1, mice adapted appropriately to both the fasted and fed state, and insulin suppressed hepatic glucose production normally. A gene expression analysis revealed that deletion of Akt in liver led to the constitutive activation of Foxo1-dependent gene expression, but again, concomitant ablation of Foxo1 restored postprandial regulation, preventing the inhibition of the metabolic response to nutrient intake caused by deletion of Akt. These results are inconsistent with the canonical model of hepatic metabolism in which Akt is an obligate intermediate for proper insulin signaling. Rather, they show that a major role of hepatic Akt is to restrain the activity of Foxo1 and that in the absence of Foxo1, Akt is largely dispensable for insulin- and nutrient-mediated hepatic metabolic regulation in vivo.

Regulation of the histone demethylase JMJD1A by hypoxia-inducible factor 1 alpha enhances hypoxic gene expression and tumor growth.

The hypoxia-inducible transcription factors (HIFs) directly and indirectly mediate cellular adaptation to reduced oxygen tensions. Recent studies have shown that the histone demethylase genes JMJD1A, JMJD2B, and JARID1B are HIF targets, suggesting that HIFs indirectly influence gene expression at the level of histone methylation under hypoxia. In this study, we identify a subset of hypoxia-inducible genes that are dependent on JMJD1A in both renal cell and colon carcinoma cell lines. JMJD1A regulates the expression of adrenomedullin (ADM) and growth and differentiation factor 15 (GDF15) under hypoxia by decreasing promoter histone methylation. In addition, we demonstrate that loss of JMJD1A is sufficient to reduce tumor growth in vivo, demonstrating that histone demethylation plays a significant role in modulating growth within the tumor microenvironment. Thus, hypoxic regulation of JMJD1A acts as a signal amplifier to facilitate hypoxic gene expression, ultimately enhancing tumor growth.