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15.5: Coordinated Regulation of Glycogen Synthesis and Breakdown - Biology

15.5: Coordinated Regulation of Glycogen Synthesis and Breakdown - Biology


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15.5: Coordinated Regulation of Glycogen Synthesis and Breakdown

Role of Glycogenolysis in Memory and Learning: Regulation by Noradrenaline, Serotonin and ATP

This paper reviews the role played by glycogen breakdown (glycogenolysis) and glycogen re-synthesis in memory processing in two different chick brain regions, (1) the hippocampus and (2) the avian equivalent of the mammalian cortex, the intermediate medial mesopallium (IMM). Memory processing is regulated by the neuromodulators noradrenaline and serotonin soon after training glycogen breakdown and re-synthesis. In day-old domestic chicks, memory formation is dependent on the breakdown of glycogen (glycogenolysis) at three specific times during the first 60 min after learning (around 2.5, 30, and 55 min). The chicks learn to discriminate in a single trial between beads of two colors and tastes. Inhibition of glycogen breakdown by the inhibitor of glycogen phosphorylase 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) given at specific times prior to the formation of long-term memory prevents memory forming. Noradrenergic stimulation of cultured chicken astrocytes by a selective β2-adrenergic (AR) agonist reduces glycogen levels and we believe that in vivo this triggers memory consolidation at the second stage of glycogenolysis. Serotonin acting at 5-HT2B receptors acts on the first stage, but not on the second. We have shown that noradrenaline, acting via post-synaptic α2-ARs, is also responsible for the synthesis of glycogen and our experiments suggest that there is a readily accessible labile pool of glycogen in astrocytes which is depleted within 10 min if glycogen synthesis is inhibited. Endogenous ATP promotion of memory consolidation at 2.5 and 30 min is also dependent on glycogen breakdown. ATP acts at P2Y1 receptors and the action of thrombin suggests that it causes the release of internal calcium ([Ca(2+)]i) in astrocytes. Glutamate and GABA, the primary neurotransmitters in the brain, cannot be synthesized in neurons de novo and neurons rely on astrocytic glutamate synthesis, requiring glycogenolysis.

Keywords: ATP astrocytes consolidation day-old chickens glycogen re-synthesis memory processing noradrenaline serotonin.


Metabolic Processes Controlled by Allosteric Enzymes (With Diagram)

An excellent example of allosteric enzyme regulation of metabolic processes is provided by the interrela­tionship in animals between the metabolic pathways that result in:

(1) The synthesis of glycogen from glu­cose and

(2) The oxidation of glucose to CO2 and water.

Nearly all of the energy-consuming processes in the body proceed at the expense of ATP and much of this ATP is derived through the oxidation of glucose. Dur­ing periods of elevated activity (e.g., exercise), glyco­gen is broken down to yield glucose, which then enters the metabolic pathway converting it to CO2 and water, with consequent generation of ATP. In contrast, dur­ing periods of rest or low energy demand, absorbed glucose is converted to glycogen.

Three of the en­zymes involved in glucose metabolism are allosteric these are phosphofructokinase (an enzyme required in the series of reactions that convert glucose-6-phosphate to CO2 and water), glycogen synthetase (involved in the incorporation of glucose-l-phosphate into glyco­gen), and glycogen phosphorylase (which removes glu­cose as glucose-l-phosphate from glycogen during glycogen catabolism).

When ATP levels are high and no major consump­tion of energy is taking place in the body, glucose is diverted into glycogen (i.e., “glycogenesis” predomi­nates). This is achieved because ATP acts as a nega­tive effector of phosphofructokinase and glycogen phosphorylase and as a positive effector, along with glucose-6-phosphate, of glycogen synthetase (Fig. 11-8a).

When the ATP level falls (e.g., during exercise) and there is an increased demand for ATP, glycogen syn­thesis is halted as absorbed glucose is directly con­sumed in the production of ATP and additional glu­cose is made available through the catabolism of glycogen (i.e., “glycogenolysis”). This pathway is acti­vated by the positive effects on phosphofructokinase and glycogen phosphorylase of the ATP precursor, AMP.

The hormone epinephrine, secreted into the blood­stream during periods of great activity, also has an ef­fect on these metabolic pathways in muscle and in liver. When epinephrine in the bloodstream reaches the muscles, it binds to the surface of the muscle cells and promotes the synthesis of cyclic AMP (cAMP) by the enzyme adeny Icy close.

The cAMP then allosterically activates a second enzyme (protein kinase), which ultimately activates glycogen phosphorylase but inactivates glycogen synthetase (Fig. 11-8b). This phe­nomenon is also considered with the functions of hormones and the role of protein phosphorylation as a metabolic regulatory mechanism.

The pathways described above illustrate the mecha­nisms for turning allosteric enzymes on and off. In the absence of such mechanisms, both pathways would simultaneously be active so that their effects cancel one another—a most unproductive state! Allosterism thus provides a basis for regulating the levels of activ­ity of related metabolic pathways.

The Regulation of Amino Acid Synthesis:

Escherichia coli provide a clear example of control of divergent metabolic pathways by feedback inhibition. An outline of the metabolic pathways for the synthesis of three amino acids is shown in Figure 11-9. Lysine, methionine, and threonine are each synthesized from aspartate and each may be utilized in protein synthe­sis.

Without metabolic controls, the consumption or utilization of any one of these amino acids would stim­ulate the pathways and cause unneeded synthesis of the unused amino acids as well as the one utilized. Such an unregulated system would consume vital re­sources and energy both factors could have survival implications to the organism and evolutionary conse­quences to the species.

However, in E. coli, the allo­steric regulatory mechanisms are most effective. The accumulation of each amino acid produces a feedback inhibition of the first enzyme in the specific branch of the pathway leading to the synthesis of that amino acid. In Figure 11-9, this negative effect is shown by dashed lines.

Moreover, an additional level of regula­tion is achieved through effects on the enzyme aspartokinase, which catalyzes and phosphorylation of aspartate. This enzyme exists in three forms (i.e., there are three isozymes), symbolized in Figure 11-9 by using three separate arrows to show the conversion of aspartate to aspartylphosphate.

One of the isozy­mes is specifically and completely inhibited by threonine the second (which is present only in small amounts) is specifically inhibited by homoserine and the third isozyme is specifically inhibited by lysine. In addition, synthesis of the latter isozyme is repressed by lysine. (Repression is a regulatory mechanism that reduces the number of enzyme molecules in the cell.


Covalent Regulation of Enzyme Activity

There are two basic types of covalent control of enzyme activity: reversible and irreversible. An example of irreversible activation is the proteolytic cleavage of proenzymes in the digestive tract that lead to the active forms of trypsin and chymotrypsin. The activation of these proteolytic enzymes outside the cell is critical, as the presence of active enzymes within the cell could lead to the unwanted degradation of cellular components, or to the complete digestion of the contents of the secretory vesicles in which the proenzymes reside prior to secretion from the cell. Another example of irreversible inhibition of enzyme activity is the action of serpins on extracellular proteases. Proteases, in addition to their action during digestion, are essential contributors to the remodeling of the extracellular matrix and in the action of the clotting cascades. Following activation of these proteases, the duration of their activity is controlled by the presence of specific inhibitors, termed serpins (serine protease inhibitors), that form tight and irreversible complexes with the proteases. This complex is then rapidly cleared from the extracellular circulation and degraded.

The key reversible covalent modification of activity is phosphorylation. In eukaryotic systems, protein kinases utilize ATP to transfer a phosphate to the protein on the side chains of either serine, threonine, or tyrasine residues in the target protein's sequence (Fig. 3.14). The specificity of kinases determines both which amino acid is phosphorylated and which protein is phosphorylated. Protein phosphatases hydrolyze the phosphate moiety off of the phosphorylated protein using water to catalyze the reaction and release free phosphate groups (Fig. 3.14). Notice that if this process is not regulated, it becomes a "futile" cycle of ATP hydrolysis. Thus, the phosphorylation and dephosphorylation reactions must be highly specific and tightly controlled, and opposing activities must be coordinated by the cell. A principal way by which changes

Figure 3.14 The phosphorylation cycle of proteins. Phosphorylation is a key reversible covalent modification of enzyme activity. Protein kinases utilize ATP to transfer a phosphate to the protein, while protein phosphatases are enzymes that remove phosphate groups. Addition or removal of phosphate changes protein activity, but does not necessarily correspond with enzyme activation or inhibition.

