Amphotericin B resistance in Leishmania mexicana: Alterations to sterol metabolism, lipid transport and oxidative stress response

Amphotericin B is increasingly used in treatment of leishmaniasis. Here, fourteen independent lines of Leishmania mexicana and one L. infantum line were selected for resistance to either amphotericin B or the related polyene antimicrobial, nystatin. Sterol profiling revealed that, in each line, the predominant ergostane-type sterol of wild-type cells was replaced by other sterol species. Broadly, two different profiles emerged among the resistant lines. Whole genome sequencing then showed that these distinct profiles were due either to mutations in the sterol methyl transferase (C24SMT) gene locus or the sterol C5 desaturase (C5DS) gene. In three lines an additional deletion of the miltefosine transporter was found. Differences in sensitivity to amphotericin B were apparent, depending on whether cells were grown in HOMEM, supplemented with foetal bovine serum, or a serum free defined medium (DM). These differences appeared to relate to the presence of lipids in the former. Metabolomic analysis after exposure to AmB showed that a large increase in glucose flux via the pentose phosphate pathway preceded cell death in cells sustained in HOMEM but not DM, indicating the oxidative stress was more significantly induced under HOMEM conditions. Several of the lines were tested for ability to infect macrophages and replicate as amastigote forms, alongside their ability to establish infections in mice. While several lines showed reduced virulence, at least one AmB resistant line displayed heightened virulence in mice whilst retaining its resistance phenotype, emphasising the risks of resistance emerging to this critical drug.

The hexokinase ″HKDC1″ interaction with the mitochondria is essential for hepatocellular carcinoma progression

Hepatocellular carcinoma (HCC) is a leading cause of death from cancer malignancies. Recently, hexokinase domain containing 1 (HKDC1), was shown to have significant overexpression in HCC compared to healthy tissue. Using in vitro and in vivo tools, we examined the role of HKDC1 in HCC progression. Importantly, HKDC1 ablation stops HCC progression by promoting metabolic reprogramming by shifting glucose flux away from the TCA cycle. Next, HKDC1 ablation leads to mitochondrial dysfunction resulting in less cellular energy which cannot be compensated by enhanced glucose uptake. And finally, we show that the interaction of HKDC1 with the mitochondria is essential for its role in HCC progression, and without this mitochondrial interaction mitochondrial dysfunction occurs. In sum, HKDC1 is highly expressed in HCC cells compared to normal hepatocytes, therefore targeting HKDC1, specifically its interaction with the mitochondria, reveals a highly selective approach to target cancer cells in HCC.

NIMG-50. DEUTERIUM METABOLIC IMAGING OF THE ALTERNATIVE LENGTHENING OF TELOMERES PATHWAY REPORTS ON TUMOR BURDEN AND PSEUDOPROGRESSION IN LOW-GRADE GLIOMAS

Glioma patient management relies heavily on magnetic resonance imaging (MRI). However, MRI is often inadequate for assessment of tumor burden and pseudoprogression. Non-invasive methods that report on molecular pathways such as telomere maintenance that drive tumor proliferation are needed. Among brain tumors, low-grade astrocytomas (LGAs) use the alternative lengthening of telomeres (ALT) pathway for telomere maintenance. The goal of this study was to identify ALT-linked metabolic alterations that can be exploited for non-invasive magnetic resonance spectroscopy (MRS)-based imaging of LGAs. We examined the patient-derived BT257 model and compared neurospheres that are ALT-dependent (BT257 ALT+) with those in which the ALT pathway has been silenced (BT257 ALT-). Our studies suggest that expression and activity of the rate-limiting glycolytic enzyme phosphofructokinase-1 are significantly higher in BT257 ALT+ neurospheres relative to ALT-, an effect that is associated with elevated glucose flux to lactate. Studies indicate that poly(ADP-ribose) polymerase inhibitors such as niraparib selectively induce telomeric fusion and cell death in ALT-dependent cells. We find that the telomeric fusion-mediated cytotoxicity of niraparib is associated with significantly reduced glycolytic flux in BT257 ALT+ neurospheres. We then examined whether 2H-MRS using [6,6’-2H]-glucose, which is a clinically translatable method of imaging glycolytic flux, can be used to monitor the ALT pathway in vivo. [6,6’-2H]-glucose flux to lactate is elevated in tumor relative to normal brain in mice bearing orthotopic BT257 tumors. Importantly, following treatment of BT257 tumor-bearing mice with niraparib, lactate production from [6,6’-2H]-glucose is significantly reduced at early timepoints when alterations in tumor volume cannot be observed by MRI, pointing to the ability of [6,6’-2H]-glucose to report on pseudoprogression in vivo. Collectively, our studies mechanistically link the ALT pathway with elevated glycolytic flux via phosphofructokinase-1 and identify deuterium metabolic imaging as a novel, non-invasive method of imaging tumor burden and treatment response in LGAs in vivo.

