AbstRAct
In Chapter 1, the origins of agriculture were traced to a period of about 10,000 years ago when the hunter-gatherer lifestyle was replaced by farming. This “green revolution” occurred in many locations and among many peoples. The diversity of foods and food compositions before and after the first green revolution suggest that fundamental genomic mechanisms exist to digest and utilize the broad spectrum of human diets (Chapters 1, 3, 4). Some of the genes that express digestive enzymes, such as amylase, are about 3.5 billion years old, as old as the first living cells. Other genes, such as enterokinase, which regulates protein digestion, are as young as 0.5 billion years old (Hedges et al., 2004). These digestive genes are part of the genome of all living organisms and are essential for maintenance, growth, and reproduction (de Duve, 2007). Their generic nature permits the present and future diversity of agriculture and food availability. As pointed out in Chapters 1 and 22, the growth of global population is already placing strains on food availability and diversity. It is my contention that greater understanding of basic human food needs, when coupled to understanding of the spectrum of food genomes, can continue to evolve and sustain global population growth and health through a new green revolution.
contents
Abstract ... 15 What Is Nutrition? ... 16 What Is the History of Life? ... 16 Genomic History of Digestion ... 18 Protein Digestion ... 18 Lipid Digestion...20 Carbohydrate Digestion ...22 Milk Sugar ...22 Table Sugar ...23 Starch Digestion ...23 Amylase Solubilization of Starch ...24 α-Glucosidase Digestion of Starch ...24 Summary ...27 References ...28
whAt Is nutRItIon?
Entropy is a universal physical quantity that defines the second law of thermody-namics: Energy dissipates, maximizing the disorder in the universe. However, liv-ing organisms defy the decay into equilibrium with the environment by feedliv-ing on negative entropy and decreasing their disorder. By living, the organism maintains itself in a stationary or low level of entropy (Schrödinger, 1956, p. 73). Nutrition is the process by which the organism is continually consuming negative entropy from the environment. In humans, the ingested negative entropy consists of foods. Here, we examine the genomic history of the processes of digesting the negative entropy contained in food macronutrients: proteins, fats, and carbohydrates. In plants, the most powerful supply of negative entropy is from sunlight. All living organisms concentrate a “stream of order” from the environment to escape the atomic chaos of entropy and display the power of maintaining self and expressing orderly events (p.
75). These interwoven events are guided by genetic mechanisms that are completely at odds with the “probabilistic mechanisms” of physics to ensure the living paradigm of orderliness. Growth and reproduction are due to an “order-from-order” principle (p. 78). The genome thus defies the disorder of the physical universe. A fundamental difference between the physical and biological universes is the harmony that bridges the genome of single with multicellular organisms. By defying the laws of physics, living cells were described as “the finest masterpiece ever achieved along the lines of the Lord’s quantum mechanics” (p. 83).
whAt Is the hIstoRy of lIfe?
Planet earth is thought to have formed about 4.5 billion years ago (bya). The com-mon ancestor of contemporary life forms populated the earth about 3.5 bya (Pollard et al., 2008, p. 17). Biochemical features stored in all present life forms suggest that this primitive microscopic cell had about 600 genes encoding DNA, protein synthetic machinery, and a plasma membrane and with mechanisms for digest-ing polymeric molecules and assimilatdigest-ing negative entropy from the environment.
Over about 1.7 bya, distinctive living species evolved. On the basis of evolutionary records, preserved in their genomes, living organisms are divided into three primary domains: archaea, bacteria, and eukarya. Genomic diversification evolved by muta-tion, duplicamuta-tion, and divergence, and lateral transfer of DNA (p. 19). Photosynthesis originated about 3 bya by symbiosis by two different families of bacteria. About 2.5 bya, a lateral transfer event brought the two genomes for photosynthesis together in cyanobacteria (blue-green algae). Sunlight energized the photosynthetic structures to activate a proton gradient used to synthesize adenosine triphosphate (ATP) and the many carbon compounds that living organisms required for negative entropy. About 2.4 bya, cyanobacteria produced most of the oxygen in the Earth’s atmosphere as the product of photosynthesis. This increase in oxygen revolutionized the chemical environment for all other species of organisms.
