Gastrointestinal
Stressing Out Over Survival: Glutamine as an Apoptotic Modulator

https://doi.org/10.1016/j.jss.2005.07.013Get rights and content

Introduction

The amino acid glutamine (GLN) has received considerable attention as a potential therapeutic adjuvant in critical illness and in improving postoperative clinical outcomes. Most studies on the role of GLN in cellular physiology have historically focused on its anabolic roles in specific cell types and its contribution to growth in cancer cells. However, an emerging body of work that examines the consequences of GLN deprivation on cellular survival and gene expression has constructed a new paradigm for this amino acid, namely, that limited extracellular GLN supplies modulate stress and apoptotic responses.

Methods

A survey of the scientific literature was conducted on GLN in cell survival signaling and apoptosis. Work from our laboratory in liver cancer cells also was included in this review.

Results

Most studies on this topic have used mammalian cell lines derived from the gut, immune system (including hybridomas), and various cancers. GLN limitation, even in the presence of an adequate glucose supply, impacts stress-related gene expression, differentially modulates receptor-mediated apoptosis, and directly elicits apoptosis through signaling mechanisms and caspase cascades that are specific to cell type. To date, GLN transporters, cellular hydration, glutaminyl-tRNA synthetase, ATP levels, mRNA stability, and glutathione economy have been variably implicated in GLN-dependent survival signaling.

Conclusion

The cell type-specific mechanisms underlying the regulatory role of GLN in cell survival continue to unfold at a steady pace through in vitro studies. These results have collectively provided testable hypotheses for further in vivo studies into their physiological relevance during GLN “nutritional pharmacology.”

Introduction

In the world of academic surgery, a desire to improve postoperative patient care and outcomes drives much of the research efforts. A major theme that has developed in this clinical realm is the concept of “nutritional pharmacology,” where specific nutrients are added in excess to patient nutritional regimens in an effort to enhance convalescence and reduce morbidity, mortality, and hospitalization time during critical illness. One of the more highly studied nutrients for this purpose is the amino acid glutamine (GLN). Classified as a “nonessential” amino acid by most biochemistry texts because of the ability of most cells to produce it, GLN has been reclassified as “conditionally essential” during the last decade because physiological demand often exceeds the cellular capacity to produce it endogenously during critical illness. The resulting GLN deficit can adversely affect cells that rely heavily on this amino acid for normal function, such as those of the gastrointestinal tract and immune system. The merits of GLN-supplemented nutritional regimens in improving outcomes has been evaluated and roundly debated. Indeed, most roundtable discussions on this topic end with the consensus that “we need more randomized prospective clinical trials” [1, 2]. Evidence for the clinical efficacy of GLN in critical illness will not be further discussed here; for very good recent reviews on this topic, the reader is referred to other sources [3, 4, 5, 6]. Instead, GLN supplementation in patient care was broached to provide context for a more academic topic: What are the cellular mechanisms by which GLN may potentially impact clinical outcomes? For clinicians, demonstrated efficacy of “glutamine therapy” would be sufficient, but as scientists, this is a topic that we are compelled to pursue.

The study of GLN in cellular physiology historically has focused on its anabolic effects, namely, its role as a metabolic precursor and physiological regulator of DNA and protein synthesis in cellular growth. GLN is particularly important for the growth, survival, and physiological health of actively dividing cells in the body such as fibroblasts [7, 8], enterocytes (intestinal epithelia) [9, 10], and lymphocytes [11, 12]. Indeed, most of these cell types have been shown to possess a GLN-intensive metabolic profile, almost to the point of auxotrophy. Not surprisingly, clinical conditions for which GLN therapy has been proposed and tested, such as after bone marrow transplant, maintenance of a “healthy bowel” after radiation therapy or resection, after burn injury and during chemotherapy, involve these cell types. Cancer cells also are avid GLN consumers [13, 14, 15, 16, 17]. In an effort to better understand the impact of GLN provision on energy metabolism, a recent study examined the effects of GLN depletion and subsequent repletion on metabolic and gene expression profiles in mouse hepatoma cells via microarray analysis and found that GLN depletion globally down-regulated metabolism [18], which is not surprising.

Just as cancer biologists have refocused their attention from growth (oncogenes) to evasion of programmed cell death (apoptosis and tumor suppressor genes) and the integration of the two processes during the last decade, recent studies have suggested that GLN may act not only to promote growth but also to suppress apoptosis and to evoke and modulate stress responses. This review therefore focuses on a new twist to an old theme, namely, the merits of GLN in supporting cellular physiology, not from an anabolic perspective, but rather as a survival factor. Hereafter, GLN metabolism and apoptosis are briefly reviewed, followed by a retrospective analysis of studies in specific cell types (enterocytes, cells of the immune system and cancer cells) showing a role for GLN in cell survival and in eliciting and modulating cellular stress responses, including implicated mechanisms for GLN effects.

