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Editorial Hepatic clearance of advanced glycation end products (AGEs)—myth or truth? Dmitri Svistounov, Ba ˚rd Smedsrød * Department of Experimental Pathology, Institute of Medical Biology, University of Tromsø, N-9037 Tromsø, Norway See Article, pages 913-919 The liver sequesters a number of circulating macromol- ecular soluble and particular waste products. This blood clearance function is carried out by the cells that line the sinusoidal wall: (i) the resident liver macrophages, or the Kupffer cells (KC), and (ii) the liver sinusoidal endothelial cells (LSEC). The KC is tuned to phagocytic uptake of large particles and aggregates, whereas LSEC are specialized on clathrin mediated endocytosis of soluble macromolecules and colloids. Until now four major endocytosis receptors have been observed to mediate waste endocytosis in LSEC. The mannose receptor, the scavenger receptor (several types are expressed by LSEC; the hyaluronan/scavenger receptor, or Stabilin-2 [1] is uniquely expressed and functionally active in LSEC), the collagen a chain receptor, and the Fc-g receptor. The waste macromolecules that are cleared by these receptors in LSEC include (i) most types of connective tissue molecules that are constantly released to the circulation as a consequence of normal turnover processes throughout the body [2]; (ii) extracellular enzymes and products of platelet-mediated coagulation [3]; (iii) intra- cellular macromolecules (for instance lysosomal enzymes [4]); (iv) soluble IgG-immune complexes [5]; (v) native macromolecules that have been modified non-enzymatically by for instance oxidation (oxidized low density lipoprotein [6]) or glycation; (vi) foreign molecules (i.e. LPS [7]). The capacity of endocytosis in LSEC is impressive: some of the waste macromolecules are turned over in quantities of several grams per day in a normal adult human individual. The process of endocytosis in these cells is the most efficient known: endocytic receptors recycle between the plasma membrane and early endosomes with a half-life of a few seconds (most other cell types perform endocytosis with a receptor recycling half-life of minutes). Most of the waste substances that are destined for uptake via receptor- mediated endocytosis in LSEC exist for a very short time in the circulation (less than 1 min in rats). During the past three decades several groups have attempted to design experiments to study the fate of circulating advanced glycation end products (AGEs). Macromolecules modified in this way are present in normal individuals throughout life, and are found in higher concentrations in older people as well as in certain diseases, most typically diabetes. Available data suggest that AGEs are eliminated from the blood mainly by scavenger receptor mediated uptake in KC and LSEC [8]. Alternative hypotheses hold that the uptake is not necessarily in the liver, but in the kidneys. One line of research (hereafter referred to as ‘the in vitro approach’) makes use of in vitro generated AGEs, that can be labelled and chased after administration in vivo. Another approach (hereafter referred to as ‘the in vivo approach’) is based on the notion that chemical analysis of blood samples, with no prior administration of in vitro generated AGEs, represents the key to solve the problem [9,10]. According to the in vivo approach it would be sufficient to perform sensitive chemical analyses to check if and to what extent AGEs are removed by any given tissue. As discussed below these approaches have distinct strong and weak sides; neither of them represents a perfect approach to determine the anatomical site of uptake of circulating AGEs. In vitro generation of AGEs involves long time (weeks or months) incubation of protein with glucose or other AGEs precursors under aseptic conditions. The in vitro approach offers the possibility of labelling AGEs with high specific radioac- tivity, enabling very low amounts of AGEs to be chased in vivo after i.v. administration. The weak side of the in vitro approach is that we do not know to what extent AGEs prepared in vitro represent the ‘native’ in vivo generated Journal of Hepatology 41 (2004) 1038–1040 www.elsevier.com/locate/jhep 0168-8278/$30.00 q 2004 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jhep.2004.10.004 * Corresponding author. E-mail address: [email protected] (B. Smedsrød).

Hepatic clearance of advanced glycation end products (AGEs)—myth or truth?

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Editorial

Hepatic clearance of advanced glycation end products(AGEs)—myth or truth?

Dmitri Svistounov, Bard Smedsrød*

Department of Experimental Pathology, Institute of Medical Biology, University of Tromsø, N-9037 Tromsø, Norway

0168-8278/$30.00 q 2004 European Association for the

doi:10.1016/j.jhep.2004.10.004

* Corresponding author.

E-mail address: [email protected] (B. Smedsrød

See Article, pages 913-919

The liver sequesters a number of circulating macromol-

ecular soluble and particular waste products. This blood

clearance function is carried out by the cells that line the

sinusoidal wall: (i) the resident liver macrophages, or the

Kupffer cells (KC), and (ii) the liver sinusoidal endothelial

cells (LSEC). The KC is tuned to phagocytic uptake of large

particles and aggregates, whereas LSEC are specialized on

clathrin mediated endocytosis of soluble macromolecules

and colloids. Until now four major endocytosis receptors

have been observed to mediate waste endocytosis in LSEC.