Genomic history indicates that the present eukaryotic lineages diverged between 2 and 1 bya (Hedges et al., 2004; Figure 2.1). Cell surface membranes characterize both eukaryotes and prokaryotes; however, internal compartmentalization is lacking
in prokaryotes. The external membrane of prokaryotes separates digestion of macro-molecules outside the cell from internal machinery requiring these nutrients. These primitive cells exert digestion by exporting enzymes, either by secretion or extension from the cell surface, to hydrolyze complex organic structures. The products of diges-tion are then processed and ingested through the membrane for metabolic processes (de Duve, 2007). Compartmentalization of internal cell structures is thought to have evolved by the regional segregation of digestive enzymes on the external surface of the plasma membrane, a feature persistent in present-day bacteria. Invagination of
Mammals
fIguRe 2.1 A timescale of eukaryote evolution. The times for each node are taken from the genomic divergence times, except for nodes 1 (310 Ma [million years ago]), 2 (360 Ma), 3 (450 Ma), and 4 (520 Ma), which are from the fossil record; nodes 8 (1450 Ma) and 16 (1587 Ma) are phylogenetically constrained and are the midpoints between adjacent nodes.
Nodes 12 to 14 were similar in time and therefore are shown as a multifurcation at 1000 Ma;
likewise, nodes 21 and 22 are shown as a multifurcation at 1967 Ma. The star indicates the occurrence of red algae in the fossil record at 1200 Ma, the oldest taxonomically identifiable eukaryote. Plastid (photosynthesis organelles) origin is shown with an arrow. (From Hedges, S.B., Blair, J.E., Venturi, M.L., and Shoe, J.L., BMC Evol Biol. 2004 Jan 28;4:2. Open Access article available at http://www.biomedcentral.com/1471-2148/4/2.)
the segregated domains of plasma membrane likely generated the membrane-bound organelles of eukarya.
One hypothesis is that cells from the prokaryotes joined in a symbiotic relationship to generate the first eukaryotes about 2.7 bya. Later, lateral transfer of the genome was surrounded by plasma membrane to become the eukaryotic nucleus. Genomic evidence has established that eukaryotes acquired mitochondria about 1.8 bya when a protobacterium became symbiotic. The bacterial genome thus contributed molecu-lar machinery for ATP synthesis by oxidation phosphorylation. Over many centuries of evolution, most symbiotic bacterial genes moved into the host cell nucleus.
The acquisition of chloroplasts began when the cyanobacterial symbiant brought photosynthetic machinery into a primitive algal cell that had mitochondria by lateral transfer about 1.6 bya. Symbiosis evolved into interdependence when the chloro-plast genes were assimilated into the nuclear genome. About 2 bya, the algal and plant branches of eukarya evolved independent strategies for multicellular existence.
Further increases in organism cell number occurred about 1.5 bya (Hedges et al., 2004). Fossils confirm that animals had evolved multicellular structures by 0.6 bya.
These primitive metazoans (multicell organisms) had mouth, intestine, and sensory structures. Evolution of genes for intercellular adhesion proteins pre-dated the meta-zoan animals. The early metameta-zoan animals resemble contemporary animal embryos in appearance. In this period, animals diverged into the three subdivisions: mol-lusks, annelid worms, bracheopods, and platyhelminths (~1.3 bya); arthropods and nematodes (~1 bya); and echinoderms and chordate (~0.5 bya, humans at ~ 0.06 bya) (Hedges et al., 2004; Pollard et al., 2008, p. 17).
genomIc hIstoRy of dIgestIon
The full scope of nutrient metabolism is discussed in Chapters 5–8. Here, we review the evolution of the major digestive processes that demark the boundary between human negative entropy intake (food) and nutrient utilization (metabolism). The macronutrients are viewed as purveyors of essential food micronutrients. We trace the genomic history (phylogenies; Huerta-Cepas et al., 2007) of the major human diges-tion enzymes. As described, digesdiges-tion was well established in the prokaryotic organ-isms at 3.5 bya and advanced to a compartmentalized system in early eukarya by 2 bya. In fossil metazoan animals, a gastrointestinal tract was evidenced by 0.6 bya.
Protein Digestion
The central dogma of molecular biology is that DNA is transcribed into RNA, and this RNA is translated into proteins (Pollard et al., 2008, p. 251). Thus, proteins are most tightly controlled by the genome. This dogma has significance for the pro-tein substrates and the digesting enzymes converting food into oligopeptides and free amino acids for absorption. The synthesis of all proteins is called translation because of the conversion of the genetic code into amino acids in the peptide chain by messenger RNA (mRNA). Small transfer RNA (tRNA) is the purveyor of specific amino acids in response to successive codons within the mRNA. The codons are made up of three nucleic acids whose sequence is transcribed from genomic DNA.
