Targeting purine synthesis in ASS1-expressing tumors enhances the response to immune checkpoint inhibitors.

Argininosuccinate synthase (ASS1) downregulation in different tumors has been shown to support cell proliferation and yet, in several common cancer subsets ASS1 expression associates with poor patient prognosis. Here we demonstrate that ASS1 expression under glucose deprivation is induced by c-MYC, providing survival benefit by increasing nitric oxide synthesis and activating the gluconeogenic enzymes pyruvate carboxylase and phosphoenolpyruvate carboxykinase by S-nitrosylation. The resulting increased flux through gluconeogenesis enhances serine, glycine and subsequently purine synthesis. Notably, high ASS1-expressing breast cancer mice do not respond to immune checkpoint inhibitors and patients with breast cancer with high ASS1 have more metastases. We further find that inhibiting purine synthesis increases pyrimidine to purine ratio, elevates expression of the immunoproteasome and significantly enhances the response of autologous primary CD8+ T cells to anti-PD-1. These results suggest that treating patients with high-ASS1 cancers with purine synthesis inhibition is beneficial and may also sensitize them to immune checkpoint inhibition therapy.

Deletion of endothelial arginase 1 does not improve vasomotor function in diabetic mice.

Endothelial arginase 1 was ablated to assess whether this prevents hyperglycemia-induced endothelial dysfunction by improving arginine availability for nitric oxide production. Endothelial Arg1-deficient mice (Arg1-KOTie2 ) were generated by crossing Arg1fl/fl (controls) with Tie2Cretg/- mice and analyzed by immunohistochemistry, measurements of hemodynamics, and wire myography. Ablation was confirmed by immunohistochemistry. Mean arterial blood pressure was similar in conscious male control and Arg1-KOTie2 mice. Depletion of circulating arginine by intravenous infusion of arginase 1 or inhibition of nitric oxide synthase activity with L-NG -nitro-arginine methyl ester increased mean arterial pressure similarly in control (9 ± 2 and 34 ± 2 mmHg, respectively) and Arg1-KOTie2 mice (11 ± 3 and 38 ± 4 mmHg, respectively). Vasomotor responses were studied in isolated saphenous arteries of 12- and 34-week-old Arg1-KOTie2 and control animals by wire myography. Diabetes was induced in 10-week-old control and Arg1-KOTie2 mice with streptozotocin, and vasomotor responses were studied 10 weeks later. Optimal arterial diameter, contractile responses to phenylephrine, and relaxing responses to acetylcholine and sodium nitroprusside were similar in normoglycemic control and Arg1-KOTie2 mice. The relaxing response to acetylcholine was dependent on the availability of extracellular l-arginine. In the diabetic mice, arterial relaxation responses to endothelium-dependent hyperpolarization and to exogenous nitric oxide were impaired. The data show that endothelial ablation of arginase 1 in mice does not markedly modify smooth muscle and endothelial functions of a resistance artery under normo- and hyperglycemic conditions.

Dietary treatment of fatty liver: High dietary protein content has an antisteatotic and antiobesogenic effect in mice.

Few studies have assessed the effect of changing ratios of dietary macronutrients on fat accumulation in adipose tissue and organs such as the liver in a 3×n(n≥3) factorial design. We investigated the effects of 7 diets from a single manufacturer containing 11-58en% protein (casein), 0-81en% carbohydrates (CHO; sucrose, maltrodextrin-10 and corn starch), and 8-42en% fat (triheptanoin, olive oil or cocoa butter) in C57BL/6J mice, a good model for diet-induced obesity and fatty liver. The diets were fed for 3weeks to wild-type and hyperlipidemic male and female mice. Caloric intake was mainly determined by dietary fat. Body weight, liver lipid and cholesterol content, NFκB activation, and fat-pad size decreased only in mice fed a high-protein diet. A high dietary protein:CHO ratio reduced plasma FGF21 concentration, and increased liver PCK1 protein content and plasma triglyceride concentration. The dietary protein:CHO ratio determined hepatic expression of Pck1 and Ppargc1a in males, and Fgf21 in females, whereas the dietary CHO:fat ratio determined that of Fasn, Acaca1, and Scd1 in females. Hepatic glycogen content was determined by all three dietary components. Both hepatic PCK1 and plasma FGF21 correlated strongly and inversely with hepatic TG content, suggesting a key role for PCK1 and increased gluconeogenesis in resolving steatosis with a high-protein diet, with FGF21 expression reflecting declining cell stress. We propose that a diet containing ~35en% protein, 5-10en% fat, and 55-60en% carbohydrate will prevent fatty liver in mice without inducing side effects.