Figure 3.14 The phosphorylation cycle of proteins. Phosphorylation is a key reversible covalent modification of enzyme activity. Protein kinases utilize ATP to transfer a phosphate to the protein, while protein phosphatases are enzymes that remove phosphate groups. Addition or removal of phosphate changes protein activity, but does not necessarily correspond with enzyme activation or inhibition.

in the phosphorylation state of proteins within the cell arise is as a response to some extracellular signal, such as hormone action (see Chap. 4 on Cellular Signaling).

A good example of coordinate regulation of a pathway is the control of glycogen synthesis and its breakdown. Glycogen synthase, as the name implies, synthesizes glycogen from glucose, and glycogen phosphorylase breaks down glycogen to glucose. Phosphorylation of glycogen synthase inactivates the enzyme, while phosphorylation of glycogen phosphorylase activates the enzyme. The cell responds to the hormone epinephrine by activating protein kinases that phosphorylate both enzymes, while the hormone insulin causes the opposite effects on the phosphorylation states of these two enzymes by activating phosphatases (Fig. 3.15).

The action of these hormones is directed to different kinases and phosphatases that are specific for their target enzymes, glycogen syn-thase and glycogen phosphorylase, in this example. This is a common theme in the regulation of cellular processes in response to changes in the external environment. In the above example, this type of coordinate regulation of activity is crucial, otherwise the activity of phosphorylase and glycogen synthase will oppose each other and no net synthesis of glycogen or net breakdown of glycogen will occur during periods of glucose excess or glucose deficiency, respectively.

Figure 3.15 Glycogen regulation. Glycogen synthesis and glycogen breakdown are regulated by the hormones epinephrine and insulin. When glucose levels are high, the organism responds by releasing insulin which signals the activation of enzymes responsible for glucose storage. The action of epinephirine opposes that of insulin and leads to the activation of phosphorylase and inhibition of glycogen synthase.


Steps involved in Glycogenesis

There are 6 major steps are involved in the Glycogenolysis:

Step 1: Glucose Phosphorylation

Glucose is phosphorylated into Glucose-6-Phosphate, a reaction that is common to the first reaction in the pathway of glycolysis from Glucose.

This reaction is catalyzed by Hexokinase in Muscle and Glucokinase in the Liver.

Glucose + ATP –> Glucose-6-P

(Enzyme: Glucokinase or Hexokinase)

Step 2: Glc-6-P to Glc-1-P conversion

Glucose-6-P is converted to Glc-1-Phosphate in a reaction catalyzed by the enzyme “Phosphoglucomutase”.

Glucose-6-P + Enz-P <—> Glucose-1,6-bis Phosphate + Enz <—> Glucose-1-Phosphate + Enzyme-P

(Enzyme: Phosphoglucomutase)

Step 3: Attachment of UTP to Glc-1-P

Glucose-1-P reacts with Uridine triphosphate (UTP) to form the active nucleotide Uridine diphosphate Glucose (UDP-Glc). The reaction is catalyzed by the enzyme “UDPGlc Pyrophosphorylase”.

UTP + Glucose-1-P <—> UDPGlc + PPi

(Enzyme: UDPGlc Pyrophosphorylase)

Step 4: Attachment of UDP-Glc to Glycogen Primer

A small fragment of pre-existing glycogen must act as a “Primer” (also called GLYCOGENIN) to initiate glycogen synthesis. The Glycogenin can accept glucose from UDP-Glc.

The hydroxyl group of the amino acid tyrosine of Glycogenin is the site at which the initial glucose unit is attached. the enzyme Glycogen initiator synthase transfers the first molecule of Glucose to Glycogenin.

Then glycogenin itself takes up a for glucose residues to form a fragment of primer which serves as an acceptor for the rest of the glucose molecules.

Step 5: Glycogen synthesis by Glycogen synthase

Glycogen synthase, the enzyme transfers the Glucose from UDP-Glc to the non-reducing end of Glycogen to form alpha 1,4-linkages.

Glycogen synthase catalyzes the synthesis of a linear unbranched molecule with alpha-1,4-glycosidic linkages.

Step 6: Glycogen Branches formation

In this step, the formation of branches is brought about by the action of a branching enzyme, namely branching enzyme (amylo-[1—>4]—>[1—>6]-transglucosidase).

This enzyme transfers a small fragment of five to eight glucose residues from the non-reducing end of the glycogen chain. to another glucose residue where it is linked by the alpha-1,6 bond.

It leads to the formation of a new non-reducing end, besides the existing one. The glycogen chain will be elongated and branched.

The overall reaction of Glycogenesis,

(Glucose)n + Glucose + 2 ATP –> (Glucose) n+1 + 2 ADP + Pi

Two ATP molecules will utilize in this process. One is required for the phosphorylation of Glucose and the other is needed for conversion of UDP to UTP.


Discussion

The coordinated regulation of glycogenolysis and glycogenesis in the liver and the skeletal muscle is dependent on a network of interacting enzymes and effectors that determine the fractional activation of GP and GS [3-6,9-12]. In the present work, the cascades involved in the regulation of glycogen synthesis and breakdown were analyzed at steady state to gain an insight into the inherent design principle of the regulatory cascades existing in muscle and liver. Using experimental data from the literature for rate and Michaelis-Menten constants, the simulation results revealed that, in muscle, the response of GP to cAMP input is more highly sensitive (

6.5), whereas in the liver, the GS sensitivities to glucose (

6.8) are high compared to that of GP (

3.2 for cAMP). The sensitivity analysis indicated that this differential performance of GS and GP in liver and muscle is due to the presence of a distinctive regulatory design and not to selection of a particular parameter set. CAPK-activated inhibitor-1 inhibits PP1, which is a major dephosphorylating enzyme in muscle, whereas GP-a inhibits GS phosphatase in liver, representing this distinctive design. The simulation results indicate that the response sensitivity of GS with respect to glucose and cAMP is highly dependent on the GP concentration in liver. Similarly, the sensitivities of the PK, GP and GS responses are dependent on inhibitor-1 concentration in muscle. The ultrasensitive response of these enzymes may be attributed to the known system-level mechanisms, namely, multistep ultrasensitivity due to cAMP, inhibitor ultrasensitivity due to phosphatase inhibitor and zero order effects due to the pyramidal relationship in enzyme component concentrations. However, the significance of this switch-like response of GP in muscle and GS in liver is unclear. It can be argued that glycogen breakdown in muscle has to be sensitive to the second messenger cAMP in order to meet the urgent requirement for glucose during exercise or the fight and flee response. Similarly, glycogen synthesis in liver has to be sensitive to blood glucose concentration, so that GS can start synthesizing glycogen whenever the blood glucose concentration increases beyond a toxic level.

In muscle, the ultrasensitive response of GP can be directly attributed to the presence of zero order effects (GP concentration about

70 μM) and compounded by the inhibitor ultrasensitivity imparted by inhibitor-1. Such a direct effect is not observed in GS owing to its minimal zero order effects (GS concentration about

3 μM). The primary stimulus, cAMP, not only increases the phosphorylation of PK, GP and GS, but also indirectly decreases their dephosphorylation through inhibitor-1. In liver, the ultrasensitive response of GS can be attributed mainly to the inhibitor ultrasensitivity caused by GP on the GS modification cycle. In this case, the zero order effect actually resides in the GP cascade, which transmits it to the GS cycle by inhibiting the dephosphorylation reaction. Furthermore, the stimulatory effect of glucose on dephosphorylation of GP-a, the inhibitory effect of glucose-6-phosphate on phosphorylation of GP-b and GS-a, and the stimulation of GS dephosphorylation by glucose-6-phosphate, enhance the sensitivity of GS. Thus, the ultrasensitivity of GS in liver is brought about by the combined action of the multistep effects of cAMP, the inhibition of GS phosphatase by active GP and the influence of glucose and glucose-6-phosphate concentration.

