HIF1α-dependent uncoupling of glycolysis suppresses tumor cell proliferation.

Hypoxia-inducible factor-1α (HIF1α) attenuates mitochondrial activity while promoting glycolysis. However, lower glycolysis is compromised in human clear cell renal cell carcinomas, in which HIF1α acts as a tumor suppressor by inhibiting cell-autonomous proliferation. Here, we find that, unexpectedly, HIF1α suppresses lower glycolysis after the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) step, leading to reduced lactate secretion in different tumor cell types when cells encounter a limited pyruvate supply such as that typically found in the tumor microenvironment in vivo. This is because HIF1α-dependent attenuation of mitochondrial oxygen consumption increases the NADH/NAD+ ratio that suppresses the activity of the NADH-sensitive GAPDH glycolytic enzyme. This is manifested when pyruvate supply is limited, since pyruvate acts as an electron acceptor that prevents the increment of the NADH/NAD+ ratio. Furthermore, this anti-glycolytic function provides a molecular basis to explain how HIF1α can suppress tumor cell proliferation by increasing the NADH/NAD+ ratio.

A palmitate-rich metastatic niche enables metastasis growth via p65 acetylation resulting in pro-metastatic NF-κB signaling.

Metabolic rewiring is often considered an adaptive pressure limiting metastasis formation; however, some nutrients available at distant organs may inherently promote metastatic growth. We find that the lung and liver are lipid-rich environments. Moreover, we observe that pre-metastatic niche formation increases palmitate availability only in the lung, whereas a high-fat diet increases it in both organs. In line with this, targeting palmitate processing inhibits breast cancer-derived lung metastasis formation. Mechanistically, breast cancer cells use palmitate to synthesize acetyl-CoA in a carnitine palmitoyltransferase 1a-dependent manner. Concomitantly, lysine acetyltransferase 2a expression is promoted by palmitate, linking the available acetyl-CoA to the acetylation of the nuclear factor-kappaB subunit p65. Deletion of lysine acetyltransferase 2a or carnitine palmitoyltransferase 1a reduces metastasis formation in lean and high-fat diet mice, and lung and liver metastases from patients with breast cancer show coexpression of both proteins. In conclusion, palmitate-rich environments foster metastases growth by increasing p65 acetylation, resulting in a pro-metastatic nuclear factor-kappaB signaling.

PHGDH heterogeneity potentiates cancer cell dissemination and metastasis.

Cancer metastasis requires the transient activation of cellular programs enabling dissemination and seeding in distant organs1. Genetic, transcriptional and translational heterogeneity contributes to this dynamic process2,3. Metabolic heterogeneity has also been observed4, yet its role in cancer progression is less explored. Here we find that the loss of phosphoglycerate dehydrogenase (PHGDH) potentiates metastatic dissemination. Specifically, we find that heterogeneous or low PHGDH expression in primary tumours of patients with breast cancer is associated with decreased metastasis-free survival time. In mice, circulating tumour cells and early metastatic lesions are enriched with Phgdhlow cancer cells, and silencing Phgdh in primary tumours increases metastasis formation. Mechanistically, Phgdh interacts with the glycolytic enzyme phosphofructokinase, and the loss of this interaction activates the hexosamine-sialic acid pathway, which provides precursors for protein glycosylation. As a consequence, aberrant protein glycosylation occurs, including increased sialylation of integrin αvß3, which potentiates cell migration and invasion. Inhibition of sialylation counteracts the metastatic ability of Phgdhlow cancer cells. In conclusion, although the catalytic activity of PHGDH supports cancer cell proliferation, low PHGDH protein expression non-catalytically potentiates cancer dissemination and metastasis formation. Thus, the presence of PHDGH heterogeneity in primary tumours could be considered a sign of tumour aggressiveness.

Palmitic acid: Enabling the tumor's nerves.

Diet can influence tumor aggressiveness. Recently in Nature, a study by Pascual et al. provided evidence that dietary palmitic acid induces an epigenetic memory by modulating particular histone methylation marks in cancer cells. This allows cancer cells to activate extracellular matrix secretion from Schwann cells of the tumor microenvironment, which ultimately potentiates metastasis initiation.

Deficiency of malate-aspartate shuttle component SLC25A12 induces pulmonary metastasis.

