Glucogenic Amino Acids Research Articles

A composite polyphenol-rich extract improved growth performance, ruminal fermentation and immunity, while decreasing methanogenesis and excretion of nitrogen and phosphorus in growing buffaloes.

The effects of a composite polyphenolic-rich extract (CPRE) on ruminal fermentation, nutrient utilisation, growth performance, excretion of nitrogen and phosphorus and methane emission were studied in growing buffaloes. Four herbal dry extracts prepared from Acacia arabica (babul; bark), Acacia catechu (cutch; bark), Punica granatum (pomegranate; peel) and Eugenia jambolana (Indian blackberry; seeds) were mixed in an equal proportion (1:1:1:1) to prepare the CPRE that contained mainly phenolic compounds (146g/kg), flavonoids (41.7g/kg) and saponins (40.5g/kg). First, in vitro tests were performed for ruminal fermentation and feed degradability using ruminal fluid as inocula and CPRE at 0 to 40g/kg substrate to decide an optimal dose of CPRE for an in vivo study on buffaloes. In the animal study, 20 buffaloes were randomly assigned to two groups (n = 10)-a control diet and a CPRE diet (control diet added with extra 20g/kg of CPRE). The in vitro tests suggested that addition of CPRE at 20g/kg substrate increased degradability of substrate, short-chain fatty acid concentration and propionate proportion, and reduced methane production, acetate proportion, acetate:propionate ratio and ammonia concentration in fermentation media, which were also noted in the rumen of buffaloes. Feeding CRPE to buffaloes did not affect feed intake, but increased daily body weight gain, dry matter and crude protein digestibility and nitrogen and phosphorus retention in the body. Total bacteria, methanogens and protozoal numbers were similar between two groups, but Fibrobacter succinogenes increased in the rumen of buffaloes fed CPRE. Concentrations of total, essential, non-essential and glucogenic amino acids were greater in the plasma of CPRE-fed buffaloes. Cell-mediated immune response improved in the CPRE-fed buffaloes compared with the control group. Estimated methane production and excretion of nitrogen and phosphorus per unit of body weight gain decreased in the CPRE group. The comprehensive results of this study clearly suggested that the composite polyphenol-rich feed additive at 20g/kg diet improved growth performance, ruminal fermentation, immunity and plasma amino acids profile, whereas it reduced indicators of environmental impacts of buffalo production.

A double-blind, placebo-controlled, single-ascending dose study to evaluate the safety, tolerability, pharmacokinetics, and pharmacodynamics of HM15136, a novel long-acting glucagon analogue, in healthy subjects.

To evaluate the safety and tolerability, pharmacokinetics, and pharmacodynamics of HM15136, a novel long-acting glucagon analogue under development, in healthy males and females presenting with no childbearing potential. A randomized, double-blind, placebo-controlled, single-ascending dose study was conducted in 56 subjects who randomly received a single subcutaneous dose of HM15136 or its matching placebo at a ratio of 6:2 at 10, 20, 30, 50, 80, 100, and 120 μg/kg. All adverse events were mild and transient. Neither serious adverse events nor discontinuation as a result of adverse events occurred. The most frequent adverse drug reaction was nausea (5.3%, only in the 100- and 120-μg/kg groups). HM15136, particularly at doses of 50 μg/kg or higher, increased fasting blood glucose, with a maximum increase and area under the curve of 1.5 mmol/L at day 10 (P= .006) and 166.3day·mmol/L (P= .022) at the dose of 80 μg/kg, while suppressing the secretion of endogenous glucagon, which continued until day 17. HM15136 also significantly reduced gluconeogenic and ketogenic amino acids. Compensatory changes in endogenous insulin and incretin hormones by HM15136 were not apparent. HM15136 was slowly but steadily absorbed and reached a peak concentration at 46-68 hours after a single subcutaneous injection. HM15136 was eliminated with a terminal phase half-life of 77.1-101.1hours. A single subcutaneous dose of HM15136 at 10-120 μg/kg was safe and well tolerated. The long half-life of HM15136, coupled with an increase in blood glucose for ~2 weeks, may warrant a weekly dosing regimen.

Comprehensive Analysis of Differentially Expressed mRNA, Non-coding RNA, and Their Competitive Endogenous RNA Network of Pacific Oyster Crassostrea gigas With Different Glycogen Content Between Different Environments

Glycogen content is a quantitative trait, its phenotype differences are found between individual oysters due to genetic effects and environmental factors which were including food, water temperature, salinity, and so on. In this study, a full sibling family of Pacific oyster Crassostrea gigas showed different phenotypes with high and low glycogen content between South Huanghai Sea (Rizhao offshore area, RZ) and North Huanghai Sea (Kongtong Dao area, KTD), respectively. At the same time, the content of 11 glucogenic amino acids and 13 fatty acids were also significant differences between RZ and KTD. RNA-seq and small RNA-seq technologies were used for transcriptome sequencing and functional enrichment analysis of differentially expressed RNA were used by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. A total of 2,084 mRNAs, 1,080 long non-coding RNAs (lncRNAs), 34 circular RNAs (circRNAs), and 7 microRNAs (miRNAs) were differentially expressed. Based on these differentially expressed genes (DEGs), miRNA target interactions (lncRNA/circRNA–miRNA pairs and miRNA–mRNA pairs) were predicted using the miRanda software. The differentially expressed mRNAs in this network were mainly shown to be involved in calcium signaling pathway and insulin signaling pathway. These findings could help to speculate that environmental factors may be epigenetically regulated by non-coding RNA in C. gigas, thereby further affecting glycogen content.

