Non-oxidative Pathway Research Articles

TKTL1 expression and its downregulation is implicated in cell proliferation inhibition and cell cycle arrest in esophageal squamous cell carcinoma.

It is well known that tumor cells mainly depend on the nonoxidative pathway of the pentose phosphate pathway (PPP), and transketolase-like 1 (TKTL1), a kind of crucial metabolism enzyme, participates in the regulation of PPP; notably, overwhelming evidence has demonstrated that TKTL1 plays pivotal roles in the development and progression of multiple tumors. However, there were no reports about the role of TKTL1 in esophageal squamous cell carcinoma (ESCC). Here, we investigated TKTL1 expression and preliminarily elucidated its underlying biological functions in ESCC. We found that TKTL1 exhibited the high expression in ESCC tissues and cells, and the survival rate of patients with negative TKTL1 expression was significantly higher than that of patients with positive TKTL1 staining (P < 0.05). Additionally, significant correlations of TKTL1 expression with histologic grade, clinical stage, and lymph node metastasis were found (P < 0.05). Subsequently, TKTL1 small interfering RNA (siRNA) significantly reduced TKTL1 messenger RNA (mRNA), and protein levels companied with the marked reduce of total transketolase activity but did not affect TKT and TKTL1 mRNA level. More importantly, TKTL1 siRNA obviously induced cell cycle arrest in G0/G1 phase and suppressed cell proliferation in vitro and in vivo coupled with the reduced cyclin D1 and cdk4 levels as well as decrease of Ki-67 proliferation index in EC1 cells. Taken altogether, our results suggest that TKTL1 as a key prognostic factor may be a novel target for therapy of the patients with ESCC.

Kocuria gwangalliensis strain SJ2에서 유래된 D-xylulose kinase 유전자의 클로닝과 특성 연구

D-Xylulose는 nonoxidative pentose phosphate 경로를 통해 glycolysis 과정으로 들어가기 전에 D-xylulose kinase에 의해서 D-xylulose-5-phosphate로 인산화 된다. K. gwangalliensis strain SJ2로부터 D-xylulose kinase (XK)를 암호화하는 유전자는 E. coli를 이용하여 서열분석 및 발현 하였으며, XK 유전자의 염기서열 1,419 bp로 구성되어 있으며 463개의 아미노산 잔기를 암호화하고 있다. 분석결과를 통해 XK 유전자가 진화과정 동안 잘 보존되었음을 보여 주었다. XK 유전자의 발현을 위해 pCold-II 발현 벡터에 클로닝 하였으며 클로닝 된 플라스미드는 E. coli strain BL21 (DE3)에 형질전환 하여 IPTG를 이용해 발현을 유도하였다. 재조합 된 XK 단백질의 크기는 약 48 kDa이었다. 이 발현된 단백질은 affinity chromatography를 이용하여 정제하였으며 D-xylulose kinase에 따른 enzymatic activity를 분석하였다. D-xylulose와 ATP로 실행한 XK enzyme kinetic 연구는 각각 250&#xB1;20 &#x3BC;M과 1,300&#xB1;50 &#x3BC;M의 Km value를 보였다. 본 연구를 통해 얻어진 결과는 분자적 수준에서 D-xylulose kinase의 특성연구의 보다 넓은 지식적 기초를 제공할 것으로 사료된다. D-Xylulose is phosphorylated to D-xylulose-5-phosphate by D-xylulose kinase before it enters glycolysis via the nonoxidative pentose phosphate pathway. A gene encoding a novel D-xylulose kinase (XK) from K. gwangalliensis strain SJ2 was sequenced and expressed in E. coli. The sequence of the isolated XK gene was 1,419 bp, encoding 472 amino acids. The XK protein was more closely related to the Arthrobacter phenanthrenivorans XK than to the Bifidobacterium catenulatum one, as reflected in the sequence identity (54.9% vs. 38.7%). The XK gene was subcloned into the pCold-II expression vector. The resulting plasmid was transformed into E. coli strain BL21 (DE3) cells and the expression of the recombinant XK protein was induced by the addition of IPTG. The resulting protein was expressed as a fusion protein of approximately 48 kDa containing a N-terminal six-histidine extension that was derived from the expression vector. The expressed protein was homogenized by affinity chromatography and showed enzymatic activity corresponding to D-xylulose kinase. XK enzyme kinetic studies with D-xylulose and ATP showed a Km of 250&#xB1;20 &#x3BC;M and 1,300&#xB1;50 &#x3BC;M, respectively. The results obtained from this study will provide a wider knowledge base for the characterization of D-xylulose kinase at the molecular level.