PSII-17 The differential expression pattern of messenger RNA for key hepatic glycogen metabolizing proteins is consistent with hepatic glycogen content in suckling pigs

The effect of developmental age (d) on expression of genes responsible for hepatic glycogen (GLY) synthesis and degradation, glucose flux, and GLY content, was determined in crossbred pigs euthanized (n = 6) at birth (d 0, pre-suckle), 1, 3, 7, 14, and 21 d. Liver GLY content and relative abundance of mRNA (RT-PCR) was determined. The relative content of liver mRNA was determined in 2 experiments, d 0, 1, 3, 7 (Experiment 1) and d 0, 1, 7, 14, 21 (Experiment 2). Within each experiment, data were analyzed using the GLM procedure of SAS. Fisher’s protected LSD procedure was used to separate treatment means. Day 0 (76.0) GLY content (mg/g) decreased (P < 0.01) 82% from d 0 to d 1, increased (P < 0.05) from d 1 (13.8) through d 14 (28.4), and did not differ (P = 0.07) between d 1 and 21. In Experiment 1, mRNA content of GLY synthesis proteins GYG1 and GYS1 was greatest (P < 0.01) at d 3 and 7; and 1 and 3; respectively, whereas mRNA content of GLY degrading proteins PGM1, PGM2, and PGM5 was greatest (P ≤ 0.01) at d 1; d 0; and d 1 and 7; respectively. In Experiment 2, mRNA content of GLY synthesis proteins GBE1 and GYS1 was greatest (P < 0.01) at d 0 and 21; and d 1 and 21; respectively, whereas mRNA content of GLY degrading proteins AGL, PGM2, PGM2L, and PGM5 was greatest (P < 0.01) at d 21; d 0; d 7, 14, and 21; and d 14 and 21; respectively. Glucose transporter SGLT1 mRNA content was greatest (P < 0.01) at d 14 and 21. These findings indicate that the pattern of mRNA content of key hepatic GLY degradation and synthesis proteins was consistent with GLY content of suckling pigs.

Neuroprotective Function of High Glycolytic Activity in Astrocytes: Common Roles in Stroke and Neurodegenerative Diseases

Astrocytes (also, astroglia) consume huge amounts of glucose and produce lactate regardless of sufficient oxygen availability, indicating a high capacity for aerobic glycolysis. Glycolysis in astrocytes is activated in accordance with neuronal excitation and leads to increases in the release of lactate from astrocytes. Although the fate of this lactate remains somewhat controversial, it is believed to fuel neurons as an energy substrate. Besides providing lactate, astrocytic glycolysis plays an important role in neuroprotection. Among the minor pathways of glucose metabolism, glucose flux to the pentose-phosphate pathway (PPP), a major shunt pathway of glycolysis, is attracting research interest. In fact, PPP activity in astrocytes is five to seven times higher than that in neurons. The astrocytic PPP plays a key role in protecting neurons against oxidative stress by providing neurons with a reduced form of glutathione, which is necessary to eliminate reactive oxygen species. Therefore, enhancing astrocytic glycolysis might promote neuronal protection during acute ischemic stroke. Contrariwise, the dysfunction of astrocytic glycolysis and the PPP have been implicated in the pathogenesis of various neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s disease, and amyotrophic lateral sclerosis, since mitochondrial dysfunction and oxidative stress trigger and accelerate disease progression.

Tribbles Homolog 3 Mediates the Development and Progression of Diabetic Retinopathy

The current understanding of molecular pathogenesis of diabetic retinopathy does not provide a mechanistic link between early molecular changes and the subsequent progression of the disease. In this study, we found that human diabetic retinas overexpressed TRIB3 and investigated the role of TRIB3 in diabetic retinal pathobiology in mice. We discovered that TRIB3 controlled major molecular events in early diabetic retinas via HIF1α-mediated regulation of retinal glucose flux, reprograming cellular metabolism, and governing inflammatory gene expression. These early molecular events further defined the development of neurovascular deficit observed in mice with diabetic retinopathy. TRIB3 ablation in STZ-induced mouse model led to significant RGC survival and functional restoration accompanied by a dramatic reduction in pericyte loss and acellular capillary formation. Under hypoxic conditions, TRIB3 contributed to advanced proliferative stages by significant upregulation of GFAP and VEGF expression, thus controlling gliosis and aberrant vascularization in OIR mouse retinas. Overall, our data reveal that TRIB3 is a master regulator of diabetic retinal pathophysiology that may accelerate the onset and progression of diabetic retinopathy to proliferative stages in humans and present TRIB3 as a potentially novel therapeutic target for diabetic retinopathy.