Effect of a very low negative dietary cation-anion difference (DCAD) diet on plasma and urine metabolomics of prepartum Holstein cows

The objectives of this cross-sectional, nonintervention, observational study were to compare urine and blood parameters between cows consuming a positive dietary cation-anion difference (DCAD) diet [early dry cows, DCAD + 250 mEq/kg of dry matter (DM), n = 15] with the same cows consuming a negative DCAD diet (−220 mEq/kg of DM) 10 d after moving them from the early dry to the prepartum group. The most remarkable finding was that cows consuming the anionic diet had very low urine pH and very low base excess in blood, suggestive of uncompensated metabolic acidosis. Importantly, the metabolomics data revealed that only urine concentrations of essential and aromatic amino acids were decreased, and that concentrations of total nonessential amino acids and glucogenic amino acids were increased in plasma and reciprocally decreased in urine, suggesting that the cows fed anionic salts were attempting to meet a high glucose demand by mobilizing gluconeogenic amino acid reserves. Notably, the dietary anionic salts exerted marked effects on glycerophospholipids, with a reduction in most phosphatidylcholine containing diacyl (PC aa) and acyl-alkyl (PC ae) moieties in plasma and urine. Further characterization of these metabolomic profiles may lead to the development of novel biomarkers to identify cows susceptible to metabolic acidosis and other metabolic diseases.

The Effect of Catabolic Transformations of Proteins and Fats on the Quality and Nutritional Value of Raw Ripened Products from Zlotnicka Spotted and Zlotnicka White Meat

Abstract The aim of the study was to compare the advancement of the ripening as well as catabolic changes in proteins and fats of Zlotnicka Spotted (ZS) and Zlotnicka White (ZW) meat and their impact on the quality and nutritional value of ready-to-eat products. The meat of the breeds ZS and ZW differed not only in the basic chemical composition but also in the susceptibility to catabolic transformations of proteins and lipids, which translated into a separate technological and nutritional quality as well as the profile of volatile odor compounds. Loins due to their compact histological structure, low pH (5.4) and decreased water activity (0.92–0.93) were characterized by a lower number of coagulase-negative cocci (3.3 log cfu/g) compared to hams. The products of both breeds differed in the content of selected neutral glucogenic amino acids with a pI in the range of 5.6–6.1 mainly. The content of biogenic amines was therefore completely dependent on the metabolic potential of acidifying bacteria. Larger number of lactic acid bacilli (7.5–7.7 log cfu/g) and lactic acid cocci (7.9–8.3 log cfu/g), as well as a higher content of saturated (55.2–53.7%) and polyunsaturated fatty acids (6.4–7.0%) shaped the final pH of hams (5.3). Presence of aldehydes, ketones and alcohols indicated existing fat oxidation despite the small values of the TBA index of hams (1.1 mg/kg) and loins (0.4–0.6 mg/kg). The volatile compounds that differentiated products of ZS and ZW formed by the oxidation and microbial activity, were, primarily: octanal, 1-hydroxypropan- 2-one, 3-methylpentan-2-one, propane-1,2-diol, 2,5-dimethylfuran and 3-hydroxybutan- 2-one, butane-2,3-dione, butane-1,2-diol, respectively.

UHPLC\u2013electrospray ionization\u2013mass spectrometric analysis of brain cell-specific glucogenic and neurotransmitter amino acid content

Astrocyte glycogen, the primary energy reserve in brain, undergoes continuous remodeling by glucose passage through the glycogen shunt prior to conversion to the oxidizable energy fuel l-lactate. Glucogenic amino acids (GAAs) are a potential non-glucose energy source during neuro-metabolic instability. Current research investigated whether diminished glycogen metabolism affects GAA homeostasis in astrocyte and/or nerve cell compartments. The glycogen phosphorylase (GP) inhibitor 1,4-dideoxy-1,4-imino-d-arabinitol (DAB) was injected into the ventromedial hypothalamic nucleus (VMN), a key metabolic-sensing structure, before vehicle or l-lactate infusion. Pure VMN astrocyte and metabolic-sensory neuron samples were obtained by combinatory immunocytochemistry/laser–catapult-microdissection for UHPLC–electrospray ionization–mass spectrometry (LC–ESI–MS) GAA analysis. DAB inhibition of VMN astrocyte aspartate and glutamine (Gln) levels was prevented or exacerbated, respectively, by lactate. VMN gluco-stimulatory nitric oxide (NO; neuronal nitric oxide synthase-immunoreactive (ir)-positive) and gluco-inhibitory γ-aminobutyric acid (GABA; glutamate decarboxylase65/67-ir-positive) neurons exhibited lactate-reversible asparate and glutamate augmentation by DAB, but dissimilar Gln responses to DAB. GP inhibition elevated NO and GABA nerve cell GABA content, but diminished astrocyte GABA; these responses were averted by lactate in neuron, but not astrocyte samples. Outcomes provide proof-of-principle of requisite LC–ESI–MS sensitivity for GAA measurement in specific brain cell populations. Results document divergent effects of decreased VMN glycogen breakdown on astrocyte versus neuron GAAs excepting Gln. Lactate-reversible DAB up-regulation of metabolic-sensory neuron GABA signaling may reflect compensatory nerve cell energy stabilization upon decline in astrocyte-derived metabolic fuel.

