insulin4

Progress in Retinal and Eye Research 22 (2003) 545–562

Functions of insulin and insulin receptor signaling in retina: possibleimplications for diabetic retinopathy

Chad E.N. Reitera,c, Thomas W. Gardnera,b,c,*aDepartment of Cellular and Molecular Physiology, The Ulerich Ophthalmology Research Center and The JDRF Diabetic Retinopathy Center at

The Penn State College of Medicine, M.S. Hershey Medical Center, 500 University Drive, Hershey, PA 17033, USAbDepartment of Ophthalmology, The Ulerich Ophthalmology Research Center and The JDRF Diabetic Retinopathy Center at The Penn State

College of Medicine, M.S. Hershey Medical Center, 500 University Drive, Hershey, PA 17033, USAcThe Penn State Retina Research Group, The Ulerich Ophthalmology Research Center and The JDRF Diabetic Retinopathy Center at

The Penn State College of Medicine, M.S. Hershey Medical Center, 500 University Drive, Hershey, PA 17033, USA

Abstract

Insulin action regulates the metabolic functions of the classically insulin-responsive tissues: liver, adipose, and skeletal muscle.

Evidence also suggests that insulin acts on neural tissue and can modulate neural metabolism, synapse activity, and feeding

behaviors. Insulin receptors are expressed on both the vasculature and neurons of the retina, but their functions are not completely

defined. Insulin action stimulates neuronal development, differentiation, growth, and survival, rather than stimulating nutrient

metabolism, e.g., glucose uptake as in skeletal muscle. Insulin receptors from retinal neurons and blood vessels share many similar

properties with insulin receptors from other peripheral tissues, and retinal neurons express numerous proteins that are attributed to

the insulin signaling cascade as in other tissues. However, undefined neuron-specific signals downstream of the insulin receptor are

likely to also exist. This review compares retinal insulin action to that of peripheral tissues, and demonstrates that the retina is an

insulin-sensitive tissue. The review also addresses the hypothesis that dysfunctional insulin receptor signaling in the retina

contributes to cell dysfunction and death in retinal diseases.

r 2003 Elsevier Science Ltd. All rights reserved.

Keywords: Insulin; Insulin receptor; Retina; Diabetic retinopathy

Contents

1. Introduction to the insulin signaling pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 546

2. Properties of retinal insulin receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 548

3. Insulin in the retina . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 550

3.1. Insulin and the blood–retina barrier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 550

3.2. Retinal insulin production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 551

3.3. Insulin signaling in Caenorhabditis elegans and Drosophila melanogaster . . . . . . . . . . . . . . 552

4. Retinal insulin signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 554

5. Insulin and insulin receptors in retinal diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 555

6. Conclusions and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 558

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 558

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 558

*Corresponding author: Department of Ophthalmology, Penn State University, College of Medicine, 500 University Drive, H097, Hershey,

PA 17033, USA. Tel.: +1-717-531-8783; fax: +1-717-531-7667.

E-mail address: [email protected] (T.W. Gardner).

1350-9462/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved.

doi:10.1016/S1350-9462(03)00035-1

1. Introduction to the insulin signaling pathway

Insulin is a potent anabolic hormone that stimulatesthe uptake and storage of carbohydrates, fatty acids,and amino acids into glycogen, fat, and protein,respectively. The signal transduction pathway (Fig. 1)by which insulin action affects various cells to accom-plish their biosynthetic roles has been extensivelyinvestigated and has increased our understanding ofmetabolism, physiology, and pathophysiology of dis-ease. It is also the subject of numerous excellent reviewsfor further details (Nystrom and Quon, 1999; Saltiel andPessin, 2002; Virkamaki et al., 1999). Insulin actionbegins with the synthesis of the mature peptide in the bcells of the pancreas. The b cells are constituents of apancreatic ‘‘organelle’’ termed the islets of Langerhanswhich also consist of a; d; and PP cells. These cells,B2%of the pancreatic mass, secrete the hormones glucagon,somatostatin, and pancreatic polypeptide, respectively,and are organized into ovoid structures dispersedthroughout the pancreas. Thus, the islets are organizedto produce the hormones that have opposing actions onblood glucose levels.The single insulin gene in humans encodes a 110

amino acid peptide, preproinsulin. The ‘‘pre-’’ portion isa 24 amino acid signal sequence that directs synthesis of