Tribbles Homolog 3 Mediates the Development and Progression of Diabetic Retinopathy

The current understanding of molecular pathogenesis of diabetic retinopathy does not provide a mechanistic link between early molecular changes and the subsequent progression of the disease. In this study, we found that human diabetic retinas overexpressed TRIB3 and investigated the role of TRIB3 in diabetic retinal pathobiology in mice. We discovered that TRIB3 controlled major molecular events in early diabetic retinas via HIF1α-mediated regulation of retinal glucose flux, reprograming cellular metabolism, and governing inflammatory gene expression. These early molecular events further defined the development of neurovascular deficit observed in mice with diabetic retinopathy. TRIB3 ablation in STZ-induced mouse model led to significant RGC survival and functional restoration accompanied by a dramatic reduction in pericyte loss and acellular capillary formation. Under hypoxic conditions, TRIB3 contributed to advanced proliferative stages by significant upregulation of GFAP and VEGF expression, thus controlling gliosis and aberrant vascularization in OIR mouse retinas. Overall, our data reveal that TRIB3 is a master regulator of diabetic retinal pathophysiology that may accelerate the onset and progression of diabetic retinopathy to proliferative stages in humans and present TRIB3 as a potentially novel therapeutic target for diabetic retinopathy.

Inhibition of eIF5A hypusination reprogrammes metabolism and glucose handling in mouse kidney

Inhibition of the eukaryotic initiation factor 5A activation by the spermidine analogue GC7 has been shown to protect proximal cells and whole kidneys against an acute episode of ischaemia. The highlighted mechanism involves a metabolic switch from oxidative phosphorylation toward glycolysis allowing cells to be transiently independent of oxygen supply. Here we show that GC7 decreases protein expression of the renal GLUT1 glucose transporter leading to a decrease in transcellular glucose flux. At the same time, GC7 modifies the native energy source of the proximal cells from glutamine toward glucose use. Thus, GC7 acutely and reversibly reprogrammes function and metabolism of kidney cells to make glucose its single substrate, and thus allowing cells to be oxygen independent through anaerobic glycolysis. The physiological consequences are an increase in the renal excretion of glucose and lactate reflecting a decrease in glucose reabsorption and an increased glycolysis. Such a reversible reprogramming of glucose handling and oxygen dependence of kidney cells by GC7 represents a pharmacological opportunity in ischaemic as well as hyperglycaemia-associated pathologies from renal origin.

BIMG-02. IMAGING IMMORTALITY: TERT EXPRESSION ALTERS GLUCOSE METABOLISM IN LOW-GRADE GLIOMAS IN A MANNER THAT CAN BE LEVERAGED FOR NONINVASIVE METABOLIC IMAGING

Telomerase reverse transcriptase (TERT) is essential for tumor immortality and uncontrolled proliferation, including in low-grade oligodendrogliomas (LGOGs). Since it is silenced in somatic cells, TERT is also a therapeutic target. Non-invasive imaging of TERT can differentiate tumor from normal brain or lesions such as gliosis and allow assessment of response to therapy. The goal of this study was to identify magnetic resonance spectroscopy (MRS)-detectable metabolic alterations associated with TERT that can be leveraged for noninvasive imaging in LGOGs. We examined patient-derived BT54 neurospheres in which TERT expression was silenced by RNA interference. 1H-MRS showed that steady-state levels of NAD(P)/H, glutathione, aspartate and AXP were elevated in BT54TERT+ neurospheres relative to BT54TERT-. Glucose flux through the pentose phosphate pathway (PPP) is essential for generating NADPH, which maintains glutathione homeostasis. 13C-MRS confirmed that [2-13C]-glucose flux through the PPP was elevated in BT54TERT+ neurospheres relative to BT54TERT-, an effect associated with higher activity of the PPP enzyme glucose-6-phosphate dehydrogenase (G6PDH). Hyperpolarized 13C-MRS is a method of increasing the signal to noise ratio of 13C-MRS such that it can monitor metabolic fluxes noninvasively in cells, animals and patients. Consistent with elevated PPP flux and G6PDH activity, hyperpolarized [U-13C]-glucose metabolism via the PPP to 6-phosphogluconate (6-PG) was elevated in BT54TERT+ neurospheres relative to BT54TERT-. Importantly, examination of an additional patient-derived LGOG model, the SF10417 model which readily forms orthotopic tumor xenografts in rats, showed that 6-PG production from hyperpolarized [U-13C]-glucose demarcated tumor from normal brain. Furthermore, LGOG patient biopsies had elevated NAD(P)/H, glutathione, aspartate, AXP and G6PDH activity relative to gliosis biopsies, confirming the clinical validity of our observations. Collectively, we have identified a metabolic signature of TERT expression that can be leveraged via hyperpolarized [U-13C]-glucose to improve diagnosis and treatment response monitoring for LGOG patients.