The odd-carbon medium-chain fatty triglyceride triheptanoin does not reduce hepatic steatosis.

BACKGROUND & AIMS: Non-alcoholic fatty-liver disease (NAFLD) is the hepatic manifestation of the metabolic syndrome. Previously, we showed that a high-protein diet minimized diet-induced development of fatty liver and even reversed pre-existing steatosis. A high-protein diet leads to amino-acid catabolism, which in turn causes anaplerosis of the tricarboxylic-acid (TCA) cycle. Therefore, we hypothesized that anaplerosis of the TCA cycle could be responsible for the high-protein diet-induced improvement of NAFLD by channeling amino acids into the TCA cycle. Next we considered that an efficient anaplerotic agent, the odd-carbon medium-chain triglyceride triheptanoin (TH), might have similar beneficial effects. METHODS: C57BL/6J mice were fed low-fat (8en%) or high-fat (42en%) oleate-containing diets with or without 15en% TH for 3 weeks. RESULTS: TH treatment enhanced the hepatic capacity for fatty-acid oxidation by a selective increase in hepatic Ppara, Acox, and Cd36 expression, and a decline in plasma acetyl-carnitines. It also induced pyruvate cycling through an increased hepatic PCK1 protein concentration and it increased thermogenesis reflected by an increased Ucp2 mRNA content. TH, however, did not reduce hepatic lipid content. CONCLUSION: The comparison of the present effects of dietary triheptanoin with a previous study by our group on protein supplementation shows that the beneficial effects of the high-protein diet are not mimicked by TH. This argues against anaplerosis as the sole explanatory mechanism for the anti-steatotic effect of a high-protein diet.

Pivotal role of glutamine synthetase in ammonia detoxification.

Glutamine synthetase (GS) catalyzes condensation of ammonia with glutamate to glutamine. Glutamine serves, with alanine, as a major nontoxic interorgan ammonia carrier. Elimination of hepatic GS expression in mice causes only mild hyperammonemia and hypoglutaminemia but a pronounced decrease in the whole-body muscle-to-fat ratio with increased myostatin expression in muscle. Using GS-knockout/liver and control mice and stepwise increments of enterally infused ammonia, we show that ∼35% of this ammonia is detoxified by hepatic GS and ∼35% by urea-cycle enzymes, while ∼30% is not cleared by the liver, independent of portal ammonia concentrations ≤2 mmol/L. Using both genetic (GS-knockout/liver and GS-knockout/muscle) and pharmacological (methionine sulfoximine and dexamethasone) approaches to modulate GS activity, we further show that detoxification of stepwise increments of intravenously (jugular vein) infused ammonia is almost totally dependent on GS activity. Maximal ammonia-detoxifying capacity through either the enteral or the intravenous route is ∼160 µmol/hour in control mice. Using stable isotopes, we show that disposal of glutamine-bound ammonia to urea (through mitochondrial glutaminase and carbamoylphosphate synthetase) depends on the rate of glutamine synthesis and increases from ∼7% in methionine sulfoximine-treated mice to ∼500% in dexamethasone-treated mice (control mice, 100%), without difference in total urea synthesis. CONCLUSIONS: Hepatic GS contributes to both enteral and systemic ammonia detoxification. Glutamine synthesis in the periphery (including that in pericentral hepatocytes) and glutamine catabolism in (periportal) hepatocytes represents the high-affinity ammonia-detoxifying system of the body. The dependence of glutamine-bound ammonia disposal to urea on the rate of glutamine synthesis suggests that enhancing peripheral glutamine synthesis is a promising strategy to treat hyperammonemia. Because total urea synthesis does not depend on glutamine synthesis, we hypothesize that glutamate dehydrogenase complements mitochondrial ammonia production. (Hepatology 2017;65:281-293).

