Gs

Glycogen synthase

M FIGURE 15-6 Integrated regulation of glycogenolysis mediated by several second messengers. (a) Neuronal stimulation of striated muscle cells or epinephrine binding to p-adrenergic receptors on their surfaces leads to increased cytosolic concentrations of the second messengers Ca2+ or cAMP respectively. The key regulatory enzyme, glycogen phosphorylase kinase (GPK), is activated by Ca2+ ions and by a cAMP-dependent protein kinase A (PKA). (b) In liver cells, p-adrenergic stimulation leads to increased cytosolic concentrations of cAMP and two other second messengers, diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). Enzymes are marked by white boxes. ( + ) = activation of enzyme activity, (—) = inhibition.

In liver cells, hormone-induced activation of phospholi-pase C also regulates glycogen breakdown and synthesis by the two branches of the inositol-lipid signaling pathway. Phospholipase C generates two second messengers, diacyl-glycerol (DAG) and inositol 1,4,5-trisphosphate (IP3) (see Figure 13-28). DAG activates protein kinase C, which phos-phorylates glycogen synthase, yielding the phosphorylated inactive form and thus inhibiting glycogen synthesis. IP3 induces an increase in cytosolic Ca2+, which activates glyco-gen phosphorylase kinase as in muscle cells, leading to gly-cogen degradation. In this case, multiple intracellular signal-transduction pathways are activated by the same signal (Figure 15-6b).

The dual regulation of glycogen phosphorylase kinase results from its multimeric subunit structure (apy8)4. The y subunit is the catalytic protein; the regulatory a and p sub-units, which are similar in structure, are phosphorylated by protein kinase A; and the 8 subunit is the Ca2 +-binding switch protein calmodulin. Glycogen phosphorylase kinase is maximally active when Ca2+ ions are bound to the calmod-ulin subunit and at least the a subunit is phosphorylated. In fact, the binding of Ca2+ to the calmodulin subunit may be essential to the enzymatic activity of glycogen phosphorylase kinase. Phosphorylation of the a and p subunits increases the affinity of the calmodulin subunit for Ca2+, enabling Ca2 + ions to bind to the enzyme at the submicromolar Ca2+ concentrations found in cells not stimulated by nerves. Thus increases in the cytosolic concentration of Ca2+ or of cAMP or of both induce incremental increases in the activity of glyco-gen phosphorylase kinase. As a result of the elevated level of cytosolic Ca2+ after neuron stimulation of muscle cells, glycogen phosphorylase kinase will be active even if it is un-phosphorylated; thus glycogen can be hydrolyzed to fuel continued muscle contraction in the absence of hormone stimulation.

Insulin and Glucagon Work Together to Maintain a Stable Blood Glucose Level

In the regulation of glycogenolysis, neural and hormonal signals regulate the same key multimeric enzyme. In contrast, the maintenance of normal blood glucose concentrations depends on the balance between two hormones that elicit different cell responses. During periods of stress, the epineph-rine-induced increase in glycogenolysis in liver cells leads to a rise in blood glucose. During normal daily living, however, the blood glucose level is under the dynamic control of insulin and glucagon.

Insulin and glucagon are peptide hormones produced by cells within the islets of Langerhans, cell clusters scattered throughout the pancreas. Insulin, which contains two polypeptide chains linked by disulfide bonds, is synthesized by the p cells in the islets; glucagon, a monomeric peptide, is produced by the a cells in the islets. Insulin reduces the level of blood glucose, whereas glucagon increases blood glucose. The availability of glucose for cellular metabolism is regulated during periods of abundance (following a meal) or

K+ channels

▲ FIGURE 15-7 Secretion of insulin from pancreatic p cells in response to a rise in blood glucose. The entry of glucose into p cells is mediated by the GLUT2 glucose transporter ( 1). Because the Km for glucose of GLUT2 is =20 mM, a rise in extracellular glucose from 5 mM, characteristic of the fasting state, causes a proportionate increase in the rate of glucose entry (see Figure 7-3). The conversion of glucose into pyruvate is thus accelerated, resulting in an increase in the concentration of ATP in the cytosol (B). The binding of ATP to ATP-sensitive K+ channels closes these channels (3), thus reducing the efflux of K+ ions from the cell. The resulting small depolarization of the plasma membrane (4|) triggers the opening of voltage-sensitive Ca2+ channels (5). The influx of Ca2+ ions raises the cytosolic Ca2+ concentration, triggering the fusion of insulin-containing secretory vesicles with the plasma membrane and the secretion of insulin ( 6). Steps 5 and 6 are similar to those that take place at nerve terminals, where a membrane depolarization induced by the arrival of an action potential causes the opening of voltage-sensitive Ca2+ channels and exocytosis of vesicles containing neurotransmitters (see Figure 7-43). [Adapted from J.-Q. Henquin, 2000, Diabetes 49:1751.]

scarcity (following fasting) by the adjustment of insulin and glucagon concentrations in the blood.

