Lactate Dehydrogenase Protein Expression Research Articles

Itaconate is a negative regulator of hepatic lipid metabolism during sepsis

Sepsis is a life‐threatening clinical implication with increased uncontrolled host immune response to an infection, mortality, morbidity, and financial burden worldwide. Due to its critical role in metabolic reprogramming in inflammation, the Irg1/itaconate has received much attention as an immunomodulator. However, understanding of itaconate’s anti‐inflammatory and immunometabolic activities in response to infections is mostly limited to immune cells. By employing multi‐omic approaches, here we show that in the context of sepsis, a disruption in TCA cycle flow drives hepatic itaconate accumulation, indicating potential non‐immune functions. However, the functional role of itaconate in this central organ critical to maintaining systemic metabolism is yet to be elucidated. To gain more insight into its physiological role in the liver during sepsis, we subjected wild‐type and whole‐body Irg1 knockout mice to sepsis via cecal slurry injection. In conjunction with our previous findings, we find wild‐type septic mice develop hepatic steatosis. Interestingly, global Irg1 knockout mice develop a more severe form of a hepatic steatotic phenotype and a significant increase in hepatic lipid burden compared to wild‐type counterparts in response to sepsis. This data demonstrates itaconate as a negative regulator of hepatic lipid accumulation in the context of sepsis. However, the exact molecular mechanism by which itaconate interacts with regulators of hepatic lipid metabolism during sepsis is yet to be uncovered. As an anti‐inflammatory metabolite, itaconate limits the glycolytic response in activated immune cells. Similarly, invitro, we find that 4‐OI, an itaconate derivative, antagonizes LPS induced glycolysis in hepatocytes. Given our findings of heightened lipid accumulation in septic Irg1 knockout mice, we further hypothesize glycolysis to be elevated and fueling de novolipogenesis via the production of acetyl‐CoA. Indeed, unbiased metabolomics data show heightened hepatic lactate levels indicative of hyperactivated glycolysis in knockout septic mice. Mechanistically, we find elevated gene and protein expression of lactate dehydrogenase, the enzyme that facilitates the conversion of pyruvate to lactate, driving this accumulation of lactate seen in septic Irg1 knockout mice. In summary, our preliminary findings thus far highlights itaconate as a negative modulator of hepatic glycolysis as well as de novo lipogenesis in restraining sepsis‐induced hepatic steatosis.

Lanthanum chloride reduces lactate production in primary culture rat cortical astrocytes and suppresses primary co-culture rat cortical astrocyte-neuron lactate transport.

Lanthanum (La) can impair learning memory and induce behavioral abnormalities in animals. However, the mechanism underlying these adverse effects of La is still elusive. It has been demonstrated that lactate derived from astrocytes is the major energy source for neurons during long-term memory (LTM) formation and the deficiency of lactate supply can result in LTM damage. However, little work has been done with respect to the impact of La on the lactate production in astrocytes and astrocyte-neuron lactate transport (ANLT). Herein, experiments were undertaken to explore if there was such an adverse effect of La. Primary culture rat cortical astrocytes and primary co-culture rat cortical astrocyte-neuron were treated with (0.125, 0.25 and 0.5mM) lanthanum chloride (LaCl3) for 24h. The results showed that LaCl3 treatment significantly downregulated the mRNA and protein expression of glucose transporter 1 (GLUT1), glycogen synthase (GS), glycogen phosphorylase (GP), lactate dehydrogenase A (LDHA), and monocarboxylate transporter 1, 2 and 4 (MCT 1 2 and 4); upregulated the mRNA and protein expression of lactate dehydrogenase B (LDHB); and decreased the glycogen level, total LDH and GP activity, GS/p-GS ratio and lactate contents. Moreover, rolipram (20, 40μM) or forskolin (20, 40μM) could increase the lactate content by upregulating GP expression and the GS/p-GS ratio, as well as antagonize the effects of La. These results suggested that La-induced learning-memory damage was probably related to its suppression of lactate production in astrocytes and ANLT. This study provides some novel clues for clarifying the mechanism underlying the neurotoxicity of La.