It is noteworthy that the simultaneous activation and deactivation of GP and GS respectively in muscle and liver results in reciprocal regulation of these enzymes by the primary stimulus. This reciprocal regulation, although identical in all tissues, still imparts a distinctive adaptive strategy in different cell types owing to subtle differences in the network. For example, the inhibition of GS phosphatase by GP in liver can compromise the reciprocal regulation in the absence of liver glycogen (i.e. starved state), while in muscle the reciprocal regulation cannot be compromised owing to an independent inhibitor-1. Our simulation of the glycogen cascade system under starved condition demonstrates that the sensitivity of GS reduces because of the reduction in inhibitor ultrasensitivity caused by GP. The percentage reduction in the inhibition of GS phosphatase is unknown. It is possible that when the liver undergoes a transition from starved to fed state, GS phosphatase can experience varying degrees of inhibition by GP. This results in a shift from a highly futile cycle with no inhibition to reciprocal regulation in the fed state. This causes GS to be always active, while GP is active only in the starved state in the presence of high glucose (see Fig. ​ Fig.5 5 ).

Hallenbeck and Walsh [40] observed that, if the quantity of phosphorylase sequestered in the glycogen particle compartment of rabbit muscle is taken into account, then the local concentration of GP can be very high (up to 2𠄵 mM). Furthermore, GP interaction with the glycogen particle is known to lower the Michaelis-Menten constants of PK and PP1, thus enhancing the zero order effects further [29,25]. Considering these observations, Meinke and Edstrom [25] estimated an apparent Hill coefficient of 51 for the activation of 3.5 mM phosphorylase. Our simulation results show that at 3.5 mM phosphorylase the system can actually show a highly ultrasensitive response with an apparent Hill coefficient as high as 200 (results not shown). This apparent Hill coefficient value is far higher than any known ultrasensitive system or any of the cooperative enzymes. Though the utility of such a highly sensitive response in vivo is unclear at present, various observations indicate that the multi-enzyme cascade system has the potential to exhibit higher sensitivity.

Signaling by hormones such as glucagon and epinephrine is known to elicit responses within a fraction of a second, incorporating amplification of the input signal and enhanced sensitivity to allosteric effectors [2,3,27,41]. It has also been shown, in the contraction of resting muscle, that GP-b is converted to GP-a within a second followed by immediate initiation of glycogenolysis [3]. Such rapid and sensitive responses are known to be the characteristic behavior of enzyme cascades with progressive increase in enzyme concentration down the cascade [2]. This effect can also be brought about by the opposing action of the same effector on modifying and demodifying enzymes [18] and the presence of a stoichiometric inhibitor [20]. It appears that living systems use these ultrasensitive regulatory mechanisms to coordinate multiple input signals, show varied responses to different signals, exhibit rapid responses at an invariably low stimulus concentration [2,3,27] and, most importantly, use negligible amount of cellular energy [42,43].

Theoretical quantification of a regulatory system as presented here reveals insights into system level properties. Ultrasensitivity, signal amplification, flexibility in operation and signal integration are all system level properties, and are not apparent in isolated components. These properties can be studied by connecting different functional units and defining the quantitative relationship between different components of a system. Our simulation results revealed that the switch-like responses of GP and GS in liver and muscle are comparable with that of the MAPK cascade in Xenopus oocytes [21]. At the metabolic level, GP and GS are also regulated by calcium levels and feedback loops constituted by effectors such as ATP, AMP, cAMP, glucose and glycogen [3-6,9-12]. Furthermore, GS and PK are known to have multiple phosphorylation sites [5,9]. Regulatory networks made up of multiple feedback loops and multiple phosphorylation cycles, as seen in the activation of maturation-promoting factor and the MAP kinase cascade during oocyte maturation [44,45], can yield multiple steady state responses. Although we have not incorporated the overall regulatory network, our analysis suggests that the enhanced sensitivity observed in the glycogen cascade system may act as a selective pressure in evolution favoring tissue-specific adaptive strategies and compartmental regulatory modules.


Introduction

Hypoxia is a strong and usually positive regulator of gene expression(D'Angio and Finkelstein,2000 Prabhakar,2001 Semenza,2001). This may be the result of selection pressures operating over millions of years to conserve essential biological functions that were acquired during anaerobic evolution. Life evolved on earth under anaerobic conditions for about 2 billion years, close to one half of the total time period of biological evolution (Barnabas et al., 1982 Papagiannis,1984). Therefore the fundamental features of biology and genetics,including DNA synthesis, transcription, translation and their regulation, were established under strictly anaerobic conditions, and perhaps as a consequence have an absolute requirement for a reducing environment in order to function(Segerer et al., 1985). Likewise it may be expected that certain biological activities, pathways and regulatory processes function preferentially under conditions of hypoxia. This may be particularly true for anaerobic bioenergetic pathways, including glycolysis, which were established as the first energy generators and ultimately became integrated with oxidative pathways. The suppression of oxidative metabolism under conditions of severe hypoxia reduces oxidative stress, decreases antioxidant levels, and simulates the primordial, reducing environment, even without molecular antioxidants(Webster et al., 2001). At least eight out of the twelve functually distinct glycolytic enzyme genes are coordinately induced by hypoxia in mammalian cells(Webster, 1987 Webster et al., 1990 Webster and Murphy, 1988). The regulation involves contributions from at least four separate molecular pathways, some of which may have been conserved through 4 billion years of evolution, dating back to the origin of life. An intriguing question that will be addressed in this review is whether the hypoxia-mediated gene induction involves activation of ancestral positive-acting factors, repression of oxygen-induced negative factors, or combinations of these. The molecular regulation of glycolysis at the level of enzyme activity has been reviewed extensively elsewhere and will not be addressed here (for reviews, see Hofer, 1996 Hardie, 2000 Romano et al.,1996 Siebers et al.,1998).

Oxygen regulation of glycolysis

Fig. 1A shows the glycolytic pathway, where 12 enzymes catalyze the anaerobic fermentation of glycogen to lactic acid, generating 3 moles of ATP per glucosyl unit. The process is an order of magnitude less efficient than oxidative metabolism, where 32 moles of ATP are generated per 2 or 3 moles of glucose, depending on whether glucose or glycogen is the substrate. The scheme shows the input and output points of the pathway. There are numerous molecular modulators of glycolytic flux, the most famous of which was discovered in 1860 by Louis Pasteur(Pasteur, 1861). Pasteur showed that oxygen inhibits fermentation and that glucose consumption is inversely proportional to the oxygen availability, i.e. that the glycolytic pathway is positively regulated by hypoxia. Pasteur received wide recognition for this stunning observation that became universally known as the `Pasteur Effect'. In 1987, our laboratory reported the observations shown in Fig. 1B(Webster, 1987). We theorized that since oxygen is a potent and ancient regulator of glycolytic flux, it might also be a regulator of glycolytic enzyme gene expression. We isolated and cloned rodent cDNAs for six glycolytic enzymes (indicated with asterisks on Fig. 1A), and we used these to measure transcription rates of the genes in muscle cells exposed to hypoxia. Fig. 1B shows a composite of the transcription of six glycolytic enzyme cDNAs compared with that of mitochondrial cytochrome c. Chronic hypoxia caused a significant and coordinated activation of transcription of these genes.

Composition and regulation of the glycolytic pathway in higher animals. (A)Linear pathway of glycolytic enzymes showing substrate input and sites of ATP utilization and generation. (B) Induction of glycolytic enzyme mRNA levels by hypoxia. Skeletal myocytes were exposed to hypoxia and mRNA transcript levels were measured at the indicated time points by the nuclear run-on technique,described in Webster (1987). The figure is a composite of six glycolytic enzyme gene transcripts using cDNAs to the enzymes indicated by the asterisks in A.

Composition and regulation of the glycolytic pathway in higher animals. (A)Linear pathway of glycolytic enzymes showing substrate input and sites of ATP utilization and generation. (B) Induction of glycolytic enzyme mRNA levels by hypoxia. Skeletal myocytes were exposed to hypoxia and mRNA transcript levels were measured at the indicated time points by the nuclear run-on technique,described in Webster (1987). The figure is a composite of six glycolytic enzyme gene transcripts using cDNAs to the enzymes indicated by the asterisks in A.