After a meal, when blood glucose rises above its normal level of 5 mM, the pancreatic p cells respond to the rise in glucose or amino acids by releasing insulin into the blood (Figure 15-7). The released insulin circulates in the blood and binds to insulin receptors on muscle cells and adipocytes (fat-storing cells). The insulin receptor, a receptor tyrosine kinase (RTK), can transduce signals through a phosphoinositide pathway leading to the activation of protein kinase B (see Figure 14-27). By an unknown mechanism, protein kinase B triggers the fusion of intracellular vesicles containing GLUT4 glucose transporters with the plasma membrane (Figure 15-8). The resulting increased number of GLUT4 on the cell surface increases glucose influx, thus lowering blood glucose. When insulin is removed, cell-surface GLUT4 is internalized by endocytosis, lower

▲ EXPERIMENTAL FIGURE 15-8 Insulin stimulation of fat cells induces translocation of GLUT4 from intracellular vesicles to the plasma membrane. In this experiment, fat cells were engineered to express a chimeric protein whose N-terminal end corresponded to the GLUT4 sequence, followed by the entirety of the GFP sequence. When a cell is exposed to light of the exciting wavelength, GFP fluoresces yellow-green, indicating the position of GLUT4 within the cell. In resting cells (a), most

GLUT4 is in internal membranes that are not connected to the plasma membrane. Successive images of the same cell after treatment with insulin for 2.5, 5, and 10 minutes show that, with time, increasing numbers of these GLUT4-containing membranes fuse with the plasma membrane, thereby moving GLUT4 to the cell surface (arrows) and enabling it to transport glucose from the blood into the cell. Muscle cells also contain insulin-responsive GLUT4 transporters. [Courtesy of J. Bogan.]

▲ EXPERIMENTAL FIGURE 15-8 Insulin stimulation of fat cells induces translocation of GLUT4 from intracellular vesicles to the plasma membrane. In this experiment, fat cells were engineered to express a chimeric protein whose N-terminal end corresponded to the GLUT4 sequence, followed by the entirety of the GFP sequence. When a cell is exposed to light of the exciting wavelength, GFP fluoresces yellow-green, indicating the position of GLUT4 within the cell. In resting cells (a), most

GLUT4 is in internal membranes that are not connected to the plasma membrane. Successive images of the same cell after treatment with insulin for 2.5, 5, and 10 minutes show that, with time, increasing numbers of these GLUT4-containing membranes fuse with the plasma membrane, thereby moving GLUT4 to the cell surface (arrows) and enabling it to transport glucose from the blood into the cell. Muscle cells also contain insulin-responsive GLUT4 transporters. [Courtesy of J. Bogan.]

ing the level of cell-surface GLUT4 and thus glucose import. Insulin stimulation of muscle cells also promotes the uptake of glucose and its conversion into glycogen, and it reduces the degradation of glucose to pyruvate. Insulin also acts on hepatocytes to inhibit glucose synthesis from smaller molecules, such as lactate and acetate, and to enhance glycogen synthesis from glucose. The net effect of all these actions is to lower blood glucose back to the fasting concentration of about 5 mM.

If the blood glucose level falls below about 5 mM, pancreatic a cells start secreting glucagon. The glucagon receptor, found primarily on liver cells, is coupled to Gs protein, like the epinephrine receptor (Chapter 13). Glucagon stimulation of liver cells activates adenylyl cyclase, leading to the cAMP-mediated cascade that inhibits glycogen synthesis and promotes glycogenolysis, yielding glucose 1-phosphate (see Figure 15-6b). Liver cells can convert glucose 1-phosphate into glucose, which is released into the blood, thus raising blood glucose back toward its normal fasting level.

