Lester Packer and Vitamin E: Editorial

The Putative Beginnings

One may assume that Lester Packer's interest in vitamin E (tocopherols) had its routes in his early morphological studies on biomembranes. The technique that became famous in the late 1950s for the study of biological specimens shows up in the literature under different terms such as freeze etching, freeze fracturing, freeze fracture, or freeze fracture electron microscopy. It provides a three-dimensional picture of a biological organelle or membrane. In 1975, some scientists, including Lester Packer, met to clarify the bewildering nomenclature of freeze etching and talked about its applicability in solving biological problems (Branton et al., 1975).

Lester and his group had already embarked on this technology before to study mitochondria and chloroplasts in different functional states (Murakami and Packer, 1970; Packer, 1970; Wrigglesworth and Packer, 1970; Wrigglesworth et al., 1970). In mitochondria of rats and rabbits, these researchers for the first time saw a clear difference between the outer and inner membranes, components associated with the inner membrane and a characteristic network in the mitochondrial matrix. The concave and convex fracture faces of the inner and outer membranes differed in density (Melnick and Packer, 1971), and the particle density changes in the outer membrane of oscillating mitochondria depended on the lipid composition (Tinberg et al., 1972).

A similar influence of the lipid composition was detected in illuminated chloroplasts (Torres-Pereira et al., 1974). Most of this had not been seen before with conventional electron microscopy, and we may state that the studies provided a more realistic view of biomembranes. The identification of membrane-associated particles, however, remained limited to a Ca²⁺ pump (Packer et al., 1974), and a later article with Rolf Mehlhorn admitted that even a numerical characterization of the membrane particle pattern might be too time-consuming (Mehlhorn and Packer, 1976).

Vitamin E, Mitochondria, and Running Rats

In the 1970s and 1980s, Lester's work adopted a more functional character. It had been reported already in 1936 that the tocopherols can act as “antioxidants” (Olcott and Mattil, 1936), and in 1971, mitochondria were shown to produce H₂O₂ (Loschen et al., 1971), which proved to be derived from superoxide radicals (Loschen et al., 1974). Lester made use of these discoveries and investigated the putative antioxidant role of vitamin E under conditions that lead to radical production in mitochondria (Maguire et al., 1989).

Davies et al. (1982) first showed that the endurance capacity in vitamin E-deficient rats was dramatically reduced and attributed the reduced endurance to a loss of mitochondria in muscle due to free radical destruction, and in a complementary study, Quintanilha et al. (1982) showed that the mitochondria of skeletal muscle and liver mitochondria were more susceptible to different types of damage, were highly susceptible to vitamin E deficiency, and clearly exhibited signs of free radical damage such as lipid peroxidation. The Packer group further excluded a diminished mitochondrial activity as cause of the reduced endurance capacity (Gohil et al., 1984). It saw an adaptation of mitochondrial function. The overwhelming effect of exhaustive exercise training, however, was a loss of vitamin E and a concomitant loss of muscle mitochondria (Gohil et al., 1985).

Lester Packer and colleagues summarized their early work on the protective effect of vitamin E on muscle mitochondria in the Annals of the New York Academy of Sciences (Packer et al., 1989a). These studies mark the beginnings of the still flourishing field of exercise physiology (for reviews see Bouviere et al., 2021; Cobley et al., 2017; Margaritelis et al., 2016). In the meantime, we have learned that it depends on the severity of exercise, muscle-damaging or not, whether we see adaptation or plain pathology (Nikolaidis et al., 2012). In light of this knowledge, the Packer experiments need a comment: They fall into the category of damaging exercise.

The early work of the Packer group has also recently gained interest in the context of ferroptosis, a lipid peroxide- and iron-mediated form of cell death that is typically precipitated by glutathione peroxidase 4-deficiency. The phenomenon can, however, also be inhibited by a glutathione-independent way: by ferroptosis suppressor protein 1 [FSP1 (Doll et al., 2019)]. The antiferroptotic activity of FSP1 depends on reduced coenzyme Q, which is guaranteed by reduced pyridine nucleotides. The Packer group had argued that loss of vitamin E is due to formation of the chromanoxyl radical. It had further demonstrated that the latter can be reduced by coenzyme Q, which thereby, however, is oxidized, while it remains reduced, as long as the electron flux from pyridine nucleotides is sufficient.

The salvage of vitamin E by reduced pyridine nucleotides had already been documented by the Packer group for mitochondria and microsomes (Kagan et al., 1992; Maguire et al., 1992; Packer et al., 1989b), and the reduction of coenzyme Q by pyridine nucleotides had been known for long. Vitamin E, in fact, also inhibits ferroptosis and might do so in a double way: by its intrinsic antioxidant activity and by keeping the coenzyme Q pool reduced and, thus, the SFP1-coenzyme Q-NAD(P)H pathway in action.

Trafficking of Vitamin E Between Membranes

That vitamin E is enriched in fat and in particular in biological membranes had been established already in the 1960s (Silber et al., 1969). The precise location (Perly et al., 1985), lateral diffusion (Gomez-Fernandez et al., 1989), the velocity of which had obviously been overestimated at that time (Gramlich et al., 2004), and flipping within membranes had become a top issue in the 1980s (Tiurin et al., 1986). The Packer group dealt with a different problem: How does vitamin E come from one membrane to another one?

