Insulin Action and Its Disturbances in Disease - part 3 pptx

Recent studies in sexually maturedminiature pigs demonstrated that when the insulin effect was determined ondifferent subfractions of muscle proteins a specific stimulatory effect on mus-

mechanisms whereby insulin selectively affects mRNA stability have not beenwell defined.

Initiation of mRNA translation into protein begins with formation of the methyl guanine cap at the 5-prime end of the RNA A number of cap-associatedproteins including eukaryotic initiation factor 4E (eIF-4E), eIF-4G and phospho-rylated heat–acid-stable protein (PHAS-1) are influenced by insulin PHAS-1binds to the eIF-4E cap binding protein, insulin enhances phosphorylation ofPHAS-1 and favours dissociation of eIF-4E and PHAS-1.14 This allows forbinding of eIF-4E to eIF-4G and hence favours association with the 40S ribo-somal subunit and translation initiation.15, 16 Also important in the binding of

7-the 40S ribosomal subunit is eIF-2, and 7-the binding of this initiation factor

is dependent on its association with GTP Controlling the recycling of theGTP/GDP-bound state of eIF-2 is eIF-2B Insulin increases the activity of eIF-2B and favours the GTP-bound (active) state of eIF-2, which in turn enhancestranslation initiation.16, 17

Protein elongation depends on the action of multiple elongation factors.Among these are elongation factor 2 (eEF-2) This factor is important for move-ment of the ribosomal complex along the mRNA and for the migration of theamino acyl-tRNA from the acceptor site to the peptidyl site of the ribosome.18Insulin enhances eEF-2 activity by reducing its phosphorylation via inhibition

of its kinase.19 A comprehensive description of the molecular mechanisms ofinsulin’s effect on translation is available in review form.20

The abundance of ribosomes and RNA content in part determines the lar capacity to synthesize protein.21 Ribosomes are made up of approximately

cellu-80 proteins and 4 ribosomal RNA (rRNA) species Production and assembly

of ribosomes takes place in the nuclei In chick embryo fibroblasts insulinhas been shown to induce a fourfold increase in the synthesis of ribosomalproteins.22Similar findings have been made in mouse myoblasts.23This appears

to in part be due to post-transcriptional events Messenger RNAs that encoderibosomal proteins appear to be preferentially associated with polysomes inmouse myoblasts treated with insulin.23 The synthesis of rRNAs has beenshown to increase after insulin treatment in a variety of cell types includingfibroblasts,22, 24 myoblasts23and hepatocytes.25Finally, insulin may also reducethe rate of ribosome degradation.25 – 27

Effect of insulin on intracellular events controlling protein breakdown

Cellular protein breakdown is a tightly controlled and highly specific process Incatabolic states such as starvation, sepsis or insulin deprivation, protein break-down can markedly increase At the intracellular level, proteins can be degradedthrough several pathways including the lysosomal pathway, the calcium-depen-dent protease pathway or the ubiquitin–proteosome path.28 The majority ofproteins in mammalian cells are degraded through the ubiquitin–proteosome

MOLECULAR MECHANISMS OF INSULIN’S EFFECT ON PROTEIN TURNOVER 109

pathway Proteins are targeted for breakdown by covalent conjugation to uitin This is an ATP-dependent process, and multiple ubiquitin molecules areadded such that a ubiquitin chain is formed.29 Proteins with a ubiquitin chainattached are degraded by the ATP-dependent 26S proteosome complex Therate-limiting step in this process is ubiquitin conjugation Indirect evidence fromanimal studies suggests that ubiquitin-dependent protein degradation is important

ubiq-in states of ubiq-insulubiq-in deprivation Proteubiq-in breakdown rates ubiq-increase markedly ubiq-inrats that are made insulinopenic by treatment with streptozotocin Treatment withselective inhibitors of the lysosomal or calcium-dependent protease pathwaysdid not affect protein breakdown When ATP synthesis was blocked, how-ever, protein breakdown declined.30 This suggests that ATP-dependent ubiqui-tin–proteosome-mediated protein breakdown is important in insulin deficiency.Others have shown that mRNAs for ubiquitin–proteosome proteins are increased

in the insulin-deficient state.31 If diabetic rats are treated with insulin, tein breakdown is reduced, and ubiquitin–proteosome mRNAs are reduced tocontrol levels.32 Acidosis and increased cortisol levels, which occur followinginsulin deprivation, stimulate protein degradation in the ubiquitin–proteosomepathway.32, 30 In summary, insulin deficiency in a diabetic animal model showscoordinate time-dependent changes in different proteolytic pathways in muscle,resulting in increased overall proteolysis Only the capacity of non-lysosomalprocesses seems to be altered in muscle in response to insulin deficiency Themany intracellular mechanisms of insulin action to affect protein turnover aresummarized in Figure 4.2

pro-Insulin effect on protein synthesis

Translation initiation -PHAS-1 phosphorylation eIF-2B activity

Elongation eEF-2 phosphorylation -

mRNA -

-Figure 4.2 Effect of insulin on protein turnover

Insulin as a regulator of protein turnover in vitro and in situ

Studying the effect of insulin on protein turnover in humans is complicated Thebody is an intricate system with many hormonal mechanisms that interact withone another Therefore, altering a single hormone such as insulin can lead tochanges in other hormones, including growth hormone, glucagon, cortisol andepinephrine to name a few These in turn can lead to changes in concentrations

of substrates, such as amino acids, glucose and fatty acids, in heart rate and

in blood flow or have more direct effects on protein synthesis and breakdown

Because of these complexities several in vitro and in situ systems have been used to study insulin’s effect on protein turnover By using an in vitro or in situ

model one can simplify the experiment by removing confounding factors likeother hormones and alterations in other parameters such as blood flow

The simplest model is an in vitro cell culture system In this system a

homoge-neous population of cells can be studied under very controlled conditions Using

a specific cell line such as L6, a rat skeletal muscle myoblast line, allows one

to determine insulin’s effect on protein turnover within a single cell type Thecomponents of the cell medium and the insulin concentration can be well defined.Using this model, it has been shown that insulin stimulates protein synthesis in L6myoblasts.33, 34This type of model is ideal for studies of signal transduction path-ways stimulated by insulin.35, 36The biggest disadvantage of a cell culture model

is that it may not be representative of the whole body system Cell lines are ally transformed in some manner and even when differentiated the cells lack some

gener-characteristics of cells in vivo For example, even when L6 myoblasts are

differen-tiated into myotubes, they do not express the same myosin heavy chain isoforms asadult skeletal muscle.37In addition, within a tissue such as skeletal muscle, thereare many different cell types such as fibroblasts, vascular muscle cells, vascularepithelium etc These may be important modulators of skeletal muscle cells andthe effect would be missed in a simple cell culture system