In vitro expansion of U87-MG human glioblastoma cells under hypoxic conditions affects glucose metabolism and subsequent in vivo growth.

Hypoxia is a characteristic feature of solid tumors leading to the over expression of hypoxia-inducible factor (HIF)-1α protein and therefore to a specific cellular behavior. However, even though the oxygen tension in tumors is low (<5 %), most of the cell lines used in cancer studies are grown under 21 % oxygen tension. This work focuses on the impact of oxygen conditions during in vitro cell culture on glucose metabolism using 1-(13)C-glucose. Growing U87-MG glioma cells under hypoxic conditions leads to a two- to threefold reduction of labeled glutamine and an accumulation of fructose. However, under both hypoxic and normoxic conditions, glucose is used for de novo synthesis of pyrimidine since the (13)C label is found both in the uracil and ribose moieties. Labeling of the ribose ring demonstrates that U87-MG glioma cells use the reversible branch of the non-oxidative pentose phosphate pathway. Interestingly, stereotactic implantation of U87-MG cells grown under normoxia or mild hypoxia within the striatum of nude mice led to differential growth; the cells grown under hypoxia retaining an imprint of the oxygen adaptation as their development is then slowed down.

How exercise may amend metabolic disturbances in diabetic cardiomyopathy.

Significance: Over-nutrition and sedentary lifestyle has led to a worldwide increase in obesity, insulin resistance, and type 2 diabetes (T2D) associated with an increased risk of development of cardiovascular disorders. Diabetic cardiomyopathy, independent of hypertension or coronary disease, is induced by a range of systemic changes and may through multiple processes result in functional and structural cardiac derangements. The pathogenesis of this cardiomyopathy is complex and multifactorial, and it will eventually lead to reduced cardiac working capacity and increased susceptibility to ischemic injury. Recent Advances: Metabolic disturbances such as altered lipid handling and substrate utilization, decreased mechanical efficiency, mitochondrial dysfunction, disturbances in nonoxidative glucose pathways, and increased oxidative stress are hallmarks of diabetic cardiomyopathy. Interestingly, several of these disturbances are found to precede the development of cardiac dysfunction. Critical Issues: Exercise training is effective in the prevention and treatment of obesity and T2D. In addition to its beneficial influence on diabetes/obesity-related systemic changes, it may also amend many of the metabolic disturbances characterizing the diabetic myocardium. These changes are due to both indirect effects, exercise-mediated systemic changes, and direct effects originating from the high contractile activity of the heart during physical training. Future Directions: Revealing the molecular mechanisms behind the beneficial effects of exercise training is of considerable scientific value to generate evidence-based therapy and in the development of new treatment strategies. Antioxid. Redox Signal. 22, 1587–1605.

Time and Demand are Two Critical Dimensions of Immunometabolism: The Process of Macrophage Activation and the Pentose Phosphate Pathway.