This phase of research was largely dominated by the young Valerian Kagan (Kagan et al., 1990a; Kagan et al., 1990b). To this end, he prepared “donor liposomes,” that is, unilamellar liposomes that were loaded with α-tocopherol to an extent that lipid peroxidation was completely inhibited. The “acceptor liposomes” were free of tocopherol and, thus, prone to lipid peroxidation. Inhibition of lipid peroxidation in the acceptor liposomes after mixing the liposome preparation was taken as evidence for an intermembrane exchange. A fusion of the membranes as cause of the tocopherol exchange was ruled out. Kagan et al. (1990a) further reported that the exchange was largely facilitated by unsaturated fatty acids in the donor liposomes.

These early experiments clearly show that an intermembrane exchange of tocopherol is possible, and their importance has still been quoted 18 years later in a comprehensive review on the role of tocopherols in membranes (Atkinson et al., 2018). Whether they mimic the in vivo situation may be doubted. Membrane-bound proteins with affinity to both tocopherols and lipids might further accelerate the exchange. However, this aspect has never been investigated in detail.

Vitamin E Protects Biomembranes. But How?

That a main function of vitamin E is membrane protection is generally accepted. However, the mechanism of this role is being debated until now. Already in 1972, Molenaar et al. (1972) summarized the possible roles of vitamin E as follows. It may act as a nonenzymatic antioxidant, as a factor affecting enzymatic lipid peroxidation, as a factor affecting biological oxidations in general, or as a membrane stabilizer through interaction of its hydrophobic side chain with membrane lipids, and most of these possibilities are still being discussed. Only a participation in the respiratory chain has been ruled out. The decision, which of the theories is most likely, depends inter alia on the precise location of vitamin E in the membrane, which is not easily assessed.

This issue is very complex and has been covered by dedicated reviews (Atkinson et al., 2021; Atkinson et al., 2018; Atkinson et al., 2010; Wang and Quinn, 2000). For the present purpose, a simplified statement may suffice. The hydrophobic tail of the tocopherols is located in the inner part of the bilayer, while the polar chromanol ring is more surface exposed and may interact with hydrophilic cellular constituents such as ascorbate and thiols (Niki, 2019).

Lester Packer's group became also involved in this delicate problem by investigating the impact of the side chain of 16 C atoms on the action of vitamin E. In this context, it should be mentioned that vitamin E is a group of compounds, all being equipped with a chromanol moiety (α-, β-, γ-, and δ-chromanol, depending on the position and number of methyl residues at the aromatic ring) and a side chain, which is a phytyl residue in RRRα configuration in natural tocopherols, a racemic one in synthetic tocopherols, and a 16 C isopentenyl side chain with three double bonds in the tocotrienols (Niki and Abe, 2019). Together, they are termed “tocols.”

The Packer group investigated tocopherols with a shortened side chain, called tocopherol homologues. Their side chain was shortened from C₁₆ (full length) to C₁₁, C₆, and C_1. Surprisingly, they displayed a higher antioxidant activity in microsomal membranes than tocopherols with a full-length phytyl side chain (Kagan et al., 1990b). The antioxidant activities of the homologues increased from C₁₆ to C1, although the isoprenoid chain was considered to be important for a proper orientation of vitamin E in the membrane. The high antioxidant potency of the shorter chain homologues may be explained by a higher recycling of vitamin E (Kagan et al., 1990b).

Recycling of vitamin E has been studied in the inner membranes of mitochondria, in microsomes, and in erythrocyte membranes. Loss of vitamin E was prevented by either the NADH-cytochrome b5 system or nonenzymatic pathways by ascorbate and lipoic acid (Constantinescu et al., 1993). Later, α-tocotrienol was compared with α-tocopherol. It displayed a higher antioxidant activity (Suzuki et al., 1993), exceeded the neuroprotective effect of α-tocopherol by three orders of magnitude and specifically interacted with c-Src kinase (Sen et al., 2000). These results reveal that the above quoted hypothesis that the side chain only affects the membrane binding of the tocols is an oversimplification. This hypothesis, first formulated by Diplock and Lucy (1973), evidently overlooked the influence of the side chain on the precise location and action of the tocols (for review see Atkinson et al., 2018).

The shortened tocopherols investigated by the Packer group were synthetic compounds purchased from the Japanese company Eisai and do not occur in nature. The in vivo metabolites of the tocols also have a shortened side chain, but with a polar group at the end. All tocols are initially oxidized by ω-oxidation at the end of the side chain and then shortened by five steps of β-oxidation, while the chromanol ring is left untouched (Brigelius-Flohé, 2019). This observation by itself is hardly compatible with a predominant role of the tocols in scavenging radicals in vivo, which always would be associated with an oxidative opening of the chromanol moiety.