Conservation of glycolytic enzyme genes

The 12 mammalian glycolytic enzyme genes are genetically unlinked and dispersed around the genome, mostly on different chromosomes(Webster and Murphy, 1988). These are some of the most ancient and highly conserved proteins and genes known, with strong conservation of both the peptide and DNA sequences even between higher mammals and bacteria(Lonberg and Gilbert, 1985 Peak et al., 1994 Poorman et al., 1984). Fig. 2 shows a Southern blot illustrating the remarkable conservation of pyruvate kinase (PK) and lactate dehydrogenase (LDH) with strong cross-homology of DNA fragments between yeast and human DNA. Glycolytic enzymes were probably among the very first enzyme pathways to appear, allowing primitive organisms to utilize simple carbohydrates as energy stores and to release energy by coupling the breakdown to high-energy phosphates(Fothergill-Gilmore and Michels,1993 Romano and Conway,1996). Although structural and functional aspects of glycolytic enzyme genes and proteins have been strongly conserved, it is not clear how gene regulatory mechanisms evolved or how the pathway established a coordinate response of widely dispersed genes to oxygen tension. Fig. 2 also illustrates a second intriguing feature of glycolytic enzyme genes, namely an apparently selective accumulation of pseudogenes in rodents, particularly mouse and rat. This is reflected in the dramatic increase of the number of hybridizing bands in these species, and was first described by Piechaczyk for the GAPDH gene(Piechaczyk et al., 1984). Our results demonstrate increased numbers of pseudogenes of PK and LDH(Fig. 2), as well as GAPDH,aldolase, triosephosphate isomerase, phosphoglycerate kinase and enolase (not shown), and suggest that the effect may be common to the entire pathway of genes. We do not know why or how this occurred.

Southern blots illustrating strong conservation of glycolytic enzyme gene sequences across species. Cells or tissues from the indicated organisms were lysed and genomic DNA was extracted by standard techniques(Webster, 1987 Webster et al., 1990 Lonberg and Gilbert, 1985). DNA was digested with restriction enzyme EcoRI, separated on agarose gels and blotted onto nitrocellulose. Membranes were probed with 32 P-cDNAs coding for pyruvate kinase (PK) and lactate dehydrogenase(LDH) as described in Webster(1987). Arrows indicate conserved DNA fragments. Note the increased number of hybridization bands for both genes in rodents (blocks indicated by vertical bars) that probably represent increased numbers of pseudogenes in these species (see text).

Southern blots illustrating strong conservation of glycolytic enzyme gene sequences across species. Cells or tissues from the indicated organisms were lysed and genomic DNA was extracted by standard techniques(Webster, 1987 Webster et al., 1990 Lonberg and Gilbert, 1985). DNA was digested with restriction enzyme EcoRI, separated on agarose gels and blotted onto nitrocellulose. Membranes were probed with 32 P-cDNAs coding for pyruvate kinase (PK) and lactate dehydrogenase(LDH) as described in Webster(1987). Arrows indicate conserved DNA fragments. Note the increased number of hybridization bands for both genes in rodents (blocks indicated by vertical bars) that probably represent increased numbers of pseudogenes in these species (see text).

Precambrian: bacterial glycolytic genes

The chart in Fig. 3 shows sections of time dating back to when life first appeared on earth. This early period is known as the Precambrian and it is divided into Hadean, Archean,Paleoproterozoic, Mesoproterozoic and Neoproterozoic. The oldest fossils include bacteria and other microorganisms that date to about 3.8 billion years ago (BYA). Glycolytic enzymes are evident in the Archean period, 2 BY before the earliest oxygen-requiring species and almost 4 BY before the present pathways (Gebbia et al., 1997 Kelly and Adams, 1994 Peak et al., 1994). Qualitative trends in the amount of global biomass are projected in Fig. 3B. Acquisition of methanogenesis by Archaebacteria probably supported an early expansion of life forms (DeLong et al., 1994 Koch, 1998 O'Callaghan and Conrad, 1992 Papagiannis, 1984 Reeve, 1992), and biomass probably increased significantly before the dip and subsequent massive expansion of the Cambrian period. Natural selection working on the expanding biomass produced increasingly high levels of biological sophistication and diversity within the anaerobic kingdoms. In fact, molecular studies of extant bacterial species such as the Archaebacteria and thermophyllic sulfur bacteria indicate complex patterns of gene expression under anoxia, including the regulation of bioenergetic gene expression by elemental sulfur and phosphorus(Brunner et al., 1998 Fardeau et al., 1996 Friedrich, 1998 Janssen and Morgan, 1992 Kelly and Adams, 1994 Ma et al., 1995 Segerer et al., 1985). There is an intriguing parallel between sulfur regulation of bioenergetic pathways in the Archean era microorganisms and oxygen regulation in eukaryotes. Oxygen replaced sulfur as the terminal electron acceptor of carbohydrate catabolism,and may simultaneously have parasitized some molecular features of the regulation over billions of years. Numerous aspects of the Archaebacteria and bacterial gene regulatory mechanisms have been conserved and elaborated in higher animals while others, including the bacterial operon, have been largely replaced. The rearrangement of primitive prokaryotic glycolytic enzyme gene operons into unlinked genes on eukaryotic chromosomes requires the parallel segregation, multiplication and/or insertion of regulatory elements with trans-acting protein factors to allow the coordinated function of the pathway (Alefounder and Perham,1989 Barnell et al.,1990 Gebbia et al.,1997).

Milestones in evolution. (A) Paleontological periods of the Precambrian era. (B) Estimates of total earth biomass as a function of time. The graph is only a qualitative representation because it is not possible to establish or extrapolate precise levels of precambrian biomass from paleontological records(Kelly and Adams, 1994 Papagiannis, 1984 DeLong et al., 1994 Koch, 1998 O'Callaghan and Conrad, 1992 Reeve, 1992). BYA, billion years ago Ph indicates initiation of the major increase in photosynthesis by cyanobacteria.

Milestones in evolution. (A) Paleontological periods of the Precambrian era. (B) Estimates of total earth biomass as a function of time. The graph is only a qualitative representation because it is not possible to establish or extrapolate precise levels of precambrian biomass from paleontological records(Kelly and Adams, 1994 Papagiannis, 1984 DeLong et al., 1994 Koch, 1998 O'Callaghan and Conrad, 1992 Reeve, 1992). BYA, billion years ago Ph indicates initiation of the major increase in photosynthesis by cyanobacteria.

The Archean period is characterized by what would be an extremely toxic atmosphere for current life forms, with methane, nitrogen and ammonia as the major components (Kasting,1993 Papagiannis,1984). Fig. 4Aillustrates an Archean coastline 3.5 BYA. The mounds in the foreground are stromatolites, multiple layers of calcified microbial colonies, mostly bacteria and fungi, dating back almost to the beginning of life(Papagiannis, 1984 Reid et al., 2000). These structures form the best record of Archean and the early Proterozoic period,known as the third domain of life (Koch,1998). Stromatolite fields can still be found in parts of South African and Western Australia. They were common throughout the Precambrian periods until about 1.0 BYA, when herbivorous predators probably featured significantly in their decline. Fig. 4B shows a piece of stroma fossil from the Bitter Springs formation of central Australia, dated at 0.85 BYA. These fossils are known as carbon films, dark compressions in the rock revealing the outlines of ancient species in the forms of spheres, circles, ribbons and leaf-like structures. The diversity represents more than 2 BY of anaerobic evolution generating complex phyla of obligate microbial anaerobes, including Archaebacteria,cyanobacteria and possibly unicellular flagellates. Studies on present day descendants of these microorganisms, in particular the obligate anaerobic hyperthermophilic Archaea, indicate that they have complex systems of bioenergetic pathways (Fardeau et al.,1996 Janssen and Morgan,1992 Kelly and Adams,1994 Ma et al.,1995). Thermoproteus tenax is an obligate anaerobic hyperthermophile and a descendent of one of the earliest Archea dating back to 3-4 BYA.

The Archean Age. (A) Stromatolite field as it may have looked 3.5 BYA (see text for details). (B) Stromatolite fossil the arrows indicate `carbon films'where microscopic details reveal microbial fossils dating back to the earliest life forms on earth. (From the University of California at Berkeley Paleontological Museum with permission).

The Archean Age. (A) Stromatolite field as it may have looked 3.5 BYA (see text for details). (B) Stromatolite fossil the arrows indicate `carbon films'where microscopic details reveal microbial fossils dating back to the earliest life forms on earth. (From the University of California at Berkeley Paleontological Museum with permission).

