SIRT3-dependent GOT2 acetylation status affects the malate–aspartate NADH shuttle activity and pancreatic tumor growth

The malate–aspartate shuttle is indispensable for the net transfer of cytosolic NADH into mitochondria to maintain a high rate of glycolysis and to support rapid tumor cell growth. The malate–aspartate shuttle is operated by two pairs of enzymes that localize to the mitochondria and cytoplasm, glutamate oxaloacetate transaminases (GOT), and malate dehydrogenases (MDH). Here, we show that mitochondrial GOT2 is acetylated and that deacetylation depends on mitochondrial SIRT3. We have identified that acetylation occurs at three lysine residues, K159, K185, and K404 (3K), and enhances the association between GOT2 and MDH2. The GOT2 acetylation at these three residues promotes the net transfer of cytosolic NADH into mitochondria and changes the mitochondrial NADH/NAD⁺ redox state to support ATP production. Additionally, GOT2 3K acetylation stimulates NADPH production to suppress ROS and to protect cells from oxidative damage. Moreover, GOT2 3K acetylation promotes pancreatic cell proliferation and tumor growth in vivo. Finally, we show that GOT2 K159 acetylation is increased in human pancreatic tumors, which correlates with reduced SIRT3 expression. Our study uncovers a previously unknown mechanism by which GOT2 acetylation stimulates the malate–aspartate NADH shuttle activity and oxidative protection.     

Dietary restriction and triiodothyronine (T3) regulation of malate-aspartate shuttle enzymes in the liver and kidney of mice

The activities of malate-aspartate shuttle enzymes viz., cytosolic and mitochondrial aspartate aminotransferase (c- and m-AsAT) and malate dehydrogenase (c- and m-MDH) were measured in liver and kidney of ad libitum (AL) and dietary-restricted (DR) mice and also on triiodothyronine (T3) treatment. The results show that the activity (U/mg protein) of c-AsAT is increased significantly in liver and the activities of c-MDH and m-AsAT are increased significantly in kidney during DR. On T3 treatment, the activities of both the isoenzymes (c- and m-) of MDH and AsAT are increased significantly in the liver of AL- and DR-fed mice. In the kidney, m-MDH showed no effect by T3 treatment, however, c-MDH increased significantly in both AL- and DR-fed mice. In contrast, m-AsAT is increased significantly in the kidney in AL-fed mice, but was not affected in DR-fed animals. In vitro reconstitution of malate-aspartate shuttle showed a higher activity in the liver and kidney of DR-fed mice, as compared to AL-fed ones and also in the T3-treated mice, compared to untreated ones. These findings suggest that malate-aspartate shuttle enzymes are differentially regulated during DR in mice, in order to adapt to the metabolic need of liver and kidney. T3 potentially regulates the shuttle enzymes, albeit to a varying degree in the liver and kidney of AL- and DR-fed mice.     

Diapause development in Bombyx eggs in relation to ‘esterase A’ activity

Diapause is broken by hydrochloric acid treatment and also terminated by long chilling of eggs in the silkworm, Bombyx mori. One of esterases in silkworm eggs named ‘esterase A’ is closely related to diapause of this insect.Hydrochloric acid treatment of diapause eggs induced a prompt elevation of esterase A activity. The elevation was observed within 30 min after treatment. This HCl treatment effectively stimulated the eggs to hatch. This indicates that the increase of esterase A activity is correlated with an active resumption of morphogenesis.The question was examined of whether chilling also increases esterase A activity or not. It was found that chilling also caused an increase of esterase A activity. This increase occurred before the re-appearance of glycogen in eggs, which indicates the termination of diapause in this insect. In fact, the establishment of hatchability in chilled eggs was observed after esterase A activity has reached the maximum level. Thus the increase of esterase A activity could be regarded as associated with the termination of diapause per se but not with the subsequent process of post-diapause development. This change in esterase A activity was observed only in chilled diapause eggs and was not observed in diapause eggs without chilling and non-diapause eggs.These results suggest that the increase of esterase A activity during chilling may be a kind of activity that occurs during the diapause stage in preparation for resumption of morphogenesis or diapause development.     