Decreased fastingserumglucogenic amino acids with a higher compared to normal protein diet during energy restrictionin women: a randomized controlled trial.

Dietary protein alters circulating amino acid (AAs) levels and higher protein intake (HP) is one means of losing weight. We examined34overweight and obese women (57 ± 4years)during6monthsofenergy restriction (7.3 ± 3.8% weight loss) divided into groups consuming either normal protein (NP;18.6 energy% protein) or HP (24.3 energy% protein). There was a reduction in fasting serumglucogenic AAs (p = 0.015) thatalso associated with greater weight loss (p < 0.05)in the HP group, but not in the NP group. These findings haveimplications for nutrient prioritization during energy restriction.

Impact of Weaning and Maternal Immune Activation on the Metabolism of Pigs

Weaning wields environmental, social, and nutritional stresses that are detectable in the blood metabolite levels of the offspring. Prenatal stress in the form of maternal immune activation (MIA) in response to infection, which is associated with health and behavior disorders, also elicits prolonged changes in blood and brain cytokine and metabolite levels of the offspring. The goal of this study was to investigate the effects of weaning and MIA on the offspring’s liver function to advance the understanding of the impact of stressors on peripheral and central nervous systems, physiology, and health. Gas chromatography–mass spectrometry analysis was used to compare the level of hepatic metabolites from 22-day-old pigs (n = 48) evenly distributed among weaning (nursed or weaned), viral MIA exposure (yes or no), and sexes. Weaning effects were detected on 38 metabolites at p-value < 0.05 (28 metabolites at FDR p-value < 0.05), and sex-dependent MIA effects were detected on 11 metabolites. Multiple intermediate and final products of the enriched (FDR p-value < 0.05) glycolysis and gluconeogenesis and pentose phosphate pathways were over-abundant in nursed relative to weaned pigs. The enriched pathways confirm the impact of weaning on hepatic metabolic shift, oxidative stress, and inflammation. Higher levels of the glucogenic amino acid histidine are observed in pigs exposed to MIA relative to controls, suggesting that the role of this metabolite in modulating inflammation may supersede the role of this amino acid as an energy source. The lower levels of cholesterol detected in MIA pigs are consistent with hypocholesterolemia profiles detected in individuals with MIA-related behavior disorders. Our findings underline the impact of weaning and MIA stressors on hepatic metabolites that can influence peripheral and central nervous system metabolic products associated with health and behavior disorders.

Bcl-2-dependent autophagy disruption during aging impairs amino acid utilization that is restored by hochuekkito

Chronic undernutrition contributes to the increase in frailty observed among elderly adults, which is a pressing issue in the sector of health care for older people worldwide. Autophagy, an intracellular recycling system, is closely associated with age-related pathologies. Therefore, decreased autophagy in aging could be involved in the disruption of energy homeostasis that occurs during undernutrition; however, the physiological mechanisms underlying this process remain unknown. Here, we showed that 70% daily food restriction (FR) induced fatal hypoglycemia in 23–26-month-old (aged) mice, which exhibited significantly lower hepatic autophagy than 9-week-old (young) mice. The liver expressions of Bcl-2, an autophagy-negative regulator, and Beclin1–Bcl-2 binding, were increased in aged mice compared with young mice. The autophagy inducer Tat-Beclin1 D11, not the mTOR inhibitor rapamycin, decreased the plasma levels of the glucogenic amino acid and restored the blood glucose levels in aged FR mice. Decreased liver gluconeogenesis, body temperature, physical activity, amino acid metabolism, and hepatic mitochondrial dynamics were observed in the aged FR mice. These changes were restored by treatment with hochuekkito that is a herbal formula containing several autophagy-activating ingredients. Our results indicate that Bcl-2 upregulation in the liver during the aging process disturbs autophagy activation, which increases the vulnerability to undernutrition. The promotion of liver autophagy may offer clinical therapeutic benefits to frail elderly patients.