The first glycolytic enzymes in the Archean period probably contributed mainly anabolic, gluconeogenic functions(Conway, 1992 Romano and Conway, 1996 Selig et al., 1997), with catabolic functions being acquired subsequently as kinases appeared to use ATP, ADP or pyrophosphate as phosphate shuttles(Romano and Conway, 1996). There are some unique characteristics of Archean era glycolysis for example catalysis of reactions by the enzymes glucokinase and phosphofructokinase(PFK) in T. tenax is dependent on ADP and pyrophosphate as cofactors. This allows these key steps to be functionally reversible, permitting gluconeogenesis as well as glycolysis, a feature not possible in the later bacterial and eukaryotic pathways(Mertens, 1991 Siebers et al., 1998 van der Oost et al., 1998). There is evidence for both divergent and convergent evolution of glycolytic genes, but not divergence from a single multifunctional glycolytic protein or gene cluster (Barnell et al.,1990 Fothergill-Gilmore,1986 Fothergill-Gilmore and Michels, 1993 Rossman,1981). Sequence and crystallographic data favor the divergent evolution of for example monophosphoglycerate mutase and diphosphoglycerate mutase, and possibly glyceraldehyde-3-P dehydrogenase and phosphoglycerate kinase from respective common ancestors, but convergence appears to have played a greater role in the development of all of the other 11 enzymes(Fothergill-Gilmore, 1986 Fothergill-Gilmore and Watson,1989). For example, there is no evidence of a common ancestor for any of the four glycolytic kinases or of the seven enzymes that bind nucleotides, with the exception of those mentioned above. Rather, it seems likely that the pathway resulted from the chance assembly of independently evolving enzymes and genes, probably in association with the co-evolution of other functions and linked pathways.

Substrate regulation by operons in bacteria

Many functionally related bacterial genes are organized into physical operons that are regulated by a master operator element, usually positioned at the 5′ end of the operon, which regulates the transcriptional rate of all genes in the operon (Alefounder and Perham, 1989 Barnell et al.,1990 Hannaert et al.,2000 Liaud et al.,2000 Unkles et al.,1997). Evidence for glycolytic enzyme gene operons include linked pyruvate kinase and PFK genes in Clostridium acetobutylicum(Belouski et al., 1998)clustered genes for phosphoglycerate kinase (PGK), triosephosphate isomerase(TPI), phosphoglycerate mutase and enolase in Baccilus subtilis(Leyva-Vazquez and Setlow,1994) linkage of GAPDH, PGK and TPI in Borrelia megaterium,Borrelia bungorferi and Borrelia hermsii(Gebbia et al., 1997 Schlapfer and Zuber, 1992)clustering of fructose 1,6-biphosphate aldolase, 3-phosphoglycerate kinase and GAPDH in E. coli (Alefounder and Perham 1989), and clustering of the glucose-6 dehydrogenase,6-phosphogluconate dehydratase and glucokinase genes with a putative glucose transporter in Zymomonas mobilis(Barnell et al., 1990). These glycolytic enzyme gene operons may be regulated independently of each other or globally. In the latter condition the multiple operons behave as a unit,termed a modulon, which is coordinately regulated by one or more wide-ranging master regulatory proteins. The best example of modulon regulation is through the cAMP receptor protein (CRP) or catabolite gene activator protein (CAP), which can activate or repress numerous regulons in response to substrate availability (Bledig et al., 1996 Kumari et al.,2000 Luesink et al.,1998 Tobisch et al.,1999). Because substrate fluctuation was the principal selection pressure for evolving Archean microorganisms, modulons became the principal mechanism for the coordinated regulation of all genes involved in carbohydrate metabolism, including glycolytic enzymes. However even in early Moneras there is evidence for fine tuning in the form of functional segregation and preferential targeting of specific genes, in particular those destined to become the `key regulatory enzymes'. For example, in E. coli an operon containing phosphofructokinase, pyruvate kinase and l -lactate dehydrogenase, all `key enzymes', is selectively regulated through a 5′ cAMP response element that binds the positive factor CcpA. Levels of CcpA in turn are determined by substrate availability(glucose, galactose, fructose) (Luesink et al., 1998 Tobisch et al.,1999). The grouping of PFK and PK is clearly significant because the operon components tend to favor contiguous functions within the glycolytic pathway.

Oxygen regulation in prokaryotes

Oxygen exerted massive selection pressures on prokaryotes and engineered cooperativity between energy storing and releasing pathways, including substrate-level phosphorylation and electron transport by dedicated carriers including cytochromes. The oxygen-regulated switching in bacteria (and possibly archaebacteria Chistoserdova et al., 1988 Iwasaki et al.,1995 Segerer et al.,1985) includes the activation and/or repression of key enzyme genes and operons involved in oxidative metabolism and glycolysis. This includes positive and negative factors regulated by oxygen tension or redox potential and involves contributions from at least three major regulatory pathways. These include the Arc and FNR systems, which regulate gene expression pre-transcriptionally in response to the redox state of the environment, and the CsrA-CsrB system, which differentially regulates the expression of glycogen synthesis, gluconeogenesis and glycolytic genes by conditionally regulating RNA stability. The latter regulation has been recently reviewed and will not be discussed here(Bunn and Poyton, 1996) we will briefly consider the oxygen-regulated Arc and FNR systems because they may be the precursors of eukaryotic glycolytic enzyme gene regulation by hypoxia.

The Arc system is involved in the repression of aerobic functions under anaerobic conditions. Arc A represses the expression of the succinate dehydrogenase, citric acid cycle and glyoxylate cycle enzyme genes, and activates cytochrome d oxidase under hypoxic conditions, thereby shutting off the succinate dehydrogenase-cytochrome oxidase pathway and activating electron transport through the d-cytochrome, which has a higher affinity for oxygen (Parkinson and Kofoid, 1992). The mechanism is a classical two-component signal-transducing system, involving a membrane-bound redox sensor and protein kinase (ArcB) and a cytoplasmic regulator (ArcA) with a DNA-binding domain(see Fig. 5). Signals from the electron transport chain (probably the redox state of heme or other iron-containing component) activate ArcB, which transmits the signal to ArcA and initiates the cascade of gene regulation. The FNR system is also involved in the anaerobic activation and repression of a wide variety of metabolic enzymes by a mechanism that parallels that of the CAP system(Chang and Meyerowitz, 1994 Parkinson and Kofoid, 1992). Expression of more than ten enzymes involved in anaerobic energy metabolism,including fumarate reductase and glycerol-3-phosphate dehydrogenase, is induced when the FNR system is activated(Spiro and Guest, 1991). Activation is believed to involve a redox switch within the FNR protein involving cysteine-bound metal ions. A conformational change of the protein creates an active DNA binding site that promotes transcription. The target sequence for activated FNR usually resides about 40 bp upstream of the transcriptional initiation site and includes the consensus sequence nTTGATnnnnATCAAn, which is a typical binding site for dimer-DNA-binding proteins containing helix-turn-helix motifs(Kiley and Reznikoff, 1991). This is significant because it may be the first example of a positive-acting transcription factor with helix-turn-helix motifs that is activated by hypoxia and involved in the coordinate regulation of genes that ultimately determine glycolytic functions. Conformational regulation by reversible binding of metals to cysteine sites is reminiscent of the redox responses of zinc finger transcription regulators, the most common regulators of gene expression in eukaryotes (Webster et al.,2001). Redox-dependent conformational changes also contribute to the transcriptional activation of mammalian glycolytic enzyme genes by specific helix-turn-helix factors (see below).

Regulation of gene expression by redox-sensitive Arc and Fnr pathways. Under aerobic conditions, ArcB and ArcA are sequentially activated by phosphorylation. ArcA negatively regulates the transcription of the cyoABCDE operon, which encodes cytochrome bo oxidase (high Vmax, low oxygen affinity) and positively regulates cydAB encoding cytochrome bd oxidase (low Vmax, high oxygen affinity). Fnr is inactive under aerobic conditions, but at low oxygen it undergoes a conformational change, probably mediated by reduction of ferric to ferrous iron at an iron-sulphur center. Conformationally activated Fnr binds DNA at sites with the inverted repeat sequences TTGAT —— ATCAA. Fnr binding represses cyoABCDE and cydAB operons and induces transcription from the operons dmsABC (dimethyl sulfoxide/trimethylamine-N-oxide reductase), frdABCD (fumarate reductase), and narGHJI(nitrate reductase). Cross-talk between the two pathways at cyoABCDE and cydAB is indicated by the broken arrow. Under microaerophilic conditions as oxygen becomes limiting, cydAB is optimally active and ArcA may successfully compete Fnr to activate the regulator under these conditions (Bunn and Poyton,1996).

