IDH2 deficiency promotes mitochondrial dysfunction and cardiac hypertrophy in mice

Cardiac hypertrophy, a risk factor for heart failure, is associated with enhanced oxidative stress in the mitochondria, resulting from high levels of reactive oxygen species (ROS). The balance between ROS generation and ROS detoxification dictates ROS levels. As such, disruption of these processes results in either increased or decreased levels of ROS. In previous publications, we have demonstrated that one of the primary functions of mitochondrial NADP⁺-dependent isocitrate dehydrogenase (IDH2) is to control the mitochondrial redox balance, and thereby mediate the cellular defense against oxidative damage, via the production of NADPH. To explore the association between IDH2 expression and cardiac function, we measured myocardial hypertrophy, apoptosis, and contractile dysfunction in IDH2 knockout (idh2^−/−) and wild-type (idh2^+/+) mice. As expected, mitochondria from the hearts of knockout mice lacked IDH2 activity and the hearts of IDH2-deficient mice developed accelerated heart failure, increased levels of apoptosis and hypertrophy, and exhibited mitochondrial dysfunction, which was associated with a loss of redox homeostasis. Our results suggest that IDH2 plays an important role in maintaining both baseline mitochondrial function and cardiac contractile function following pressure-overload hypertrophy, by preventing oxidative stress.     

Reduced antioxidant capacity and diet-induced atherosclerosis in uncoupling protein-2-deficient mice

Vascular dysfunction in response to reactive oxygen species (ROS) plays an important role in the development and progression of atherosclerotic lesions. In most cells, mitochondria are the major source of cellular ROS during aerobic respiration. Under most conditions the rates of ROS formation and elimination are balanced through mechanisms that sense relative ROS levels. However, a chronic imbalance in redox homeostasis is believed to contribute to various chronic diseases, including atherosclerosis. Uncoupling protein-2 (UCP2) is a mitochondrial inner membrane protein shown to be a negative regulator of macrophage ROS production. In response to a cholesterol-containing atherogenic diet, C57BL/6J mice significantly increased expression of UCP2 in the aorta, while mice lacking UCP2, in the absence of any other genetic modification, displayed significant endothelial dysfunction following the atherogenic diet. Compared with wild-type mice, Ucp2(-/-) mice had decreased endothelial nitric oxide synthase, an increase in vascular cell adhesion molecule-1 expression, increased ROS production, and an impaired ability to increase total antioxidant capacity. These changes in Ucp2(-/-) mice were associated with increased aortic macrophage infiltration and more numerous and larger atherosclerotic lesions. These data establish that in the vasculature UCP2 functions as an adaptive antioxidant defense to protect against the development of atherosclerosis in response to a fat and cholesterol diet.     

CD36-deficient mice are resistant to alcohol- and high-carbohydrate-induced hepatic steatosis

CD36 is a scavenger receptor with multiple ligands and cellular functions, including facilitating cellular uptake of free fatty acids (FFAs). Chronic alcohol consumption increases hepatic CD36 expression, leading to the hypothesis that this promotes uptake of circulating FFAs, which then serve as a substrate for triglyceride (TG) synthesis and the development of alcoholic steatosis. We investigated this hypothesis in alcohol-fed wild-type and Cd36-deficient (Cd36^−/−) mice using low-fat/high-carbohydrate Lieber-DeCarli liquid diets, positing that Cd36^−/− mice would be resistant to alcoholic steatosis. Our data show that the livers of Cd36^−/− mice are resistant to the lipogenic effect of consuming high-carbohydrate liquid diets. These mice also do not further develop alcoholic steatosis when chronically fed alcohol. Surprisingly, we did not detect an effect of alcohol or CD36 deficiency on hepatic FFA uptake; however, the lower baseline levels of hepatic TG in Cd36^−/− mice fed a liquid diet were associated with decreased expression of genes in the de novo lipogenesis pathway and a lower rate of hepatic de novo lipogenesis. In conclusion, Cd36^−/− mice are resistant to hepatic steatosis when fed a high-carbohydrate liquid diet, and they are also resistant to alcoholic steatosis. These studies highlight an important role for CD36 in hepatic lipid homeostasis that is not associated with hepatic fatty acid uptake.     