An atypical distribution of lactate dehydrogenase isoenzymes in the hooded seal (Cystophora cristata) brain may reflect a biochemical adaptation to diving.

The brains of some diving mammals can withstand periods of severe hypoxia without signs of deleterious effects. This may in part be due to an enhanced cerebral capacity for anaerobic energy production. Here, we have tested this hypothesis by comparing various parameters of the lactate dehydrogenase (LDH) in the brain of the hooded seal (Cystophora cristata) with those in the brains of the ferret (Mustela putorius furo) and mouse (Mus musculus). We found that mRNA and protein expression of lactate dehydrogenase a (LDHA) and lactate dehydrogenase b (LDHB), and also the LDH activity were significantly higher in the ferret brain than in brains of the hooded seal and the mouse (p < 0.0001). No conspicuous differences in the LDHA and LDHB sequences were observed. There was also no difference in the buffering capacities of the brains. Thus, an enhanced capacity for anaerobic energy production likely does not explain the higher hypoxia tolerance of the seal brain. However, the brain of the hooded seal had higher relative levels of LDHB isoenzymes (LDH1 and LDH2) compared to the non-diving mammals. Moreover, immunofluorescence studies showed more pronounced co-localization of LDHB and glial fibrillary acidic protein in the cortex of the hooded seal. Since LDHB isoenzymes primarily catalyze the conversion of lactate to pyruvate, this finding suggests that the contribution of astrocytes to the brain aerobic metabolism is higher in the hooded seal than in non-diving species. The cerebral tolerance of the hooded seal to hypoxia may therefore partly rely on different LDH isoenzymes distribution.

Dietary effect of green tea extract on epidermal levels of skin pH related factors, lactate dehydrogenase protein expression and activity in UV-irradiated hairless mice

Purpose: Skin pH, an indicator of skin health, is maintained by various organic factors, which include lactate, free amino acid (FAA), and free fatty acid (FFA). As skin ages or with illness, skin pH becomes less acidic, and functional food has been developed to maintain the acidic pH of skin. In this study, we determined the dietary effect of green tea extract (GTE) on skin pH of photo-aged mice, as measured by epidermal levels of lactate, FAA, and FFA. The protein expression and activity of lactate dehydrogenase (LDH), an enzyme of pyruvate reduction for lactate generation, was further determined. Methods: Albino hairless mice were fed a control diet (group UV+) or a diet with 1% GTE (group GTE) in parallel with UV irradiation for 10 weeks. A normal control group was fed a control diet without UV irradiation for 10 weeks (group UV-). Results: Skin pH was higher (less acidic) in group UV+ than in group UV-. In parallel, epidermal levels of lactate and FFA, as well as of LDH protein expression and activity, were reduced in group UV+. Dietary supplementation of GTE (group GTE) reduced skin pH to similar to the level of group UV-, and inversely increased epidermal levels of lactate, LDH protein expression and activity, but not of FFA. Although epidermal levels of FAA were similar in groups UV- and UV+, it was increased in group GTE to a level higher than in group UV-. In further analysis of major FFA, epidermal levels of palmitic acid [16:0], oleic acid [18:1(n-9)], and linoleic acid [18:2(n-6), but not of stearic acid [18:0] in group GTE were similar to or lower than those in group UV+. Conclusion: Dietary GTE normalized skin pH with increased levels of lactate and FAA, as well as with increased protein expression and activity of LDH in the epidermis of UVB irradiated hairless mice.