Abstract 2453: Resistance of triple-negative breast cancer cells to EGFR-targeted tyrosine kinase inhibitors is associated with Annexin A6-dependent metabolic reprogramming

Abstract The ability of cancer cells to alter their metabolism is one of the major mechanisms underlying therapeutic resistance in multiple tumor types, including triple-negative breast cancer (TNBC). Triple-negative breast cancer (TNBC) remains a hard-to-treat breast cancer subtype. Significant effort has thus far been devoted to epidermal growth factor receptor (EGFR) targeted therapies as 70% of TNBC tumors overexpress EGFR. However, clinical trials utilizing anti-EGFR therapies have shown great success in various cancer types including non-small cell lung cancer but have been largely unsuccessful in TNBC due to the lack of understanding of TKI-acquired resistance. We have shown that the expression status of annexin-A6 (AnxA6), a calcium-dependent membrane binding tumor suppressor, stabilizes activated EGFR on the cell surface. We have also shown that chronic treatment of AnxA6-low TNBC cells with Lapatinib induces AnxA6 expression and accumulation of cholesterol in late endosomes suggesting a novel mechanism for resistance of TNBC to EGFR-TKIs. Other studies have revealed that AnxA6 expression contributes to impaired fat storage and adiponectin release in adipose tissues, and that lack of AnxA6 compromises alanine-dependent gluconeogenesis and liver regeneration in mice. However, whether the EGFR-TKI induced upregulation of AnxA6 is associated with metabolic reprogramming in TNBC cells remains unclear. Using the Seahorse XF Analyzer, we assessed the live-cell oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of TNBC cells. We demonstrate that the basal mitochondrial respiration and glycolytic rates classified TNBC cells into two distinct metabolic phenotypes. Those with higher OCR/ECAR ratios and cellular ATP production capacity were mostly AnxA6 high cells (energetic phenotype), while those with lower OCR/ECAR ratios and cellular ATP production capacity were mostly AnxA6 low cells (quiescentphenotype). Induction of AnxA6 expression following chronic treatment of AnxA6-low TNBC cells with Lapatinib (LapR) reverted these otherwise quiescent metabolic phenotypes to the energetic phenotype associated with higher OCR/ECAR ratios and cellular ATP production capacity. However, downregulation of AnxA6 in AnxA6 high TNBC cells attenuated the mitochondrial and glycolytic function with reduced OCR/ECAR ratios and decreased cellular ATP production capacity. NMR-based metabolomics revealed that AnxA6 depleted and/or lapatinib-resistant TNBC cells have a greater dependency on gluconeogenic amino acids including alanine, cysteine, glycine, and proline. These data suggest that altered expression of AnxA6 is accompanied by significant bioenergetic adaptations and provide novel insights into the failure of EGFR-targeted therapies as therapeutic options for TNBC. Citation Format: Stephen D. Williams, Sarrah E. Widatalla, Amos M. Sakwe. Resistance of triple-negative breast cancer cells to EGFR-targeted tyrosine kinase inhibitors is associated with Annexin A6-dependent metabolic reprogramming [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2453.

English

BACKGROUND Hyperglycemia, observed in type 2 diabetus mellites (T2DM) patients, is the primary root cause for the development of life threatening - diabetes induced micro- &amp; macrovascular complications. Generally, in fasting state, gluconeogenesis accounts for about 30 % of overall hepatic glucose output and the increase in type 2 diabetic subjects may be much more than this level. The majority of gluconeogenic precursors may originate from muscle glycolysis and in the form of glucogenic amino acids including alanine. We wanted to assess fasting plasma total amino acid levels in T2DM patients and correlate the levels with the glycaemic status of T2DM patients in this study. METHODS The observational comparative study was undertaken at Research and Development department, from October 2019 to November 2020 and it consisted of 157 subjects, including 75 normal control subjects, and 82 T2DM patients. The T2 diabetic patients attending medical OPD of Subbaiah Institute of Medical Sciences, Purle, Shivamogga (SUIMS) were randomly selected and were sub-grouped in to 3 groups depending on their fasting plasma glucose (FPG) levels. The group 1 patients were having FPG level in the range of 150 – 200 mg / dl, group 2 patients having in the range of 201 - 250 mg / dl and the group 3 patients were having FPG above 251 mg / dl. RESULTS It is evident from the present study that FPG levels as well as AAN levels are significantly (P &gt; 0.001) elevated in T2DM patients as compared to normal control subjects and there is a proportional rise in AAN levels in relation to FPG Levels in T2dm patients. CONCLUSIONS The rise in plasma AN levels is proportional to glycemic status in T2DM patients and can be employed to assess the glycemic status in these patients. KEY WORDS Amino Acids, Gluconeogenesis, Type 2 Diabetes Mellitus

277-OR: Use of Noncarbohydrate Fuels Is Associated with Materno-Fetal Complications in Type 1 Diabetes Pregnancy: Metabolomics Analysis of the CONCEPTT Trial