Diabetes mellitus results from a deficiency in the amount of insulin released from the pancreas in response to glucose (type I) or from a decrease in the ability of muscle and fat cells to respond to insulin (type II). In both types, the regulation of blood glucose is impaired, leading to persisent hyperglycemia and numerous other possible complications in untreated patients. Type I diabetes is caused by an autoimmune process that destroys the insulin-producing p cells in the pancreas. Also called insulin-dependent diabetes, this form of the disease is generally responsive to insulin therapy. Most Americans with diabetes mellitus have type II, but the underlying cause of this form of the disease is not well understood. I

Oxygen Deprivation Induces a Program of Cellular Responses

In glycogenolysis, the activity of preexisting proteins was regulated by the integration of multiple signals. Organisms that require oxygen respond to oxygen deprivation, a single stimulus, in multiple ways, some occurring rapidly and others taking longer to develop. In addition, over evolutionary time, animals that live at high altitude (e.g., llamas, guanacos, alpacas) became adapted to low oxygen. This adaptation entailed single amino acid changes in the p-globin chain that increased the oxygen affinity of hemoglobin in these animals compared with that of hemoglobin in other animals.

Among the rapid responses to low oxygen (hypoxia) is dilation of blood vessels, permitting increased blood flow. This response is regulated by nitric oxide, cyclic GMP, and protein kinase G (see Figure 13-30). A rapid shift in metabolism, called the Pasteur effect, also occurs when cells are deprived of adequate oxygen. First observed in yeast cells, this response accelerates the anaerobic metabolism of glucose when aerobic metabolism and oxidative phosphorylation slows owing to low oxygen. Burning more carbohydrates compensates for the reduced ATP yield from anaerobic metabolism. Phosphofructokinase 1, the third enzyme in gly-colysis, is inhibited by ATP and stimulated by AMP; so, when the cell is short on energy, glycolysis increases (see Figure 8-12). The adjustment is rapid, inasmuch as it does not require the synthesis of new molecules.

Slow adaptive responses to low oxygen at the level of the whole organism include increasing the production of ery-throcytes, which is stimulated by erythropoietin produced in the kidney (see Figure 14-7). Transcription of the erythro-poietin gene is regulated primarily by hypoxia-induced factor 1 (HIF-1), a transcriptional activator. The amount of HIF-1 increases drastically as the partial pressure of oxygen decreases from 35 mm Hg to zero, a range typical of normal fluctuations. The nature of the oxygen sensor that causes the increased expression of HIF-1 is not yet known, but it probably requires a protein that has a heme-containing oxygen-binding site somewhat like that in hemoglobin. In addition to regulating the erythropoietin gene, HIF-1 coor-dinately activates the transcription of several other genes whose encoded proteins help cells respond to hypoxia (Figure 15-9). One of these proteins, vascular endothelial growth factor (VEGF), is secreted by cells lacking oxygen and promotes local angiogenesis, the branching growth of blood vessels. Expression of VEGF requires not only HIF-1 but also a Smad transcription factor, which is activated by a TGFp signal. The ability of HIF-1 to control different genes in different cell types presumably results from this type of combinatorial action.

The results of recent studies revealed that the degradation of HIF-1 is controlled by an oxygen-responsive prolyl hy-droxylase. HIF-1 is a dimer composed of two subunits, a and p. The p subunit is abundant in the cytosol under high or low oxygen conditions but, when oxygen is plentiful, the a subunit (HIFa) is ubiquitinated and degraded in proteasomes (Chapter 3). Ubiquitination is promoted by the von Hippel-Lindau protein (pVHL), which binds to a conserved "degradation domain" of HIF-1a. The binding of pVHL in turn is facilitated by hydroxylation of a proline in the pVHL-binding site on HIF-1a. The prolyl hydroxylase catalyzing this reaction requires iron and is most active at high oxygen, leading to degradation of the a subunit and no transcrip-tional activation by HIF-1. At low oxygen, when hydroxy-lation does not occur, active dimeric HIF-1 forms and is translocated to the nucleus. The hypoxia-response pathway mediated by HIF-1 and its regulation by pVHL have been conserved for more than half a billion years, given that it is the same in mammals, worms, and insects.

H Hypoxia affects the growth of blood vessels, particularly the small capillaries, whose exact pattern, unlike that of major blood vessels like the aorta, is not genetically determined. Angiogenesis, the branching growth of the vasculature, is stimulated by hypoxia, thus ensuring that all cells are in adequate proximity to the oxygenated blood supply. Growing tumors stimulate angiogenesis to ensure their own blood supply (Chapter 23). Understanding the signals that control angiogenesis could potentially lead to the development of therapeutic agents that stimulate angiogenesis in a transplanted or diseased organ that is receiving insufficient blood or that inhibit angiogenesis in developing tumors, thereby suffocating them. I

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