The genetic code converting the information from codons into amino acid sequence is almost universal. Thus, the four nucleotides in the genome are expanded to 64 different triplet codons, resulting in 20 specific amino acid translations. One codon specifies the start, and three specify the stop of coding. The mRNA translation takes place in ribosomes in the cytoplasm of prokaryotes and the endoplasmic reticulum (ER) of eukaryotes. Resulting soluble proteins are folded by mechanisms encoded in the sequence but are sensitive to cytoplasmic physical conditions. Transmembrane protein folding is frequently assisted by molecular chaperones that inhibit aggrega-tion and assist sorting to cellular organelles and membrane domains such as the lumen of the digestive tract. Many cell surface proteins are glycosylated in the Golgi as a processing step; more than 200 enzymes orchestrate the addition of sugar resi-dues to peptides. Glycoproteins are important for cell adhesion and are highly resis-tant to digestion.
Adult humans consume about 50 g/d of protein. About 90% of this intake is digested to absorbable peptides and amino acids in the upper intestine (Erickson and Kim, 1990; see Chapter 7). A series of different peptidases participate in this diges-tion process (Erickson and Kim, 1990). Gastric pepsin is an endoprotease hydrolyz-ing an aspartic residue in the peptide sequence (Whitcomb and Lowe, 2007). This human chromosome 11 paralogous gene with multiple isoforms is limited in expres-sion throughout animal phylogeny, suggesting a common ancestor before differen-tiation of the arthropods about 1.5 bya (Benson et al., 2008). A bovine homologue, renin, is an enzyme used for making cheese by precipitation of milk caseins.
The pancreatic peptidases include trypsin, chymotrypsin, and elastin (Whitcomb and Lowe, 2007). The trypsins are a family of secreted serine endoproteases cod-ing from nine genes on chromosome 7 and one on chromosome 9. The genes are embedded within T-cell receptor beta (TCR-B) loci of both chromosomes. The mul-tiple-protein isoforms from chromosome 7 trypsins have redundant secreted activi-ties; however, that from chromosome 9 is more resistant to inhibitors (Benson et al., 2008). An association with TCR-B is present throughout mammals from about 0.2 bya, but the gene itself is rooted in many bacterial species and thus was present from as long as 1.1 bya. Chymotrypsin is another secreted pancreatic serine endoprotease.
It is coded from a duplicated gene located on chromosome 16 and is a neighbor of two more pancreatic elastase serine endoprotease genes on chromosome 1. The phylogeny of these chymotrypsin and elastase genes is parallel to that of trypsin, with a secreted presence in bacteria and conservation of associated genes on chro-mosomes on mammalian species. The pancreas also produces a family of secreted carboxypeptidase exoproteases. Carboxypeptidase A is expressed as two isoforms and cleaves terminal aromatic amino acids; carboxypeptidase B cleaves terminal aliphatic amino acids. Both are zinc-requiring metalloproteases. These reside on human chromosome 7 within a cluster of four genes, two of which are specific for the pancreas. The genomic context of these human pancreatic proteolytic enzymes is conserved within rodent chromosomes, which diverged about 0.3 bya, and the phylogenic roots extend back to bacteria that evolved about 2 bya. These secreted digestive gene products are likely rooted in the genome of the common ancestor of all cell lineages (de Duve, 2007).
The final steps of digestion of food proteins to absorbable peptides and free amino acids take place at the lumenal membrane of small intestinal enterocytes (Sterchi and Woodley, 1980a, 1980b; Sterchi, 1981; Rawlings et al., 2008). The lumenal brush border membrane anchors a series of peptidases, most of which hydrolyze terminal amino acids (Table 2.1). Enterokinase has the specialized function of activating the secreted pancreatic proteases described. It has a younger phylogeny than the remain-ing enzymes. The remainremain-ing peptidases are expressed in many tissues, includremain-ing T cells, and are identified by CD antigen numbers. These membrane enzymes often play a role in the regulation of peptide hormone blood levels and are targets for pharmacologic inhibitors. γ-Glutamyl peptidase is a key enzyme in the glutathione cycle and plays a critical role in xenobiotic detoxification. The last five enzymes diverged before bacterial emergence (2.5 bya) and likely are descendants from membrane-bound digestive genes expressed by the first common ancestor of life (de Duve, 2007).