Amelioration of hepatic steatosis by dietary essential amino acid-induced ubiquitination

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IDH1 and IDH2 mutations are frequent in Chinese patients with acute myeloid leukemia but rare in other types of hematological disorders

Frequent mutations in the isocitrate dehydrogenase 1 and 2 genes (IDH1 and IDH2) have been identified in gliomas and acute myeloid leukemia (AML). Our aim is to assess whether IDH mutations were presented in Chinese patients with various hematological disorders. In this study, we screened the IDH1 and IDH2 mutations in a cohort of 456 Chinese patients with various hematological malignancies and disorders. We found three missense (p.R132C, p.R132G, and p.I99M; occurred in five patients) and one silent mutation (c.315C>T; occurred in two patients) in the IDH1 gene and two missense mutations (p.R140Q and p.R172K; occurred in four AML patients) and one silent mutation (c.435G>A) in the IDH2 gene. Except for one non-Hodgkin lymphoma (NHL) patient harboring IDH1 mutation p.R132C, all IDH1 and IDH2 missense mutations were observed in patients with AML. Intriguingly, the IDH2 mutation p.R140Q and novel IDH1 mutation p.I99M co-occurred in a 75-year-old patient with AML developed from myelodysplastic syndromes (MDS). The frequency of IDH1 and IDH2 missense mutations in Chinese AML patients reached 5.9% and 8.3%, respectively. Our results supported the recent findings that IDH gene mutations were common in AML. Conversely, IDH mutations were rather rare in Chinese patients with other types of hematological disorders.     

IDH1基因突变在胶质瘤中的研究进展

胶质瘤是颅内最常见的恶性肿瘤,发病率逐年增高。越来越多的证据表明IDH1基因突变与胶质瘤密切相关。本文就近年来有关胶质瘤与IDH1基因突变的研究作一综述。IDH1基因编码胞浆内NADP依赖的异柠檬酸脱氢酶,后者能够对异柠檬酸进行催化生成α-酮戊二酸。在40%的胶质瘤中存在IDH1突变,在继发性胶质母细胞瘤中变异率最高。作为一种代谢的关键酶,IDH1突变后可以将α-KG转变成2-HG,后者具有促进细胞增殖和促进肿瘤发生的作用。而且,IDH1突变可以导致胶质瘤代谢和表观遗传学方面的改变。同时,IDH1突变可以通过增加HIF-1α水平及活性增加血管生成。目前在不同级别的胶质瘤当中,IDH1突变已经成为一个与预后密切相关的独立预测因素。对IDH1突变的研究有助于深入了解胶质瘤病因及干预措施的具体机制,有助于胶质瘤的分子水平分类和治疗。     

Membrane-bound sn-1,2-diacylglycerols explain the dissociation of hepatic insulin resistance from hepatic steatosis in MTTP knockout mice

