There is no need to starve animals for mitochondria purification. It does not change properties of the organelle. All it potentially could do is affect substrate composition and availability which you can control much better by supplementing your buffers with those. Don't complicate you life unnecessary - there are plenty of complications you can't envision. Good luck!

Dear Jiang:

I have posted an excerpt from my book "Practical Mitochondriology", which answers most of your question. However, you should remember that mice are very small animals and therefore metabolic changes associated with feeding and starvation run much faster than in rats. Everything depends on what is your Goal. If simply want reproducible results, than letting mice to starve 6-7 hrs or overnight will bring animals to definite metabolic state of starvation. But the results will be more reproducible. It is another matter that liver mitochondria will not oxidize pyruvate since glycolysis is inhibited. They will though oxidize glutamate. The common practice to use succinate + rotenon is obsolete and should not be used. If you want simply to study respiratory activity per se, than use succinate 5 mM +glutamate 5 mM + malate 2 mM. Liver mitochondria will respire well in all metabolic states. however, the latter substrate mixture will not reflect the actual metabolic state of the liver. Liver consumes fatty acids. In general, I say that to study properly liver mitochondria is much more difficult than, say, brain or heart mitochondria. Some more details you can find in my book, which is posted in Research Gate account and is free to downloading.

Good luck,

Alexander Panov.

Isolation of rat and mouse liver mitochondria.

Most of our current knowledge about mitochondria was obtained in experiments with liver mitochondria. The major reason for this was the simple fact that just using one animal, in a short time the researcher had plenty of mitochondria for experimentation. However, the indiscriminate usage of methods suitable for liver mitochondria for mitochondria from other organs, often led to erroneous results. Take, for example, the usage of succinate + rotenone as a respiratory substrate.

Close relationships between the functions of an organ and the mitochondria.

One of the major ideas, which I will try to unveil in this Manuscript, is a seemingly “simple” idea that mitochondrial structure and functions closely relate to the structure and functions of the organ and cells from which these mitochondria originated. Although this idea seems obvious, to this day many researchers think that there is no principal difference between brain, heart or liver mitochondria. And there is a belief, that liver mitochondria are so simple. In fact, after I worked for many years with mitochondria from different organs and cell types, I came to conclusion that of all mitochondria, the liver mitochondria are the most difficult to study. They are in many ways unique and distinct from say, heart, brain or kidney mitochondria. And this is because metabolically and functionally liver stands apart from the energy metabolism of any other organ.

The methods of isolation and measuring the respiratory activities of liver mitochondria were generally considered as simple and fundamental. However, looking back at the old papers, published during 60s and 70s, you can see that the quality of mitochondria isolated even at the leading laboratories was usually not good. There were many pitfalls unknown at the time, which I will explain gradually in the course of this Manual. In this chapter I will share my experiences and thoughts regarding the liver mitochondria.

The metabolic properties of liver mitochondria strongly depend on the metabolic state of the liver.

This is in contrast to the heart or brain, where properties of mitochondria do not depend so much on the metabolic state of the body or liver.

According to the British Liver Trust, liver has more than 500 functions. The most important of them are:

Processing digested food from the intestine;

Controlling levels of fats, amino acids and glucose in the blood, and thus maintaining the metabolic (substrate) homeostasis for other organs and tissues;

Neutralizing and destroying drugs and toxins;

Making enzymes and proteins, which are responsible for most chemical reactions in the body, for example those involved in blood clotting and repair of damaged tissues, blood serum albumin;

Manufacturing bile;

Breaking down food and turning it into energy;

Storing iron, vitamins and other essential chemicals;

Manufacturing, breaking down and regulating numerous hormones including sex hormones;

Production of large quantities of purine nucleotides for bone marrow and neuronal tissue;

Combating infections in the body;

Clearing the blood of particles and infections including bacteria; etc.