The growth pattern of the human intestine and its mesentery.

BACKGROUND: It remains unclear to what extent midgut rotation determines human intestinal topography and pathology. We reinvestigated the midgut during its looping and herniation phases of development, using novel 3D visualization techniques. RESULTS: We distinguished 3 generations of midgut loops. The topography of primary and secondary loops was constant, but that of tertiary loops not. The orientation of the primary loop changed from sagittal to transverse due to the descent of ventral structures in a body with a still helical body axis. The 1st secondary loop (duodenum, proximal jejunum) developed intraabdominally towards a left-sided position. The 2nd secondary loop (distal jejunum) assumed a left-sided position inside the hernia before returning, while the 3rd and 4th secondary loops retained near-midline positions. Intestinal return into the abdomen resembled a backward sliding movement. Only after return, the 4th secondary loop (distal ileum, cecum) rapidly "slid" into the right lower abdomen. The seemingly random position of the tertiary small-intestinal loops may have a biomechanical origin. CONCLUSIONS: The interpretation of "intestinal rotation" as a mechanistic rather than a descriptive concept underlies much of the confusion accompanying the physiological herniation. We argue, instead, that the concept of "en-bloc rotation" of the developing midgut is a fallacy of schematic drawings. Primary, secondary and tertiary loops arise in a hierarchical fashion. The predictable position and growth of secondary loops is pre-patterned and determines adult intestinal topography. We hypothesize based on published accounts that malrotations result from stunted development of secondary loops.

High-protein diets prevent steatosis and induce hepatic accumulation of monomethyl branched-chain fatty acids.

The hallmark of nonalcoholic fatty liver disease is steatosis of unknown etiology. To test how dietary protein decreases steatosis, we fed female C57BL/6 J mice low-fat (8 en%) or high-fat (42 en%) combined with low-protein (11 en%), high-protein (HP; 35 en%) or extra-high-protein (HPX; 58 en%) diets for 3 weeks. The 35 en% protein diets reduced hepatic triglyceride, free fatty acid, cholesterol and phospholipid contents to ~50% of that in 11 en% protein diets. Every additional 10 en% protein reduced hepatic fat content ~1.5 g%. HP diets had no effect on lipogenic or fatty acid-oxidizing genes except Ppargc1α (+30%), increased hepatic PCK1 content 3- to 5-fold, left plasma glucose and hepatic glycogen concentration unchanged, and decreased inflammation and cell stress (decreased Fgf21 and increased Gsta expression). The HP-mediated decrease in steatosis correlated inversely with plasma branched-chain amino-acid (BCAA) concentrations and hepatic content of BCAA-derived monomethyl branched-chain fatty acids (mmBCFAs) 14-methylpentadecanoic (14-MPDA; valine-derived) and, to a lesser extent, 14-methylhexadecanoic acid (isoleucine-derived). Liver lipid content was 1.6- to 1.8-fold higher in females than in males, but the anti-steatotic effect of HP diets was equally strong. The strong up-regulation of PCK1 and literature data showing an increase in phosphoenolpyruvate and a decline in tricarboxylic acid cycle intermediates in liver reveal that an increased efflux of these intermediates from mitochondria represents an important effect of an HP diet. The HP diet-induced increase in 14-MPDA and the dietary response in gene expression were more pronounced in females than males. Our findings are compatible with a facilitating role of valine-derived mmBCFAs in the antisteatotic effect of HP diets.