Aims: We assessed metabolomic signatures in maternal and cord blood associated with suboptimal outcomes in the continuous glucose monitoring in women with type 1 diabetes in pregnancy trial (CONCEPTT). Methods: Serum samples from 162 mothers (12, 24 and 34 weeks’ gestation) and 93 cord blood samples were analysed for 1049 metabolites and 1041 lipids using ultra-performance liquid chromatography-tandem mass spectroscopy. We used adjusted and unadjusted logistic regression of metabolomic variables using adjudicated outcomes: extremely-large-for-gestational-age (ELGA; &amp;gt;97.5th centile), pre-eclampsia and neonatal hypoglycaemia with modified Bonferroni false discovery rate p≤0.001. Results: All materno-fetal complications studied were associated with reliance on non-carbohydrate sources of fuel. Lipids through beta oxidation were the main fuel source in ELGA (24 and 34 weeks), neonatal hypoglycaemia (12 weeks only) and pre-eclampsia (12 and 24 weeks). Marked protein catabolism was evident in neonatal hypoglycaemia (34 weeks) and pre-eclampsia (24 and 34 weeks). Cord blood in ELGA infants showed evidence of simultaneous beta oxidation and de novo lipogenesis, a biologically futile cycle of creating and destroying lipids, which consumes excess energy and substrate. Cord blood from infants with neonatal hypoglycaemia showed evidence of pronounced protein catabolism providing glucogenic amino acids for gluconeogenesis. Conclusions: Reliance on lipid or protein sources for fuel was associated with ELGA, neonatal hypoglycaemia and pre-eclampsia. Carbohydrate metabolism was insufficient to meet cellular energy demands, possibly due to insufficient insulin, insufficient dietary carbohydrate or both. Improving outcomes in type 1 diabetes pregnancy may require greater focus on normalising carbohydrate metabolism through optimal carbohydrate intake and matched insulin dosing. Disclosure C. L. Meek: None. Z. Stewart: None. S. Furse: None. J. M. Yamamoto: None. D. Feig: Advisory Panel; Self; Novo Nordisk. A. Koulman: None. H. R. Murphy: None. Funding JDRF (17/2011/533, 80/2010/585); Diabetes UK (DUK-HKF 17/0005712, DUK-PG 17/0005633.BBSRC BB/M027252/1)

Elucidating the Antimycobacterial Mechanism of Action of Ciprofloxacin Using Metabolomics.

In the interest of developing more effective and safer anti-tuberculosis drugs, we used a GCxGC-TOF-MS metabolomics research approach to investigate and compare the metabolic profiles of Mtb in the presence and absence of ciprofloxacin. The metabolites that best describe the differences between the compared groups were identified as markers characterizing the changes induced by ciprofloxacin. Malic acid was ranked as the most significantly altered metabolite marker induced by ciprofloxacin, indicative of an inhibition of the tricarboxylic acid (TCA) and glyoxylate cycle of Mtb. The altered fatty acid, myo-inositol, and triacylglycerol metabolism seen in this group supports previous observations of ciprofloxacin action on the Mtb cell wall. Furthermore, the altered pentose phosphate intermediates, glycerol metabolism markers, glucose accumulation, as well as the reduction in the glucogenic amino acids specifically, indicate a flux toward DNA (as well as cell wall) repair, also supporting previous findings of DNA damage caused by ciprofloxacin. This study further provides insights useful for designing network whole-system strategies for the identification of possible modes of action of various drugs and possibly adaptations by Mtb resulting in resistance.

The Role of Annexin‐A6 in the Metabolic Reprogramming and EGFR‐TKI resistance in Triple‐negative breast cancer cells

The ability of cancer cells to alter their metabolism is one of the major mechanisms underlying therapeutic resistance in multiple tumor types, including triple-negative breast cancer (TNBC). Triple-negative breast cancer (TNBC) remains a hard-to-treat breast cancer subtype. Significant effort has thus far been devoted to epidermal growth factor receptor (EGFR) targeted therapies as 70% of TNBC tumors overexpress EGFR. However, clinical trials utilizing anti-EGFR therapies have shown great success in various cancer types including non-small cell lung cancer but have been largely unsuccessful in TNBC due to the lack of understanding of TKI-acquired resistance. We have shown that the expression status of annexin-A6 (AnxA6), a calcium-dependent membrane binding tumor suppressor, stabilizes activated EGFR on the cell surface. We have also shown that chronic treatment of AnxA6-low TNBC cells with Lapatinib induces AnxA6 expression and accumulation of cholesterol in late endosomes suggesting a novel mechanism for resistance of TNBC to EGFR-TKIs. Other studies have revealed that AnxA6 expression contributes to impaired fat storage and adiponectin release in adipose tissues, and that lack of AnxA6 compromises alanine-dependent gluconeogenesis and liver regeneration in mice. However, whether the EGFR-TKI induced upregulation of AnxA6 is associated with metabolic reprogramming in TNBC cells remains unclear. Using the Seahorse XF Analyzer, we assessed the live-cell oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of TNBC cell lines. We demonstrate that the basal mitochondrial respiration and glycolytic rates classified TNBC cell lines into two distinct metabolic phenotypes. Those with higher OCR/ECAR ratios and cellular ATP production capacity were mostly AnxA6 high cells (energetic phenotype), while those with lower OCR/ECAR ratios and cellular ATP production capacity were mostly AnxA6 low cells (quiescent phenotype). Induction of AnxA6 expression following chronic treatment of AnxA6-low TNBC cells with Lapatinib (LapR) reverted these otherwise quiescent metabolic phenotypes to the energetic phenotype associated with higher OCR/ECAR ratios and cellular ATP production capacity. However, down regulation of AnxA6 in AnxA6 high TNBC cells attenuated the mitochondrial and glycolytic function with reduced OCR/ECAR ratios and decreased cellular ATP production capacity. NMR-based metabolomics revealed that AnxA6 depleted and/or lapatinib-resistant TNBC cells have a greater dependency on gluconeogenic amino acids including alanine, cysteine, glycine, and proline. These data suggest that altered expression of AnxA6 is accompanied by significant bioenergetic adaptations and provide novel insights into the failure of EGFR-targeted therapies as therapeutic options for TNBC.