the peptide into the endoplasmic reticulum (ER), whereit is cleaved. This remaining peptide is termed proinsu-lin. In the ER, proinsulin is folded and three disulfidebonds stabilize the insulin A and B chains of 21 and 30amino acids, respectively. Proinsulin is then stored insecretory granules within the b cell until the b cellreceives signals to release the contents into circulation.While in the secretory granule, the C (connecting) chain(35 amino acids) that links the A and B chains togetheris removed to form the mature insulin molecule. Insulin,C peptide, and uncleaved proinsulin are all secreted intocirculation via the portal vein (Davis and Granner,1996; Ganong, 1997). In the liver, insulin worksreciprocally with glucagon to control the balance ofglucose and lipid metabolism.Insulin action at the cellular level begins with the

localization of the insulin receptor (IR) into plasmamembranes. The IR is a heterotetramer comprised oftwo extracellular a subunits and two transcellular bsubunits linked by disulfide bonds. The diverse effects ofinsulin commence when the receptor binds ligand on thea subunits, which induces a conformational change andactivates the intrinsic tyrosine kinase activity in the bsubunits in the cytoplasm. The IR autophosphorylates itsb subunit on tyrosine residues within its tyrosine kinasedomain and initiates a cascade of phosphorylation,

Fig. 1. Comparison of the conserved insulin-like signaling pathway among flies, worms, and mammals. Insulin-like peptides signal through

structurally similar receptor tyrosine kinases, and the signal is perpetuated down a conserved pathway. IRS proteins (termed CHICO in Drosophila

and unidentified in C. elegans) recruit PI3K and lead to Akt activation in response to insulin-like hormones. Not discussed in the text is PTEN, a lipid

phosphatase which negatively regulates PI3K activity by reducing phosphatidylinositol 30-OH phosphate (PIP3) content in cell membranes, and

mutations of have been described in tumors. Conservation of this central growth-promoting pathway suggests numerous effectors have evolved

around it to add specificity between different cell types within one organism. Dashed lines indicate hypothesized interactions. Adapted from Garofalo

RS, Trends in Endocrinology and Metabolism, 13, 156–162.

C.E.N. Reiter, T.W. Gardner / Progress in Retinal and Eye Research 22 (2003) 545–562546

dephosphorylation, and translocation events within thecell. Immediate protein substrates of the IR kinaseinclude, among others, the insulin receptor substrates(IRS 1-4), Grb-2 associated binder (Gab-1), Shc, andCbl, which in turn recruit other adapter and enzymaticproteins to propagate the signal and produce abiological effect.Specifically, the IR-IRS- phosphatidylinositol 30-

OH kinase ðPI3KÞ-Akt pathway has received muchattention as it is well conserved (Fig. 1) and plays a rolein many cellular processes including glucose uptake,glycogen synthesis, protein synthesis, and cellularsurvival.The IRS proteins interact with the IR b subunit and

are phosphorylated on numerous residues with insulinstimulation (White, 2002a; Yenush and White, 1997).Four IRS proteins derived from four independent genes,and a similar protein, Gab-1, have been described. Thesemolecules all contain similar domains and numeroussites for both tyrosine and serine phosphorylation. IRSand Gab proteins possess no catalytic activity before orafter phosphorylation, but serve as docking proteins toconcentrate other signaling molecules at the plasmamembrane by the binding of its pleckstrin homology(PH) domain to the inner leaflet. At various phosphory-lated tyrosine residues, proteins that contain Srchomology 2 (SH2) domains bind and are recruited tothe plasma membrane. The tissue distribution andpresumed function of the IRS proteins are not fullyredundant despite their similarities. This is illustrated ingenetic ablation studies in which IRS-1�=� mice showsignificantly reduced growth, while in IRS-2�=� mice,growth is only diminished by 10%, but they developovert diabetes leading to death (Araki et al., 1994;Tamemoto et al., 1994; Withers et al., 1998). It iscurrently believed that IRS-2 may play a moreprominent role in brain function which may also bemediated by other growth factors that signal throughIRS-2 (White, 2002a).The regulatory subunit of class IA PI3K contains two