Microsomal triglyceride transfer protein (MTTP) deficiency results in a syndrome of hypolipidemia and accelerated NAFLD. Animal models of decreased hepatic MTTP activity have revealed an unexplained dissociation between hepatic steatosis and hepatic insulin resistance. Here, we performed comprehensive metabolic phenotyping of liver-specific MTTP knockout (L-Mttp^−/−) mice and age-weight matched wild-type control mice. Young (10–12-week-old) L-Mttp^−/− mice exhibited hepatic steatosis and increased DAG content; however, the increase in hepatic DAG content was partitioned to the lipid droplet and was not increased in the plasma membrane. Young L-Mttp^−/− mice also manifested normal hepatic insulin sensitivity, as assessed by hyperinsulinemic-euglycemic clamps, no PKCε activation, and normal hepatic insulin signaling from the insulin receptor through AKT Ser/Thr kinase. In contrast, aged (10-month-old) L-Mttp^−/− mice exhibited glucose intolerance and hepatic insulin resistance along with an increase in hepatic plasma membrane sn-1,2-DAG content and PKCε activation. Treatment with a functionally liver-targeted mitochondrial uncoupler protected the aged L-Mttp^−/− mice against the development of hepatic steatosis, increased plasma membrane sn-1,2-DAG content, PKCε activation, and hepatic insulin resistance. Furthermore, increased hepatic insulin sensitivity in the aged controlled-release mitochondrial protonophore-treated L-Mttp^−/− mice was not associated with any reductions in hepatic ceramide content. Taken together, these data demonstrate that differences in the intracellular compartmentation of sn-1,2-DAGs in the lipid droplet versus plasma membrane explains the dissociation of NAFLD/lipid-induced hepatic insulin resistance in young L-Mttp^−/− mice as well as the development of lipid-induced hepatic insulin resistance in aged L-Mttp^−/− mice.     

Lysine acetylation restricts mutant IDH2 activity to optimize transformation in AML cells

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GCN2 deficiency protects against high fat diet induced hepatic steatosis and insulin resistance in mice

Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic lipid deposition and oxidative stress. It has been demonstrated that general control nonderepressible 2 (GCN2) is required to maintain hepatic fatty acid homeostasis under conditions of amino acid deprivation. However, the impact of GCN2 on the development of NAFLD has not been investigated. In this study, we used Gcn2^?/? mice to investigate the effect of GCN2 on high fat diet (HFD)-induced hepatic steatosis. After HFD feeding for 12?weeks, Gcn2^?/? mice were less obese than wild-type (WT) mice, and Gcn2^?/? significantly attenuated HFD-induced liver dysfunction, hepatic steatosis and insulin resistance. In the livers of the HFD-fed mice, GCN2 deficiency resulted in higher levels of lipolysis genes, lower expression of genes related to FA synthesis, transport and lipogenesis, and less induction of oxidative stress. Furthermore, we found that knockdown of GCN2 attenuated, whereas overexpression of GCN2 exacerbated, palmitic acid-induced steatosis, oxidative & ER stress, and changes of peroxisome proliferator-activated receptor gamma (PPARγ), fatty acid synthase (FAS) and metallothionein (MT) expression in HepG2 cells. Collectively, our data provide evidences that GCN2 deficiency protects against HFD-induced hepatic steatosis by inhibiting lipogenesis and reducing oxidative stress. Our findings suggest that strategies to inhibit GCN2 activity in the liver may provide a novel approach to attenuate NAFLD development.     

Reductive carboxylation and 2-hydroxyglutarate formation by wild-type IDH2 in breast carcinoma cells