Distributions of aspartate aminotransferase and malate dehydrogenase activities in rat retinal layers

Aspartate aminotransferase (AAT), an enzyme interconverting glutamate and aspartate, has been suggested to be a marker for glutamatergic and/or aspartatergic neurons. However, AAT, glutamate, and aspartate are also involved in cellular metabolism, e.g., the malate-aspartate shuttle. To investigate the extent to which AAT might be involved in these several functions in retina, the distribution of AAT activity in rat retinal layers was compared to that of malate dehydrogenase (MDH), an enzyme of aerobic metabolism proposed to be physically complexed with AAT in the malate-aspartate shuttle mechanism. The distribution of AAT activity in retinal layers closely paralleled that of MDH (correlation coefficient AAT versus MDH = 0.93). AAT activity was proportionately higher than MDH in the photoreceptor inner segments, containing a high density of mitochondria, and in the outer plexiform layer (OPL), containing photoreceptor terminals and bipolar and horizontal cell processes. The amount of total AAT activity in the inner segments related to the mitochondrial isoenzyme is almost twice that in the other layers tested, including the OPL. The correlation between AAT and MDH activities is consistent with AAT involvement in retinal energy metabolism, although other functions, such as neurotransmission, are possible.     

Characterization of shuttle mechanisms for the transport of reducing equivalents into mitochondria

The malate-aspartate, fatty acid, and α-glycerophosphate shuttles for the transport of reducing equivalents into mitochondria were reconstituted, using isolated hepatic mitochondria and the extramitochondrial components of the shuttles. Clofibrate and thyroxin increased, while propylthiouracil treatment decreased, the activity of mitochondrial α-glycerophosphate dehydrogenase. Despite these changes, the activity of the reconstituted α-glycerophosphate shuttle was similar in mitochondria from control rats and those from rats treated with clofibrate and propylthiouracil. There was an increase in the activity of the shuttle using mitochondria from thyroxin-treated rats. Rotenone caused 60–90% inhibition of this shuttle, suggesting that rotenone-sensitive NADH dehydrogenase participates in the pathway of oxidation of extramitochondrial hydrogen. Palmitate, oleate, and octanoate were equally effective in reconstituting a cyclic fatty acid shuttle. The shuttle was inhibited by various compounds affecting mitochondrial metabolism, including oligomycin, dinitrophenol, cyanide, rotenone, atractyloside, and α-bromopalmitate. Carnitine and several dicarboxylic and tricarboxylic acids which stimulate fatty acid elongation, augmented fatty acid shuttle activity. The malate-aspartate shuttle was inhibited by cycloserine, amino-oxyacetic acid, and hydrazine, and stimulated by pyridoxal phosphate, at the same concentrations which affected the activities of cytoplasmic and mitochondrial glutamic oxalacetic transaminase. This shuttle was inhibited by uncouplers, antimycin, azide, cyanide, rotenone, amobarbital, oligomycin, and several inhibitors of anion transport including iodobenzylmalonate and avenaciolide. The reconstituted shuttle is sufficiently active to provide about 70–80% of the oxalacetate required for maximal rates of gluconeogenesis. Extrapolations based on the rates of mitochondrial oxidation of acetaldehyde and the activity of the microsomal ethanol oxidizing system suggest that any one of the shuttles could account for the rate of ethanol metabolism in vitro by the alcohol dehydrogenase pathway.     

Characterization of the Kinetics of Cardiac Cytosolic Malate Dehydrogenase and Comparative Analysis of Cytosolic and Mitochondrial Isoforms

Because the mitochondrial inner membrane is impermeable to pyridine nucleotides, transport of reducing equivalents between the mitochondrial matrix and the cytoplasm relies on shuttle mechanisms, including the malate-aspartate shuttle and the glycerol-3-phosphate shuttle. These shuttles are needed for reducing equivalents generated by metabolic reactions in the cytosol to be oxidized via aerobic metabolism. Two isoenzymes of malate dehydrogenase (MDH) operate as components of the malate-aspartate shuttle, in which a reducing equivalent is transported via malate, which when oxidized to oxaloacetate, transfers an electron pair to reduce NAD to NADH. Several competing mechanisms have been proposed for the MDH-catalyzed reaction. This study aims to identify the pH-dependent kinetic mechanism for cytoplasmic MDH (cMDH) catalyzed oxidation/reduction of MAL/OAA. Experiments were conducted assaying the forward and reverse directions with products initially present, varying pH between 6.5 and 9.0. By fitting time-course data to various mechanisms, it is determined that an ordered bi-bi mechanism with coenzyme binding first followed by the binding of substrate is able to explain the kinetic data. The proposed mechanism is similar to, but not identical to, the mechanism recently determined for the mitochondrial isoform, mMDH. cMDH and mMDH mechanisms are also shown to both be reduced versions of a common, more complex mechanism that can explain the kinetic data for both isoforms. Comparing the simulated activity (ratio of initial velocity to the enzyme concentration) under physiological conditions, the mitochondrial MDH (mMDH) activity is predicted to be higher than cMDH activity under mitochondrial matrix conditions while the cMDH activity is higher than mMDH activity under cytoplasmic conditions, suggesting that the functions of the isoforms are kinetically tuned to their individual physiological roles.     