Cellular prion protein directly interacts with and enhances lactate dehydrogenase expression under hypoxic conditions

Although a physiological function of the cellular prion protein (PrPc) is still not fully clarified, a PrPc-mediated neuroprotection against hypoxic/ischemic insult is intriguing. After ischemic stroke prion protein knockout mice (Prnp0/0) display significantly greater lesions as compared to wild-type (WT) mice. Earlier reports suggested an interaction between the glycolytic enzyme lactate dehydrogenase (LDH) and PrPc. Since hypoxic environment enhances LDH expression levels and compels neurons to rely on lactate as an additional oxidative substrate for energy metabolism, we examined possible differences in LDH protein expression in WT and Prnp0/0 knockout models under normoxic/hypoxic conditions in vitro and in vivo, as well as in a HEK293 cell line.While no differences are observed under normoxic conditions, LDH expression is markedly increased after 60-min and 90-min of hypoxia in WT vs. Prnp0/0 primary cortical neurons with concurrent less hypoxia-induced damage in the former group. Likewise, cerebral ischemia significantly increases LDH levels in WT vs. Prnp0/0 mice with accompanying smaller lesions in the WT group. HEK293 cells overexpressing PrPc show significantly higher LDH expression/activity following 90-min of hypoxia as compared to control cells. Moreover, a cytoplasmic co-localization of LDH and PrPc was recorded under both normoxic and hypoxic conditions. Interestingly, an expression of monocarboxylate transporter 1, responsible for cellular lactate uptake, increases with PrPc-overexpression under normoxic conditions. Our data suggest LDH as a direct PrPc interactor with possible physiological relevance under low oxygen conditions.

Melatonin alters the glycolytic profile of Sertoli cells: implications for male fertility.

Melatonin co-operates with insulin in the regulation of glucose homeostasis. Within the testis, glucose metabolism in the somatic Sertoli cells (SCs) is pivotal for spermatogenesis. Since the effects of melatonin on male reproductive physiology remain largely unknown, we hypothesized that melatonin may affect spermatogenesis by modulating SC metabolism, interacting with insulin. To test our hypothesis, rat SCs were maintained in culture for 24 h in the presence of insulin, melatonin or both and metabolite production/consumption was determined by proton nuclear magnetic resonance ((1)H-NMR). Protein levels of glucose transporters (GLUT1 and GLUT3), phosphofructokinase 1, lactate dehydrogenase (LDH) and monocarboxylate transporter 4 were determined by western blot. LDH activity was also assessed. SCs treated with melatonin showed an increase in glucose consumption via modulation of GLUT1 levels, but decreased LDH protein expression and activity, which resulted in lower lactate production. Moreover, SCs exposed to melatonin produced and accumulated less acetate than insulin-exposed cells. The combined treatment (insulin plus melatonin) increased acetate production by SCs, but intracellular acetate content remained lower than in insulin exposed cells. Finally, the intracellular redox state, as reflected by intracellular lactate/alanine ratio, was maintained at control levels in SCs by melatonin exposure (i.e. melatonin, alone or with insulin, increased the lactate/alanine ratio versus cells treated with insulin). Furthermore, SCs exposed to insulin plus melatonin produced more lactate and maintained the protein levels of some glycolysis-related enzymes and transporters at control levels. These findings illustrate that melatonin regulates SCs metabolism, and thus may affect spermatogenesis. Since lactate produced by SCs provides nutritional support and has an anti-apoptotic effect in developing germ cells, melatonin supplementation may be an effective therapy for diabetic male individuals facing subfertility/infertility.

Proposed cytotoxic mechanisms of the saffron carotenoids crocin and crocetin on cancer cell lines

We investigated the cytotoxic activities of crocin and crocetin, 2 major carotenoids isolated from the stigma of Crocus sativus (saffron), on 5 human cancer cell lines and proposed their possible anticancer mechanisms. Crocetin, a glycosylated carotenoid, showed approximately 5- to 18-fold higher cytotoxicity than crocin, a carboxylic carotenoid (IC50 of 0.16-0.61 mmol/L for crocetin vs. 2.0-5.5 mmol/L for crocin). This suggests that structural differences account for the different efficacies between them. Fluorescence-activated cell sorting (FACS) analysis showed that crocetin induced a significant level of cellular reactive oxygen species (ROS) in HeLa cells, whereas crocin did not. This ROS induction supported the cytotoxicity of crocetin, but not of crocin. A significant activation of nuclear factor erythroid 2-related factor 2 (Nrf2) was observed in both HeLa cells treated with crocin and crocetin: a 3.0-fold increase by 1 mmol/L crocetin and a 1.6-fold increase by 0.8 mmol/L crocin compared to the control. Furthermore, both crocetin and crocin reduced the protein expression of lactate dehydrogenase A (LDHA), one of the targets for chemoprevention in cancer cells, by 34.2% and 10.5%, respectively, compared to the control in HeLa cells. These findings suggest that crocetin and crocin have different mechanisms for their observed cytotoxicity in cancer cell lines.