The mannose receptor, the scavenger receptor (several types

are expressed by LSEC; the hyaluronan/scavenger receptor,

or Stabilin-2 [1] is uniquely expressed and functionally

active in LSEC), the collagen a chain receptor, and the Fc-greceptor. The waste macromolecules that are cleared by

these receptors in LSEC include (i) most types of connective

tissue molecules that are constantly released to the

circulation as a consequence of normal turnover processes

throughout the body [2]; (ii) extracellular enzymes and

products of platelet-mediated coagulation [3]; (iii) intra-

cellular macromolecules (for instance lysosomal enzymes

[4]); (iv) soluble IgG-immune complexes [5]; (v) native

macromolecules that have been modified non-enzymatically

by for instance oxidation (oxidized low density lipoprotein

[6]) or glycation; (vi) foreign molecules (i.e. LPS [7]).

The capacity of endocytosis in LSEC is impressive: some

of the waste macromolecules are turned over in quantities of

several grams per day in a normal adult human individual.

The process of endocytosis in these cells is the most efficient

known: endocytic receptors recycle between the plasma

membrane and early endosomes with a half-life of a few

seconds (most other cell types perform endocytosis with

Study of the Liver. Pub

).

a receptor recycling half-life of minutes). Most of the waste

substances that are destined for uptake via receptor-

mediated endocytosis in LSEC exist for a very short time

in the circulation (less than 1 min in rats).

During the past three decades several groups have

attempted to design experiments to study the fate of

circulating advanced glycation end products (AGEs).

Macromolecules modified in this way are present in normal

individuals throughout life, and are found in higher

concentrations in older people as well as in certain diseases,

most typically diabetes. Available data suggest that AGEs

are eliminated from the blood mainly by scavenger receptor

mediated uptake in KC and LSEC [8]. Alternative

hypotheses hold that the uptake is not necessarily in the

liver, but in the kidneys. One line of research (hereafter

referred to as ‘the in vitro approach’) makes use of in vitro

generated AGEs, that can be labelled and chased after

administration in vivo. Another approach (hereafter referred

to as ‘the in vivo approach’) is based on the notion that

chemical analysis of blood samples, with no prior

administration of in vitro generated AGEs, represents the

key to solve the problem [9,10]. According to the in vivo

approach it would be sufficient to perform sensitive

chemical analyses to check if and to what extent AGEs

are removed by any given tissue. As discussed below these

approaches have distinct strong and weak sides; neither of

them represents a perfect approach to determine the

anatomical site of uptake of circulating AGEs. In vitro

generation of AGEs involves long time (weeks or months)

incubation of protein with glucose or other AGEs precursors

under aseptic conditions. The in vitro approach offers the

possibility of labelling AGEs with high specific radioac-

tivity, enabling very low amounts of AGEs to be chased in

vivo after i.v. administration. The weak side of the in vitro

approach is that we do not know to what extent AGEs

prepared in vitro represent the ‘native’ in vivo generated

Journal of Hepatology 41 (2004) 1038–1040

www.elsevier.com/locate/jhep

lished by Elsevier B.V. All rights reserved.

D. Svistounov, B. Smedsrød / Journal of Hepatology 41 (2004) 1038–1040 1039

AGEs. One knows for certain that the AGE adducts that are

present in in vitro generated AGEs are also detected on

AGEs formed in vivo. But it is unlikely that the extent of

AGE modification is as high in the in vivo formed specimen

as in the in vitro generated molecules. Using the in vitro

approach it was found that i.v. administered AGEs are very

rapidly taken up in KC and LSEC [8].

The advantage of the in vivo approach is that only native

AGEs are measured. However, using only the in vivo

approach one will not be able to detect the most interesting

AGEs, namely those that are rapidly cleared from the

circulation. Even the most sensitive analytical tools

presently available will not be able to show significant

differences in the concentration of AGEs in blood samples

taken from the portal and hepatic veins, simply because the

speed and efficiency of uptake greatly exceed the rate of

AGEs formation. The speed of blood circulation must also

be considered: in average the recycling time for blood

through liver in humans is 3.6 min, meaning that all the

blood contents are monitored by the liver scavenger

receptors every few minutes. This would make it impossible

for AGEs modified to a ‘high physiological degree’ to

accumulate to a detectable level above the background in

the blood. Moreover, the natural formation of AGEs in the

blood is certainly slower than a few minutes. One of the

authors (P.J.T.) of the presently discussed article previously

used a 24 h incubation schedule to prepare CML-albumin

and methylglyoxal-derived hydroimidazolone-proteins (two

AGEs) with minimal degree of modification [11,12].

Understanding this dynamics is crucial for the appreciation

that AGEs modified to a ‘high physiological degree’ escape

detection due to (i) their very slow formation, (ii) their very

rapid uptake, and (iii) the very efficient blood recycling.