In vitro diapause substance in the silkworm, Bombyx mori

By means of in vitro studies, in which isolated suboesophageal ganglions of the Bombyx silkworm were cultured, it was shown that at least two kinds of substances are biosynthesized and exert independent effects on determination of diapause or non-diapause in silkworm eggs. They are referred to as the diapause and non-diapause substance, respectively. Whether diapause or non-diapause eggs are laid may depend upon the different quality of these substances.     

Changes in amino acid pools in the silkworm,Bombyx mori during embryonic life: Alanine accumulation and its conversion to proline during diapause

Alanine accumulated in silkworm eggs at the onset of diapause. When the eggs were kept at 4°C during diapause, this alanine was converted to glutamate, glutamine and especially proline. On resumption of development at 25°C after diapause, proline was used as an energy source for protein synthesis. In HCl-treated diapause eggs, which develop like non-diapause eggs, most amino acids showed similar developmental changes to those in eggs in resumption of embryogenesis after diapause. However, the proline level increased until the middle of embryonic development and then decreased. Continuous incubation of diapause eggs at 25°C after day 10 of oviposition caused a decrease in alanine with increases in glutamine and proline, while the levels of most other amino acids either decreased slightly or remained unchanged until day 80, when most eggs died. These results show that diapause eggs have a metabolic complex coupled with carbohydrate and amino acid metabolism inclusive of the 2-oxoglutarate-glutamate shuttle. Under conditions when embryogenesis proceeded, the level of phosphoethanolamine decreased rapidly.     

The function of redox shuttles during aerobic glycolysis in two strains of Ehrlich ascites tumor cells

The function of glycerophosphate and malate-aspartate shuttles during glucose metabolism in two strains of Ehrlich ascites tumor cells was evaluated by several experimental approaches. The activities of the enzymes involved in these shuttle systems were assayed in the cytosolic and mitochondrial compartments after cell fractionation by the digitonin method. The glycerophosphate shuttle can be ruled out because of the lack of relevant enzymatic activities, and the failure of glucose to increase rotenone-inhibited respiration. Analysis of glycolytic flux in the presence of aminooxyacetate indicates that the activity of malate-aspartate shuttle may be very low. Balance studies of glucose uptake and lactate production suggest the existence of other pathways for the reoxidation of cytosolic NADH, which are acetyl-CoA dependent. Estimation of citrate synthase and ATP citrate lyase, in addition to the observed high activity of malate dehydrogenase, suggests a malate-citrate shuttle.     

苹果酸-天冬氨酸穿梭关键酶和肺腺癌临床特征的相关性

目的:探讨苹果酸-天冬氨酸穿梭途径中的关键酶和肺腺癌临床特征的相关性。方法:首先从GEO数据库、TCGA平台中获取肺腺癌的转录组数据和相应的临床信息,通过非参数检验分析苹果酸脱氢酶1/2和天冬氨酸氨基转移酶1/2这四种关键酶在肿瘤组织和正常组织之间的表达差别,再进一步分析其和肺腺癌患者总生存期、人口学特征、TNM参数以及肿瘤恶性生物学标志物之间的关系。结果:肺腺癌中苹果酸脱氢酶1/2和天冬氨酸氨基转移酶1/2均呈高表达;天冬氨酸氨基转移酶2 (Glutamic-oxaloacetic transaminase 2,GOT2)和肺腺癌的生存概率有关,高表达GOT2的病人往往具有较短的总生存期;GOT2的表达和肺腺癌的人口学特征、TNM参数、临床分期均无明显统计学关联,与肿瘤恶性标志物PCNA呈显著正相关(r=0.2,P0.05)。结论:肺腺癌组织中苹果酸-天冬氨酸穿梭途径中关键酶GOT2呈高表达,并可能促进肺腺癌的发生和恶性进展。     