Effect of an intensified individual nutrition therapy on serum metabolites in critically ill patients - A targeted metabolomics analysis of the ONCA study.

The effect of medical nutrition on serum metabolomics has been poorly explored. The aim of the study was to investigate the relation between energy supply and metabolic profiles in critically ill patients. Twenty mechanically ventilated patients on enteral nutrition (EN) or enteral/parenteral nutrition (EN/PN) were randomized into two groups. One group received an individual energy supply based on indirect calorimetry (IC group, n=9), the other group received a standard energy supply based on a formula, the standard care group (SC group, n=11). Targeted metabolomics was performed in early-, late- and post-acute metabolic phase. Individual versus standard care energy supply resulted in a metabolite class separation between the IC and the SC group (P<0.001). In the SC group concentrations of four glucogenic amino acids and three biogenic amines increased between the early- and late-acute metabolic phase (P<0.05). The metabolomics pattern differed between the routes of nutrition administration (P<0.01). The amount of energy supply by EN or PN, besides other factors, seems to modulate serum metabolites. Nutrition therapy based on individualized energy supply is associated with a reduction of metabolites reflecting catabolism. Therefore, metabolomics could be a new tool to determine metabolic phases in critically ill patients.

Functional partnership between carbonic anhydrase and malic enzyme in promoting gluconeogenesis in Leishmania major.

Leishmania has a remarkable ability to proliferate under widely fluctuating levels of essential nutrients, such as glucose. For this, the parasite is heavily dependent on its gluconeogenic machinery. One perplexing aspect of gluconeogenesis in Leishmania is the lack of the crucial gene for pyruvate carboxylase (PC). PC-catalyzed conversion of pyruvate to oxaloacetate is a key entry point through which gluconeogenic amino acids are funneled into this pathway. The absence of PC in Leishmania thus raises question about the mechanism of pyruvate entry into the gluconeogenic route. In the present study, we report that this task is accomplished in Leishmania major through a novel functional partnership between its mitochondrial malic enzyme (LmME) and carbonic anhydrase 1 (LmCA1). Using a combination of pharmacological inhibition studies with genetic manipulation, we show that both of these enzymes are necessary for promoting gluconeogenesis and supporting parasite growth under glucose-limiting conditions. Functional cross-talk between LmME and LmCA1 was evident when it was observed that the growth retardation caused by inhibition of any one of these enzymes could be protected to a significant extent by overexpressing the other enzyme. We also found that, although LmCA1 exhibited constitutive expression, the LmME protein level was strongly upregulated under low glucose conditions. Notably, both LmME and LmCA1 were found to be important for survival of Leishmania amastigotes within host macrophages. Taken together, our results indicate that LmCA1 by virtue of its CO2 concentrating ability stimulates LmME-catalyzed pyruvate carboxylation, thereby driving gluconeogenesis through the pyruvate-malate-oxaloacetate bypass pathway. Additionally, our study establishes LmCA1 and LmME as promising therapeutic targets.

Milk Production and Energetic Metabolism of Heat-Stressed Dairy Goats Supplemented with Propylene Glycol.