LiPiD Digestion
A surrounding membrane contributes to the defiance of entropy by the living cell.
This is a planar structure composed of phosphodiglycerides oriented to display hydrophilic PO4 external extensions coating a core of hydrophobic diglyceride tails (Pollard et al., 2008, p. 113). Other lipids are also embedded into the external faces, and hydrophobic membrane proteins and transporters traverse the lipid core. The membrane is stable and relatively impermeable to ions and electrons. It is believed that the earliest life forms evolved the lipid membrane to decrease local entropy within a living cell about 3.5 bya. All sequenced genotypes have conserved enzymes that catalyze the synthesis of coenzyme A and mevalonate in the synthetic pathway to phosphodiglycerides and cholesterol (Friesen and Rodwell, 2004). By contrast, only eukaryotes synthesize neutral triglycerides, which are stored as intracellular hydrophobic droplets (Turkish and Sturley, 2007). These membrane and stored lipid classes are major contributors to food negative entropy. The disorders of atheroscle-rosis and obesity are thought to be influenced by quality and quantity of lipids in foods (see Chapter 6). The glycerides are not primary products of DNA transcrip-tion, as are proteins, but are products of regulated multienzyme metabolic pathways;
the consequence is the synthesis of a family of di- and triglycerides with fatty acids ranging from 14 to 20 carbons in length with variable degrees of saturation.
The adult Western diet contains about 100 g/day of fat, of which more than 90%
exists as triglycerides. Virtually all food triglycerides and diglycerides require lume-nal small intestilume-nal digestion before more than 95% absorption (Lowe 1997, 2002).
This is accomplished by a series of lipase enzymes. Lipases are esterases that can hydrolyze acyltriglycerides into di- and monoglycerides, glycerol, and free fatty acids at a water-lipid interface. Gastric lipase is the initial digesting activity hydrolyzing position 3 (sn-3) ester linkages of the glycerides. This is a developmentally important enzyme for normal young human infants, who have a physiological delay in matura-tion of pancreatic lipase. The human enzyme is transcribed from chromosome 10, where it resides within a cluster of five paralogous genes. This gastric lipase chro-mosomal grouping is conserved in rodents. The gastric expressed gene is only found
tAble 2.1 selected membrane-bound Peptidases Active in food digestion in human small Intestine nameAbbreviationchromosome subunitsfunctionsdivergenceec EnterokinaseENTK21q21YesActivates pancreatic proteasesBacteria ~ 2 bya3.4.21.9 Dipeptidyl-peptidase IVDPP IV2q24NoDegrades GLP-1 CD26Archaea ~ 3.5 bya3.4.14.5 Aminopeptidase NAMPN15q25Yes; ZnDegrades enkephalins CD13Archaea ~ 3.5 bya3.4.11.2 Aminopeptidase AAPA4q25Yes; ZnDegrades ACE CD249Archaea ~ 3.5 bya3.4.11.7 X-Pro aminopeptidase 2XPNPEP2Xq25Yes; Mn isoformsDegrades bradykininArchaea ~ 3.5 bya3.4.11.9 γ-Glutamyl transpeptidaseGGT1-620p11.1, 20q11.22, 22q11.21, 22q11.23Yes; isoformsDegrades GSH CD224Archaea ~ 3.5 bya2.3.2.2 Note: Named lymphocyte antigens are shown in bold. bya, billion years ago. GLP-1, glucagon-like peptide 1; Enkephalins, endogenous opioids; ACE, angio- tensin1-converting enzyme; GSH, L-gamma-glutamyl-L-cysteinyl-glycine.
in eukaryotes. Pancreatic triglyceride lipase (PTL) is the second and major lipase that hydrolyzes all sn-1 and sn-3 esters of acylglycerides but not membrane lipids.
Pancreatic lipase is also on human chromosome 10 within a locus of four paralogs whose relationship is conserved in rodents. One of these paralogous genes produces an 80% homologous pancreatic lipase-related protein (PLRP2), which hydrolyzes acyltriglycerides and all membrane lipids. Both pancreas-expressed genes are only found in eukaryotes. Both proteins have two domains: N-terminal and C-terminal.