Reduction of Reactive Oxygen Species Ameliorates Metabolism-Secretion Coupling in Islets of Diabetic GK Rats by Suppressing Lactate Overproduction

We previously demonstrated that impaired glucose-induced insulin secretion (IS) and ATP elevation in islets of Goto-Kakizaki (GK) rats, a nonobese model of diabetes, were significantly restored by 30–60-min suppression of endogenous reactive oxygen species (ROS) overproduction. In this study, we investigated the effect of a longer (12 h) suppression of ROS on metabolism-secretion coupling in β-cells by exposure to tempol, a superoxide (O2−) dismutase mimic, plus ebselen, a glutathione peroxidase mimic (TE treatment). In GK islets, both H2O2 and O2− were sufficiently reduced and glucose-induced IS and ATP elevation were improved by TE treatment. Glucose oxidation, an indicator of Krebs cycle velocity, also was improved by TE treatment at high glucose, whereas glucokinase activity, which determines glycolytic velocity, was not affected. Lactate production was markedly increased in GK islets, and TE treatment reduced lactate production and protein expression of lactate dehydrogenase and hypoxia-inducible factor 1α (HIF1α). These results indicate that the Warburg-like effect, which is characteristic of aerobic metabolism in cancer cells by which lactate is overproduced with reduced linking to mitochondria metabolism, plays an important role in impaired metabolism-secretion coupling in diabetic β-cells and suggest that ROS reduction can improve mitochondrial metabolism by suppressing lactate overproduction through the inhibition of HIF1α stabilization.

Metabolic and proteomic differentials in head and neck squamous cell carcinomas and normal gingival tissue

A high lactate content in malignant head and neck cancer (Head and neck squamous cell carcinomas, HNSCC) is associated with a higher risk of metastatic spread and lower overall patient survival. However, until present, the underlying mechanisms are not clearly understood. Here, a systematic comparison of glucose metabolism in HNSCC and homologous normal tissue is presented for the first time. The concentrations of glucose, lactate and ATP were measured in cryobiopsies of 29 human HNSCC and of 9 normal mucosa using bioluminescence imaging. The protein expression of lactate dehydrogenase (LDH) was analyzed by Western blotting. Tumors own a higher content of lactate and LDH in comparison with normal tissues. However, within the tumor group, the grade of LDH expression shows substantially strong variation and overlap with normal values. Furthermore, LDH expression was not correlated with tumor lactate content. Investigating a small subpopulation, patients with a short-term survival had significantly higher tumor lactate levels compared to patients with long-term survival. The data provide clear evidence of an enhanced glycolysis in tumors compared to normal tissue. This may partially but not completely attributable to an elevated expression of LDH. High tumor lactate levels may be predictive for restricted patient survival. In conclusion, lactate measurements, for example non-invasively with MRT, should be advanced for use in clinical routine as a supportive tool for tumor diagnosis and prognosis.

Intratumoral lactate dehydrogenase 5 (LDH5) protein expression is associated with expression of angiogenesis markers and hypoxia in patients with colorectal cancer (CRC)