That the chromanol moiety of the tocols can undergo uni- and bimolecular redox reactions is undoubted. This ability is widely accepted as the role of tocols in protecting the unsaturated lipids in biological bilayers against oxidative destruction. Whether this “antioxidant” effect of vitamin E is its only function is another question. When reading articles from the Packer laboratory, one might get the impression that the antioxidant role of vitamin E is its only one. However, there is no unequivocal statement supporting this assumption. These publications often took the antioxidant function or lipid peroxidation as a convenient readout. However, Lester Packer was too open-minded for dogmatic statements such as: “Vitamin E, an antioxidant and nothing more” (Traber and Atkinson, 2007). He always remained open to novel aspects of vitamin E research, as is evident from Traber and Packer (1995).

Herein the authors already described the evidence for structure-specific roles of vitamin E in cellular metabolism, signal transduction, and incorporation into nascent lipoproteins. They discussed the impact of vitamin E on arachidonic acid metabolism, cell proliferation and differentiation, and highlighted the lacking correlation of these effects and antioxidant potencies of the vitamin E forms investigated. They saw profound differences in the effects of closely related forms of vitamin E. Thus, already in 1995, a new and exciting area was promised (see The New Horizons).

The New Horizons

Already before the beginning of the new century, a variety of inspiring ideas on new vitamin E functions were born, and Lester Packer was one of the first embarking on them. In 1993, Lester Packer coauthored a publication showing that α-tocopherol inhibited smooth muscle cell proliferation and protein kinase C activity (Chatelain et al., 1993) and another one on the inhibition of NF-κB activation (Suzuki and Packer, 1993). These early publications on a role of tocopherols in cellular signaling are not necessarily conflicting with the antioxidant function of tocopherols, since NF-κB is known to be redox-regulated (Flohé et al., 1997). The same holds true for the inhibition of protein kinase C, because practically all phosphorylation cascades are inhibited by antioxidants.

However, a huge number of enzyme activities have meanwhile been shown to be positively or negatively affected by tocopherols [for review see (Zingg, 2019)]. These effects are usually interpreted as resulting from redox-regulated signaling pathways. However, altered gene expression may also be considered.

In 2003, Lester Packer together with the groups of Kishorchandra Gohil and Maret Traber looked for tocopherol-sensitive genes in liver and brain of mice. They took advantage of mice that were deficient in the α-tocopherol transfer protein, an animal model of human ataxia with vitamin E deficiency (AVED). As AVED patients, the mice are suffering from a systemic vitamin E deficiency since their birth (Gohil et al., 2003). In their liver preferentially, genes regulating vasculogenesis were affected, whereas in the cortex, genes responsible for the assembly of synaptic vesicles were repressed. This pattern of gene expression correlates with behavior and cognitive deficits in the animals but was again explained as a consequence of oxidative stress.

In a follow-up study (Gohil et al., 2004), they highlighted the repression of genes affecting synaptic function, myelination, the repression of the retinoic acid-related orphan receptor alpha (ROR alpha) in the cortex and the adrenal glands, and discussed the similarity with the pathological symptoms in AVED patients. The induction of genes indicating neurodegeneration in the motor cortex was also reported and all these phenomena were interpreted as consequences of oxidative stress due to vitamin E deficiency. In sharp contrast, however, Gerald Rimbach and colleagues (quoted in Brigelius-Flohé, 2021) concluded from a similar study on long-term alimentary vitamin E deficiency that the gene expression pattern under vitamin E deficiency depends on time, the tissue under consideration, and can hardly be explained by a single mechanism.

In a roundtable discussion under the guidance of the late Lester Packer in 2004, even the possible existence of a nuclear vitamin E receptor, as is known for other lipophilic vitamins, was raised (Packer et al., 2004).

More than one surprise also came from the investigation of the metabolic fate of the tocols. Not only did the detection of metabolites with an intact chromanol ring shed doubt on an exclusive in vivo antioxidant function of the tocols, these metabolites appear to have their own activities that may explain some of the tocol functions [for review see (Birringer and Lorkowski, 2019)].

…and the Man

He also was a charming host and perfect group leader. Each time I or we, that is, myself and my husband, visited the Packer laboratory, we saw it overcrowded with people from all over the world, mostly young ones working with unbelievable enthusiasm. I guess over the decades they came from all continents. And Lester used to invite his guests and coworkers to wild parties, sailing trips on the San Francisco Bay, to fancy restaurants, or to his cozy home north of Berkeley, where his wife Anne offered tasty dinners with fresh sockeye salmon or delicious oyster snacks. It always was an exciting adventure to be there.

Lester Packer organized many conferences. He also founded the Oxygen Club of California (OCC). Although the name reminds of a local organization, Lester changed it to an international one that held its scientific meetings all over the world and keeps the memory of this highly stimulating scientist alive (Fig. 1).

OPEN IN VIEWER

FIG. 1.

The late Lester Packer (right) talking to Regina Brigelius-Flohé (left), the author of the present minireview, and Josiane Cillard (middle), who co-organized the international symposium “Nutrition, Biologie d'Oxygène et Médicine” in Paris in June 2013. The meeting was jointly organized by the Societé Francaise de Recherche sur les Radicaux Libres and the Oxygen Club of California. The photograph was taken and kindly provided by Leopold Flohé. Color images are available online.