In order to account for these parameters but to still maintain a very

con-trolled system, several investigators have used in situ methods to study the

effect of insulin on protein turnover These models have utilized perfused animaldiaphragm, heart, skeletal muscle, or whole limbs.38 – 40, 16 Consistently, insulin

reduces tissue protein breakdown Although in situ studies provide a

simpli-fied system that may be optimal for understanding mechanisms behind insulin’s

effect on protein metabolism, they too may not fully represent the in vivo

situa-tion Ultimately, to fully understand insulin’s regulation of protein metabolism

in humans, one must study an in vivo system A number of methods have been

used to measure protein turnover in animal models and in human subjects

Animal studies

Rodent models have been extensively used to study insulin effect on proteinmetabolism In studies performed in growing rodents indicated that insulin

MEASUREMENT OF PROTEIN METABOLISM 111

deficiency was associated with reduced synthesis rates of muscle proteins,

where-as in fully grown rodents insulin failed to stimulate muscle protein synthesis.41Similarly, in piglets insulin stimulates muscle protein synthesis rates42 and withincreasing age the magnitude of synthesis rates decreases.43, 44 These mea-surements were performed on mixed tissue proteins, representing the averagefractional synthesis rates of many proteins Recent studies in sexually maturedminiature pigs demonstrated that when the insulin effect was determined ondifferent subfractions of muscle proteins a specific stimulatory effect on mus-cle mitochondrial protein synthesis was observed, with no significant effect onsynthetic rates of sarcoplasmic and myosin heavy chain proteins.45 In contrast,the insulin effect on liver proteins in mini-pigs is variable, showing no effect onliver tissue protein synthesis whereas synthesis rate of fibrinogen was inhibited.46Since human adult life is much longer than that of rodents and pigs it is impor-tant to study the insulin effect on adult humans to understand the regulation ofprotein turnover in humans after the genetic potential for growth is passed

breakdown or turnover) in human subjects

Measurement of protein turnover

Net protein turnover, a result of both synthesis and breakdown, can be fied using a number of different methods Some of the more global techniquesinclude whole body nitrogen balance, 3-methylhistidine excretion (specificallyfor myofibrillar protein breakdown), regional amino-acid balance and systemicamino-acid tracer incorporation By using biopsies or separation techniques,protein synthesis can also be measured within a specific tissue or for a specificprotein Ultimately, the regulation of protein concentrations may be a result ofmany factors including changes in gene expression, mRNA stability and trans-lation efficiency Assessment of changes in protein turnover induced by insulincan take place at many levels: (1) the cellular level, where one may observethe mRNA changes and changes in translation efficiency; (2) the tissue level,where one can study the effect of insulin on a specific tissue or set of proteins(such as skeletal muscle on myofibrillar proteins); (3) the regional or wholebody level, where one can more globally assess insulin’s effects (Figure 4.3) Inthis section, the various methods of studying protein turnover in human subjectswill be discussed Following the description of each method, we shall reviewthe use of the method to assess the effect of insulin on protein turnover

quanti-Whole body nitrogen balance

When protein is broken down, free amino acids and their metabolites are releasedinto the circulation All amino acids contain at least one nitrogen molecule.Transamination is a critical process necessary to transfer nitrogen for synthesis

Whole body

Regional

Tissue

Amino-acid availability

Specific proteins

Transcriptional and translational regulation

DNA and mRNA

Figure 4.3 Sites to assess insulin effect on protein metabolism

of non-essential amino acids Amino acids that are oxidized or transaminatedcan give rise to ammonia Most of this circulating ammonia is converted to urea

in the liver via the ornithine cycle and can be excreted in the urine Urinarynitrogen is composed of 80–85 per cent urea and ammonia Another 5–10 percent of urinary nitrogen is accounted for by creatine, creatinine, uric acid andfree amino acids.47 By collecting urine and stool for 24 hours, one can quantifytotal body nitrogen loss To determine net nitrogen loss daily nitrogen intake alsohas to be measured This reflects the summation of multiple processes includingchanges in protein breakdown, protein synthesis, dietary protein intake, andalterations in the recycling of amino acids

Although this method seems straightforward in concept, there are severalproblems with it First, results can be affected by changes in renal function,hydration status, certain medications and the amount of protein that is ingested.Generally, subjects are asked to maintain a specific diet (normalized for proteinintake) for several days before a study This reduces the variability in nitro-gen generated by dietary protein intake In diabetic patients with reduced renalfunction, proteinuria or renal tubular acidosis, the results of whole body nitrogenbalance can be unreliable

Insulin effect as measured by nitrogen balance and free amino-acid concentrations

Early studies on diabetic patients used whole body nitrogen balance to assessthe effect of insulin on whole body protein metabolism Withdrawal of insulin

MEASUREMENT OF PROTEIN METABOLISM 113

treatment has been shown to increase urinary nitrogen losses and to increasethe concentrations of several essential amino acids, especially branched chainamino acids.48, 1, 49 Insulin treatment normalizes the increased urinary nitrogen

loss and the increased circulating amino-acid concentrations.5, 49, 50 In 1976,Walsh and colleagues51 studied 18 uncontrolled diabetic patients before andafter 6–8 weeks of treatment This group was a mixture of type 1 and type 2diabetic patients In subjects who were given insulin to control blood sugars,there was an average weight gain of 8.7 per cent and average nitrogen balance

of+13 per cent In the diabetic patients treated with diet alone or with diet and

an oral agent there was no change in weight, and only a+3.8 per cent nitrogenbalance.51 This increase in body mass and a positive nitrogen balance showsthat in patients who are relatively insulin deficient (diabetic patients) treatmentwith insulin has an anabolic effect

on the effect of insulin on myofibrillar protein breakdown

Insulin effect as measured by 3-MH

In healthy volunteers, insulin infusion does not change the flux of 3-MH acrossthe leg or forearm.52, 53 In contrast, in a study of poorly controlled diabetic