Using the in vivo approach, the high resolution analytical

tool LC-MS/MS has been used to determine the presence of

specified AGEs in peripheral and hepatic venous blood

(control human subjects), or portal venous and hepatic

venous blood (cirrhotic subjects) [13]. With this state-of-

the-art methodology the authors observed no or only minute

differences in the level and type of AGEs that enter and

leave the liver. From this, the authors conclude that liver

does not contribute to extraction of in vivo formed AGEs

from the blood. They also put forward a hypothesis that the

kidneys represent the major site of elimination of AGEs

from the blood. At first glance, this may seem like a

plausible interpretation. However, a closer look at the

premises makes it clear that more solid data is required for a

shift of the current paradigm of hepatic elimination of

AGEs.

The authors state that in vivo formation of proteins

highly modified by AGEs is unlikely considering the

kinetics of albumin glycation under physiological con-

ditions. If ‘highly modified’ means AGE-modification to the

same extent as in vitro modified AGEs prepared by

traditional methods [14], this statement by the authors

would be agreed upon by most AGE-researchers. There is a

general concensus that highly modified AGE-albumin

prepared in vitro is not a perfect model for studies of

AGEs turnover in vivo. Nevertheless, one has to ask the

following question: do in vivo formed AGEs have to be

modified to the same extent as the commonly used in vitro

highly modified AGE albumin in order to be recognized by

receptors for endocytic uptake? In fact, one of the authors

(P.J.T.) of the presently discussed article previously

reported that HSA minimally modified by methylglyoxal

(MGmin-HSA) (1.4–2.4 modified arginine residues per

molecule) is taken up by receptor-mediated endocytosis

and degraded by the monocytic cell line THP-1 [11]. The

degree of AGE-modification was indeed much lower in this

MGmin-HSA (1–2 arginines per albumin molecule) com-

pared with conventionally used in vitro formed highly

modified AGE-BSA (in a typical batch of highly modified

AGE-BSA 37 of 59 lysines and 10 of 23 arginines are

modified). Furthermore, it is logical to assume that LSEC,

which exhibit a higher endocytic activity and express more

scavenger receptors compared to monocytes, would rep-

resent a more efficient site of uptake of MGmin-HSA. The

authors reported previously that approximately 2% of total

HSA contains MG-derived imidazolon (MG-H1) in normal

control subjects [9]. Assuming a total content of 250 g of

albumin in the blood of a normal adult individual, 2% would

correspond to 5 g of MG-H1-albumin, which will be present

in the circulation at any time. There is another important

consideration to take into account when comparing AGE-

modification of protein in vitro and in vivo: due to the fact

that the same individual protein molecules are present

during the in vitro generation of AGE-albumin, each of the

albumin molecules present will bind AGE-adducts with the

same probability. In contrast, since the probability of AGE-

modification increases with increasing life time of any

individual protein in vivo, the generation of AGEs in vivo

will result in differently modified proteins, spanning from

‘young’, newly synthesized proteins containing no or very

few AGE-modifications, to ‘old’ proteins containing most

of the modifications. On this basis, one would come closer

to reality by assuming that only albumin molecules that

have existed for more than one half-life will carry all the

MG-H1 groups. Using this assumption, along with the result

published previously by P.J.T. [11], it follows that 4% of the

albumin molecules older than one half-life carry MG-H1.

Moreover, applying combinatorics analysis it can be

calculated that the probability for any one of these albumin

molecules to carry more than 1 MG-H1 is 5.9%. This means

that at any time 300 mg of albumin molecules in the

circulation of normal humans will have more than 1 MG-H1

residues. According to the paper by P.J.T. cited above [11],

this degree of modification is sufficient to bring about

receptor-mediated uptake in scavenger cells. Of note, this

calculation most likely represents an underestimation, since

it was based on only one type of AGE-modification. In

fact, 12 different AGE-species are presently known, and it

is known that formation of AGEs can results in products

D. Svistounov, B. Smedsrød / Journal of Hepatology 41 (2004) 1038–10401040

that contain several AGE-adducts on the same protein

molecule. On this basis one can safely assume that the

amount of protein sufficiently modified to bring about

endocytic uptake and degradation will be significantly

higher than that calculated on the basis of only MG-H1-

mofication. It should be noted that this calculation applies

to healthy humans. In the diabetic state, for example, the

protein modification will be much higher than in normals

due to the increased levels of glucose, along with increased

serum concentrations of many types of AGE-precursors,

such as glyoxal, methylglyoxal and 3-deoxyglucosone,

that increase 1.2, 3.4 and 3.1 times, respectively [15].

Conceivably, these circumstances when taken together,

generate AGEs with a high enough degree of modification

for scavenger receptor-mediated uptake in liver. The

likelihood for this to happen in the diabetic patient is

even greater. But alas, concluding from the considerations

discussed above it is practically impossible to detect these

AGE-modified proteins in the blood because they will

disappear almost immediately after reaching the modifi-

cation threshold for uptake in the liver RES.

From the above considerations it is conceivable that

more solid evidence is needed if the current paradigm of

elimination of AGEs in liver scavenger cells be exchanged

with a new paradigm stating that AGEs are eliminated

mainly in extrahepatic tissues.

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