BACKGROUND: Aspartate biosynthesis and its delivery to the cytosol can be crucial for tumor growth in vivo. However, the impact of intracellular aspartate levels on metastasis has not been studied. We previously described that loss-of-aspartate glutamate carrier 1 (SLC25A12 or AGC1), an important component of the malate-aspartate shuttle, impairs cytosolic aspartate levels, NAD+/NADH ratio, mitochondrial respiration, and tumor growth. Here, we report the impact of AGC1-knockdown on metastasis. RESULTS: Low AGC1 expression correlates with worse patient prognosis in many cancers. AGC1-knockdown in mouse lung carcinoma and melanoma cell lines leads to increased pulmonary metastasis following subcutaneous or intravenous injections, respectively. On the other hand, conventional in vitro metastasis assays show no indication of increased metastasis capacity of AGC1-knockdown cells. CONCLUSION: This study highlights that certain branches of metabolism impact tumor growth and tumor metastasis differently. In addition, it also argues that commonly known metastasis indicators, including EMT genes, cell migration, or colony formation, do not always reflect metastatic capacity in vivo.

Detection of twin pregnancies in ewes by pregnancy-associated glycoprotein assay and transabdominal ultrasonography.

The objectives of the present study were to evaluate and compare the accuracy of transabdominal ultrasonography and pregnancy-associated glycoprotein (PAG) assay in the diagnosis of twin pregnancies in ewes and to evaluate the utility of the PAG assay for predicting fetal gender in singleton pregnancies. The animals in the study consisted of 179 pregnant ewes. The number of fetuses in the ewe was determined using transabdominal ultrasonography between days 40 and 60 (on days 40, 45, 50, 55, and 60). Blood samples were collected from all the ewes on the same day to determine the PAG concentrations. The results found were highly sensitive for the detection of twin pregnancies by transabdominal ultrasonography. The accuracy of transabdominal ultrasonography in detecting twin pregnancies was found to be higher on day 60 than on other days (P < 0.05). The sensitivities of PAG assay in detecting twin pregnancies on days 40, 45, 50, 55, and 60 were 91.67%, 66.67%, 81.82%, 88.89%, and 33.33%, respectively. The accuracies of the PAG assay in detecting twin pregnancies on days 40, 45, and 50 were found to be statistically significant higher than other days (P < 0.05). The PAG assay had low sensitivity and specificity for predicting fetal gender. It was concluded that twin pregnancies in ewes can be diagnosed with high accuracy using transabdominal ultrasonography on day 60 of pregnancy and as well as using the PAG assay during the early days of pregnancy (on days 40, 45, and 50). The PAG assay is not useful for predicting fetal gender.

Maintaining cytosolic aspartate levels is a major function of the TCA cycle in proliferating cells.

Cancer cells rely on glutamine to fuel mitochondria, however it remains unclear whether this is needed for bioenergetic or biosynthetic pathways. Our study suggests that an essential function of mitochondrial glutamine metabolism is to provide aspartate to the cytosol where it can be used for nucleotide and protein synthesis.

Cytosolic Aspartate Availability Determines Cell Survival When Glutamine Is Limiting.

Mitochondrial function is important for aspartate biosynthesis in proliferating cells. Here, we show that mitochondrial aspartate export via the aspartate-glutamate carrier 1 (AGC1) supports cell proliferation and cellular redox homeostasis. Insufficient cytosolic aspartate delivery leads to cell death when TCA cycle carbon is reduced following glutamine withdrawal and/or glutaminase inhibition. Moreover, loss of AGC1 reduces allograft tumor growth that is further compromised by treatment with the glutaminase inhibitor CB-839. Together, these findings argue that mitochondrial aspartate export sustains cell survival in low-glutamine environments and AGC1 inhibition can synergize with glutaminase inhibition to limit tumor growth.

Transgenic overexpression of VEGF-C induces weight gain and insulin resistance in mice.

Obesity comprises great risks for human health, contributing to the development of other diseases such as metabolic syndrome, type 2 diabetes and cardiovascular disease. Previously, obese patients were found to have elevated serum levels of VEGF-C, which correlated with worsening of lipid parameters. We recently identified that neutralization of VEGF-C and -D in the subcutaneous adipose tissue during the development of obesity improves metabolic parameters and insulin sensitivity in mice. To test the hypothesis that VEGF-C plays a role in the promotion of the metabolic disease, we used K14-VEGF-C mice that overexpress human VEGF-C under control of the keratin-14 promoter in the skin and monitored metabolic parameters over time. K14-VEGF-C mice had high levels of VEGF-C in the subcutaneous adipose tissue and gained more weight than wildtype littermates, became insulin resistant and had increased ectopic lipid accumulation at 20 weeks of age on regular mouse chow. The metabolic differences persisted under high-fat diet induced obesity. These results indicate that elevated VEGF-C levels contribute to metabolic deterioration and the development of insulin resistance, and that blockade of VEGF-C in obesity represents a suitable approach to alleviate the development of insulin resistance.