Section snippets

GLN metabolism

As the most abundant amino acid in the plasma at levels around 0.6 mm, GLN exhibits the most rapid intracellular turnover rate of all amino acids [19]. Because of its abundance and rapid metabolism, GLN has been described as the major intercellular nontoxic ammonia shuttle in the body. GLN also serves as a metabolic intermediate contributing carbon and nitrogen for the synthesis of other amino acids, nucleic acids, fatty acids and proteins [20, 21]. Because GLN is a major source for cellular

Apoptosis

Programmed cell death (PCD) is an evolutionarily conserved biochemical pathway resulting in a characteristic morphological cell death termed apoptosis [30]. Hallmarks of apoptosis include membrane blebbing, cell shrinkage, chromatin condensation, and endonucleolytic cleavage of DNA [31]. Unlike necrosis, apoptosis is an energy (ATP)-dependent process that is highly regulated and avoids eliciting an inflammatory response from cell death. Apoptosis is required for successful organogenesis during

Enterocytes

The importance of GLN in maintaining gut homeostasis and health has long been established [58]. GLN is the major oxidative energy source for intestinal epithelial cells [59, 60]. Animal studies have shown the necessity of GLN for the synthesis of enterocyte nucleotides [61] and maintenance of intestinal glutathione levels [62]. Prolonged total parenteral nutrition (TPN) without GLN is known to result in whole-body GLN depletion and gut mucosal atrophy which can be ameliorated with GLN

Immune system-derived cells

The importance of GLN to cells of the immune system is well established [76, 77]. For example, GLN is required for the late events of T-cell activation, lymphocyte progression through the cell cycle [78], and protection of activated human T cells from apoptosis [79]. To determine the role of GLN in activation-induced T-cell death, Chang et al. stimulated Jurkat T cells, a CD4+ human lymphoblastoid cell line, with phorbol myristate acetate (PMA, 20 ng/mL), a protein kinase C activator, and

Hybridoma cells

Fusion of immortalized myeloma cells with spleen-derived lymphocytes to create monoclonal antibody – producing hybridoma cells has been standard practice and a watershed to the biotechnology industry since this innovative technique was conceived in the 1970s [90]. The exhaustion of nutrients, especially GLN, leads to apoptotic cell death during large-scale mammalian cell culture in bioreactors [91, 92]. Apoptosis can have devastating effects on the production of biopharmaceuticals like

Cancer cells

Some of the studies discussed earlier regarding “enterocytes” and “immune system-derived cells” used cancerous cell lines (HT-29, lymphoma, leukemia, myeloma), so those results may also be interpreted as cancer-related responses to GLN deprivation in addition to the underlying premise that they retain properties of the normal parent tissue. The propensity of cancerous cells to exhibit heightened GLN consumption has been long established, and has led to reluctance to use GLN-supplemented

Conclusions and future directions

In summary, overt GLN deprivation ultimately elicits apoptosis by intrinsic and/or extrinsic pathways, depending on cell type. Conversely, an enhanced GLN supply curbs death receptor-mediated apoptosis in certain cell types, but may actually enhance it in some cancer cells. Prior to the onset of apoptosis, GLN limitation promotes adaptive stress response pathways that aid in survival such as cell cycle arrest, ER stress (also known as the unfolded protein response) and angiogenesis, again

References (143)

  • I. Vermes et al.

    A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V

    J. Immunol Methods

    (1995)
  • P.X. Petit et al.

    Mitochondria and programmed cell deathBack to the future

    FEBS Lett.

    (1996)
  • E.S. Alnemri et al.

    Human ICE/CED-3 protease nomenclature

    Cell

    (1996)
  • C.A. Smith et al.

    The TNF receptor superfamily of cellular and viral proteinsactivation, costimulation, and death

    Cell

    (1994)
  • H. Zou et al.

    An APAF-1.cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9

    J. Biol. Chem.

    (1999)
  • P. Li et al.

    Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade

    Cell

    (1997)
  • M. Tewari et al.

    Yama/CPP32 beta, a mammalian homolog of CED-3, is a CrmA-inhibitable protease that cleaves the death substrate poly(ADP-ribose) polymerase

    Cell

    (1995)
  • O. Bussolati et al.

    Characterization of apoptotic phenomena induced by treatment with L-asparaginase in NIH3T3 cells

    Exp. Cell Res.

    (1995)
  • H.G. Windmueller et al.

    Intestinal metabolism of glutamine and glutamate from the lumen as compared to glutamine from blood

    Arch. Biochem. Biophys.