A process is a function of time; in immunometabolism, this is reflected by the stepwise adaptation of metabolism to sustain the bio-energetic demand of an immune-response in its various states and shades. This perspective article starts by presenting an early attempt to investigate the physiology of inflammation, in order to illustrate one of the basic concepts of immunometabolism, wherein an adapted metabolism of infiltrating immune cells affects tissue function and inflammation. We then focus on the process of macrophage activation and aim to delineate the factor time within the current molecular context of metabolic-rewiring important for adapting primary carbohydrate metabolism. In the last section, we will provide information on how the pentose phosphate pathway may be of importance to provide both nucleotide precursors and redox-equivalents, and speculate how carbon-scrambling events in the non-oxidative pentose phosphate pathway might be regulated within cells by demand. We conclude that the adapted metabolism of inflammation is specific in respect to the effector-function and appears as a well-orchestrated event, dynamic by nature, and based on a functional interplay of signaling- and metabolic-pathways.

The role of fat and alcohol in acute pancreatitis: A dangerous liaison.

Excessive alcohol consumption is a major trigger for severe acute pancreatitis which may lead to multi-organ dysfunction and premature death of the individual. Hyperlipidaemia is a risk factor for both acute and chronic pancreatitis and the role of fatty acids in mediating damage has received increasing attention in recent years. In the pancreas ethanol is metabolised by both oxidative and non-oxidative pathways. The latter, predominant route generates fatty acid ethyl esters (FAEEs) from fatty acid substrates via the action of diverse enzymes called FAEE synthases, including carboxylester lipase an enzyme synthesized and secreted by the acinar cells. Inhibition of the oxidative pathway promotes formation of FAEEs which induce sustained elevations of cytosolic calcium leading to inhibition of mitochondrial function, loss of ATP and necrosis of isolated pancreatic acinar cells. Furthermore, FAEEs undergo hydrolysis in the mitochondria releasing free fatty acids that exert toxic effects. Our recent work has shown that pharmacological inhibition of carboxylester lipase ameliorated detrimental effects of non-oxidative ethanol metabolism in isolated pancreatic acinar cells invitro and in a new invivo experimental model of alcoholic acute pancreatitis, revealing a specific enzyme target for ethanol-induced injury. Strategies that prevent FAEE synthesis, protect mitochondria, reduce calcium overload or sustain calcium homeostasis by ATP provision may provide promising therapeutic avenues for the treatment of alcoholic acute pancreatitis.

The coupling of glycolysis and the Rubisco-based pathway through the non-oxidative pentose phosphate pathway to achieve low carbon dioxide emission fermentation

In this study, Rubisco-based engineered Escherichia coli, containing two heterologous enzymes of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and phosphoribulokinase (PrkA), has been shown to be capable of the in situ recycling of carbon dioxide (CO2) during glycolysis. Two alternative approaches have been proposed to further enhance the carbon flow from glycolysis to a Rubisco-based pathway through the non-oxidative pentose phosphate pathway (NOPPP). The first is achieved by elevating the expression of transketolase I (TktA) and the second by blocking the native oxidation–decarboxylation reaction of E. coli by deleting the zwf gene from the chromosome (designated as JB/pTA and MZB, respectively). Decreases in the CO2 yield and the CO2 evolution per unit mole of ethanol production by at least 81% and 40% are observed. It is demonstrated in this study that the production of one mole of ethanol using E. coli strain MZB, the upper limit of CO2 emission is 0.052mol.

Gain of glucose-independent growth upon metastasis of breast cancer cells to the brain.

Breast cancer brain metastasis is resistant to therapy and a particularly poor prognostic feature in patient survival. Altered metabolism is a common feature of cancer cells, but little is known as to what metabolic changes benefit breast cancer brain metastases. We found that brain metastatic breast cancer cells evolved the ability to survive and proliferate independent of glucose due to enhanced gluconeogenesis and oxidations of glutamine and branched chain amino acids, which together sustain the nonoxidative pentose pathway for purine synthesis. Silencing expression of fructose-1,6-bisphosphatases (FBP) in brain metastatic cells reduced their viability and improved the survival of metastasis-bearing immunocompetent hosts. Clinically, we showed that brain metastases from human breast cancer patients expressed higher levels of FBP and glycogen than the corresponding primary tumors. Together, our findings identify a critical metabolic condition required to sustain brain metastasis and suggest that targeting gluconeogenesis may help eradicate this deadly feature in advanced breast cancer patients.