Regulation of gene expression by redox-sensitive Arc and Fnr pathways. Under aerobic conditions, ArcB and ArcA are sequentially activated by phosphorylation. ArcA negatively regulates the transcription of the cyoABCDE operon, which encodes cytochrome bo oxidase (high Vmax, low oxygen affinity) and positively regulates cydAB encoding cytochrome bd oxidase (low Vmax, high oxygen affinity). Fnr is inactive under aerobic conditions, but at low oxygen it undergoes a conformational change, probably mediated by reduction of ferric to ferrous iron at an iron-sulphur center. Conformationally activated Fnr binds DNA at sites with the inverted repeat sequences TTGAT —— ATCAA. Fnr binding represses cyoABCDE and cydAB operons and induces transcription from the operons dmsABC (dimethyl sulfoxide/trimethylamine-N-oxide reductase), frdABCD (fumarate reductase), and narGHJI(nitrate reductase). Cross-talk between the two pathways at cyoABCDE and cydAB is indicated by the broken arrow. Under microaerophilic conditions as oxygen becomes limiting, cydAB is optimally active and ArcA may successfully compete Fnr to activate the regulator under these conditions (Bunn and Poyton,1996).

Notably, none of the aerobic/anaerobic regulatory systems described above directly regulate glycolytic enzyme genes, although cross talk between the Arc, FNR and CcpA networks causes changes in the transcription rates of glycolytic enzyme operons in response to carbohydrate. The absence of a direct regulation of glycolytic enzyme gene transcription (by substrates, alternative pathways such as sulfur, or oxygen) in the Archean era and subsequently in bacteria would be predicted if such regulation was acquired during the selection and gene shuffling that accompanied the transition to oxidative metabolism. The establishment of direct oxygen regulation of glycolytic enzyme genes may in fact have paralleled mitochondrial symbiosis and the establishment of compartmented energy pathways. Photosynthetic cyanobacteria underwent a major expansion 1.5 BYA, producing >1000 different variants and initiating a rapid increase of atmospheric oxygen(Kasting, 1993 Reid et al., 2000). Atmospheric oxygen during the Archean period was less than 1% of the current level, but by about 1.8 BYA it was 15%, and probably increased to the current level by 0.5 BYA. This accumulated oxygen had a major impact on life. It has been estimated that as much as 99% of the existing anaerobic life forms were extinguished by the toxic byproducts of reactive oxygen (Cannio, 2000). Oxygen allowed the rapid diversification and expansion of survivors because of the increased energy made available from oxidative metabolism. The main expansion occurred within the eukaryotic kingdom, stimulated by the high energy-producing potential of mitochondria. Mitochondria contributed a highly efficient energy production system that was partially insulated from other cellular functions. Metabolic and gene regulatory pathways, including responses to hypoxia, arose in parallel to coordinate mitochondrial and glycolytic function (Webster et al.,1990).

Eukaryotic glycolytic genes

The archeological period known as the `Vendian' is thought to include the earliest species that survived the oxygen explosion(Li et al., 1998 Rasmussen et al., 2002 Seilacher et al., 1998). Organisms within this period bridge the gap between the late Precambrian and early Cambrian periods and represent the ancestors of most if not all eukaryotes. Rich deposits of Vendian fossils have been discovered in three major locations: the Ediacara Hills in Southeast Australia, the Russian Winter coast, and Misty Point in Newfoundland, Canada. Examples of these fossils are shown in Fig. 6. Vendian life forms representing the transition to eukaryotic organisms include sponges,hydra, filamentous algae and fungi. Estimates of the start of the Vendian period vary from about 0.8 to more than 1 BYA. Yeasts belong to the Fungi, and are all facultative anaerobes capable of growth with or without functional mitochondria (Ferguson and von Borstel.,1992).


Steps of glycogenolysis

Glycogenolysis begins by the action of glycogen phosphorylase (EC 2.4.1.1), a homodimer that for its activity requires the presence of pyridoxal-5-phosphate, a derivative of pyridoxine or vitamin B6. The enzyme catalyzes the phosphorolytic cleavage of α-(1,4) glycosidic bond, releasing glucose molecules one at a time from the non-reducing ends, that is, the ends with a free 4’-OH group, of the external branches. This reaction, which does not consume ATP but an orthophosphate, yields glucose 1-phosphate.

Glycogen(n glucose residues) + Pi → Glucose 1-Phosphate + Glycogen(n-1 glucose residues)

Note: in the small intestine, pancreatic α-amylase (EC 3.2.1.1) catalyzes the hydrolytic cleavage of the α-(1,4) glycosidic bonds of the starch, and yields glucose molecules.

In vivo, glycogen phosphorylase catalyzes an irreversible phosphorolysis, a particularly advantageous reaction for skeletal muscle and heart (see below). The irreversibility of the reaction is ensured by the ratio [Pi]/[glucose 1-phosphate], which is usually greater than 100. Conversely, the reaction is easily reversible in vitro.
Glycogen phosphorylase acts repetitively on the non-reducing ends of branches, coming to a halt when the glucose unit that is 4 residues away from the branch point is reached: this is the outer limit of the limit dextrin. At this point, two enzymatic activities, present on the same polypeptide chain, complete glycogen breakdown: the α-(1,4)-glucan-6-glycosyltransferase (EC 2.4.1.24) and the amylo-α-(1,6)-glucosidase or debranching enzyme (EC 3.2.1.33). The first enzymatic activity transfers three of the remaining four glucose units from the branch to the non-reducing end of another branch, leaving in the first chain only a single glucose unit, that is attached to the chain by an α-(1,6)-glycosidic bond. The second enzymatic activity hydrolyzes this α-(1,6)-glycosidic bond, releasing glucose and an unbranched chain of α-(1,4)-linked glucose units.
Without the branch, glycogen phosphorylase can continue to remove glucose units until it reaches the next limit dextrin.

Therefore, the products of the reactions catalyzed by the three enzymatic activities are:

  • glucose 1-phosphate (about 90% of the glucose molecules released)
  • a small amount of free glucose, the remaining 10% [these are the 1,6 linked residues in the muscle, hexokinase activity (EC 2.7.1.1) is so high that any free glucose molecule is phosphorylated to glucose 6-phosphate, and therefore activated, and metabolized within the cell]
  • a smaller and less branched glycogen molecule.

Metabolic fate of glucose 1-phosphate in muscle and liver

Glucose 1-phosphate is a charged molecule, and therefore it is trapped within the cell.
It is converted to glucose 6-phosphate in the reaction catalyzed by phosphoglucomutase (EC 5.4.2.2), the same enzyme that also intervenes in glycogen synthesis converting glucose 6-phosphate to glucose 1-phosphate. This enzyme catalyses a reversible reaction: the direction is determined by the relative concentrations of the two molecules, and in this case moves the phosphate group from C1 to C6.

Glucose 1-Phosphate ⇄ Glucose 6-Phosphate

In the muscle, and in most of the other organs and tissues, glucose from glycogenolysis enters glycolysis as glucose 6-phosphate, bypassing the activation step catalyzed by hexokinase. Therefore, glycogen phosphorylase, releasing an already “activated” glucose molecule, saves an ATP. An ATP molecule is required to synthesize another glycolytic intermediate, the fructose 1,6-bisphosphate.
In this way, some of the activation energy required for glycogen synthesis is conserved: the net yield of ATP per glucose molecule by glycolysis to lactate is 3 rather than 2, an advantage for the working muscle. The overall equation is:

Glycogen(n glucose residues) + 3 ADP + 3 Pi → Glycogen(n-1 glucose residues) + 2 Lactate + 3 ATP

In the liver, glucose 6-phosphate from glycogen is dephosphorylated by glucose 6-phosphatase (EC 3.1.3.9), and then released into the bloodstream. These are the steps in the removal of glucose units, in the form of phosphorylated glucose, by hepatic glycogenolysis:

Glycogen(n glucose residues) + Pi → Glucose 1-Phosphate + Glycogen(n-1 glucose residues)

glucose-6-phosphate + H2O → glucose + Pi

Glycogen(n glucose residues) + H2O → Glycogen(n-1 glucose residues) +Gglucose


15.5: Coordinated Regulation of Glycogen Synthesis and Breakdown - Biology

C2006/F2402 '04 OUTLINE OF LECTURE #15

(c) 2004 Dr. Deborah Mowshowitz, Columbia University, New York, NY . Last update 03/11/2004 12:46 PM .