The effects of repeated filling cystometries on cystometric variables in spinal cord-injured patients with overactive detrusor, who utilize different type of urine drainage methods.

STUDY DESIGN: Cross-sectional study. OBJECTIVES: Our aim was to compare the effects of repeated cystometric measurements in spinal cord injury (SCI) patients with neurogenic detrusor overactivity (NDO) who use indwelling catheters (IDC) or intermittent catheterization (IC). SETTING: Turkey. METHODS: A total of 20 SCI patients with NDO, 9 patients on IC and 11 on IDC for at least two consecutive months were included. After emptying the bladder, first involuntary detrusor contraction volume (1stIDCV), cystometric bladder capacity (CC), bladder compliance and maximum detrusor pressure (MPdet) were assessed by filling it with sterile physiological saline at room temperature at a continuous rate of 30 ml min(-1). The bladder was re-emptied after the process and a second filling cystometry was performed in the same way. RESULTS: When all study population were taken into account, 1stIDCV and CC measures were significantly increased in the second cystometry compared with the first cystometry (P=0.001 and P=0.022, respectively), whereas there was no statistically significant difference on bladder compliance and MPdet measures between the first and the repeated cystometry. There was no statistically significant difference on 1stIDCV, CC and bladder compliance measures between the first and the repeated cystometries for IC group, whereas there was statistically significant increase on these measures in the IDC group (P=0.003, P=0.008 and P=0.022, respectively). In addition there was no statistically significant difference on MP(det) measures between the first and the repeated cystometries for both the urine drainage methods. When IC and IDC groups were compared according to mean values of differences in 1stIDCV, CC and bladder compliance measures between the two cystometries, the IDC group had a statistically significant increase in all parameters when compared with the IC group in the second cystometry performed (P=0.001, P=0.003 and P=0.048, respectively). CONCLUSION: Repeated cystometric measurements in SCI patients with NDO lead to an increase in 1stIDCV and CC. However, when the type of urine drainage method is taken into account, although repeated filling cystometry leads to an increase in 1stIDCV, MCC and bladder compliance in patients with IDC, it does not cause a difference in patients on IC.

Blockade of VEGF-C and VEGF-D modulates adipose tissue inflammation and improves metabolic parameters under high-fat diet.

OBJECTIVE: Elevated serum levels of the lymphangiogenic factors VEGF-C and -D have been observed in obese individuals but their relevance for the metabolic syndrome has remained unknown. METHODS: K14-VEGFR-3-Ig (sR3) mice that constitutively express soluble-VEGFR-3-Ig in the skin, scavenging VEGF-C and -D, and wildtype (WT) mice were fed either chow or high-fat diet for 20 weeks. To assess the effect of VEGFR-3 blockage on adipose tissue growth and insulin sensitivity, we evaluated weight gain, adipocyte size and hepatic lipid accumulation. These results were complemented with insulin tolerance tests, FACS analysis of adipose tissue macrophages, in vitro 3T3-L1 differentiation assays and in vivo blocking antibody treatment experiments. RESULTS: We show here that sR3 mice are protected from obesity-induced insulin resistance and hepatic lipid accumulation. This protection is associated with enhanced subcutaneous adipose tissue hyperplasia and an increased number of alternatively-activated (M2) macrophages in adipose tissue. We also show that VEGF-C and -D are chemotactic for murine macrophages and that this effect is mediated by VEGFR-3, which is upregulated on M1 polarized macrophages. Systemic antibody blockage of VEGFR-3 in db/db mice reduces adipose tissue macrophage infiltration and hepatic lipid accumulation, and improves insulin sensitivity. CONCLUSIONS: These results reveal an unanticipated role of the lymphangiogenic factors VEGF-C and -D in the mediation of metabolic syndrome-associated adipose tissue inflammation. Blockage of these lymphangiogenic factors might constitute a new therapeutic strategy for the prevention of obesity-associated insulin resistance.