patients there was a substantially greater excretion of urinary 3-MH as pared with healthy volunteers When the same diabetic patients were restudiedafter achieving satisfactory glycemic control, urinary 3-MH excretion was notdifferent from that of healthy volunteers.54 This suggests that insulin deficiencyresults in increased muscle (we cannot differentiate between skeletal and smooth)protein breakdown and that replacement of insulin inhibits this breakdown Theavailable techniques to measure 3-MH have widely varying coefficients of vari-ation, which makes these measurements insensitive to small differences

com-4.4 Whole body and regional protein turnover

The effect of amino-acid availability

Amino acids are the building blocks of proteins The availability of these ing blocks can determine whether protein synthesis can take place Based onthekm value of amino-acyl tRNA ligase it was argued that normal physiologi-cal changes in free amino acids have little effect on protein synthesis However,recent studies have clearly demonstrated that amino acids by themselves enhancetranslational efficiency of gene transcripts.55, 56 Amino acids can be provided

build-by reuse of amino acids provided build-by protein breakdown or they can be provided

in the form of a meal or infusion Amino-acid availability is of great tance when considering insulin’s effect on protein turnover, because amino-acidavailability has been shown to be a major factor controlling muscle proteinsynthesis.57 – 59 Systemic or regional infusion of insulin has been shown toreduce blood concentrations of amino acids (hypoaminoacidemia).60, 48, 61, 62

impor-A reduced rate of protein breakdown by insulin is the likely cause of thisinsulin-induced hypoamino acidaemia Another potential site of the insulin effect

is on transmembrane transport of amino acids Transmembrane transport ofneutral amino acids in skeletal muscle is mediated by at least four different sys-tems (A, ASC, L and Nm) Regional studies of forearm skeletal muscle usingmethylaminoisobutyric acid (MeAIB), a non-metabolizable amino-acid analoguespecific for system A amino-acid transport, showed that physiologic hyperinsuli-naemia stimulates the activity of system A amino-acid transport.63 This effectmay play a role in determining the response of muscle amino-acid transportand protein metabolism in response to insulin When trying to reconcile theresults of whole body and regional studies in humans, it is important to notewhether blood amino-acid concentrations were monitored and/or clamped duringthe study When discussing results below, we shall note this

Amino-acid tracer techniques

Use of a labelled amino-acid tracer allows simultaneous determination of tein synthesis and breakdown rates at the whole body level and across tissuebeds Quantifying incorporation of the tracer into a specific protein or proteinfraction or mixed proteins can yield the synthesis rate Measuring the dilution

pro-of the tracer (provided it is a labelled essential amino acid) in the free tracee(amino-acid) pool in the steady state is extensively used for calculation of pro-tein breakdown rates During a steady state condition the rate of appearance

of an essential amino acid such as leucine is the same as its disappearancerate Therefore, in a fasted state, rate of appearance is equivalent to proteinbreakdown because essential amino acids only appear from protein breakdown,and rate of disappearance (sum of catabolism and incorporation into protein)can be estimated Once the catabolic rate (e.g leucine oxidation, phenylalanine

WHOLE BODY AND REGIONAL PROTEIN TURNOVER 115

hydroxylation to tyrosine etc.) and flux (appearance or disappearance rate) aremeasured, rate of incorporation of amino acid into protein (protein synthesis)can be calculated by subtracting the catabolic rate of the amino acid from itsflux7(Figure 4.3)

In addition, from tracer and tracee measurements in artery and vein (e.g.femoral vein for leg or hepatic vein for splanchnic bed) as well as blood flowmeasurements (usually based on indicator dye dilution) the kinetics of protein(breakdown and synthesis) and net balances can be estimated in the respectivetissue beds.49 In addition, serial needle biopsy of skeletal muscle and infusion

of an isotopic tracer and measurements of isotopic abundance of the tracer

in muscle protein or proteins will allow the estimation of fractional synthesisrates of mixed proteins or specific proteins.64Similar approaches can be applied

to measure fractional synthesis rates of circulating plasma proteins.65, 66 Thetracer technique, therefore, can be used to determine whole body, regional andspecific protein (such as myosin heavy chain) synthesis rates In most cases, ifthe appropriate samples are taken (including blood, breath samples and tissuebiopsies), a single experiment can determine all of these parameters Two tracermethods are widely used for determination of tissue protein synthesis rates inhumans – flooding dose and continuous infusion

The flooding dose technique

With the flooding dose a large amount of unlabelled amino acid (tracee) isinjected as a bolus along with the labelled amino acid (tracer).67 The goal ofinfusing this large dose is to quickly achieve an equilibrium of tracer concentra-tion between the plasma and the intracellular ‘precursor pool’ The obligatory

‘precursor pool’ is the amino acid acylated to its transfer RNA (amino-acyltRNA) This is the step just prior to incorporation of the amino acid into aprotein To accurately calculate synthesis rates based on extracellular tracerenrichment, the extracellular tracer enrichment and intracellular ‘precursor pool’enrichment must be in equilibrium

The primary advantage of this technique is that protein synthesis rates can bedetermined in a short period of time (10–30 minutes) Since a large amount oftracer is infused, it will make up a greater percentage of the amino acids incor-porated into protein This is particularly useful in studies of acute interventionssuch as short term infusion of a compound

The main disadvantage of this technique is that a number of assumptionsneed to be made First, the large bolus of amino acid must be assumed tohave no effect on protein dynamics Second, in order for rates of synthesis andbreakdown to be calculated, one must assume that enrichment is at steady stateduring the study period, which may not be the case during a declining phase ofboth tracer and tracee These assumptions can be incorrect if certain requirements

of the flooding dose condition are not met, particularly if the concentration of

the ‘flooding dose’ is too low or the study period is too long Either of thesecan cause non-equilibrium conditions in tracer enrichment between extracellularand intracellular compartments The tracer in this approach is not truly in the

‘tracer amount’ and the high concentration of ‘tracer’ may affect the proteinsynthesis measurements.68 The advantages and disadvantages of this techniquehave been described in detail elsewhere.69 – 74

The continuous infusion technique

With the continuous infusion technique, a continuous lower level infusion oftracer is given In order to reach a steady state more quickly, the continuousinfusion is typically preceded by a priming bolus of tracer.75 The continuousinfusion technique allows study over a long period of time (several hours).Hence, this technique is better suited to the study of proteins that have aslow rate of turnover Most skeletal muscle proteins fall into this category