Handouts: Need 14C (Homeostasis), 14D (Temperature Regulation), 15 A -- Glucose Homeostasis 15 B -- Lactation & Stress Response

I. Homeostasis, cont. How are components of the internal milieu regulated?

A. Regulation of body temperature (in humans) -- the see-saw view (14C)

1. Features not found in glucose case:

a. Multiple sensors in different places (for core and skin temp.)

b. Need separate integrative center (IC).

(1). Role of IC: Compares set-point to actual value, sends appropriate message to effectors.

(2). Sensor/IC function may be combined, as in Glucose example.

(3). Separate IC needed if there are multiple sensors, as in this case.

(4). In this example, IC = hypothalamus (HT)

(5). Role of separate IC: Co-ordinates incoming (afferent) information from sensors compares set-point to actual value, sends appropriate outgoing (efferent) information to effectors.

2. Different body systems involved as effectors

3. Cooling vs. Heating -- What can effectors do? Effectors can increase or decrease heat loss can only increaseheat generation. (No air conditioner.) Therefore ability of humans to cope with very cold environments is better than their ability to cope with excessively hot environments.

Try Problem 5-2, c. & 5-5.

4. Metabolic rate and temperature control in homeotherms (organisms with aprox. const. internal temp.) that are endotherms (generate own heat internally) as vs. poikilotherms/ectotherms. (See Purves p. 700 [p. 817] for further discussion of these terms.) See handout 14D, top.

a. Constriction/dilation of blood vessels uses relatively little energy. This allows adjustment of body temperature without changing metabolic rate in range of external temperature called the "neutral zone."

b. Both heating (by shivering) and cooling (by sweating) require lots of energy. Therefore MR (metabolic rate) increases outside neutral zone at both high and low temperatures.

c. Overall how MR (metabolic rate) changes with external temperature (see handout 14D, top or Purves, fig. 40.15 [37-19]). Thermo-neutral zone is bounded by critical temperatures = points at which shivering or sweating occur = set points for shivering or sweating.

B. Body Temperature and the General Case -- The Circuit View -- see handout 14D, bottom.

  • Shift curve of MR vs external temp to right shivering and sweating both kick in at higher temps. (You don't have to cool off as much to start shivering and you need to heat up more to stop sweating.) Raises set point (desired level) & actual level of internal body temperature.

  • Why fevers? High temperatures prevent bacteria from obtaining iron from host & improve immune function.

(2). Feedforward or anticipation -- Planning ahead. Altering set points and/or critical points to adjust to anticipated factors. (Or you can think of it as just ignoring the usual critical points.) Examples:

Body temperature: Skin temperature affects critical temperature/set points for generating heat and/or shivering. If body is cold, but it's warm outside, shivering can be postponed, saving energy, and you'll still warm up. This is equivalent to lowering (or ignoring) set point/critical points for shivering, not changing set point of internal body temperature. Changes what effectors and what controlled processes you use to warm up, but not the end result. (See Purves, fig. 40. 19 [37.23]).

Secreting insulin when you start to digest food in the stomach, but before the digestion products (glucose, amino acids etc.) reach the blood. This way tissues will be ready to take up the glucose as soon as it enters the blood.

C. What other components of internal milieu are regulated besides glucose, temperature? Many nutrients like amino acids concentrations of water, salts and ions (Na+, K+ etc.), gases (CO2, O2), waste products, volume & pressure of blood, and pH.

Try Problems 5-3, 5-4, & 5-9.

II. Matching circuits and signaling -- an example: How the glucose circuit works at molecular/signaling level

A. Re-consider the circuit diagram for homeostatic control of blood glucose levels -- what goes along the arrows, and what happens in the black boxes? (See handouts 14C & 14D or Purves 50.20 [47.19])

B. Mechanism of Action of hormones Involved

1. Insulin

a. Receptor: Insulin works through a special type of tyrosine kinase linked receptor See Purves 15.7. Insulin has many affects on cells and the mechanism of signal transduction is complex (activating multiple pathways). In many ways, insulin acts like a GF (it has GF-like effects on other cells is in same family as ILGF's).

b. Effect on GLUT 4: In some tissues (muscle, adipose), insulin mobilizes transporter for facilitated diffusion (of glucose) -- GLUT 4 protein -- promotes fusion of vesicles containing the transporters with plasma membrane. No other hormone can cause this effect.

c. Other Effects: In other tissues, insulin promotes utilization of glucose -- activates appropriate enzymes for glycogen, fat storage.

d. Overall: promotes uptake & utilization of glucose.

a. Receptor: Glucagon works through a G protein linked receptor that triggers the cAMP pathway (as for epinephrine).

b. Effects: Effects on tissues vary generally promotes production/release of glucose, not uptake or utilization.

c. Receptor triggers same pathway as epinephrine. Note that the same signaling pathway can be used for two different hormones (epinephrine & glucagon).

(1). Epi. & glucagon bind to different receptors, but both receptors activate the same G protein and trigger the same series of events --> cAMP --> etc. so get same response to both hormones in same tissue.

(2). Receptors present on cell surface determine which tissues will respond to each hormone. Muscle has Epi receptors and responds to Epi but not glucagon liver has receptors for both and responds to both.

(3). Two hormones control same process (glycogen metabolism) for different purposes -- Epi to respond to stress glucagon to respond to low blood sugar (maintain homeostasis).

(4). Same hormones give different response in liver vs adipose tissue. How? Both hormones trigger production of cAMP and activation of PKA. But different enzymes and processes (glycogen metabolism vs. fat metabolism) available to be controlled by same kinase.

C. Absorptive vs Postabsorptive State -- A more complex view of the circuit (See Purves fig. 50.21 [47.20]) & handout 15A.

1. What is really being regulated by insulin & glucagon? Really two different things:

a. Maintenance of glucose homeostasis

b. Managing an episodic event (eating) -- this can be considered just another example of homeostasis -- here the 'episodic' nature of eating generates two basic states that must be controlled differently to maintain homeostasis.

2. There are two main states of food (not just glucose) supply:

a. Absorptive -- anabolic --> synthesis & storage of macromolecules glucose is primary energy source. In this state, right after you eat, the risk is that blood glucose levels will rise too much.

b. Postabsorptive -- catabolic --> breakdown of macromolecules. and synthesis of glucose from smaller stuff (gluconeogenesis) fatty acids are primary energy source (except in brain). In this state, between meals, the risk is that blood glucose levels will fall too much.

3. Roles of Effectors = target organs (see handout 15A) in raising/lowering glucose levels.

a. Liver -- carries out both storage and release of glucose so acts as buffer only organ that can release glucose into blood and does gluconeogenesis takes up glucose without insulin (does not use GLUT 4).

b. Muscle -- stores or releases energy and protein.

c. Adipose -- stores or releases fat/ fatty acids.

d. All three organs co-operate -- have pathway in post-absorptive state that uses all three involves traffic of components from one tissue to another.

4. Roles of the Hormones

a. Insulin. Absorptive state is absolutely dependent on insulin. What does insulin do?

(1). In liver

(a). Insulin promotes glycogen synthesis (& breakdown of glucose for energy) -- uses glucose up.

(b). Insulin inhibits glycogen breakdown (& gluconeogenesis) -- inhibits production and release of glucose.

(c). Insulin also promotes glucose uptake, but not directly. Insulin promotes uptake by increasing phosphorylation and utilization of glucose.

(2). In other tissues(adipose tissue, resting skeletal muscle)

(a). Insulin promotes synthesis of storage forms of metabolites -- fat (triglycerides), glycogen, &/or protein

(b). Insulin inhibits breakdown of stores of fat, glycogen etc.

(c) Insulin directly stimulates glucose uptake. Insulin mobilizes the glucose transporter (GLUT4) so glucose uptake can occur. Insulin is required for uptake of glucose into these tissues.