Mitochondrial NADPH-dependent isocitrate dehydrogenase, IDH2, and cytosolic IDH1, catalyze reductive carboxylation of 2-oxoglutarate. Both idh2 and idh1 monoallelic mutations are harbored in grade 2/3 gliomas, secondary glioblastomas and acute myeloid leukemia. Mutant IDH1/IDH2 enzymes were reported to form an oncometabolite r-2-hydroxyglutarate (2HG), further strengthening malignancy. We quantified CO₂-dependent reductive carboxylation glutaminolysis (RCG) and CO₂-independent 2HG production in HTB-126 and MDA-MB-231 breast carcinoma cells by measuring ¹³C incorporation from 1-¹³C-glutamine into citrate, malate, and 2HG. For HTB-126 cells, ¹³C-citrate, ¹³C-malate, and ¹³C-2-hydroxyglutarate were enriched by 2-, 5-, and 15-fold at 5 mM glucose (2-, 2.5-, and 13-fold at 25 mM glucose), respectively, after 6 h. Such enrichment decreased by 6% with IDH1 silencing, but by 30–50% upon IDH2 silencing while cell respiration and ATP levels rose up to 150%. Unlike 2HG production RCG declined at decreasing CO₂. At hypoxia (5% O₂), IDH2-related and unrelated ¹³C-accumulation into citrate and malate increased 1.5–2.5-fold with unchanged IDH2 expression; whereas hypoxic 2HG formation did not. ¹³C–2HG originated by ∼50% from other than IDH2 or IDH1 reactions, substantiating remaining activity in IDH1&2-silenced cells. Relatively high basal ¹²C–2HG levels existed (5-fold higher vs. non-tumor HTB-125 cells) and ¹³C–2HG was formed despite the absence of any idh2 and idh1 mutations in HTB-126 cells. Since RCG is enhanced at hypoxia (frequent in solid tumors) and 2HG can be formed without idh1/2 mutations, we suggest 2HG as an analytic marker (in serum, urine, or biopsies) predicting malignancy of breast cancer in all patients.     

IgE isotype switch and IgE production are enhanced in IL-21-deficient but not IFN-gamma-deficient mice in a Th2-biased response

IgE plays a critical role in the pathogenesis of allergy and asthma. Therefore, suppression of IgE production would provide therapeutic benefits to patients suffering from these diseases. We have reported that the production of IgE is regulated differently in the spleen vs. the draining lymph nodes (LN). IgE isotype switch and IgE producing B cell expansion occur in the draining LN after antigen (Ag) immunization, but do not happen in the spleen. In addition, a population of pre-existing IgE+ cells is observed in the spleen of normal or sham immunized mice, but is not present in the draining LN. To further understand the regulation of IgE production in different lymphoid organs, and the potential inhibitory factors of IgE isotype switch in the spleen, the involvement of IL-21 and IFN-gamma in regulating IgE production was investigated by using the IL-21 and the IFN-gamma deficient mice. We found that in the absence of IL-21 IgE isotype switch and IgE+ cell clonal expansion were dramatically enhanced in the spleen and IgE isotype switch was partially increased in the draining LN. In addition, IgE production of the pre-existing CD19-CD5+B220(low) IgE+ cells in the spleen was also increased in the absence of IL-21 under physiological conditions. In contrast, using the IFN-gamma deficient mice, we did not observe a negative impact of IFN-gamma on either IgE isotype switch or IgE production. Our data suggest that IL-21 appears to be a critical cytokine to keep low IgE levels under physiological and pathological conditions.     

血清成纤维细胞生长因子2、21、23与妊娠期糖尿病患者新生儿结局的关系

摘要 目的：探讨血清成纤维细胞生长因子2（FGF2）、成纤维细胞生长因子21（FGF21）、成纤维细胞生长因子23（FGF23）与妊娠期糖尿病（GDM）患者新生儿结局的关系。方法：选取2021年1月～2022年12月期间于我院产检的妊娠24～28周孕妇147例,均进行口服葡萄糖耐量试验（OGTT）,根据OGTT结果分为GDM组（n=86）和非GDM组（n=61）。其中GDM组根据新生儿结局分为不良组（n=21）和良好组（n=65）。对比非GDM组、GDM组的血清FGF2、FGF21、FGF23水平及新生儿结局情况。对比不良组和良好组的血清FGF2、FGF21、FGF23水平。单因素及多因素Logistic回归分析影响GDM患者新生儿结局的影响因素。结果：GDM组的血清FGF2、FGF21、FGF23水平均高于非GDM组（P<0.05）。GDM组的不良新生儿结局总发生率高于非GDM组（P<0.05）。不良组的血清FGF2、FGF21、FGF23水平均高于良好组（P<0.05）。单因素分析显示,GDM患者不良新生儿结局与年龄、孕前体质量指数（BMI）、分娩前BMI、空腹血糖（FPG）、餐后2 h血糖（2hPG）、空腹胰岛素（FINS）、胰岛素抵抗指数（HOMA-IR）有关（P<0.05）。多因素分析结果显示,年龄偏高、FPG偏高、孕前BMI偏高、2hPG偏高、分娩前BMI偏高、HOMA-IR偏高、FGF2偏高、FINS偏高、FGF21偏高、FGF23偏高均是GDM患者不良新生儿结局的危险因素（P<0.05）。结论：GDM患者血清FGF2、FGF21、FGF23水平升高,其与年龄、孕前BMI、分娩前BMI、FPG、2hPG、FINS、HOMA-IR偏高均是导致GDM患者不良新生儿结局的危险因素。     