Ribose-5-phosphate isomerase A regulates hepatocarcinogenesis via PP2A and ERK signaling.

The deregulated nonoxidative pentose phosphate pathway (PPP) is known to promote oncogenesis, but the molecular mechanism remains unknown. Here, we report that human ribose-5-phosphate isomerase A (RPIA) plays a role in human hepatocellular carcinoma (HCC). A significant increase in RPIA expression was detected both in tumor biopsies of HCC patients and in a liver cancer tissue array. Importantly, the clinicopathological analysis indicated that RPIA mRNA levels were highly correlated with clinical stage, grade, tumor size, types, invasion and alpha-fetoprotein levels in the HCC patients. In addition, we demonstrated that the ability of RPIA to regulate cell proliferation and colony formation in different liver cancer cell lines required ERK signaling as well as the negative modulation of PP2A activity and that the effects of RPIA could be modulated by the addition of either a PP2A inhibitor or activator. Furthermore, the xenograft studies in nude mice revealed that the modulation of RPIA in liver cancer cells regulated tumor growth and that NIH3T3 cells overexpressing RPIA exhibited increased proliferation, enhanced colony formation, elevated levels of p-ERK1/2 and accelerated tumor growth. This study provides new insight into the molecular mechanisms by which RPIA overexpression can induce oncogenesis in HCC. Furthermore, it suggests that RPIA can be a good prognosis biomarker and a potential target for HCC therapy.

Biodegradation of Nonylphenol Monopropoxyethoxylates.

Aerobic biodegradation behavior of nonylphenol monopropoxyethoxylates was investigated in two tests with different inocula-sewage sludge and river water. Both primary biodegradation and formation of different biodegradation intermediates were studied. Primary biodegradation of nonylphenol monopropoxyethoxylates was relatively fast and complete with the sewage sludge as the inoculum. On the other hand, biodegradation with river water as the inoculum was slower and primary biodegradation in this test reached only about 60 % during almost 50 days. The biodegradation intermediates from both oxidative and non-oxidative pathways were found. In the non-oxidative route monopropoxy poly(ethylene glycol)s were observed which indicate existence of the central fission biodegradation pathway. In the oxidative pathway carboxylic acids were identified. The biodegradation intermediates identified with the use of high performance liquid chromatography with mass spectrometric detection persisted for many days in both tests.

Metabolomic and (13)C-metabolic flux analysis of a xylose-consuming Saccharomyces cerevisiae strain expressing xylose isomerase.

Over the past two decades, significant progress has been made in the engineering of xylose-consuming Saccharomyces cerevisiae strains for production of lignocellulosic biofuels. However, the ethanol productivities achieved on xylose are still significantly lower than those observed on glucose for reasons that are not well understood. We have undertaken an analysis of central carbon metabolite pool sizes and metabolic fluxes on glucose and on xylose under aerobic and anaerobic conditions in a strain capable of rapid xylose assimilation via xylose isomerase in order to investigate factors that may limit the rate of xylose fermentation. We find that during xylose utilization the flux through the non-oxidative Pentose Phosphate Pathway (PPP) is high but the flux through the oxidative PPP is low, highlighting an advantage of the strain employed in this study. Furthermore, xylose fails to elicit the full carbon catabolite repression response that is characteristic of glucose fermentation in S. cerevisiae. We present indirect evidence that the incomplete activation of the fermentation program on xylose results in a bottleneck in lower glycolysis, leading to inefficient re-oxidation of NADH produced in glycolysis.