On the other hand, this technique is not ideal for quick turn over proteinsbecause of the amino-acid recycling that can occur over a prolonged time period.Another disadvantage is that in most cases a surrogate measure of the oblig-atory precursor (amino-acyl tRNA) has to be used for calculation of proteinsynthesis This results in underestimation of protein synthesis calculation.76For whole body measurements surrogate measures of intracellular pool, such

as ketoisocaprioate in the case of leucine tracer, have been used with somestrong theoretical reasons.77 However, this approach is not practical with everyamino-acid tracer

Amino-acid tracers

In the past, radiolabelled amino acids were used as tracers More recently, ble isotope amino-acid tracers have been more widely used, which has manytheoretical advantages and is more acceptable for volunteers for studies andinstitutional ethical committees The incorporation of the tracer into protein canthen be quantified by mass spectrometry The amount of incorporated tracer is

sta-a reflection of the sta-amount of newly synthesized protein over the time of theinfusion.7The amino acid chosen for the tracer varies from study to study, and

it is not uncommon to use more than one tracer within a single study.7

For whole body studies tracers such as L[1-13C] leucine and labelled lalanine (e.g L[15N] phenylalanine L[2H5] phenylalanine) are extensively used.For regional studies involving skeletal muscle bed phenylalanine has manyadvantages, which include its small intracellular pool and thus the shorter periodneeded to equilibrate with the free amino-acid pool Within skeletal muscleand the other tissues of the forearm or leg, phenylalanine is not metabolized.Protein synthesis rates can be determined by measuring the rate of disappear-ance of phenylalanine However, if one is studying the splanchnic bed (liverand intestine), it is important to account for the conversion of phenylalanine

pheny-WHOLE BODY AND REGIONAL PROTEIN TURNOVER 117

to tyrosine within the liver using an independent tracer of tyrosine.7 Recentstudies have also demonstrated that phenylalanine is converted to tyrosine inthe kidney78 besides in the liver Therefore, for regional studies involving kid-ney as well, phenylalanine and tyrosine tracers have to be used to measureprotein turnover

Leucine is an essential amino acid that composes 6–8 per cent of protein.Because leucine concentrations are high within most proteins, it provides largeenrichment when used as a tracer This is particularly useful when studyingsynthesis rates of proteins that have slow rates of turnover Use of leucine inregional studies, however, can make calculations more complicated because itcan be either directly incorporated into protein (non-oxidative metabolism) orreversibly transaminated to form ketoisocaproic acid (KIC) KIC can then befurther oxidized to carbon dioxide and isovaleryl CoA (Figure 4.3) or reami-nated back into leucine If leucine is labelled at the carboxyl carbon (e.g 13C)and the amino group with15N, it is possible to quantify leucine transaminationrates Leucine tracers with both labels have been used to measure transami-nation rates at the whole body79 and regional levels.80, 49 In order to account

for the metabolic products one must collect breath samples for measurement oflabel within expired carbon dioxide or 13CO2 production across tissue beds inregional studies.80 Measurement of 13C-KIC enrichment can be a useful sur-rogate of the precursor pool leucyl-tRNA enrichment Measurement of thiscompound requires far less muscle tissue and labour than does direct mea-surement of leucyl tRNA It has been demonstrated in human studies to be agood surrogate81 although muscle tissue fluid is closer to tRNA enrichment.81For studies involving liver proteins (plasma proteins such as albumin, fibrino-gen, APOB100etc.) plasma [13C] KIC is an excellent surrogate measure of liverleucyl-tRNA enrichment For skeletal muscle, muscle tissue fluid leucine enrich-ment is a better indicator of leucyl tRNA Amino-acid tracers can thus be used

to study protein kinetics of the whole body, of a region, of a certain tissue or

malized leucine flux, providing strong evidence that insulin suppresses proteinbreakdown Somewhat surprisingly, whole body protein synthesis also increased

with insulin deprivation and was suppressed with insulin treatment The nitude of synthesis suppression is less than breakdown suppression In wholebody leucine flux studies that also included an amino-acid infusion to maintainlevels, there was a further decrease in protein breakdown and leucine oxida-tion in response to insulin treatment.82, 90, 84Results are mixed regarding wholebody protein synthesis when amino acids were infused and leucine flux wasmeasured Some studies showed an increase in whole body protein synthesis(as measured by non-oxidative leucine flux)90, 84 and others did not.83 Duringinsulin deprivation there is also a marked increase in amino-acid oxidation,which is largely due to a substantial increase in the transamination process inthe case of leucine.7 Not all of the changes in type 1 diabetic patients during

mag-insulin deficiency are directly related to mag-insulin deficiency per se There are

many secondary events that occur following insulin withdrawal in type 1 betic patients Such secondary changes include increase in levels of circulatingamino acids (especially branched chain amino acids), glucagon, non-esterifiedfatty acids and β hydroxybutyrate levels Increased amino-acid levels (due toincreased protein breakdown) contribute substantially to the large increase inleucine transamination and leucine oxidation Increased amino acid levels alsocause increased protein synthesis in splanchnic bed.59Increased glucagon levelsalso cause increased oxidation of leucine, which has been shown in patientswith type 1 diabetes.91 Beta-hydroxybutyrate has been shown to stimulate syn-thesis of muscle protein synthesis.7 This effect of β-hydroxybutyrate and theinhibitory effect of fatty acids on protein breakdown may limit the cataboliceffect of insulin deficiency

dia-Regional protein turnover

Within a local area – across the forearm, across the leg or across the nic bed – protein turnover can be assessed by amino-acid balance and tracermeasurements Amino-acid balance and tracer enrichment are determined byinfusing, systemically, a labelled amino-acid tracer to achieve a steady state inthe plasma and precursor pools The amount of tracer enrichment and amino-acid concentration present in the venous and arterial sides are then determined.Amino-acid balance is the arterio-venous difference in amino acid multiplied

splanch-by blood flow Rate of appearance (protein breakdown) and rate of ance (synthesis and catabolism) can be estimated by mathematical equations.92, 7

disappear-The estimation of catabolic rate and synthesis rates are possible using multipleamino-acid tracers The details of the models used for these measurements aregiven elsewhere.93, 49, 7 Measurement of protein turnover in a region accounts

for turnover in all of the tissues of that region In the leg or forearm this wouldinclude skin, connective tissue, adipose tissue and skeletal muscle althoughskeletal muscle accounts for the major portion when deep veins such as femoralvein are used to sample In the splanchnic bed, tissues of the abdomen includingliver and intestine are the main contributors to protein turnover

