Overview

Metabolic pathways are separated not only by virtue of different enzyme systems that catalyze their forth and back reactions (anabolism and catabolism), but also by multiple compartmentalization. Compartments represent aqueous solutions like the cytoplasm, extracellular fluid, the mitochondrial matrix, or the endoplasmatic reticulum lumen all separated from each other by membranes or phospholipid bilayers. Weakly hydrophobic molecules (e.g. molecular oxygen O₂, carbon dioxide CO₂, steroid hormones, thyroxin) rapidly diffuse across these membranes by free diffusion. Water H₂O has a slow rate of diffusion and its flow is facilitated by the presence of aquaporins (water channels).

This is a typical situation for polar and charged molecules. Membrane proteins are the 'enzymes' which facilitate the transport of metabolic intermediates between cell compartments as well as into and out of the cell. All transport systems pertaining to metabolites can be understood as mediated transport promoting either facilitated diffusion or active transport. Protein mediated diffusion differs from non-mediated diffusion of O₂ and CO₂ in four distinct ways:

1. the speed and specificity of the transport
2. the transport capacity is finite, i.e., exhibits saturation kinetics
3. transport can be competitively inhibited (antagonists)
4. transport can be chemically inactivated (heat, high salt)

Transporter or ion channel?

Although chemical potentials (gradients) determine the direction of diffusion across membranes (from a compartment with high concentration  to a compartment with low concentration, i.e. the [electro-] chemical potential gradient in the case of ions), thermodynamics cannot tell us how fast the diffusion proceeds. The difference in transport rate between ion channels and transporters is at least 10,000 fold. Transporters other than channels are called carriers and are subdivided into passive (facilitators) and active (pumps) systems. Carriers are also referred to as permeases, antiporters and symporters and translocases. The low flux rate associated with carriers is the prize the membrane transport system pays in exchange for substrate specificity. Facilitated transport is tightly coupled to the chemical potential of metabolites or a group of metabolites (representing a pathway). They are also coupled to the energy charge of the cell in the case of pumps. Active transport is either coupled to ATP hydrolysis or ion gradients. The latter uses a mechanism called flux coupling where the energy requiring transport of the substrate (against its gradient) is coupled to the exergonic flux of an ion (Na+; K+; H+) or in exchange for an other metabolite. These systems work either as symporters (co-directional flux) or antiporters (counter-flow).
 

Facilitated glucose transport in humans

Glucose transporters in humans come in five subtypes called GLUT1 (gene name SLC2A1; solute carrier family 2), through GLUT5. They are located in the plasma membrane of cells and have glucose binding sites outside and inside the membrane. The relative affinities of these two binding sites may differ. Different cells express different glucose transport genes.

 

GLUT1 and 3	These two types are found in all cells (except liver, intestinal epithelia and pancreatic beta cells) and are responsible for a basic glucose uptake. The Michaelis-Menten constant Km = 1mM and is slightly lower than the lower range of healthy blood glucose concentration of 4 - 8mM. Thus, glucose is efficiently absorbed by cells even during fasting and starving conditions. Liver, kidney and intestinal cells can synthesize glucose and release it into the blood circulation, a process known as gluconeogenesis (see GLUT2).
GLUT2:	A low affinity glucose transporter that is typically expressed in liver, intestinal epithelia and beta cells (insulin secretion) of the pancreas. Its Km = 15-20mM is several fold higher than average blood glucose levels of 4 - 8mM glucose. As result glucose entry into liver cells (and beta cells) is normally slow, but proportional to the glucose level in blood, while GLUT1/3 systems are saturated and promote a steady glucose influx into cells (e.g. in  neurons). In the small intestine, glucose absorption into mucosal epithelial cells occurs via a Na/glucose symporter. Glucose is pumped into the cells using the energy of the Na gradient (flux coupling) where both substrates accumulate. Glucose subsequently diffuses via GLUT2 into the extracellular fluid reaching the portal vein blood circulation. Intracellular Na+ is kept low by the Na-K-ATPase on the same basal membrane as GLUT2. The Na/glucose transporter is the primary uptake system of intestinal glucose and galactose during fasting conditions, when sugar levels are low. During and after a meal, GLUT2 is also found in the apical membrane supporting the efficient glucose uptake from the intestinal lumen.
GLUT4	is found in adipocytes and skeletal muscle cells and has an affinity of Km = 5mM, right at blood serum levels of glucose (4-8mM). This receptor is upregulated by insulin. High glucose levels, which will saturate the muscle transporter (but not the liver/pancreas type), cause the secretion of insulin. Insulin activates the glut4 gene and more transporter proteins are synthesized and incorporated into the muscle cell membrane, increasing the capacity for glucose transport in this system (increase of Jmax; see below).
GLUT5:	has a preference for fructose, the monosaccharide found in fruit sugar together with glucose. GLUT5 is found in the apical membrane of intestinal enterocytes allowing the diffusion driven absorption of fructose into the enterocytes. Fructose is thought to leave the enterocytes on the basolateral side via the GLUT2 transporter or may be metabolically converted to glucose before leaving the enterocytes.