CARBOHYDRATE METABOLISM IN THE EGGS OF THE SILKWORM, BOMBYX MORI. I. ABSENCE OF PHOSPHOFRUCTOKINASE IN THE DIAPAUSING EGG

In the diapausing eggs of the silkworm, Bombyx mori , glycogen is rapidly converted to sorbitol and glycerol, and this conversion is reversed at termination of the diapause (C hino , 1958). To elucidate the pathway leading to this polyol formation and its regulatory mechanisms, enzymes concerning carbohydrate metabolism were surveyed in diapausing as well as in developing eggs of the silkworm.
Most of the enzyme activities concerning citric acid cycle are low at the beginning of the embryogenesis and during diapause, but increase at the later stages of the development. Making an exception, reduction rate of malate and fumarate was rather high from the onset of the embryonic development. Several glycolytic enzymes were also studied. Most remarkable fact is that phosphofructokinase activity could not be demonstrated in the diapausing and also in the early stages of the developing eggs. Other enzymes, viz. α-glycerophosphate dehydrogenase, aldolase, glyceraldehyde-3-phosphate dehydrogenase were detected from the beginning of the embryogenesis.
Absence of phosphofructokinase, together with the high activity in glucose-6-phosphate dehydrogenase, suggests that predominant pathway in carbohydrate metabolism in the early stages of embryogenesis and in the diapause period is by way of pentose phosphate pathway. This supposition is confirmed by the experiments using labeled glucose. Incorporation of the label into glycerol of the diapausing eggs was three to four fold when G-6-¹⁴C was injected into pupae as compared with the case of G-1-¹⁴C injection. The above experiments provide evidence supporting the theory that glycogen is converted into sorbitol and glycerol mostly by way of the pentose phosphate pathway in the diapausing eggs.     

Methotrexate: studies on cellular metabolism. IV. Effect on the mitochondrial oxidation of cytosolic-reducing equivalents in HeLa cells

The effect of methotrexate (MTX) on the mitochondrial oxidation of cytosolic-reducing equivalents in HeLa cells was studied. MTX inhibited (100 per cent) malate dehydrogenase activity, but no effect was observed on that of GOT. MTX (0.5 mM) inhibited (100 per cent) the activity of reconstituted enzymatic system MDH-GOT, probably as a consequence of inhibition of malate dehydrogenase activity. MTX decreased pyruvate production (54 per cent), demonstrating its inhibitory action on the malate-aspartate shuttle. Blockage of the malate-aspartate shuttle by MTX accounts for the decrease in cellular energetic gain. The results obtained are consistent with the view that in HeLa cells, as well as in other tumour cells, the transport of reducing equivalents from cytoplasmic NADH into the respiratory chain of mitochondria is via the malate-aspartate shuttle.     

Regulation of malate dehydrogenases from neonatal,adolescent, and mature rat brain

Since the malate-aspartate shuttle in brain has been shown to be closely linked to brain energy metabolism and neurotransmitter synthesis, the activity of MDH, one of the enzymes of the malateaspartate shuttle, was studied in cortical non-synaptic mitochondria (mMDH) and cytosol (cMDH) in 1–4 day, 18–20 day and 7–8 week old rats. The mean mMDH activity (nmol/min/mg protein) was 10,517±734 (mean±SEM), 8,882±241 and 10,323±561 and cMDH activity was 2,453±99, 4,673±152 and 6,821±205 in 1–4 day, 18–20 day and 7–8 week old rats, respectively. While cMDH activity increased with age (p<0.0001), mMDH activity showed no change. This study also determined if endogenous compounds, previously shown to alter malate metabolism, affected MDH activities. Lactate inhibited only cMDH activity, by a competitive mechanism. Oxaloacetate inhibited mMDH by partial non-competitive inhibition and cMDH by competitive inhibition. Alpha-ketoglutarate competitively inhibited both enzymes; however, the inhibition of mMDH activity was more pronounced than that of cMDH activity. Citrate inhibited mMDH via an uncompetitive mechanism and cMDH via a noncompetitive mechanism. The mechanisms of inhibition of mMDH and cMDH by each of the effectors were the same over the three ages. The results suggest mMDH and cMDH activities show a dissimilar developmental pattern and may be regulated differently by endogenous effectors. The greater sensitivity of mMDH, compared to cMDH, to certain effectors may be related to the dual role of mMDH in the tricarboxylic acid cycle and the malate-aspartate shuttle.These data were presented in part at the meeting of the Federation of American Societies for Experimental Biology in Atlanta, Georgia, April 1991. This work was performed in partial fulfillment of the requirements for the M.S. Degree in Nutritional Sciences (P.M.)     