Simple SummaryHeat stress (HS) causes significant milk production losses and negatively impacts animal health. Previous research indicated that heat-stressed goats eat less and degrade their skeletal muscles to cover shortages in nutrient requirements. Propylene glycol (PG) is a glucogenic precursor and may be a remedy for energy shortage in HS situations. In the current study, dairy goats were exposed to thermoneutral (TN; 15 to 20 °C) or HS (12 h/d at 37 °C and 12 h/d at 30 °C) conditions. In each ambient temperature, goats were fed a control diet (CO) or the same diet supplemented with PG. Goats supplemented with PG gained more body weight, and experienced greater blood glucose and insulin levels compared to CO goats. However, supplementation with PG depressed feed intake and milk fat content. In conclusion, feeding propylene glycol was useful to reduce body weight losses typically observed under HS conditions, but did not improve milk yield or milk composition.Heat-stressed dairy animals increase their reliance on glucose. This elevated glucose demand is partially met by increasing the conversion of glucogenic amino acids (AA) in the liver. Propylene glycol (PG) is a glucogenic precursor and was not tested in dairy goats under thermoneutral (TN) and heat stress (HS) conditions simultaneously. We hypothesize that if HS-goats are fed with PG, they would get more glucose and consequently spare more glucogenic AA for milk protein synthesis rather than gluconeogenesis. Eight multiparous dairy goats (40.8 ± 1.1 kg body weight; 84 ± 1 days in milk) were used in a replicated 4 × 4 Latin square design of 4 periods; 21 d each (14 d adaptation, 5 d for measurements, and 2 d of transition). Goats were allocated to one of 4 treatments in a 2 × 2 factorial arrangement. Factors were control (CO) without PG or 5% of PG, and thermoneutral (TN; 15 to 20 °C) or heat stress (HS; 12 h/d at 37 °C and 12 h/d at 30 °C) conditions. Feed intake, rectal temperature, respiratory rate, milk yield, milk composition, and blood metabolites were measured. Compared to TN, HS goats had lower (p < 0.01) feed intake (–34%), fat-corrected milk (–15%), and milk fat (–15%). Heat-stressed goats also tended (p < 0.10) to produce milk with lower protein (–11%) and lactose (–4%) contents. Propylene glycol increased blood glucose (+7%; p < 0.05), blood insulin (+37%; p < 0.10), and body weight gain (+68%; p < 0.05), but decreased feed intake (–9%; p < 0.10) and milk fat content (–23%; p < 0.01). Furthermore, blood non-esterified fatty acids (–49%) and β-hydroxybutyrate (–32%) decreased (p < 0.05) by PG. In conclusion, supplementation of heat-stressed dairy goats with propylene glycol caused milk fat depression syndrome, but reduced body weight loss that is typically observed under HS conditions. Supplementation with lower doses of PG would avoid the reduced feed intake and milk fat depression, but this should be tested.

Sex-dimorphic neuroestradiol regulation of ventromedial hypothalamic nucleus glucoregulatory transmitter and glycogen metabolism enzyme protein expression in the rat

BackgroundVentromedial hypothalamic nucleus (VMN) gluco-regulatory transmission is subject to sex-specific control by estradiol. The VMN is characterized by high levels of aromatase expression.MethodsThe aromatase inhibitor letrozole (LZ) was used with high-resolution microdissection/Western blot techniques to address the hypothesis that neuroestradiol exerts sex-dimorphic control of VMN neuronal nitric oxide synthase (nNOS) and glutamate decarboxylase65/67 (GAD) protein expression. Glycogen metabolism impacts VMN nNOS and GAD profiles; here, LZ treatment effects on VMN glycogen synthase (GS) and phosphorylase brain- (GPbb; glucoprivic-sensitive) and muscle (GPmm; norepinephrine-sensitive) variant proteins were examined.ResultsVMN aromatase protein content was similar between sexes. Intracerebroventricular LZ infusion of testes-intact male and ovariectomized, estradiol-replaced female rats blocked insulin-induced hypoglycemic (IIH) up-regulation of this profile. LZ exerted sex-contingent effects on basal VMN nNOS and GAD expression, but blocked IIH-induced NO stimulation and GAD suppression in each sex. Sex-contingent LZ effects on basal and hypoglycemic patterns of GPbb and GPmm expression occurred at distinctive levels of the VMN. LZ correspondingly down- or up-regulated baseline pyruvate recycling pathway marker protein expression in males (glutaminase) and females (malic enzyme-1), and altered INS effects on those proteins.ConclusionsResults infer that neuroestradiol is required in each sex for optimal VMN metabolic transmitter signaling of hypoglycemic energy deficiency. Sex differences in VMN GP variant protein levels and sensitivity to aromatase may correlate with sex-dimorphic glycogen mobilization during this metabolic stress. Neuroestradiol may also exert sex-specific effects on glucogenic amino acid energy yield by actions on distinctive enzyme targets in each sex.

ChREBP downregulates SNAT2 amino acid transporter expression through interactions with SMRT in response to a high-carbohydrate diet.

Carbohydrate responsive element-binding protein (ChREBP) has been identified as a primary transcription factor that maintains energy homeostasis through transcriptional regulation of glycolytic, lipogenic, and gluconeogenic enzymes in response to a high-carbohydrate diet. Amino acids are important substrates for gluconeogenesis, but nevertheless, knowledge is lacking about whether this transcription factor regulates genes involved in the transport or use of these metabolites. Here, we demonstrate that ChREBP represses the expression of the amino acid transporter sodium-coupled neutral amino acid transporter 2 (SNAT2) in response to a high-sucrose diet in rats by binding to a carbohydrate response element (ChoRE) site located -160 bp upstream of the transcriptional start site in the SNAT2 promoter region. Additionally, immunoprecipitation assays revealed that ChREBP and silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) interact with each other, as part of the complex that repress SNAT2 expression. The interaction between these proteins was confirmed by an in vivo chromatin immunoprecipitation assay. These findings suggest that glucogenic amino acid uptake by the liver is controlled by ChREBP through the repression of SNAT2 expression in rats consuming a high-carbohydrate diet.NEW & NOTEWORTHY This study highlights the key role of carbohydrate responsive element-binding protein (ChREBP) in the fine-tuned regulation between glucose and amino acid metabolism in the liver via regulation of the amino acid transporter sodium-coupled neutral amino acid transporter 2 (SNAT2) expression after the consumption of a high-carbohydrate diet. ChREBP binds to a carbohydrate response element (ChoRE) site in the SNAT2 promoter region and recruits silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) corepressor to reduce SNAT2 transcription. This study revealed that ChREBP prevents the uptake of glucogenic amino acids upon the consumption of a high-carbohydrate diet.