(3). Note:Insulin is not required for uptake of glucose in brain, liver or working skeletal muscle. Liver and brain use different transporters (GLUT 1, 2 & 3) located permanently in the plasma membrane, and exercise mobilizes GLUT4 in skeletal muscle.

b. Glucagon . Postabs. state largely caused by lack of insulin also utilizes glucagon but stress hormones (cortisol and epinephrine) can fill in for glucagon. What steps are affected by Glucagon?

(1). In liver: Promotes catabolism (breakdown) of glycogen, promotes gluconeogenesis and inhibits synthesis of glycogen (all through cAMP).

(2). In adipose: Inhibits synthesis and promotes breakdown of fats (through cAMP).

(3). In muscle: No effects -- muscle lacks glucagon receptors.

For questions on this topic see problem set 7 -- additional problems.

III. Lactation: Example of Positive Feedback & Use of Neuronal signals See 15B.

A. Overall Loop : suckling by baby ---> milk ejection ("letdown) --> more suckling --> more milk ejection etc. until baby stops nursing.

B. Signaling Pathway : Suckling by baby stimulates nerve endings in nipple ---> nerve signal to HT --> release of oxytocin from post. pit. --> contraction of myoepithelial cells (similar to smooth muscle) surrounding alveolus (milk producing section of mammary gland) ---> milk ejection from lumen of alveolus ---> etc.

At same time, HT neurons stimulate ant. pit to release prolactin (PL) ---> stimulates inner layer of cells surrounding lumen of alveolus --> promotes milk production and secretion of milk into lumen of gland.

Question to think about: what's the circuit look like here? What's the IC? The effector? Etc.

Try problems 7-14 & 7-18.

To look at a more complex case using nerves & hormones helps to look at basic organization of nervous system first. This section is FYI -- not covered in class -- will be discussed after nerve cell function.

IV. How is Nervous System organized overall?

1. CNS = brain + spinal cord

2. PNS -- Part of nervous system outside of CNS. See Purves 46.2 [43.2] -- Names of Divisons

a. Two divisions of PNS: Afferent (carrying info into the CNS) vs Efferent (carrying info away from the CNS)

b. Two divisions of Efferent: Somatic (controls skeletal muscle = voluntary actions) vs autonomic (controls all unconscious responses = automatic actions )

c. Two divisions of autonomic: Parasympathetic (PS) vs Sympathetic (S)

B. How do PS and S co-operate? (See Purves 46.11 [3.11])

1. What do they innervate (what organs to they connect to)?

a. Many organs innervated by both

b. Some organs innervated by only one

(1). liver, sweat glands -- S only

(2). tears -- PS only

2. What results does stimulation (signal from nerve) produce?

a. Not always S excites PS inhibits. Ex: salivation -- S inhibits PS excites

b. Usually S --> response needed in a crisis PS --> response needed to return to relaxed state.

c. Examples:

(1). Effects of S -- heart rate increases liver releases glucose bladder relaxes (to hold more).

(2). Effects of PS -- heart rate decreases, digestion & salivation increase.

V. Stress response -- How do hormones and nerves act together to respond to stress? See handout 15B.

A. Phase one -- Sympathetic activity stimulates target organs (that are not glands) --> Direct response of heart, liver, lungs, etc.

B. Phase two -- Sympathetic activity --> activation of glands

1. Stimulate pancreas ---> glucagon increases insulin decreases --> additional stimulation of some target organs

2. Stimulate adrenal medulla --> release of epinephrine --> stimulation of same targets as sympathetic activity & some additional targets -- hormones can reach where nerves can't go.

C. Phase three -- stimulate HT/AP axis to produce cortisol

HT in brain ---> releases CRH --> AP ---> releases ACTH --> adrenal cortex ---> produces cortisol --> target organs ---> stimulation of breakdown of fats & protein (instead of glucose) inhibition of immune system.

Note that each additional phase is slower but involves additional degrees of amplification due to second messengers, transcription, etc.


15.5: Coordinated Regulation of Glycogen Synthesis and Breakdown - Biology

C2006/F2402 '07 OUTLINE OF LECTURE #18

(c) 2007 Dr. Deborah Mowshowitz, Columbia University, New York, NY . Last update 04/11/2007 01:41 PM .

Handouts: Need 17B, 18A (Homeostasis) -- Seesaw view for Glucose and Temperature Regulation 18 B -- Lactation & Typical Circuit

I. Organization -- How are cells set up to co-operate in a multicellular organism? See last lecture & 17B.

II. How is a component of the internal milieu regulated?

A. Let's look at a specific example, namely blood glucose. The see-saw view. See handout 18A or Purves 50.19 (50.20).

1. Have a regulated variable -- glucose level in blood.

2. Need a sensor (or receptor) -- to measure levels of "regulated variable" (glucose). Here, sensor is in pancreas.

3. Need effector(s) -- to control levels of regulated variable (glucose) -- usually have one or more effectors that respond in opposing ways. In this case, effectors for uptake of glucose are liver, adipose tissue, and skeletal muscle effector for release of glucose is liver.

Note: Some of the terms discussed here are used differently in molecular biology and in physiology. Fortunately, the meaning is usually obvious from the context. For example, the terms "effector" and "negative feedback" are used differently in the two contexts. In physiology, "effector" usually means "a tissue or organ (like muscle or liver) that carries out an action and thus produces an effect." In this example, the effectors = organs that act to raise or lower the blood glucose. In molecular biology, the term "effector" is usually used to mean "a modulator of protein function." A modulator = a small molecule (like an inducer, enzyme activator etc.) that binds to a protein, alters the shape and/or function of the protein, and thus triggers an effect. See below for comments on 'negative feedback.'

4. Have a set point -- the level the regulated variable (blood glucose) should be. Set point is also sometimes used to mean the level at which corrections (to raise or lower the value) kick in.

In most cases, there is no significant difference between these two definitions of set point. In some cases, the desired value (first definition) and the value at which corrections occur (second definition) may be different. For example, there may be two cut-off points-- upper and lower, that bracket the desired level of a regulated variable. At levels above or below the respective cut-off points, messages are sent to the appropriate effectors to take corrective action. The term "critical values" is sometimes used instead of "set points" to describe the cut-off point(s).

5. Signaling -- need some signal system to connect the sensor(s) and the effector(s). Can be nervous &/or hormonal. In this case, primary (but not only) signal is hormonal & primary hormones (signals) are insulin & glucagon.

6. Negative Feedback -- the system responds to negate deviations from the set point. Important features:

a. Works to stabilize blood glucose levels

b. System is self-correcting -- Deviations in either direction (if blood glucose is either too high or too low) are corrected back to standard.

c. There are two opposing actions by effectors, not just one.

(1). If [G] gets too high, effectors take G up from blood. (top half of seesaw diagram)

(2). If blood [G] gets too low, effector releases G to blood. (bottom half of seesaw diagram)

d. Negative feedback is not always inhibition. In this case, an increase in glucose uptake is used to help lower high blood sugar levels. The deviation from the set point was fixed by accelerating, not inhibiting, a process. In negative feedback, deviations from the set point can be corrected either by speeding up a process (such as glucose uptake) or slowing down a process (such as glycogen breakdown to glucose).

e. How is this different from positive feedback? In positive feedback, the system responds to increase deviations from the set point -- a small deviation triggers a bigger one, which triggers a bigger one and so on. The deviations get bigger and bigger until → boom! (See lactation, below, for an example.)

f. Terminology: In physiology, negative feedback means the system is self correcting as in b & d above. It doesn't matter whether the corrections are achieved by inhibition (turning off the heater) or acceleration (turning on the air conditioner). In biochemistry, negative feedback usually means inhibition of an earlier step.

7. Value of regulated variable does not remain exactly constant , but stays within narrow limits.

See problem 5-1 & 5-2 a & b.

B. Example #2 -- Regulation of body temperature (in humans) -- the see-saw view (handout 18A)

1. Note many features are same as in glucose case.

2. Features not found in glucose case:

a. Multiple sensors in different places (for core and skin temp.)

b. Nature of Signal -- Signals are neuronal, not hormonal

c. Integrative center (IC)

(1). Role of IC: Compares set-point to actual value, sends appropriate message to effectors.

(2). Type of IC

(a). Sensor/IC function may be combined, as in Glucose example.

(b). Separate IC needed if there are multiple sensors, as in this case. IC co-ordinates incoming information from multiple sensors

(3). In this example, IC = hypothalamus (HT)


Watch the video: Ελπίζουν σε ρύθμιση (November 2022).