Endothelium-dependent hyperpolarization-related relaxations diminish with age in murine saphenous arteries of both sexes.

BACKGROUND AND PURPOSE: We investigated the effects of aging on the contributions of NO and endothelium-dependent hyperpolarization (EDH) to endothelium-dependent relaxation in saphenous arteries of male and female C57BL/6J mice aged 12, 34 and 64 weeks. EXPERIMENTAL APPROACH: Vasomotor responses of saphenous arteries were analysed by wire myography in the absence and presence of stimuli of the endothelium, inhibitors of NOS, and inhibitors and stimulants of small (KCa 2.3) and intermediate (KCa 3.1) conductance calcium-activated potassium channels. KEY RESULTS: Arterial relaxing responses to sodium nitroprusside and to ACh in the absence of pharmacological inhibitors (indomethacin and L-NAME), were similar in all age groups and sexes, but those mediated by endothelium-derived NO were slightly but significantly increased in 64-week-old male mice. In the presence of inhibitors, 12-week-old animals showed pronounced ACh-induced relaxation, which was significantly reduced in 34- and 64-week-old mice of both sexes. The EDH-related component of ACh-induced relaxations was abolished by TRAM-34 (KCa 3.1 blocker) or UCL 1684 (KCa 2.3 blocker). Although the maximal relaxation induced by NS309 (KCa activator) was not affected by aging, the sensitivity for NS309 significantly decreased with aging. The presence of SKA-31 (KCa modulator) potentiated relaxations induced by ACh in arteries of 12-week-old but not older mice. CONCLUSION AND IMPLICATIONS: In a small muscular artery of mice of either sex, total endothelium-dependent relaxation is not affected by age. However, possibly due to changes in KCa channel function, the contribution of EDH to endothelium-dependent relaxations decreased with age. The contribution of endothelium-derived NO increases in old male mice.

Prevention and reversal of hepatic steatosis with a high-protein diet in mice.

UNLABELLED: The hallmark of NAFLD is steatosis of unknown etiology. We tested the effect of a high-protein (HP)(2) diet on diet-induced steatosis in male C57BL/6 mice with and without pre-existing fatty liver. Mice were fed all combinations of semisynthetic low-fat (LF) or high-fat (HF) and low-protein (LP) or HP diets for 3weeks. To control for reduced energy intake by HF/HP-fed mice, a pair-fed HF/LP group was included. Reversibility of pre-existing steatosis was investigated by sequentially feeding HF/LP and HF/HP diets. HP-containing diets decreased hepatic lipids to ~40% of corresponding LP-containing diets, were more efficient in this respect than reducing energy intake to 80%, and reversed pre-existing diet-induced steatosis. Compared to LP-containing diets, mice fed HP-containing diets showed increased mitochondrial oxidative capacity (elevated Pgc1α, mAco, and Cpt1 mRNAs, complex-V protein, and decreased plasma free and short-chain acyl-carnitines, and [C0]/[C16+C18] carnitine ratio); increased gluconeogenesis and pyruvate cycling (increased PCK1 protein and fed plasma-glucose concentration without increased G6pase mRNA); reduced fatty-acid desaturation (decreased Scd1 expression and [C16:1n-7]/[C16:0] ratio) and increased long-chain PUFA elongation; a selective increase in plasma branched-chain amino acids; a decrease in cell stress (reduced phosphorylated eIF2α, and Fgf21 and Chop expression); and a trend toward less inflammation (lower Mcp1 and Cd11b expression and less phosphorylated NFκB). CONCLUSION: HP diets prevent and reverse steatosis independently of fat and carbohydrate intake more efficiently than a 20% reduction in energy intake. The effect appears to result from fuel-generated, highly distributed small, synergistic increases in lipid and BCAA catabolism, and a decrease in cell stress.