SH2 domains, which flanks their p110 interactiondomain, and binds to IRS proteins following insulinstimulation. Like IRS proteins, these proteins also act asdocking molecules, and five proteins are in this class:p85a; p50a; AS53 ðp55a), p85b; and p55g: These proteinsrecruit the catalytic subunits of PI3K, p110a; p110b; orp110d (Fruman et al., 1998; Myers et al., 1992). Theprimary function of p110 proteins is to phosphorylatethe 30-OH position of inositol rings of the plasmamembrane to form phosphatidylinositol 3-phosphateðPIP3Þ; but they also contain serine phosphorylationactivity. Membrane regions rich in PIP3 aid in therecruitment of PH-domain containing proteins.Further along in the signaling sequence, Akt-1, -2,

and -3 (or PKB-a; -b; and -g; respectively), via their PHdomains, are recruited from the cytosol to plasma

membranes enriched in 30 phosphorylated inositol ringswhere the kinase becomes maximally active by phos-phorylation on conserved threonine and serine residues.The Akt kinases were also termed RAC kinase for

%Related to

%A and

%C kinase because of structural

similarities among the three (Kandel and Hay, 1999).A consensus amino acid sequence has been identified forefficient phosphorylation of substrates by Akt, but thereis significant overlap among other kinases as well suchas structural, localization, and tissue-specific considera-tions in determining whether a protein is a substrate ofthe Akt isoforms. Akts are involved in numerousinsulin-induced cellular events listed above. For exam-ple, the glucose transporter 4 (GLUT4) is the insulin-responsive transporter in skeletal muscle and adiposetissue; with insulin, intracellular vesicles containingGLUT4 translocate and fuse with the plasma mem-brane. A dominant negative form of Akt, when over-expressed in adipocytes, blocks insulin-stimulatedGLUT4 translocation, and overexpression of a domi-nant active form of Akt is sufficient for GLUT4exocytosis in the absence of insulin (Cong et al., 1997;Kohn et al., 1996). The targets of Akt which promoteGLUT4 translocation have yet to be identified. Akt alsophosphorylates and inhibits glycogen synthase kinase 3bðGSK-3bÞ; which allows glycogen storage to occur inliver and muscle as glycogen synthase is not phosphory-lated by GSK-3b and, therefore, remains active (Crosset al., 1995). It has been postulated that Akt regulatesprotein synthesis. Various proteins of the proteinsynthesis initiation factor complex are phosphorylatedby Akt, as well as mTOR/FRAP and p70 S6K.Together, mTOR/FRAP and p70 S6K further regulatethe initiation rates of protein synthesis by modulatingthe activity of the mRNA cap-binding complex and S6ribosomal protein phosphorylation (Schmelzle and Hall,2000).Akt has also been shown to exert a strong anti-

apoptotic effect on cells. Indeed the viral form of Akt, v-akt, has transforming effects on cells, suggesting anoverabundance of Akt signaling may override apoptoticsignals within cells, contributing to an oncogenicphenotype (Bellacosa et al., 1991; Testa and Bellacosa,2001). Numerous targets of Akt signaling have beenidentified that contribute to the prosurvival effect ofinsulin. Caspases (cysteine–aspartic acid proteases) areresponsible for cleavage of cellular proteins resulting inapoptosis of the cell. Caspases are cleaved and activatedby numerous cellular signals and other caspases in acascading manner (Earnshaw et al., 1999). The phos-phorylation of caspase 9 by Akt inhibits this activationto promote cellular survival (Cardone et al., 1998). TheBcl family member protein, Bad, has also beenimplicated in regulating apoptosis in an Akt-dependentmanner (Datta et al., 1997). When unphosphorylated,Bad antagonizes pro-survival Bcl proteins such as Bcl-2

C.E.N. Reiter, T.W. Gardner / Progress in Retinal and Eye Research 22 (2003) 545–562 547

and Bcl-XL in mitochondria, but when phosphorylatedby Akt, Bad is inactivated. This step is believed tomaintain the integrity and functionality of the mito-chondria for the production of ATP. Bcl-2 and Bcl-XL

dimerize and function to maintain mitochondrialintegrity, and following phosphorylation by Akt, Badis sequestered by chaperone 14-3-3 proteins in thecytosol to promote cell survival. It is also hypothesizedthat the tumor supressor, p53, is also a target of Aktactivity. Rat hippocampal neurons that were exposed tohypoxic conditions or nitric oxide toxicity to induceapoptosis were rescued by IGF-1 treatment and Aktactivation, which was also dependent on p53 expression(Yamaguchi et al., 2001). Clearly other factors, such ashypoxia inducable factor (HIF) proteins, can alsomediate a protective effect against hypoxia, but thisobservation is consistent with others that Akt translo-cates to the nucleus where it can regulate other proteins(Andjelkovic et al., 1997). Forkhead family transcrip-tion factors are also targeted by Akt in the nucleus(Brunet et al., 1999). Upon phosphorylation by Akt,Forkhead is transported out of the nucleus where it issequestered in the cytoplasm by 14-3-3 proteins in aninactive state. Forkhead family proteins regulate ex-pression of genes that induce apoptosis such as FasL;conversely, Akt may regulate pro-survival genes byinfluencing other transcription factors such as NF-kBand CREB (Brunet et al., 2001). This function of Aktmay be a key contrast between the classically insulin-responsive tissues and retina, or neurons in general, inwhich Akt plays a greater role in promoting cellularsurvival (Dudek et al., 1997) and insulin-mediatedsynapse integrity (Puro and Agardh, 1984) than nutrientstorage. Further specificity may involve specific Aktisoform expression in the retina, which has not yet beenfully elucidated.In general, the current understanding of insulin action