即时浸酸显著降低滞育家蚕卵的还原型与氧化型谷胱甘肽比值

即时浸酸在阻止家蚕Bombyx mori卵滞育发动的同时, 显著提高了家蚕卵H₂O₂含量。还原型谷胱甘肽（reduced glutathione, GSH）与氧化型谷胱甘肽（oxidized glutathione, GSSG）的比值是一种氧化胁迫状态的动态指标。为了调查即时浸酸是否造成滞育家蚕卵氧化胁迫, 本研究利用分光光度法分别测定了滞育家蚕卵和5 min即时浸酸滞育家蚕卵中GSH和GSSG含量以及谷胱甘肽转移酶（glutathione-S-transferase, GST）活性。结果表明: 处理后24 h, 即时浸酸处理家蚕卵的总谷胱甘肽（GSH+2GSSG）含量、 GSH含量、 GSSG含量、 GSH/GSSG比值和GST活性分别相当于同期滞育家蚕卵的204%, 78%, 550%, 14%和97%。据此推测, 即时浸酸在阻止滞育发动的同时, 可能通过促进GSH氧化为GSSG, 而显著降低了GSH/GSSG比值, 使家蚕卵处于过氧化状态。     

Efficient strategies for changing the diapause character of silkworm eggs and for the germline transformation of diapause silkworm strains

Abstract To overcome the disadvantages of current silkworm Bombyx mori transgenic technology, such as costly and time‐consuming to maintain non‐diapause transgenic silkworms, we report here on the development of treatments for the germline transformation of diapause silkworm strains. Our results showed that HCl treatment within 3 h of oviposition was able to prevent the diapause of eggs from Japanese lineage diapause silkworm strains and was also suitable for germline transformation of the same strains. By incubating developing mother eggs from Chinese lineage diapause silkworm strains at 15°C (15°C‐IME), we were able to prevent the diapause of their daughter eggs; a similar strategy (15°C‐IMES) for the germline transformation of the same strains was that the mother eggs were incubated at 15°C, and the daughter eggs were then microinjected according to the conventional microinjection methods used for non‐diapause eggs. By combining temperature and light controls, the improved 15°C‐IMES strategy prevented diapause in daughter eggs, and also enabled the germline transformation of both Japanese and Chinese lineage diapause silkworm strains. Although each of the strategies developed here has advantages and disadvantages, we suggest that the 15°C‐IMES strategy is a good reference for the establishment of germline transformation technologies of other egg diapause insects. These new strategies for the efficient germline transformation of diapause silkworm strains are likely to improve the practical use of silkworm transgenic lines in sericulture and also highlight silkworm functional genomics research and its modeling.     

Metabolism of hydrogen peroxide between diapause and non‐diapause eggs of the silkworm,Bombyx Mori during chilling at 5°C

When diapause and non‐diapause eggs of the same bivoltine strain of Bombyx mori were chilled at 5°C for more than 30 days, the hatchability of diapause eggs increased while that of non‐diapause eggs decreased, respectively. To investigate the relationship between effects of chilling on the hatchability and the metabolism of hydrogen peroxide (H₂O₂), content of H₂O₂ and activities of superoxide dismutase (SOD), xanthine oxidase (XO), and catalase (CAT) between diapause and non‐diapause eggs were determined during the chilling at 5°C. The significant enhancement of H₂O₂ occurred prior to the quick increase of the hatchability in diapause eggs and coincided with the quick decline of the hatchability in non‐diapause eggs, respectively. Diapause eggs contained significantly higher H₂O₂ and XO activity and lower CAT activity compared to non‐diapause eggs. Our results showed that there were significant differences in the metabolism of H₂O₂ between diapause and non‐diapause eggs during chilling and that significant enhancement of H₂O₂ may be involved in the diapause termination of diapause eggs and the cell damage of non‐diapause eggs. © 2010 Wiley Periodicals, Inc.     