In order to maintain in the blood the metabolic homeostasis for other organs and tissues, the liver must convert one type of metabolites into other metabolites. For example, carbohydrates can be converted into fats and amino acids; fatty acids and amino acids can be converted into glucose. There are several metabolic states of the liver, the appearance of which depends on the type of food and the time passed after food consumption. Of course, the metabolic properties of the liver will depend also what type of animal you study: herbivorous (rabbits), carnivorous, or omnivorous (human, rats, mice and pigs). It should be also kept in mind that everything, what enters the blood from the stomach and gut, goes directly into the liver through the portal vein. In an animal, such as a rat or a mouse, we can distinguish metabolically several states, depending on the time passed after the last food consumption: i) the absorption period, ii) the fed state, and iii) the starved metabolic state, which have different degrees of metabolic changes depending on the longevity of fasting.

1. The Absorption period. During absorption period the metabolites (carbohydrates, fatty acids, amino acids), derived by digestion of food, are delivered through the portal vein to the liver cells. Fatty acids become activated to acyl-CoAs, carbohydrates are phosphorylated, and amino acids become converted into proteins or other metabolites. In rodents (rats & mice), the liver mitochondria are capable to transport and activate fatty acids inside mitochondria in the matrix without participation of carnitine. Activation of fatty acids results in formation of adenosine monophosphate:

Fatty acyl + ATP + CoA  Fatty-acyl-CoA + AMP

Thus, AMP can be accumulated in the cytosol and the mitochondrial matrix. Therefore, if the liver mitochondria were isolated within 1-2 hr post feeding of the animal, the rate of oxidative phosphorylation will be very low because of the low content in the matrix of exchangeable mitochondrial adenine nucleotides ([ADP] + [ATP]). We have shown that liver mitochondria isolated during absorption period contain large amount of AMP. The AMP content in the mitochondrial matrix strongly depends on the type of the diet. If the food was rich in fats, the level of AMP will be high. Because long-chain acyl-CoAs are powerful inhibitors of adenine nucleotide translocase (ANT), oxidative phosphorylation will be inhibited and the hepatocyte’s [ATP]/[ADP] and the phosphate potential ([ATP]/[ADP] + [Pi]) ratios also will be low.

Figure 3.5A shows that liver mitochondria (LM) from the fed rats at the absorption period showed a remarkable inhibition of succinate oxidation both in the state 4 (resting respiration) and state 3 (phosphorylating respiration). However, upon addition of uncoupler the state 3U was similar to that of LM from the fasted rats. Consecutive additions of ADP gradually increased the rates of the state 3 respiration. But even after the 5th addition, the state 3 rate remained inhibited by 40-50% as compared with the state 3U. Both carnitine (Fig. 3.5B) and BSA (Fig. 3.5C) had no effect on the state 3 respiration of LM from rats at the absorbtion period. This is because limitation of the state 3 respiration was associated with the low exchangeable pool of adenine nucleotide in the matrix ([ADP] + [ATP]) due to high concentration of AMP ([AMP]).

In the experiment in vitro, the rate of oxidative phosphorylation can be restored by incubation of mitochondria with α-ketoglutarate, which produces ATP during the substrate phosphorylation step and restores the exchangeable pool of adenine nucleotides in the transphosphorylation reaction: ATP + AMP  2ADP. (Panov A.V., Konstantinov Yu.M., Lyakhovich V.V. The possible role of palmitoyl-CoA in the regulation of the adenine nucleotide transport in mitochondria under different metabolic states. J. Bioenergetics.,7 (1975) 75-85).

2. The Fed metabolic state. 2-3 hours after feeding, the isolated liver mitochondria showed the highest rates of oxidative phosphorylation, low State 4 respiration and therefore the highest respiratory control ratios (RCR). This is the so called “fed” metabolic state of the liver. In this metabolic state the liver had high content of carbohydrates, therefore the pyruvate dehydrogenase complex was not inhibited, and the level of long-chain acyl-CoAs esters was low (Panov et al., 1975, J. Bioenergetics, 7, 75-85). In the “fed” metabolic state, the liver mitochondria readily oxidized pyruvate + malate, citrate, isocitrate and α-ketoglutarate, as well as glutamate, or succinate.