    (1975)
  • H.G. Windmueller et al.

    Uptake and metabolism of plasma glutamine by the small intestine

    J. Biol. Chem.

    (1974)
  • R.R. van der Hulst et al.

    Glutamine and the preservation of gut integrity

    Lancet

    (1993)
  • H.T. Papaconstantinou et al.

    Glutamine deprivation induces apoptosis in intestinal epithelial cells

    Surgery

    (1998)
  • H.T. Papaconstantinou et al.

    Prevention of mucosal atrophyRole of glutamine and caspases in apoptosis in intestinal epithelial cells

    J. Gastrointest. Surg.

    (2000)
  • M. Garcia-Calvo et al.

    Inhibition of human caspases by peptide-based and macromolecular inhibitors

    J. Biol. Chem.

    (1998)
  • M.E. Evans et al.

    Glutamine prevents cytokine-induced apoptosis in human colonic epithelial cells

    J. Nutr.

    (2003)
  • A. Chow et al.

    Glutamine reduces heat shock-induced cell death in rat intestinal epithelial cells

    J. Nutr.

    (1998)
  • M. Coeffier et al.

    Acute enteral glutamine infusion enhances heme oxygenase-1 expression in human duodenal mucosa

    J. Nutr.

    (2002)
  • P. Newsholme

    Why is L-glutamine metabolism important to cells of the immune system in health, postinjury, surgery or infection?

    J. Nutr.

    (2001)
  • W.K. Chang et al.

    Glutamine protects activated human T cells from apoptosis by up-regulating glutathione and Bcl-2 levels

    Clin. Immunol.

    (2002)
  • G. Weingartmann et al.

    HSP70 expression in granulocytes and lymphocytes of patients with polytraumaComparison with plasma glutamine

    Clin. Nutr.

    (1999)
  • R. Exner et al.

    Glutamine deficiency renders human monocytic cells more susceptible to specific apoptosis triggers

    Surgery

    (2002)
  • C.Y. Li et al.

    Heat shock protein 70 inhibits apoptosis downstream of cytochrome c release and upstream of caspase-3 activation

    J. Biol. Chem.

    (2000)
  • M. Zellner et al.

    Glutamine starvation of monocytes inhibits the ubiquitin-proteasome proteolytic pathway

    Biochim. Biophys. Acta

    (2003)
  • M. Al-Rubeai et al.

    Apoptosis in cell culture

    Curr. Opin. Biotechnol.

    (1998)
  • F. Hesse et al.

    Developments and improvements in the manufacturing of human therapeutics with mammalian cell cultures

    Trends Biotechnol.

    (2000)
  • A. Tinto et al.

    The protection of hybridoma cells from apoptosis by caspase inhibition allows culture recovery when exposed to non-inducing conditions

    J. Biotechnol.

    (2002)
  • H. Li et al.

    Activation of caspase-2 in apoptosis

    J. Biol. Chem.

    (1997)
  • Y. Guo et al.

    Caspase-2 induces apoptosis by releasing proapoptotic proteins from mitochondria

    J. Biol. Chem.

    (2002)
  • R.J. Weil

    The future of surgical research

    Plos. Med.

    (2004)
  • T.R. Ziegler

    Glutamine supplementation in bone marrow transplantation

    Br. J. Nutr.

    (2002)
  • D. Kelly et al.

    Role of L-glutamine in critical illnessnew insights

    Curr. Opin. Clin. Nutr. Metab. Care

    (2003)
  • F. Novak et al.

    Glutamine supplementation in serious illnessA systematic review of the evidence

    Crit. Care Med.

    (2002)
  • J. Wernerman

    Glutamine and acute illness

    Curr. Opin. Crit Care

    (2003)
  • W. Engstrom et al.

    The relationship between purines, pyrimidines, nucleosides, and glutamine for fibroblast cell proliferation

    J. Cell Physiol.

    (1984)
  • A. Zetterberg et al.

    Glutamine and the regulation of DNA replication and cell multiplication in fibroblasts

    J. Cell Physiol.

    (1981)
  • V.S. Klimberg et al.

    The importance of intestinal glutamine metabolism in maintaining a healthy gastrointestinal tract and supporting the body’s response to injury and illness

    Surg. Annu.

    (1990)
  • M.S. Ardawi et al.

    Glutamine metabolism in lymphocytes of the rat

    Biochem. J.

    (1983)
  • K. Brand

    Glutamine and glucose metabolism during thymocyte proliferation. Pathways of glutamine and glutamate metabolism

    Biochem. J.

    (1985)
  • J.C. Aledo

    Glutamine breakdown in rapidly dividing cellswaste or investment?

    Bioessays

    (2004)
  • B.P. Bode et al.

    Glutamine and cancer

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