WHOLE BODY AND REGIONAL PROTEIN TURNOVER 119

Insulin and regional protein metabolism in type 1 diabetic patients and healthy controls

The results of regional studies across the leg or forearm using either nine or leucine as tracers in type 1 diabetic patients are mixed Some show areduction in protein breakdown after insulin infusion83, 49, 94while others show

phenylala-no effect.95None of these studies showed any effect on protein synthesis ever, the relative fraction of muscle protein synthesis to whole body proteinsynthesis increases after insulin replacement.92

How-Results from the splanchnic bed are interesting In the insulin deprived state,splanchnic bed protein synthesis exceeds breakdown49 (Figure 4.4) Figure 4.4shows that insulin deprivation I(−) results in increased protein breakdown par-ticularly in skeletal muscle (sk muscle) I(−) also increases protein synthesis(only in the splanchnic bed) but to a lesser degree Insulin treatment I(+) results

in suppressed protein breakdown, particularly in skeletal muscle and protein thesis (only in splanchnic bed) Insulin levels are the lowest in the fasting statebetween meals It has been suggested that since there is a net breakdown inskeletal muscle protein during insulin deficient states muscle may serve as areservoir of amino acids Figure 4.5 shows that between meals, when insulinlevels are low, there is a preservation of splanchnic bed protein synthesis whilethere is a net degradation of muscle protein After a meal, when insulin levelsare high, there is a net gain in muscle protein and a decline in splanchnic bedprotein The regulatory sites of insulin and amino acids are indicated in thefigure During the fasting state, expendable muscle proteins could be brokendown in order to provide the necessary amino-acid supply to the splanchnic

other splanchnic

Figure 4.4 Effect of insulin on protein breakdown and synthesis in type 1 diabetic patients

Circulating proteins

AA efflux

AA efflux

Insulin PB Circulating proteins

PB PS

(a)

(b)

Figure 4.5 Sites of protein accretion between and after meals – based on studies with insulin and AA infusion 59

bed for synthesis of crucial proteins such as clotting factors Studies performed

in non-diabetic people demonstrated that protein synthesis in splanchnic bed ishigher than protein breakdown in the fasted state.96Muscle releases amino acids

in the fasted state because muscle protein breakdown is higher than muscle tein synthesis Therefore muscle is a provider of amino acids to the splanchnicbed to maintain synthesis of proteins Recent studies have shown the kidney is

pro-a net producer of tyrosine pro-and contributes to the systemic circulpro-ation.78 Wheninsulin levels increase muscle protein breakdown decreases and the output ofamino acids decreases This occurs in association with a decrease in splanchnicprotein synthesis However, if amino acids are infused along with insulin, there

is a further reduction in muscle protein breakdown and there is an increase inmuscle protein synthesis.59 Amino acids have an independent effect on splanch-nic protein synthesis and increase in splanchnic protein synthesis While insulin

is the major regulator of muscle protein turnover with minimal effect on nic protein turnover, amino acids have major effects on both these two tissuebeds Based on these studies, it is proposed that reduced insulin levels during

splanch-WHOLE BODY AND REGIONAL PROTEIN TURNOVER 121

the fasted state result in an increased output of amino acids from muscle bedand these amino acids are vital for synthesis of essential proteins in the liver.Following a mixed meal the amino acids from meal enhance protein accretion

in both splanchnic and muscle beds Insulin plays a key facilitative role in allthese processes

In type 1 diabetic patients insulin deficiency causes a substantial increase

in muscle and splanchnic protein breakdown (Figure 4.4) While muscle tein synthesis is not significantly affected by short term insulin deficiency intype 1 diabetic patients, splanchnic protein synthesis increases The net effect

pro-is that both protein breakdown and protein synthespro-is increase at the wholebody level However the greater increase in protein breakdown results in netprotein catabolism The increased splanchnic protein synthesis and prevention

of decline in muscle protein synthesis during short term insulin deficiency isthought to be related to increased circulating amino acids, based on studies innon-diabetic people.59

Tissue-specific protein synthesis

Biopsy of a specific tissue during an infusion or flooding dose of a tracer is ticularly useful when one wishes to study a tissue with a slow rate of turnoversuch as skeletal muscle A biopsy is taken at baseline and then at some pointafter an intervention In these biopsy samples one can measure the synthesisrates of particular groups of proteins by determining the amount of tracer incor-porated over the intervention time from a defined precursor pool In skeletalmuscle samples our laboratory typically determines the tracer incorporation intomixed muscle protein (a mixture of the proteins present in the muscle), intosarcoplasmic protein (protein present within the cytosol) and into myosin heavychain (a key structural and contractile protein) In addition, mitochondria areisolated and the amount of tracer incorporated into mitochondrial proteins isdetermined The purification of these fractions and measurement techniques isdescribed elsewhere.97Similarly, fractional synthesis rates of circulating proteinscan be measured after purifying specific proteins.65, 98, 66, 99

par-Insulin and mixed muscle protein in type 1 diabetic patients

Several groups have looked at the effect of insulin on the synthesis of mixedmuscle protein in type 1 diabetic patients after insulin treatment In none of thesewas there a change in the mixed muscle protein synthesis,82, 91, 85, 86 even with

amino-acid infusion.82

Fractional synthesis rate of a specific protein

If a specific protein can be purified from a biopsy specimen it is possible todetermine the fractional synthesis rate of the protein.100, 97 In our laboratory

we use SDS PAGE protein electrophoresis to purify myosin heavy chain frommuscle myofibrillar protein fraction separated by ultracentrifugation.101 Massspectrometry is used in measuring the isotopic abundance of a protein such

as myosin heavy chain102 that has a very slow turnover rate After protein orproteins are purified one has to hydrolyse the protein into amino acids anduse gas chromatography/combustion/isotope ratio mass spectrometry to deter-mine the change of isotopic enrichment between two biopsy periods Knowingthe precursor pool isotopic enrichment (amino-acyl tRNA or its surrogate mea-sures), fractional synthesis rates of specific protein or protein subfractions (e.g.mitochondrial proteins or sarcoplasmic proteins) can be measured.7

