Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in Tissue

Summary

We describe a protocol for mapping the spatial distribution of enzymatic activity for enzymes that generate nicotinatmide adenine dinucleotide phosphate (NAD(P)H) + H+ directly in tissue samples.

Abstract

Mapping enzymatic activity in space and time is critical for understanding the molecular basis of cell behavior in normal tissue and disease. In situ metabolic activity assays can provide information about the spatial distribution of metabolic activity within a tissue. We provide here a detailed protocol for monitoring the activity of the enzyme lactate dehydrogenase directly in tissue samples. Lactate dehydrogenase is an important determinant of whether consumed glucose will be converted to energy via aerobic or anaerobic glycolysis. A solution containing lactate and NAD is provided to a frozen tissue section. Cells with high lactate dehydrogenase activity will convert the provided lactate to pyruvate, while simultaneously converting provided nicotinamide adenine dinucleotide (NAD) to NADH and a proton, which can be detected based on the reduction of nitrotetrazolium blue to formazan, which is visualized as a blue precipitate. We describe a detailed protocol for monitoring lactate dehydrogenase activity in mouse skin. Applying this protocol, we found that lactate dehydrogenase activity is high in the quiescent hair follicle stem cells within the skin. Applying the protocol to cultured mouse embryonic stem cells revealed higher staining in cultured embryonic stem cells than mouse embryonic fibroblasts. Analysis of freshly isolated mouse aorta revealed staining in smooth muscle cells perpendicular to the aorta. The methodology provided can be used to spatially map the activity of enzymes that generate a proton in frozen or fresh tissue.

Introduction

Understanding the locations within tissues in which enzymes have high or low activity is essential for understanding development and physiology. Transcript or protein levels are often used as surrogates for enzymatic activity. While such studies can be informative, they do not provide information that can be critical for determining an enzyme's activity, such as post-translational modifications, the presence of protein complexes, or the enzyme's subcellular localization. When enzymatic activity is directly measured, it is often monitored in homogenized protein lysates that no longer contain information about individual cells within the mixture or the spatial distribution of the cells with high or low activity within a tissue.

We provide here a detailed protocol for mapping the spatial distribution of enzymatic activity within a tissue sample. The methodology is based on earlier studies demonstrating that tetrazolium salts can be used to localize the activity of dehydrogenases, reductases, and oxidases in frozen tissue¹. With these methods, a water-insoluble formazan is formed when protons are transferred to a tetrazolium salt^2,3. Glucose-6-phosphate dehydrogenase generates NADPH and a proton, and has been detected with tetrazolium activity. Glucose-6-phosphate dehydrogenase has been monitored in in European flounder hepatocytes⁴, in alveolar type 2 cells of the lungs⁵ and nephrons of the kidney⁵. Tetrazolium salts have also been used to monitor transketolase activity in frozen tissue⁶. A similar approach was recently used to monitor the activity of multiple dehydrogenases in the same tissue on adjacent slides⁷.

We describe here a method to use tetrazolium salts to monitor the spatial distribution of lactate dehydrogenase activity (Figure 1). Lactate dehydrogenase can convert pyruvate generated by glycolysis to lactate, and the reverse reaction. Lactate dehydrogenase activity is consequently an important determinant of pyruvate's entrance into the tricarboxylic acid cycle versus its secretion as lactate. Lactate levels in the blood are often used to diagnose a range of diseases, including cancer^8,9,10, because it can signal that illness or injury has damaged cells and the enzyme has been released.

There are four lactate dehydrogenase genes: LDHA, LDHB, LDHC, and LDHD¹¹. LDHA and LDHB are thought to have arisen from duplication of an early LDHA gene¹². LDH is active as a tetramer and LDHA and LDHB can form homotetramers and heterotetramers with each other. LDHA is reported to have higher affinity for pyruvate, while LDHB is reported to have higher affinity for lactate, and to preferentially convert lactate to pyruvate¹³. The LDHA promoter contains binding sites for the HIF1α, cMYC and FOXM1 transcription factors¹¹. In addition, like many other glycolytic enzymes^14,15, LDH can be modified by post-translational modifications. Fibroblast growth factor receptor 1 can phosphorylate LDHA at Y10, which promotes tetramer formation, or Y83, which promotes NADH substrate binding¹⁶. LDHA can also be acetylated¹⁷. For these reasons, a complete understanding of LDH activity requires monitoring not only LDH protein levels, but the enzyme's activity as well.