Why are there differences in the effective glucose transport mediated by distinct GLUT transporters? Differences can be assessed (quantified) based on the dose-response analysis where the uptake rate is measured at increasing glucose concentration (measured as flux J_B of substrate B; J_max indicates saturation and and constant K_m is defined as substrate concentration where  Jmax  is half maximal).  GLUT1&3 are saturated at physiological blood glucose levels while GLUT2 in liver still slows down the glucose equilibration because of its much lower binding affinity for the substrate. The binding curve for GLUT2 at physiological conditions is essentially linear. This simply means that glucose uptake rates in liver are directly proportional to blood glucose levels, exactly what one expects from a glucostat. Muscle glucose uptake is not saturated at physiological (resting) conditions, but will be saturated after a diet high in carbohydrates. To ensure maximal glucose absorption by muscle cells, insulin triggers GLUT4 protein biosynthesis (gene expression regulation) effectively increasing the transport capacity of muscle plasma membranes to over 200% normal.

Mitochondrial carriers and shuttles

The inner mitochondrial membrane is an important selective barrier that controls the coupling of the central respiratory energy producing pathways  -  citric acid cycle and oxidative phosphorylation  -  with carbohydrate, fat, and amino acid metabolism. Several transport system have been identified that couple the citric acid cycle intermediates with the cytoplasm, transfer reducing equivalent from cytoplasmic NADH to matrix NADH (or FADH₂), and couple the energy state of a cell, i.e., its ADP/ATP ratio with the electron transport chain. During glycolysis NADH is produced and its reducing power utilized by the electron transport process in the mitochondrial inner membrane. The nicotinamide-adenosine dinucleotide molecule cannot cross neither the outer nor the inner mitochondrial membrane in order to bind to complex I (binding site on matrix side of membrane). Mammalian systems have two shuttle systems that extract the reducing equivalent from cytoplasmic NADH and funnel it into oxidative phosphorylation.

The first system is the glycerol-P-shuttle where dihydroxyacetonephosphate (DHAP) is reduced by NADH/H⁺ to glycerole-3-phosphate (C00093) and NAD⁺ by Glycerol-3-phosphate dehydrogenase (EC 1.1.1.8). The small, reduced and negatively charged C3 compound readily diffuses across the outer membrane porin channels (VDAC; voltage dependent anion channel). It binds to a dehydrogenase complex where is delivers its reducing equivalent to an FAD coenzyme to form FADH₂ and DHAP. The reduced FADH₂ donates the electron/hydrogen pair to ubiquinone much like complex II, although this system is different from complex II. The glycerol-P shuttle thus transports NADH equivalents against an NADH gradient. Because the process is equivalent to the matrix compartment succinate dehydrogenase complex (complex II) using FAD as coenzyme, the resulting proton pumping (via quinone pool) is only sufficient to produce two mols of ATP for every mol of cytoplasmic NADH/H⁺.

The second system is slightly more complex, its intermediates tightly coupled to the levels of citric acid cycle intermediates, but is able to transport the full reducing equivalent of cytoplasmic NADH/H⁺ into the mitochondrial matrix. This system is known as malate-aspartate shuttle. Here NADH is oxidized using oxaloacetate reduction to malate. Malate diffuses across the outer mitochondrial membrane (using porin). From the intermembrane space it is transported into the matrix via the malate-alpha-ketogluatarate antiporter in the inner membrane (for every malate entering the matrix compartment one molecule of alpha-ketoglutarate is expelled). Malate is subsequently reoxidized to oxaloacetate reducing NAD⁺ to NADH/H⁺. The full reducing equivalent of cytoplasmic NADH is thus transporter into the mitochondria.