The N-terminal is associated with interfacial activation, the process of becoming active at the lipid-water interface. The function of the C-terminal is to mediate inter-action with lipids. Many food components inhibit pancreatic lipase, and the pan-creas secretes colipase, which conserves activity by functioning as a PTL cofactor.
Colipase binds to the bile-salt covered triacylglycerol interface thus allowing the PTL enzyme to anchor itself to the water-lipid interface. Colipase gene is located on chromosome 6 and only found in eukaryotes. The locus is conserved on the mouse chromosome. In mouse knockout (KO) studies, PTL deficiency was asymptomatic, but colipase deficiency was associated with steatorrhea.
Bile salt-dependent lipase is also secreted by the pancreas. This enzyme has broad substrate specificity for all fat and membrane lipids. It is also secreted in human milk. This enzyme codes from human chromosome 9 and is conserved in archael and bacterial genomes. Phospholipase A2 (PLA2) is a secretory pancreatic enzyme that cleaves the sn-2 position of the glycerol backbone of membrane phospholipids.
It is clustered with two paralogs on chromosome 1 and is also expressed on placenta, synovial membranes, and platelets. PLA2 is only expressed in eukaryotes. Knockout of the last two specific genes in mice was not associated with steatorrhea, suggesting that the overlapping substrate specificities of the various lipases are redundant, and that individual deficiencies are compensated by the remaining lipases.
CarbohyDrate Digestion
The carbohydrates are a major source of negative energy in the human diet; some rural agricultural workers consume more than 500 g/day (Robayo-Torres et al., 2006, and see Chapter 5). The amount digested in the small bowel is dependent on the spe-cies of carbohydrate fed; the range is from more than 95% down to less than 30%
with digestion-resistant starches (described in the section α-Glucosidase Digestion of Starch). Food carbohydrates exist as glycosides bound to membrane proteins and lipids and as sugar units of disaccharides and glucose polymers. In the first category, the carbohydrates provide stability to the lipid-water plasma membrane (Pollard et al., 2008, p. 113). The second case provides stored energy-rich foods that ensure adequate glucose for prandial metabolism.
milk sugar
Lactose is the principal carbohydrate in milk (Robayo-Torres et al., 2006). It is a disaccharide composed of glucose and galactose linked as 1- β-D-galactopyranosyl-4-α-D-glucopyranose. N-Acetyllactosamine synthase is highly conserved, with seven paralogous genes on various chromosomes and is a component of lactose synthase along with α-lactalbumin (chromosome 12). The N-acetyllactosamine synthase is a
highly conserved gene in all genomes. In contrast, the α-lactalbumin gene is lim-ited to mammalian genomes but has an ancestral root as a lysosomal enzyme gene.
N-Acetyllactosamine synthase is expressed in seven isoforms and plays a crucial role in protein N-glycosylations. As lactose synthase complex, these two genes are cen-tral to human and bovine lactation, for which lactose production drives the volume of milk produced.
Small intestinal mucosal lactase is the hydrolase that digests lactose to the mono-saccharide units. The lactase protein is an internally duplicated enzyme that belongs to the glycosyl hydrolases family GH 1 (Benson et al., 2008). The enzyme is bound at the C-terminal to enterocyte lumenal plasma membrane and has both lactase and phlorizin hydrolase activity. The second activity also hydrolyzes β-glucosides of lip-ids and micronutrients such as pyridoxine-5′-β-D-glucoside and other glycosylated phytochemicals. The enzyme has a glutamic acid proton donor and glutamic acid nucleophile and a (β/α)8 barrel structure. In the human, the gene for lactase activity is downregulated at about 4 years of age, resulting in symptomatic lactose intolerance. A
Small intestinal mucosal lactase is the hydrolase that digests lactose to the mono-saccharide units. The lactase protein is an internally duplicated enzyme that belongs to the glycosyl hydrolases family GH 1 (Benson et al., 2008). The enzyme is bound at the C-terminal to enterocyte lumenal plasma membrane and has both lactase and phlorizin hydrolase activity. The second activity also hydrolyzes β-glucosides of lip-ids and micronutrients such as pyridoxine-5′-β-D-glucoside and other glycosylated phytochemicals. The enzyme has a glutamic acid proton donor and glutamic acid nucleophile and a (β/α)8 barrel structure. In the human, the gene for lactase activity is downregulated at about 4 years of age, resulting in symptomatic lactose intolerance. A