Discovery of DC_H31 as potential mutant IDH1 inhibitor through NADPH-based high throughput screening

IDH1 mutations are early events in the development of IDH-mutant gliomas and leukemias and are associated with various regulation of molecular process. Mutations of active site in IDH1 could lead to high levels of 2-HG and the suppression of cellular differentiation, while these changes can be reversed by molecule inhibitors target mutant IDH1. Here, through in-house developed enzymatic assay-based high throughput screening platform, we discovered DC_H31 as a novel IDH1-R132H/C inhibitor, with the IC₅₀ value of 0.41 μmol/L and 2.7 μmol/L respectively. In addition, saturable SPR binding assay indicated that DC_H31 bound to IDH1-R132H/C due to specific interaction. Further computational docking studies and structure-activity relationship (SAR) suggest that DC_H31 could occupy the allosteric pocket between the two monomers of IDH1-R132H homodimer, which accounts for its inhibitory ability. And it is possible to conclude that DC_H31 acts via an allosteric mechanism of inhibition. At the cellular level, DC_H31 could inhibit cell proliferation, promote cell differentiation and reduce the production of 2-HG with a dose-dependent manner in HT1080 cells. Taken together, DC_H31 is a potent selective inhibitor of IDH1-R132H/C both in vitro and in vivo, which can promote the development of more potent pan-inhibitors against IDH1-R132H/C through further structural decoration and provide a new insight for the pharmacological treatment of gliomas.     

Synthesis and evaluation of radiolabeled AGI-5198 analogues as candidate radiotracers for imaging mutant IDH1 expression in tumors

Mutations in the metabolic enzyme isocitrate dehydrogenase 1 (IDH1) are commonly found in gliomas. AGI-5198, a potent and selective inhibitor of the mutant IDH1 enzyme, was radiolabeled with radioiodine and fluorine-18. These radiotracers were evaluated as potential probes for imaging mutant IDH1 expression in tumors with positron emission tomography (PET). Radioiodination of AGI-5198 was achieved using a tin precursor in 79?±?6% yield (n?=?9), and ¹⁸F-labeling was accomplished by the Ugi reaction in a decay-corrected radiochemical yield of 2.6?±?1.6% (n?=?5). The inhibitory potency of the analogous nonradioactive compounds against mutant IDH1 (IDH1-R132H) was determined in enzymatic assays. Cell uptake studies using radiolabeled AGI-5198 analogues revealed somewhat higher uptake in IDH1-mutated cells than that in wild-type IDH1 cells. The radiolabeled compounds displayed favorable tissue distribution characteristics in vivo, and good initial uptake in IDH1-mutated tumor xenografts; however, tumor uptake decreased with time. Radioiodinated AGI-5198 exhibited higher tumor-to-background ratios compared with ¹⁸F-labeled AGI-5198; unfortunately, similar results were observed in wild-type IDH1 tumor xenografts as well, indicating lack of selectivity for mutant IDH1 for this tracer. These results suggest that AGI-5198 analogues are not a promising platform for radiotracer development. Nonetheless, insights gained from this study may help in design and optimization of novel chemical scaffolds for developing radiotracers for imaging the mutant IDH1 enzyme.