Insulin’s effect on specific proteins

When the synthesis rate of myosin heavy chain was studied in type 1 diabeticpatients before and following insulin treatment, there was no change observed.91Insulin, however, has a specific effect on muscle mitochondrial protein syn-thesis When insulin was infused at high physiological levels while replacingglucose and amino acids, muscle mitochondrial protein synthesis was stimulated(Figure 4.3).10 This increase in muscle mitochondrial protein synthesis occurred

in association with an increase in muscle mitochondrial enzyme activity andATP production This important finding demonstrated a pivotal role of insulinand amino acids in the regulation of mitochondrial oxidative phosphorylation

in skeletal muscle Certain liver proteins appear to be responsive to changes

in insulin concentration De Feo and colleges65, 98 have studied the fractional

synthesis rates of albumin, antithrombin III, fibrinogen and apoB-100 in healthyvolunteers and in type 1 diabetic patients In type 1 diabetic patients deprived

of insulin, the synthesis of albumin was reduced but the synthesis of fibrinogenwas increased.98 The authors proposed that the increase in fibrinogen repre-sented an acute phase response, because in healthy subjects insulin infusionstimulated the synthesis of albumin but reduced the synthesis of fibrinogen andantithrombin III

Effect of insulin in healthy volunteers

Some of the insulin effect on protein metabolism has already been discussed

in comparison with type 1 diabetes Healthy volunteers are typically studied inthe post-absorptive (fasting) state Baseline insulin levels are low in this stateand calculations of amino-acid flux are simplified because there is no dilution oftracer due to dietary intake of amino acid With only a few exceptions,52, 103–105

there seems to be good agreement that insulin inhibits protein breakdown innormal subjects Using a variety of tracers, many regional and whole bodystudies have reached the same conclusion.82, 106–112, 53, 113 However, there is

not good agreement regarding the effect of insulin on protein synthesis in healthyvolunteers – about half of the studies show no effect of insulin on protein

WHOLE BODY AND REGIONAL PROTEIN TURNOVER 123

synthesis.52, 82, 106, 108, 110, 112, 114, 96, 104 Several others show an increase inregional or whole body synthesis with insulin.103, 109, 115, 105 These differences

may be related to insulin dose, methodologies, duration of insulin infusion andlevels of circulating amino acids and other substrates On the whole, there isquite convincing data to suggest that insulin can inhibit protein breakdown inhealthy volunteers If protein synthesis rates are unchanged or increased, theoverall effect of insulin would be a whole body accretion of protein Based onrecent studies it is clear that plasma amino acids have a critical role in stim-ulating muscle protein synthesis and insulin alone reduces circulating amino

acids in vivo, which may explain some of the discrepancies between in vivo and

in vitro studies Insulin also has a specific effect on synthesis of certain

mus-cle protein fractions such as mitochondrial proteins and plasma proteins such

as albumin

Effect of insulin in type 2 diabetics patients

Type 2 diabetic patients are insulin resistant and as a result, at least early in thecourse of the disease, they have chronically high insulin levels The effect ofthis insulin resistance to glucose metabolism and hyperinsulinaemia on proteinturnover is not well defined Oral hypoglycemic agents (glyburide) have beenshown to reduce endogenous glucose production in type 2 diabetic patientsbut have no effect on protein turnover.116 When type 2 diabetic patients havebeen infused with relatively high dose insulin over a short three hour clampperiod, there is a suppression of protein breakdown similar to that seen inmatched control subjects.107, 117 However, intensive insulin treatment had no

effect on protein turnover in comparison with less stringent glycemic controlwith insulin.118 Interestingly, after a longer term infusion (overnight) after 10days of subcutaneous insulin there appears to be a resistance to the insulin effect

on protein breakdown Under these conditions, insulin does not significantly press protein breakdown.119 This resistance to the effect of insulin to suppressprotein breakdown is important Since type 2 diabetic patients have chronicallyhigh insulin levels, normal tissue turnover could not take place if protein break-down continued to be suppressed A resistance to the action of insulin on glucosedisposal is not necessarily coupled with resistance to insulin’s suppression ofprotein breakdown This allows for normal protein turnover to take place even

sup-in the face of high sup-insulsup-in levels At this posup-int, it is unclear whether the sup-cellular mechanisms behind the resistance to insulin’s effect on glucose disposaland protein metabolism are similar or whether they are independent

intra-As in control subjects, the fractional synthesis rates of mixed muscle tein, myosin heavy chain and mitochondrial protein were unchanged in diabeticpatients infused with insulin.120 It is interesting that intensive insulin treatment

pro-in type 2 diabetic patients did not stimulate mitochondrial protepro-in synthesis,which is consistent with the recent report that increasing insulin levels to the

same extent failed to increase muscle mitochondrial ATP production in type 2diabetic patients, in contrast with non-diabetic control subjects.10

Summary

Considering all of the data, including those from intracellular studies, in situ

studies and human subject studies, there is good evidence that insulin can ulate protein turnover in many ways Insulin can (1) selectively enhance thetranscription of certain mRNAs through insulin response elements in promoters,(2) selectively enhance the stability of certain RNAs, (3) enhance translationinitiation and elongation and (4) enhance ribosomal abundance Insulin canalso selectively inhibit the degradation of some proteins through the ubiqui-tin–proteosome system Studies in the whole organism help us to understandthe relative influence of changes in protein synthesis and breakdown in the over-all protein balance They also help us understand the differential effects withincertain tissue beds

reg-Studies in type 1 diabetic patients and non-diabetic people indicate that insulinhas differential effects on skeletal muscle protein turnover from those on thesplanchnic bed protein turnover This is particularly important when consider-ing the fluctuation in insulin levels in relation to meals The effects observed

in type 1 diabetic patients are more dramatic than those in healthy controlsbecause they can be studied in the insulin deficient state It may be that thissame paradigm is true in healthy subjects but that it is more difficult to detectthe differences Insulin stimulates muscle mitochondrial protein synthesis andmitochondrial biogenesis when amino acids are provided Insulin-induced fall

in circulating amino acids blunts the stimulatory effect of insulin on synthesis

of muscle proteins Insulin’s primary effect on muscle appears to be an tion of protein breakdown While amino acids have a key role in modulatinginsulin effect on muscle protein synthesis, amino acids are the main regulators

inhibi-of splanchnic protein synthesis Insulin, however, has highly specific effects oncertain liver proteins and muscle mitochondrial proteins and more research inthe area is warranted