Endometrial echotexture parameters in Turkish Saanen Goats (Akkeci) during oestrus and early pregnancy.

This experiment was conducted to evaluate endometrial echotexture in oestrus and early pregnancy and its association with ovarian hormones and foetal count in goats. Akkeci goats (Saanen×Kilis crossbreed, n=40) were randomly divided into two groups. Ten does (NAT) were mated on natural oestrus and 30 does (SYN) were subjected to synchronisation-prior to mating. The uterus was scanned on the days of sponge insertion (d -14), sponge removal (d -2) and mating (d 0) as well as 17 (d 17) and 30 (d 30) days after mating. Mean gray level (MGL), homogeneity (HOM) and contrast (CON) values were calculated. Blood samples were collected on days ultrasonography was performed. Data were analyzed by Chi-square, ANOVA, regression tests. HOM value reached the highest level on the mating day and then continuously decreased (P<0.0001). Overall, HOM values were greater for SYN does than for NAT does after mating. CON values were virtually stable during the experimental period. MGL value fluctuated during the breeding period (P<0.03) at a similar fashion in NAT and SYN does. Foetal count was not correlated with plasma hormones and echotexture parameters. Plasma progesterone concentration was correlated with echotexture parameters (r=-0.28 for HOM; r=0.29 for CON; r=0.25 for MGL; P<0.05 for all) during post-mating. In conclusion, echotexture parameters changed during the breeding period, in association with plasma progesterone concentration. Future studies should test if the echotextural changes during embryonic fixation days can be used as a marker for early detection of pregnancy in does.

Liposomes prepared dynamically by interactions between bile salt and phospholipid molecules.

Quasi-elastic light scattering (QELS) studies showed that vesicles can spontaneously form from sodium glycocholate-egg phosphatidylcholine mixed micelles upon dilution with physiological saline buffer (pH 7.5). A water-soluble drug, cytosine arabinoside, did not affect the transition pattern. Transitions were also obtained when dilutions were performed using diluted serum solutions (5% and 10% serum in buffer). However, if intermicellar bile salt monomer concentration (imc) was kept constant in diluent media, the transition was completely suppressed. Approximately 10% of the cytosine arabinoside was trapped inside vesicles which were formed in buffer, although this value decreased to approx. 2.5% when diluted serum was used. These results suggest that the mixed micelle to vesicle transition can be an alternative means to sonication in loading drugs into in vitro vesicles. A good correlation was found between the transmission electron microscopy (TEM) diameter and the mean hydrodynamic diameter obtained by QELS.

Toxicity and clearance of a combination of liposome-encapsulated ganciclovir and trifluridine.

New Zealand albino rabbits were given intravitreal injections of liposome-encapsulated ganciclovir and trifluridine. Preoperatively and postoperatively, the eyes were evaluated by indirect ophthalmoscopy at different time intervals up to 14 days after injection. At intervals up to 14 days postinjection, the animals were sacrificed, the eyes enucleated, vitreous antiviral levels determined in one group of eyes, and histopathological examination conducted in the other group. The results of this study demonstrated prolonged intravitreal drug levels above the mean inhibitory dose for many strains of virus belonging to the herpes simplex virus family. No evidence of gross retinal toxicity was found by clinical or light microscopic examination of the treated eyes.

Intravitreal liposome-encapsulated trifluorothymidine in a rabbit model.

Two groups of albino rabbits received an intravitreal injection of liposome-encapsulated trifluorothymidine. One group underwent a clearance study using high-performance liquid chromatography. The results of this study demonstrated a prolonged vitreal drug level within the range of ID50 for many strains of herpesvirus and human cytomegalovirus (CMV) at 28 days after injection. The eyes of another group were evaluated with preoperative and postoperative indirect ophthalmoscopy, electroretinography, and histologic examination. No retinal toxicity was found.

Intravitreal injection of liposome-encapsulated ganciclovir in a rabbit model.

The authors determined the intravitreal clearance of liposome-encapsulated ganciclovir. Liposome-encapsulated ganciclovir (84.1 micrograms/0.1 ml) was injected into the vitreous cavity of New Zealand rabbits, which were killed at 24 hours and 7, 14, and 28 days after injection. Total ganciclovir concentrations in the vitreous, up to 28 days, were higher than ID50 (50% inhibitory dose) for different clinical and laboratory strains of viruses belonging to the herpes simplex family.