The longevity of the “fed” metabolic state is different for different species of rats (Panov et al., 1991, Int. J. Biochem. 23 (9), 875-879), as well as between rats and mice. The smaller the animal, the shorter is the fed metabolic state. For different strains of rats the longevity of the fed state may vary from 4 to 12 hours passed from the time of the last feeding (Panov et al., 1991). The time of transition from the fed to the starved metabolic state was characteristic for each strain of rats. In mice, the transition is much faster than in rats, because they are much smaller animals (the Kleiber’s Law). Though, I never estimated the exact transition times for mice.

Figure 3.5. Influence of carnitine and BSA on respiration of mitochondria from the livers of rats 1.0 hour after feeding (absorption period). Note: in the paper this figure was erroneously indicated as the “fed” state.

The incubation medium contained: 100 mM KCl, 50 mM sucrose, 20 mM Tris-HCl (pH 7.4), 5 mM phosphate, 0.5 mM MgCl2, 3 µg rotenone. Additions: 5 mM succinate, 150 µM ADP, 0.5 µM CCCP (chlorocarbonylcyanide). (A) Control; (B) 0,5 mM carnitine; (C) preincubation with 0.1% BSA. (From: Panov et al., J. Bioenergetics, vol. 5, 75-85, 1975).

Information: According to the Kleiber's Law, the metabolic rate for all organisms follows exactly the 3/4 power-law of the body mass, i.e., q0 = M3/4. It holds well from the smallest bacterium to the largest animal. The relation remains valid even down to the individual components of a single cell such as the mitochondrion, and the respiratory complexes (References: Wang et al. 2001; Rau 2002).

3. The Starved metabolic state. The fed state is followed by the “starved” metabolic state, when the liver switches from oxidation of carbohydrates to predominant oxidation of fatty acids and gluconeogenesis. In this metabolic state, hepatocytes accumulate long-chain acyl-CoAs, which inhibit oxidative phosphorylation and slightly uncouple mitochondria due to inhibition of the mitochondrial ANT and fixation of ANT in the “c” conformational state, which increased the conductivity for H+ and K+ (Panov et al., 1975, 1980).

Figure 3.6. Influence of carnitine and BSA on respiration of liver mitochondria from starved (12 hrs) rats. (A) Control; (B) 0,5 mM carnitine; (C) preincubation with 0.1% BSA. (From: Panov et al., J. Bioenergetics, vol. 5, 75-85, 1975).

There are large variations in the mitochondrial activity due to variations in the longevity of the absorption and fed metabolic states, which also depend on the type of a diet and the species metabolic phenotype. Therefore most researchers isolate liver mitochondria from the animals starved overnight that is when the animals are far into the “starved” metabolic state.

In the starved metabolic state, the mitochondrial pyruvate dehydrogenase complex is inhibited, therefore the tricarboxylic acids (TCA) cycle does not function normally, and the liver mitochondria do not oxidize pyruvate, citrate, and poorly oxidize α-ketoglutarate. The only remaining substrates for respiration are glutamate + malate, succinate and fatty-acids. To study oxidation of fatty acids by liver mitochondria is not a simple issue. Therefore, traditionally researchers, working with the liver mitochondria, used glutamate + malate or, more commonly, succinate + rotenone as the substrates. Oxidation of succinate in the absence of rotenone becomes gradually inhibited by accumulating oxaloacetate. In order to avoid this inhibition the researchers used (and still use) succinate + rotenone. This is highly unphysiological and misleading. It is much more correctly to utilize a mixture of succinate 5 mM + glutamate 5-10 mM + malate 2 mM. In this case respiration of LM from the fasted animals will be at maximum and linear in all mitochondrial metabolic states.

Figures 3.6A and 3.6B show that in the presence of carnitine there was a significant increase in the rate of the state 3 respiration. This is because carnitine removed long chain acyl-CoAs, which are powerful inhibitors of ADP/ATP carrier (ANT).

The increase of the state 4 respiration in the presence of carnitine may be explained by the fact that transport of acyl-carnitines is the energy-dependent process, but mostly because acyl-CoA dehydrogenase feeds electrons to the membrane’s pool of CoQ, exactly as does Complex II during oxidation of succinate. As a result, the energy-dependent reverse electron transport and ROS production are stimulated and this increases the state 4 respiration.