In type 2 diabetic patients, there is a resistance to insulin’s effect on glucosedisposal, and there is also a resistance to insulin-induced suppression of proteinbreakdown The mechanism is unclear; however, this resistance is important inmaintenance of normal protein turnover in type 2 diabetic patients Insulin has nostimulatory effect on muscle mito-protein synthesis in type 2 diabetic patients.The question of how insulin might regulate protein turnover, and hence tissuemass, is complicated Currently, the limiting factor in more thoroughly under-standing this process is one of technology We have been limited to studying,for the most part, groups of proteins For example, the measurement of mixedmuscle protein or mitochondrial protein fractional synthesis rates may be thesummation of results from hundreds of different proteins The currently available

REFERENCES 125

techniques make it difficult to analyse the effect of insulin on a large number

of specific proteins As techniques become more refined for the purification andprofiling of many proteins simultaneously (proteomics), we will gain a detailedunderstanding of the regulation of the entire network of proteins within specificcell types in response to insulin

Acknowledgements

This work was supported by NIH grants RO1 DK41973 and MO1RR00585, theDavid Murdock-Dole Professorship (K S Nair) and the Mayo Clinic ClinicianInvestigator Training Program (L J S Greenlund)

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15, 136 – 142.

Genetically Modified Mouse

Models of Insulin Resistance

Gema Medina-Gomez, Christopher Lelliott

and Antonio J Vidal-Puig

Insulin resistance is a syndrome characterized by a diminished ability of insulin

to perform its normal physiological functions Insulin resistance is the mainfeature of type 2 diabetes and is the key factor in the development of themetabolic syndrome Thus, it is very important to determine the mechanismsregulating insulin sensitivity After initial attempts to characterize insulin sig-

nalling pathways in vitro it became evident that the system was too complex, and to establish any physiological correlates it was necessary to develop in vivo

models This complexity is derived from the fact that insulin not only regulatesglucose homeostasis but also exerts effects on lipid and protein metabolism thatmay be affected in insulin-resistant states Furthermore, it was clear that uponactivation of the insulin receptor, specific yet divergent signals and signallingpathways were generated that could regulate multiple metabolic pathways andthat defects at different locations in these pathways may lead to apparentlyparadoxical effects A further degree of complexity resulted from tissue-specificpeculiarities of the insulin signalling network that may confer different degrees

of susceptibility to the defects leading to insulin resistant states This may createheterogeneity in the insulin sensitivity in different tissues, which may ultimatelyaffect the partitioning of energy between organs Moreover, the possibility ofcross talk between the insulin and other signalling networks (e.g insulin-likegrowth factors, IGFs) added further complications to the mechanisms control-

ling energy homeostasis Thus, it was important to have an in vivo system to

Insulin Resistance. Edited by Sudhesh Kumar and Stephen O’Rahilly

 2005 John Wiley & Sons, Ltd ISBN: 0-470-85008-6

explore how a complete organism reacted and adapted to states of insulin tance Genetically modified animal models have emerged as extremely valuabletools to assist in this task In this chapter we review how the use of geneticallymodified mice has helped to elucidate the physiological role of insulin and the

resis-defects associated with insulin resistance in vivo.

mechanisms leading to insulin resistance

Transgenesis and homologous recombination technologies offer powerful tools

for the study of the molecular mechanism of disease in vivo Using these

tech-nologies almost any genetic modification can be introduced into a given genome.For instance, mice can be engineered to overexpress specific genes in selectedtissues constitutively (transgenic models), or at a particular stage within devel-opment (inducible models) Similarly, mice can also be engineered lacking aspecific gene (knockout mice), either in the whole animal (global knockout),

or in a specific tissue (tissue specific knockout), at a specific time Geneticmanipulation can be used to recreate human diseases in mice by introduc-ing mutated human genes into the mouse genome (humanized knockin mice).These mice are extremely useful tools to test the beneficial effects of drugs andensure that their effects in rodents may be more readily extrapolated to humans.Breeding strategies can concentrate in the same mouse several genetic modi-fications in a heterozygous state, recreating models close to polygenic forms

of the disease Thus it is clear that molecular biology technology allows thedevelopment of well defined genetic manipulations to answer very specific bio-logical questions

of insulin resistance

For a gene to be considered a candidate to mediate insulin resistance directly, it isrequired that (a) this gene encodes a protein known to be involved in the insulinsignalling pathway, and/or (b) there is solid physiological evidence to supportthe view that this protein may interfere with the normal events of the insulinsignalling cascade The number of reasonable candidates is increasing in parallelwith our understanding of the molecular mechanisms controlling insulin sensitiv-ity This initial candidate list focused on genes integrated in the insulin signallingnetwork More recently, genes related to lipid and cytokine metabolism havebeen demonstrated to interfere with insulin sensitivity Rodents harbouring mod-ifications in those genes provide crucial information about their relevance forinsulin sensitivity, their role in specific tissues and how insulin resistance in aspecific tissue affects whole organism energy homeostasis (Table 5.1)

CANDIDATE GENES INVOLVED IN INSULIN RESISTANCE 135 Table 5.1 Genetically modified mouse models of insulin resistance

No diabetes β-cell hyperplasia Growth retarded

β-cell hypoplasia Diabetes

61

Tissue-specific knockout mice

Obesity, dyslipidaemia

Altered reproduction.