In addition to the method we present here, other approaches have been used to monitor lactate dehydrogenase activity. Lactate dehydrogenase activity can be monitored spectrophotometrically in homogenized protein lysate. The generation of NADH as lactate is converted to pyruvate can be measured based on absorbance at 340 nm, while the disappearance of NADH can be monitored as pyruvate is converted to lactate¹⁸. Lactate dehydrogenase activity has also been monitored with magnetic resonance imaging (MRI). ¹³C- pyruvate can be administered and the conversion of pyruvate to lactate can be monitored as the ratio of [1-¹³C]lactate/[1-¹³C]pyruvate. Elevated ratios of [1-¹³C]lactate/[1-¹³C]pyruvate have been observed in cancer tissue¹⁹. While MRI-based approaches can provide information on lactate dehydrogenase activity in normal and disease tissues, the methodologies do not have the resolution needed to determine the activity level in specific cells. The methodology provided here can provide information on lactate dehydrogenase activity in tissue areas and even in single cells.

Using in situ activity assays, we found that the activity of lactate dehydrogenase is high in the hair follicle stem cells of mouse skin²⁰. We also used the method to monitor the activity of lactate dehydrogenase in cultured embryonic stem cells and found the activity is higher in the stem cells than the feeder layer. Finally, we monitored the activity of lactate dehydrogenase in fresh mouse aorta and observed staining in smooth muscle cells. We describe here a detailed protocol for monitoring lactate dehydrogenase activity in frozen mouse skin.

Protocol

All experiments described were approved by the Animal Care Committee at the University of California, Los Angeles.

1. Generate Slides of Frozen Mouse Skin

1. Euthanize mice in accordance with institution policy.
   Note: Follow institutional policy for protective clothing. All protocols involving animals must be approved by the Institutional Animal Care Committee.
2. Remove the mouse’s hair with an animal hair trimmer.
3. Make incisions in the skin using scissors. Using forceps, lift the skin away from the mouse. Using scissors, cut the skin section away.
4. Fill the cryomold with freezing reagent compound (see Table of Materials).
5. Place skin sections into filled cryomolds using needle-nosed forceps. Orient skin so that tissue slices will generate a cross section of the skin from the epidermis to the dermis, hypodermis and muscle (Figure 2).
   Note: If possible, mount experimental and control mice together in the same cryomold so they can be sectioned onto the same slide and processed together. Take care not to create bubbles.
6. Place cryomold on the flat surface of a block of dry ice in an ice bucket and let freeze. Continue to observe the orientation of the skin. Adjust if necessary.
   Note: Wear cryogenic gloves when handling dry ice.
7. Transfer cryomolds into a -80 °C freezer for storage. Samples can be maintained in the freezer for approximately 3 months without significant loss of enzymatic activity. Do not let frozen sections dry out.
8. Using a cryostat at -20 °C, slice tissue to create sections 7 - 10 µm thick for the best skin morphology²¹.

2. Preparing Slides for Staining

1. Establish the set of slides to be tested. Include a control slide on which NAD will be withheld and another control slide on which the substrate, lactate, will be withheld. Include a positive control slide from a frozen tissue block previously investigated.
2. Briefly fix slides containing skin sections with 4% formalin for 5 min either by pipetting 1 mL of 4% formalin onto the slide, or when processing multiple slides, by dipping slides into a container containing 4% formalin.
   Note: The fixation will ensure the skin does not peel off from the slides during the following procedures and preserve the skin morphology.
   Caution: Formalin is a carcinogen and hazardous; wear gloves, do not let it touch your skin and perform this step in a chemical fume hood.
3. Wash slides with at least 1 mL phosphate buffered saline, pH 7.4, per slide either by pipetting or dipping.