NADH/H⁺ interacts with complex I (to reduce ubiquinone Q_B) and three mols of ATP are synthesized for every mol of malate transported into the matrix. Oxaloacetate cannot cross the inner membrane because their is no transport system (carrier) for it. Instead, oxaloacetate is converted to aspartate by the oxaloacetate-aspartat transferase ( a transamination reaction; see nitrogen metabolism). Aspartate is transported out of the mitochondria in exchange for glutamate where the aspartate is converted back to its keto acid form oxaloacetate by a cytoplasmic oxaloacetate-aspartate transferase. The alpha-ketoglutarate and glutamate undergo an analogous transamination reaction in both the cytoplasm and mitochondrial matrix, thus closing the malate-aspartate shuttle system and providing a net transport of NADH form the cytoplasm to the mitochondrial matrix.

This shuttle system is fully reversible, unlike the glycerol-P shuttle, where the membrane bound glycerol dehydrogenase (FAD coenzyme) cannot catalyze the DHAP reduction to glycerol-3-phosphate (for thermodynamic reasons; see reduction potentials). The transport system is not coupled to an ATPase or ion gradient, however, the substrate availability determines if NADH/H⁺ reducing equivalents are imported or exported from the mitochondrial matrix. Under aerobic conditions, the citric acid cycle is fully active producing a steady-state throughput of NADH into complex I keeping its concentration in the matrix low and NAD⁺ high. As a result, cytoplasmic NADH reducing equivalent is imported providing the necessary oxidized NAD⁺ coenzyme for glycolysis.  If the energy state of the cell is high (ATP accumulates) the electron transport chain is suppressed and accumulating NADH/H⁺ reducing equivalents are exported from rather than imported into the matrix and both citric acid cycle and glycolysis are allosterically inhibited.

This malate-aspartate shuttle also links amino acid metabolism with the energy charge and citric acid cycle of the cell. It depends on the presence of aminotransferases. High levels of citric acid cycle intermediates increase the capacity of the malate-aspartate shuttle with high transfer rates of cytoplasmic reducing equivalent into the mitochondrial matrix. Note that the carbon carriers of the reducing equivalent are not used up in this reversible, cyclic carrier mechanism. The malate-aspartate shuttle couples the energy state of the cell in terms of reducing power to the metabolic rate of the mitochondria (ATP synthesis).

ATP-ADP translocase

A third system that uses membrane bound proteins to 'read' the metabolite concentrations is the ATP-ADP translocase in the inner mitochondrial membrane. It is an antiporter that exchanges one molecule of ADP for every molecule of ATP moving into the mitochondria effectively coupling cytoplasmic ADP/ATP ratios with mitochondrial ADP/ATP ratios. In contrast to the malate-aKG and aspartate-glutamate antiporters, the ATP-ADP translocase is an electrogenic antiporter because the 1:1 exchange of a dinucleotide for a trinucleotide produces a charge difference. ATP is usually exported into the cytoplasm and ADP imported for further ATP synthesis. ATP carries 4 negative charges while ADP carries only 3. The result is a net export of one negative charge. This charge imbalance is coupled to the proton gradient, which in the first place makes the inner mitochondrial membrane outside positive and the matrix side negatively charged. In effect, the membrane potential component of the proton gradient is the energy source to drive the ATP-ADP translocase to secrete ATP^4- from the mitochondrial matrix while importing ADP^3-. Despite similar binding affinities of the two nucleotides for the translocase, ATP experiences a stronger driving force than ADP resulting in a 30 fold higher ATP efflux than ADP. One might expect that inorganic phosphate equilibrates this charge imbalance because it is a necessary substrate for ATP synthesis. Phosphate uptake into the mitochondrial matrix, however, is mediated by a H⁺/P_i symporter. This transporter carries inorganic phosphate along protons in an electroneutral manner pushing the negatively charged phosphate against the electrochemical gradient.

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