Other knockout mice

FABPs (−/−) Obesity/maintained insulin sensitivity

5.4 Insulin signalling network

The insulin signalling network has been discussed in Chapter 1 Thus it is notthe objective of this chapter to describe in detail all its different components Insummary, this network is composed of an insulin receptor1that, when activated

by insulin, phosphorylates and activates soluble intracellular adaptor molecules(e.g IRS and Shc family) Once activated, these adaptors interact with down-stream signal transduction molecules Two main pathways emerge from theinsulin receptor: (a) the mitogenic Grb2/Sos and the Ras-MAP kinase path-way and (b) the PI3kinase pathway, which exerts most of the metabolic actions

of insulin Figure 5.1 shows a representative scheme of insulin signalling ways Upon insulin binding to the insulin receptor there is a divergent cascade ofcoupled phosphorylation/dephosphorylation processes modulated by kinase andphosphatase enzymes These signalling pathways elicit specific responses span-ning not only metabolic pathways (e.g PI3kinase) but also pathways controllingcell proliferation (e.g MAP kinases) The cell-specific response to insulin isdetermined by the repertoire of substrates Thus alteration in selected moleculesmay influence some tissues more specifically than others and may result inparadoxical effects

path-JKN

Shc Raf-1

p70S6 kinase

GLUCOSE TRANSPORT I

PI3K

GLYCOGENESIS

SOS/Grb2

PP-1G

Glycogen synthase

SHIP

p90 rsk

PTP-1B

Transcription factors (e.g FOXO1)

Figure 5.1 Insulin signalling pathways

INSULIN SIGNALLING AND GLOBAL KNOCKOUTS 137

5.5 Factors leading to insulin resistance

Insulin resistance may arise from abnormalities in the molecules involved inthe insulin signalling cascade Alternatively, insulin resistance may also becaused by factors that, although they are not strictly part of the insulin net-work, are capable of interfering with or modifying some of these molecules.This is the type of insulin resistance typically associated with obesity In thissituation, cytokines such as tumour necrosis factor-alpha (TNFα) produce insulinresistance by inactivating molecules of the insulin signalling network throughseveral mechanisms, including phosphorylation of key serine residues, down-regulation of the insulin receptor (IR), defects of the IR tyrosine kinase activityand decreased activity of IRS1 and PI3 kinase.2, 3 TNFα also may promoteinsulin resistance through down-regulation of other molecules such as peroxi-some proliferator-activated receptor gamma (PPARγ)4or leptin The search forinsulin sensitivity modulators has identified adiponectin/ACRP30 and resistin

as new potential candidate molecules to mediate insulin resistance.5 – 7 In fact,defects in ACRP30 and resistin have been identified as potential links betweenobesity and insulin resistance It is unclear as yet whether the development ofinsulin resistance in the context of obesity is a specifically designed adaptivemechanism to counteract the excess of fuel, or the unexpected effect of lipo-toxicity on a system that was primarily designed to survive conditions of fueldeprivation Alternatively, it may be possible that under conditions of lipotox-icity mechanisms primarily designed for other purposes, such as apoptosis orhost defence, are inappropriately activated

through global knockouts

The first approach usually performed to determine whether a specific gene isrelevant for insulin signalling is probably the generation of a global knockout

An early conclusion of this type of study was, in the case of insulin receptor(IR) knockout mice, that the IR is required for survival outside the maternaluterus Homozygous IR knockouts are born live, but die within the first week oflife with marked signs of ketoacidosis.8, 9 However, heterozygous IR knockout

are viable and around 10 per cent become diabetic.10

The second line of events in the insulin signalling transduction cascadeinvolves a family of four types of insulin receptor substrate (IRS) Ablation ofIRS1 (IRS1KO) results in small mice with skeletal muscle insulin resistance thatwas compensated byβ-cell hyperplasia.11, 12 However, the pancreatic islets of

the IRS1KO mice had defective insulin production and secretion, albeit enough

to keep these mice euglycemic Other interesting phenotypes of the IRS1KOmice were the development of hypertension due to defects in vascular relaxation,and hypertriglyceridaemia secondary to defective lipoprotein lipase activity inadipose tissue Thus a global defect in IRS1 reproduces some of the metabolic

syndrome features The global knockout of IRS2 produced a strong phenotypewith insulin resistance in liver and frank diabetes at a young age Unlike IRS1KOmice, in this animal model defects in PDX, aβ-cell transcription factor regulated

by IRS2, prevented β-cell compensatory hyperplasia.13 This results in cient insulin secretion to compensate for the insulin resistance The knockouts

insuffi-of IRS3 and IRS4 did not result in any phenotype that suggested that thesesubstrates of the insulin receptor are involved in carbohydrate metabolism.14, 15The third line in the insulin signalling cascade, regulating more especially car-bohydrate metabolism, involves phosphoinositide 3 kinases (PI3kinase) This is

a group of enzymes that phosphorylate inositol rings within membrane lipids, which then act as secondary message molecules PI3kinases form dimers

phospho-of regulatory and catalytic subunits Several forms phospho-of the regulatory (p85α, p55α,p50α, p85β, p55γ) and catalytic subunits (p110α, β) exist As indicated before,PI3kinase is a key element in the metabolic response to insulin, specifically mod-ulating glucose transport, antilipolysis, fatty acid synthesis or glycogen synthesis.Complete ablation of all p85 regulatory subunits results in early death afterbirth.16, 17 However, heterozygous mice, with a decrease in the dosage of theregulatory subunits p85α, β55 and 50α, show increased insulin sensitivity, lead-ing to hypoglycaemia.18, 19It seems that ablation of regulatory subunits increases

the availability of the catalytic subunits, indicating that the stoichiometry of p85

to p110 may be a key factor regulating signal transduction via PI3kinase

3
-phosphorylated lipids generated by PI3 kinase serve as a membrane ment signal for the following kinase in the signalling pathway, PDK-1 (3
-phosphoinositide-dependent protein kinase) The global knockout of PDK1 islethal at embryonic day 9.5 due to multiple abnormalities However hypomor-phic PDK1 mice, expressing only 10 per cent of PDK1, are viable These miceare markedly smaller (40–50 per cent) than their wild type littermates Interest-ingly, their decrease in size is due to diminished cell volume, while cell numberand proliferation are conserved.20 This animal model also provides evidencethat the expression of PDK1 is required to normally activate PKB/Akt, S6K andRSK kinases

recruit-The next level of insulin signalling regulation after PDK1 is PKB/Akt Threedifferent isoforms of this enzyme exist but only Akt2 seems to mediate insulinsensitivity in skeletal muscle and liver.21Indeed, genetic ablation of Akt2 producesinsulin resistance in liver and skeletal muscle that results in a diabetic phenotype.Conversely, ablation of Akt1/PKBα does not result in insulin resistance or glucoseintolerance, although these animals’ growth is severely compromised Thus theseanimal models establish that Akt2 is the only essential isoform for the maintenance

of normal glucose homeostasis

This cascade of kinases finally phosphorylates several transcription factors,modifying their transcriptional activity The transcriptional effects of insulininvolve positive and negative effects on gene expression One of the mostrecently identified insulin-regulated transcription factors is the forkhead factor