Figure 3.6C shows that addition of defatted BSA into incubation medium caused a slight decrease in the state 4 respiration rate. BSA had no influence on the state 3 respiration. This indicates that inhibition of the state 3 respiration in LM from fasted rats was not caused by fatty acids, but rather by l.c. acyl-CoAs, which have much higher binding affinity to ANT than to BSA. In addition, there is evidence that fatty acids do not uncouple rat liver mitochondria (Panov et al. 2010).

I used to isolate mitochondria from the livers of rats that were sacrificed 4-6 hours after feeding that is in the fed metabolic state. For this, we controlled feeding of the animals by providing food twice a day for 1 hour at 8 AM and 8 PM. Rat liver mitochondria (RLM) from the fed rats oxidized pyruvate, and also oxidized succinate (rotenone not added) in State 4 and State 3 without noticeable inhibition by oxaloacetate (OAA). The OAA-dependent inhibition of succinate oxidation developed only after addition of an uncoupler. Subsequent addition of glutamate accelerated the State 3U until the oxygen was fully consumed.

It should be kept in mind that liver mitochondria contain less respiratory complexes as compared with the heart or brain mitochondria. In addition, for the liver mitochondria maintenance of the high ATP/ADP level in the cytosol or the high rate of ATP synthesis are not the major functions. In hepatocytes many key ATP-consuming reactions in the anabolic or anaplerotic (gluconeogenesis, synthesis of purines), and catabolic (urea synthesis) metabolic pathways occur in the mitochondrial matrix. Furthermore, liver and liver mitochondria can withstand up to 45 minutes of total ischemia if the animal was in the fed metabolic state, and 30 minutes, if the animal fasted 12 hrs before the experiment.

Figure 3.7. The electron microscopic picture of a liver cell. This picture was given to me in 1971 by Lars Ernster, my Scientific Father. It is a classic picture of a hepatocyte obtained by R. Gustafsson.

It should be also remembered that metabolic features of the liver cells and liver mitochondria are subjects to strong species and strain variations within the same species.

Figure 3.7 shows that in hepatocytes, mitochondria are predominantly located close to the nucleus, which reflects the high synthetic activities in the liver. For anabolic synthetic reactions, which occur in hepatocytes, the energetic value of ATP or NAD(P)H are much more important than the rates of ATP and NAD(P)H production. The energetic values are determined by the ratios: [ATP]/[ADP] ratio; [ATP]/ [ADP] + [Pi] - phosphate potential, which reflect oxidative phosphorylation; the Energy Level, or the Energy Potential: [ATP] + ½[ADP]/ [ATP] + [ADP] + [AMP], which depends on both oxidative phosphorylation and adenylate kinase reaction (Atkinson D.E. 1968, 1977, 1978).

Since liver has high regenerative potential and contains young hepatocytes with young mitochondria, as well as older mitochondria at different stages of fusion/fission, the total mitochondrial fraction of the liver can be subdivided into several fractions according to their size and density. Some authors identified 5 fractions. From the practical point of view, you have to discriminate the following 4 fractions: very heavy, heavy, middle and light fractions.

Very heavy liver mitochondria are large; probably because of fusion of several smaller mitochondria (you can see one on Fig. 3.7 in the left low corner). This fraction will be partly sedimented together with the tissue debris at 900-1000 g. The heavy fraction of large mitochondria will be sedimented between 1200 – 3000 g, the middle fraction is the largest in the quantity, will be sedimented up to 9000 g. Mitochondria that are sedimented at accelerations higher than 9000 g belong to the light fraction and contain, beside smaller mitochondria, lysosomes and peroxisomes. Lysosomes and peroxisomes, if they are present, cause mitochondria loose integrity sooner than mitochondria in the middle fraction that do not contain these subcellular particles.

Hi everyone,

Thank you so much for your detailed explanation. Plenty to learn. I have got functional liver mitochondria with your comments in mind. Thanks a lot!