4107 Background: Recent clinical trials (CONFIRM 1 and CONFIRM 2) have shown that metastatic CRC patients (pts) with high serum lactate dehydrogenase (LDH) derive the greatest therapeutic benefit from PTK787/ZK 222584 (PTK/ZK). PTK/ZK is a novel, oral tyrosine kinase inhibitor (TKI), which blocks all known VEGF receptors (VEGFR). From previous studies, total LDH and isoenzyme LDH5 have been associated with tumor aggressiveness and hypoxia. In the present study, we tested whether CRC pts with high levels of tumor LDH5 have increased expression of proteins involved with hypoxia (hypoxia inducible factors [HIF1a and 2a], pyruvate dehydrokinase [PDHK]), increased vessel density (VD), angiogenesis (VEGFA; phosphorylated VEGFR2 [pKDR]), acidity (carbonic anhydrase 9 [CA9]), and others. Methods: Baseline sections from either primary or metastatic tumor sites from 42 pts of the CONFIRM 1 and 2 trials were analysed with immunohistochemistry utilizing an established nominal scoring system. The degree of association between the scores of protein expressed was estimated by the phi-coefficient (correlation coefficient) and assessed by means of p-values from pairwise Fisher’s exact test (two- sided). Results: Associations were observed between LDH5 and the following: pKDR (Phi=.53; p&lt;.001), VEGF (Phi=.41; p=.006), HIF-1a (Phi=.56; p&lt;.001), VD (Phi=.34; p=.052), and PDHK (Phi=.58; p=.014), respectively. HIF-1a associated with pKDR (Phi=.38; p=.027), VD (Phi=.34; p=.045), and VEGFA (Phi=.33; p=.067). VEGFA associated with PDHK (Phi=.52; p=.035). Conclusions: Our results demonstrate that mCRC patients with high level of intratumoral protein expression of LDH5 have elevated HIF-1a, pKDR, VEGFA, PDHK expression and VD. These results support the concept that tumor hypoxia and angiogenesis are associated and that elevated LDH protein expression may serve as a surrogate marker for activated HIF-1a pathway. No significant financial relationships to disclose.

Lactate dehydrogenase expression at the onset of altered loading in rat soleus muscle.

Both functional overload and hindlimb disuse induce significant energy-dependent remodeling of skeletal muscle. Lactate dehydrogenase (LDH), an important enzyme involved in anaerobic glycolysis, catalyzes the interconversion of lactate and pyruvate critical for meeting rapid high-energy demands. The purpose of this study was to determine rat soleus LDH-A and -B isoform expression, mRNA abundance, and enzymatic activity at the onset of increased or decreased loading in the rat soleus muscle. The soleus muscles from male Sprague-Dawley rats were functionally overloaded for up to 3 days by a modified synergist ablation or subjected to disuse by hindlimb suspension for 3 days. LDH mRNA concentration was determined by Northern blotting, LDH protein isoenzyme composition was determined by zymogram analysis, and LDH enzymatic activity was determined spectrophotometrically. LDH-A mRNA abundance increased by 372%, and LDH-B mRNA abundance decreased by 43 and 31% after 24 h and 3 days of functional overload, respectively, compared with that in control rats. LDH protein expression demonstrated a shift by decreasing LDH-B isoforms and increasing LDH-A isoforms. LDH-B activity decreased 80% after 3 days of functional overload. Additionally, LDH-A activity increased by 234% following 3 days of hindlimb suspension. However, neither LDH-A or LDH-B mRNA abundance was affected following 3 days of hindlimb suspension. In summary, the onset of altered loading induced a differential expression of LDH-A and -B in the rat soleus muscle, favoring rapid energy production. Long-term altered loading is associated with myofiber conversion; however, the rapid changes in LDH at the onset of altered loading may be involved in other physiological processes.

Nandrolone Deconate??s Effect on Lactate Dehydrogenase Expression in Aged Fast-Type Skeletal Muscle