3. Preparing Staining Solution

1. Vortex together reagents for lactate dehydrogenase activity staining (50 mM Tris pH 7.4, 750 µM NADP, 80 µM phenazine methosulfate (PMS), 600 µM nitrotetrazolium blue chloride, and 30 mM lactate) in an appropriately sized tube depending on the amount of staining solution required.
   Note: 1 mL is sufficient to completely cover one slide.
2. Prepare a second solution in which all reagents are present except NAD. Prepare a third solution in which all reagents are present except lactate.

4. Incubate Slides in Staining Solution

1. Gently pipet the staining solution or control solutions onto the prepared slides covering the section in its entirety (1 mL is sufficient for one slide). If multiple slides are being processed together, dip all slides into the staining solution at the same time (make sure the container has enough solution to completely cover the tissue section).
2. Incubate the slides in a humidified environment at 37 °C in the dark. If staining solution is on top of the slide, place the slide in a humidified environment to prevent evaporation.

5. Monitor the Slides

1. Monitor the conversion of the staining solution from clear to blue by visual inspection. When the samples have reached the desired level of blueness, stop the reaction by removing the staining solution.
   Note: For skin, 10 min is sufficient. The time will depend on the organ and the enzymatic activity.
2. Rinse slides with phosphate buffered saline by pipetting (1 mL is sufficient for each slide) or dipping.
   Note: Any samples that will be compared to each other must be maintained in staining solution for the same amount of time.

6. Counterstain and mount

1. Pipet a counterstain onto the slides when the slides have reached an appropriate level of blueness.
   Note: Counterstains that turn the nuclei red or green will provide good contrast with the blue created by the formazan precipitant.
2. Mount with aqueous or non-aqueous mounting medium²². One to three drops (40 - 50 µL) of mounting medium is sufficient depending on the size of the cover slip.

7. Image slides and quantify

1. Image slides under a light microscope. Take photographs of experimental and control samples and all negative controls at 10X, 20X, and 40X magnification.
2. Determine the intensity of blue stain in different regions with image analysis software.

Results

We have previously reported results for in situ activity assays in mouse skin²⁰. As shown in Figure 3, we observed high levels of lactate dehydrogenase activity in the hair follicle stem cells at the base of the hair follicle when the procedures described above were followed. These findings were corroborated by fluorescence activated cell sorting of skin for hair follicle stem cells and confirming high lactate dehydrogenase ac...

Discussion

The method described here can be used to monitor the activity of lactate dehydrogenase or other metabolic enzymes that generate NADH or NADPH, in different cell types within a tissue or within different portions of a tissue over time. Lactate dehydrogenase is an important enzyme for understanding the biology of stem cells and tumors, and the ability to monitor lactate dehydrogenase activity in individual cells is likely to provide important insights into the function of this enzyme.

One import...

Materials

Name	Company	Catalog Number	Comments
Surgical instruments			For collecting skin from euthanized mice
Tissue-tek cryomold 25 mm x 20 mm x 5 mm	Fisher Scientific	NC9511236	For freezing mouse skin
Tissue-Tek O.C.T. compound	Fisher Scientific	NC9638938	For mounting cryomolds
Ice bucket	Fisher Scientific	07-210-106
Dry Ice
Polysine Adhesion Slide	Fisher Scientific	12-545-78
4% formalin	Fisher Scientific	23-245-684	Dilluted in water
phosphate buffered saline, pH 7.4
vortex
Tris base	Fisher Scientific	23-245-684
NAD	Sigma-Aldrich	N7004
Phenazine methosulfate	Sigma-Aldrich	P9625
Nitrotetrazolium blue chloride	Sigma-Aldrich	N6876
Lithium L-lactate	Sigma-Aldrich	L2250	Substrate
37°C incubator (or tissue culture incubator)
Braziliant! Counter stain	Anatech	861	Counter stain
Mounting medium	Vector Laboratories	H-5000
Cover slips for slides	Fisher Scientific	12-544D
Light microscope