Pyruvate Dehydrogenase Inhibition Leads to Decreased Glycolysis and Increased Reliance on Gluconeogenesis in Acute Myeloid Leukemia

Background: Acute myeloid leukemia (AML) is an aggressive disease characterized by poor outcomes and chemotherapy resistance. Devimistat (CPI-613®) is a novel agent that inhibits two key tricarboxylic acid (TCA) cycle enzymes, pyruvate dehydrogenase (PDH) and α-ketoglutarate dehydrogenase complexes (KGDH). Our lab has shown that genetic and pharmacologic inhibition of PDH sensitizes cells to chemotherapy and recent clinical trials of chemotherapy plus devimistat have been promising. However, the means by which AML cells adapt to PDH inhibition during devimistat treatment is unknown. Methods: Murine AML cells were used to knockout PDH through the CRISPR-CAS9 system. Viability assays were done using EZQuant or Cell-Titer Glo assays. Glycolytic flux was assessed by measurement of the extracellular acidification rate (ECAR) using the XF24 Extracellular Flux Analyzer. Glucose uptake was measured by Glucose Uptake-Glo assay. Protein levels were assessed by Western blot. Gene expression was measured by qPCR. Amino acid dependencies were assessed by incubating cells in Hank's Balanced Salt Solution with devimistat and either asparagine or glutamate for 4 hours followed by return to complete media and viability assayed after 72-hours. Results: Previous studies have shown that glycolysis can compensate for mitochondrial inhibition so we determined glycolytic rates during PDH inhibition. We have shown previously that genetic PDH loss decreases ECAR levels consistent with decreased glycolytic flux. Devimisat treatment also decreased ECAR levels in K562 and OCI cells in a dose dependent manner. We next examined glucose uptake. PDH knockout cells showed decreased glucose uptake compared to control. This could be due to reduced glucose import or reduced glucose retention. The expression of glucose importer GLUT1 was significantly decreased in cells with genetic or pharmacologic inhibition of PDH. LDHA expression was also reduced in PDH KO cells and devimistat treated cells, contributing to the ECAR decrease. Hexokinase II (HKII) traps glucose in AML cells and docks at the mitochondrial membrane. It can be dislodged with mitochondrial stress and subsequently degraded via the proteasome. PDH knockout cells and devimistat treated cells demonstrated reduced levels of HKII that could be rescued with the proteasomal inhibitor bortezomib. Taken together this data shows that genetic and pharmacologic inhibition of PDH decreases glycolysis by decreasing glucose import and retention. To assess the necessity of the diminished glycolytic activity we treated PDH knockout and devimistat treated cells with the glycolytic inhibitor 2-deoxy-D-glucose. Both devimistat treated and PDH KO cells were exquisitely sensitive to glycolysis inhibition. We next assessed the importance of gluconeogenic amino acids. We specifically looked at glutamate and asparagine as they have been shown to be highly utilized in cancers. Asparagine but not glutamate rescued AML cells from devimistat treatment. Asparaginase which catalysis the conversion of asparagine to aspartic acid, thus limiting the amount of asparagine in the cells sensitizes cells to both genetic and pharmacologic PDH inhibition. Asparagine can be converted into oxaloacetate which can be converted into phosphoenolpyruvate (PEP) for gluconeogenesis. Phosphoenolpyruvate carboxykinase (PCK2) catalyzes the rate limiting step in gluconeogenesis converting oxaloacetate to PEP which can travel backwards through glycolysis. We found that PCK2 was highly upregulated in both the PDH knock cells and devimisat treated cells. Indicating that with the reduced glucose import and retention the cells rely on gluconeogenesis from amino acids to maintain needed glycolytic intermediates. Conclusions: This data shows that PDH inhibition leads to diminished glycolytic rate, increased reliance on amino acid metabolism and increased gluconeogenesis. This in turn suggests that glycolysis inhibition and amino acid deprivation in combination with devminisat mediated PDH inhibition should be explored in clinical trials. Disclosures Pardee: Amgen: Honoraria, Speakers Bureau; Pharmacyclics: Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Rafael: Research Funding; Karyopharm: Research Funding; BMS: Consultancy, Honoraria, Speakers Bureau; Genentech, Inc.: Consultancy; AbbVie: Consultancy; Rafael Pharmaceuticals: Consultancy. OffLabel Disclosure: Devimistat for the treatment of AML