Gas-phase Oxidation of Alcohols with O2 and N2O Catalyzed by Au/TiO2: A Comparative Study

Gas-phase oxidation of alcohols (EtOH, PrOH, i-PrOH, BuOH) to their carbonyl derivatives was used as test reaction to elucidate the mechanism of a low-temper- ature catalytic activity of Au/TiO2. The reactions were carried out in the presence of molecular oxygen, nitrous oxide as well as in the absence of the gas-phase oxidants. The relative contribution of oxidative and non-oxidative dehydrogenation pathways was thus estimated. The pre- sence of oxygen in the feed brought about to a double peak profile of catalytic activity as a function of temperature for all the alcohols. The low-temperature peak fells on 120-130 C. In contrast, the use of N2O as an oxidant gave rise to usual profile of catalytic activity, which is similar to that of anaerobic dehydrogenation of alcohols. The results obtained allowed to suggest the mechanism of the alcohols oxidation. The low temperature peak is probably related to participation of active oxygen species, generated from O2 on the catalyst surface. Oxidation with N2O is interpreted by preliminary dehydrogenation of alcohols to corre- sponding carbonyl derivatives followed by H2 oxidation.

Counterion-induced modulation in biochemical properties of nitrile functionalized silver(I)-N-heterocyclic carbene complexes

The new Ag(I) complexes (2–4) with proligand 1 were prepared and characterized as prospective antibacterial drug candidates. These compounds consist of a Ag(I) center linearly coordinated to two identical N-heterocyclic carbenes (NHCs). Crystal structures were solved for complexes 3 and 4, the resulting structural data being in good agreement with expectations. We wondered whether the presence of different counter ions in these compounds might lead to modulation of their biological properties. Notably, in spite of their similar structural pattern, Ag(I)–NHC complex 2 with Br− manifested better antibacterial actions than complexes 3 and 4 with and , respectively. Stability study over time in bacterial solutions revealed that these complexes have better stability than AgNO3. Their interaction with DNA and RNA was explored, cleavage activity indicates that all Ag(I)–NHC complexes exhibited interesting nuclease activity via non-oxidative pathway.

Abstract CT338: Combination of a PI3K- and a PARP-inhibitor to treat high-grade serous ovarian or triple-negative breast cancer

Abstract Clinical trials have shown promising responses of BRCA-linked breast and ovarian cancers to PARP inhibitor therapy, but remissions are often short-lived and incomplete. The PI3K pathway is frequently activated in these malignancies. Recently, we reported in vivo synergy of a PI3K- and a PARP-inhibitor in a mouse model of BRCA1-related breast cancer. While the PARP-inhibitor olaparib alone attenuated tumor growth modestly, a dramatic reduction in tumor growth was observed when olaparib was combined with the PI3K-inhibitor BKM120. In BRCA1-mutant HCC1937 BC cells, PI3K- (but not akt-) inhibition increased indicators of DNA damage, such as poly-ADP-ribosylation and γH2AX, suggesting a critical role of PI3K activity for the maintenance of genomic stability. Here, we report on the molecular mechanism underlying this synergy, on treatment outcomes in an improved mouse model system and the development of an early-phase clinical study. Pre-clinical modeling was done in BRCA1-mutant HCC1937 cells and in a mouse model based on the syngenic transplantation of tumors derived on the K14-Cre BRCA1f/fp53f/f background. Metabolic profiling in vitro and in vivo showed that PI3K-inhibition decreased flux through glycolysis and specifically through the non-oxidative pentose-phosphate pathway, the main source of ribose-5-phosphate required for the de novo synthesis of nucleotides in HCC1937 cells. Within 3 hours and preceding cell cycle changes, BKM120 caused a decrease in nucleotide pools that was further exacerbated by the addition of olaparib. Nucleotide shortage led to to replication stress with the appearance of γH2AX and increased poly-ADP-ribosylation. Tumors with complete loss of BRCA1 and p53 proved particularly vulnerable to this treatment strategy. We were able to induce complete and durable remissions of murine tumors arising on the K14-Cre BRCA1f/fp53f/f background with a 28-day course of BKM120 and Olaparib. These preclinical data have served as the rationale for a phase I, multi-center study (NCT01623349) combining the oral PARP inhibitor olaparib with the oral PI3-kinase inhibitor BKM120 in patients with recurrent HGSC or recurrent TNBC. The study is being conducted through the Stand Up to Cancer (SU2C)'s Targeting PI3-kinase in Women's Cancers Dream Team. It has a 3 + 3 design, escalating if 0/3 or 1/6 participants have a DLT during the first cycle of therapy. The study objectives are to determine the recommended phase II dose (RP2D) of daily continuous oral olaparib (using the tablet formulation) and BKM120, assess toxicities, safety, and preliminary activity of this combination, and determine pharmacokinetic profiles of both agents as well as translational endpoints. The study serves as an example for the development of a clinical trials concept for TNBC and HGSC based on a close collaboration of basic and clinical scientists through the SU2C mechanism. Citation Format: Gerburg M. Wulf, Ashish Juvekar, Costas A. Lyssiotis, Hai Hu, Sina Yadegarynia, Hui Liu, Baek Kim, Eric Winter, Ralph Scully, John Asara, Lewis C. Cantley, Ursula Matulonis. Combination of a PI3K- and a PARP-inhibitor to treat high-grade serous ovarian or triple-negative breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr CT338. doi:10.1158/1538-7445.AM2014-CT338