Changes in glutathione redox cycle during diapause determination and termination in the bivoltine silkworm,Bombyx moil

To explore whether glutathione regulates diapause determination and termina tion in the bivoltine silkworm Bombyx mori, we monitored the changes in glutathione redox cycle in the ovary of both diapanse and nondiapauseegg producers, as well as those in dia pause eggs incubated at different temperatures. The activity ofthioredoxin reductase （TrxR） was detected in ovaries but not in eggs, while neither ovaries nor eggs showed activity of glutathione peroxidase. A lower reduced glutathione/oxidized glutathione （GSH/GSSG） ratio was observed in the ovary of diapauseegg producers, due to weaker reduction of oxidized glutathione （GSSG） to the reduced glutathione （GSH） catalyzed by glutathione reductase （GR） and TrxR. This indicates an oxidative shift in the glutathione redox cy cle during diapause determination. Compared with the 25℃treated diapause eggs, the 5℃treated diapause eggs showed lower GSH/GSSG ratio, a result of stronger oxidation of GSH catalyzed by thioredoxin peroxidase and weaker reduction of GSSG catalyzed by GR. Our study demonstrated the important regulatory role of glutathione in diapause determination and termination of the bivoltine silkworm.     

A time-interval activation of esterase A4 by cold: Relation to the termination of embryonic diapause in the silkworm Bombyx mori

The inactive esterase A4 (Ease A4) purified from the diapausing eggs of the silkworm, Bombyx mori, was chilled in vitro. The enzyme activity was very low during the early chilling period and it was suddenly elevated at a certain time of the chilling (2 weeks or less after chilling), depending upon when the chilling period began, and was followed by a rapid fall. The sudden elevation of the Ease A4 activity in vitro is equivalent to that observed in vivo and is coincident with the chilling period, the latter being indispensable for diapause termination.Data are also presented that suggest that the cold-induced activation of the Ease A4 may result from an autonomous structural change of the enzyme molecule which proceeds gradually in the cold.     

Effects of L-malate on physical stamina and activities of enzymes related to the malate-aspartate shuttle in liver of mice

L-malate, a tricarboxylic acid cycle (TCA) intermediate, plays an important role in transporting NADH from cytosol to mitochondria for energy production and may be involved in the beneficial effects of improving physical stamina. In the present study, we investigated the effects of L-malate on the performance of forced swimming time and blood biochemical parameters related to fatigue - blood urea nitrogen (BUN), glucose (Glc), creatine kinase (CK),total protein (TP) and lactic acid (LA). To investigate the effects of L-malate on the malate-aspartate shuttle and energy metabolism in mice, the activities of enzymes related to the malate-aspartate shuttle were measured. L-malate was orally administered to mice continuously for 30 days using a feeding atraumatic needle. The swimming time was increased by 26.1 % and 28.5 %, respectively, in the 0.210 g/kg and 0.630 g/kg L-malate-treated group compared with the control group. There were no differences in the concentrations of Glc, BUN and TP between the L-malate-treated groups and the control groups. However, the levels of CK were significantly decreased in the L-malate-treated groups. The results predict a potential benefit of L-malate for improving physical stamina and minimizing muscle damage during swimming exercise. The activities of cytosolic and mitochondrial malate dehydrogenase were significantly elevated in the L-malate-treated group compared with the control group. These enzymatic activities may be useful indicators for evaluating changes affecting the malate-aspartate shuttle and energy metabolism in the liver of mice.     

低温冷藏提高家蚕滞育卵NAD含量和胞质苹果酸脱氢酶活性

为了调查5℃低温处理是否改变家蚕Bombyx mori卵滞育NAD代谢, 本研究利用HPLC和分光光度法测定了经25℃和5℃分别处理的滞育卵中NADH 含量、 NAD⁺含量、 乳酸脱氢酶（LDH）活性和胞质苹果酸脱氢酶（cMDH）活性。结果表明： 5℃处理的NAD（NADH + NAD⁺）含量和cMDH活性分别增加了106%和53%, 并且显著高于25℃处理（P< 0.01）; 但是两种处理的NADH/NAD⁺比值和LDH活性没有显著差异（P> 0.05）。据此推测, 5℃低温处理加强了家蚕滞育卵NAD⁺合成和再生能力。