1399 Aging is associated with a decreased plantaris muscle mass and glycolytic and oxidative enzyme activity. Lactate Dehydrogenase (LDH) is an important regulatory enzyme involved in anaerobic glycolysis, and catalyzes the interconversion of lactate to pyruvate. The mechanisms inducing differential LDH expression with advanced age are not well understood. PURPOSE: This study's purpose was to examine aging and anabolic steroid administrations effect on LDH activity and expression in fast-type skeletal muscle. METHODS: Eight 25-month old male Fischer 344XF1 Brown Norway rats were used in this study. Four rats were administered nandrolone deconate (ND), an anabolic steroid with a long biological half-life, every seven days and sacrificed at the end of 4 weeks. RESULTS: LDH-A and LDH-B activity was 71% (p< .001) and 69% (p< .001) lower in aged plantaris muscle compared to young plantaris muscle. LDH-A and LDH-B activity increased 31% (p< .01) and 33% (p< .01) respectively following ND administration when compared to age-matched controls not receiving steroid. LDH protein expression shifted towards the faster LDH isoforms following ND administration in aged plantaris muscle when compared to age-matched controls not receiving ND. CONCLUSIONS: In summary, age induced differential expression of LDH A&B in the rat plantaris muscle, which is partially reversed by ND administration. Supported by a grant from NSF/EPSCOR and NIH/BRIN

Nandrolone Deconateʼs Effect on Lactate Dehydrogenase Expression in Aged Fast-Type Skeletal Muscle

1399 Aging is associated with a decreased plantaris muscle mass and glycolytic and oxidative enzyme activity. Lactate Dehydrogenase (LDH) is an important regulatory enzyme involved in anaerobic glycolysis, and catalyzes the interconversion of lactate to pyruvate. The mechanisms inducing differential LDH expression with advanced age are not well understood. PURPOSE: This study's purpose was to examine aging and anabolic steroid administrations effect on LDH activity and expression in fast-type skeletal muscle. METHODS: Eight 25-month old male Fischer 344XF1 Brown Norway rats were used in this study. Four rats were administered nandrolone deconate (ND), an anabolic steroid with a long biological half-life, every seven days and sacrificed at the end of 4 weeks. RESULTS: LDH-A and LDH-B activity was 71% (p< .001) and 69% (p< .001) lower in aged plantaris muscle compared to young plantaris muscle. LDH-A and LDH-B activity increased 31% (p< .01) and 33% (p< .01) respectively following ND administration when compared to age-matched controls not receiving steroid. LDH protein expression shifted towards the faster LDH isoforms following ND administration in aged plantaris muscle when compared to age-matched controls not receiving ND. CONCLUSIONS: In summary, age induced differential expression of LDH A&B in the rat plantaris muscle, which is partially reversed by ND administration. Supported by a grant from NSF/EPSCOR and NIH/BRIN

LACTATE DEHYDROGENASE EXPRESSION AT THE ONSET OF ALTERED LOADING IN THE RAT SOLEUS MUSCLE

Both functional overload and disuse atrophy induce a phenotype shift in the primarily slow phenotype rat soleus toward a fast phenotype. Lactate Dehydrogenase (LDH) is an important regulatory enzyme involved in anaerobic glycolysis and catalyzes the interconversion of lactate to pyruvate. The mechanisms inducing differential LDH expression at the onset of increased or decreased loading is not well understood. PURPOSE The purpose of this study was to determine LDH A&B mRNA and protein concentrations at early time points of altered loading in the rat soleus muscle. METHODS Male Sprague-Dawley rat soleus muscle was functionally overloaded for 3 days by synergist ablation or subjected to disuse atrophy by hindlimb suspension for 3 days. LDH mRNA concentration was determined by northern blotting, and LDH isoenzyme composition was determined by zymogram analysis. RESULTS LDH-A mRNA abundance increased 69% (.4±1 vs. 1.3±3, p < 0.05) and LDH-B mRNA abundance decreased 31% (4.82±.56 vs. 3.3±45, p < 0.05) after 3 days of functional overload when compared to control rats. LDH protein expression demonstrated a shift from the slower LDH isoforms to the faster LDH isoforms. There were no changes observed in either LDH-A (2.16±29 vs. 2.27±.19) or LDH-B (1.97±34 vs. 2.08±.21) mRNA abundance following 3 days of hindlimb suspension. CONCLUSIONS In summary, loading induces differential expression of LDH A&B in the rat soleus muscle, which is related to whether loading activity is increased or decreased. Supported by a grant from the South Carolina Space Grant Consortium and ACSM/NASA.