The effects of thiamine treatment on pre-diabetic versus overt diabetic rat hearts: Role of non-oxidative glucose pathways

The etiology of insulin resistance is complex and multi-faceted. For example, metabolic perturbations associated with obesity and/or increased consumption of energy-dense foodstuffs, coupled with a sedentary lifestyle may fuel this process [1,2]. Nutrient excess, and more specifically excursions of acute hyperglycemia, can play a significant role in the development of insulin resistance and the associated onset of cardiovascular diseases [3,4].

Akt Phosphorylation and Regulation of Transketolase Is a Nodal Point for Amino Acid Control of Purine Synthesis

The phosphatidylinositol 3-kinase (PI3K)/Akt pathway integrates environmental clues to regulate cell growth and survival. We showed previously that depriving cells of a single essential amino acid rapidly and reversibly arrests purine synthesis. Here we demonstrate that amino acids via mammalian target of rapamycin 2 and IκB kinase regulate Akt activity and Akt association and phosphorylation of transketolase (TKT), a key enzyme of the nonoxidative pentose phosphate pathway (PPP). Akt phosphorylates TKT on Thr382, markedly enhancing enzyme activity and increasing carbon flow through the nonoxidative PPP, thereby increasing purine synthesis. Mice fed a lysine-deficient diet for 2 days show decreased Akt activity, TKT activity, and purine synthesis in multiple organs. These results provide a mechanism whereby Akt coordinates amino acid availability with glucose utilization, purine synthesis, and RNA and DNA synthesis.

Employing a combinatorial expression approach to characterize xylose utilization in Saccharomyces cerevisiae

Fermentation of xylose, a major constituent of lignocellulose, will be important for expanding sustainable biofuel production. We sought to better understand the effects of intrinsic (genotypic) and extrinsic (growth conditions) variables on optimal gene expression of the Scheffersomyces stipitis xylose utilization pathway in Saccharomyces cerevisiae by using a set of five promoters to simultaneously regulate each gene. Three-gene (xylose reductase, xylitol dehydrogenase (XDH), and xylulokinase) and eight-gene (expanded with non-oxidative pentose phosphate pathway enzymes and pyruvate kinase) promoter libraries were enriched under aerobic and anaerobic conditions or with a mutant XDH with altered cofactor usage. Through characterization of enriched strains, we observed (1) differences in promoter enrichment for the three-gene library depending on whether the pentose phosphate pathway genes were included during the aerobic enrichment; (2) the importance of selection conditions, where some aerobically-enriched strains underperform in anaerobic conditions compared to anaerobically-enriched strains; (3) improved growth rather than improved fermentation product yields for optimized strains carrying the mutant XDH compared to the wild-type XDH.

Metabolic network reconstruction, growth characterization and 13C-metabolic flux analysis of the extremophile Thermus thermophilus HB8

Thermus thermophilus is an extremely thermophilic bacterium with significant biotechnological potential. In this work, we have characterized aerobic growth characteristics of T. thermophilus HB8 at temperatures between 50 and 85°C, constructed a metabolic network model of its central carbon metabolism and validated the model using 13C-metabolic flux analysis (13C–MFA). First, cells were grown in batch cultures in custom constructed mini-bioreactors at different temperatures to determine optimal growth conditions. The optimal temperature for T. thermophilus grown on defined medium with glucose was 81°C. The maximum growth rate was 0.25h−1. Between 50 and 81°C the growth rate increased by 7-fold and the temperature dependence was described well by an Arrhenius model with an activation energy of 47kJ/mol. Next, we performed a 13C-labeling experiment with [1,2-13C] glucose as the tracer and calculated intracellular metabolic fluxes using 13C–MFA. The results provided support for the constructed network model and highlighted several interesting characteristics of T. thermophilus metabolism. We found that T. thermophilus largely uses glycolysis and TCA cycle to produce biosynthetic precursors, ATP and reducing equivalents needed for cells growth. Consistent with its proposed metabolic network model, we did not detect any oxidative pentose phosphate pathway flux or Entner-Doudoroff pathway activity. The biomass precursors erythrose-4-phosphate and ribose-5-phosphate were produced via the non-oxidative pentose phosphate pathway, and largely via transketolase, with little contribution from transaldolase. The high biomass yield on glucose that was measured experimentally was also confirmed independently by 13C–MFA. The results presented here provide a solid foundation for future studies of T. thermophilus and its metabolic engineering applications.

The detrimental effects of acute hyperglycemia on myocardial glucose uptake

AimsAlthough acute hyperglycemic (AHG) episodes are linked to lower glucose uptake, underlying mechanisms remain unclear. We hypothesized that AHG triggers reactive oxygen species (ROS) production and increases non-oxidative glucose pathway (NOGP) activation, i.e. stimulation of advanced glycation end products (AGE), polyol pathway (PP), hexosamine biosynthetic pathway (HBP), PKC; thereby decreasing cardiac glucose uptake. Main methodsH9c2 cardiomyoblasts were exposed to 25mM glucose for 24h vs. 5.5mM controls±modulating agents during the last hour of glucose exposure: a) antioxidant #1 for mitochondrial ROS (250μM 4-OHCA), b) antioxidant #2 for NADPH oxidase-generated ROS (100μM DPI), c) NOGP inhibitors — 100μM aminoguanidine (AGE), 5μM chelerythrine (PKC); 40μM DON (HBP); and 10μM zopolrestat (PP). ROS levels (mitochondrial, intracellular) and glucose uptake were evaluated by flow cytometry. Key findingsAHG elevated ROS, activated NOGPs and blunted glucose uptake. Transketolase activity (pentose phosphate pathway [PPP] marker) did not change. Respective 4-OHCA and DPI treatment blunted ROS production, diminished NOGP activation and normalized glucose uptake. NOGP inhibitory studies identified PKCβII as a key downstream player in lowering insulin-mediated glucose uptake. When we employed an agent (benfotiamine) known to shunt flux away from NOGPs (into PPP), it decreased ROS generation and NOGP activation, and restored glucose uptake under AHG conditions. SignificanceThis study demonstrates that AHG elicits maladaptive events that function in tandem to reduce glucose uptake, and that antioxidant treatment and/or attenuation of NOGP activation (PKC, polyol pathway) may limit the onset of insulin resistance.

11th Euro Fed Lipid Congress, New Strategies for a High Quality Future (27–30 October 2013, Antalya, Turkey)

11th Euro Fed Lipid Congress, New Strategies for a High Quality Future (27–30 October 2013, Antalya, Turkey)