is derived from studies involving the classically ‘‘insulin-responsive’’ tissues or cells and cell lines derived fromthem. The isoforms of the IR, IRS proteins, PI3Ksubunits, and Akt vary by tissue and cell type. Theseexpression characteristics probably confers locally spe-cific responses. It has been demonstrated that the IR isexpressed abundantly in brain, retina, and otherneurons, and its function probably differs from pre-viously studied systems (Heidenreich et al., 1988; Reiteret al., 2003). Thus, further study is required to under-stand the similarities and differences of IR signaling inthe central nervous system and retina compared to otherinsulin-sensitive tissues.

2. Properties of retinal insulin receptors

Insulin binding characteristics have been described inthe mammalian brain and other regions of the central

nervous system (CNS), including the retina (Havranko-va et al., 1978a). However, our understanding of insulinaction in the retina and CNS has lagged considerablycompared to other insulin-responsive tissues in whichinsulin signaling is perturbed by diseases such asdiabetes and polycystic ovarian syndrome (Gambineriet al., 2002; Virkamaki et al., 1999). Comprehensivestudies by Waldbillig and Rodrigues et al. answeredmany questions about basic mammalian retinal IRcharacteristics, which are summarized within Table 1(Rodrigues et al., 1988; Waldbillig et al., 1991, 1987a, b,1988). They demonstrated specific insulin binding inpurified bovine rod outer segments, which was specifi-cally competed with excess insulin as opposed to 100-fold greater amounts of proinsulin. Similar to brain andliver, 1 nanomolar insulin stimulated phosphate incor-poration into the IRb subunit (Waldbillig et al., 1987a).Furthermore, the retinal IR, when stimulated withinsulin, possesses tyrosine kinase activity towards anexogenous substrate (Waldbillig et al., 1987b). Thesesimilarities among IRs in vitro suggest that retinal IRsmay function in a cell-specific manner in vivo.Our lab has recently demonstrated that the rate of

retinal IR activity in vivo also resembles brain IRactivity (Reiter et al., 2003). In contrast, the tyrosinephosphorylation and kinetic activity of the IR from liverand skeletal muscle fluctuate with circulating insulinlevels during periods of fasting and feeding, which inturn affects the physiology of downstream mediators ofinsulin signaling. Within 90 min of re-feeding fastedrats, IRb and IRS-1 tyrosine phosphorylation and p85association with IRS-1 are elevated concomitant withplasma insulin (Ito et al., 1997). Likewise, PI3K activityassociated with phosphotyrosine-containing proteins orShc is reduced with fasting in chicken liver and restoredupon refeeding (Dupont et al., 1998). Our observationsare consistent with those on brain IR kinetic activity(Simon et al., 1986) in which retinal IR tyrosinephosphorylation is unaltered between fasted and fedrats. Likewise, the kinetic activity of the IR in retinamirrors brain IR activity and is resistant to changesassociated with fasting, which diminishes liver IRactivity. Furthermore, in fasted rats, this tonic state ofactivity was greater than liver IR activity (Reiter et al.,2003). Taken together with the above studies byWaldbillig and Rodrigues, the IR in retina behavessimilarly to liver IR in vitro, while retinal IR activity ismore similar to brain IR activity in vivo where the IR ismaintained in a tonic state of activity. This differencemay result from how circulating insulin is delivered totissues of the body, and suggests that the blood-retinalbarrier (BRB) stabilizes insulin access to the retina.One major structural difference between IRs ex-

pressed in retina/neurons and IRs expressed in liver,skeletal muscle, or adipose is the extent of glycosylation.Both a and b subunits are glycosylated, but in general,


the extent of glycosylation of IRs expressed on neuronalcells is less than IRs expressed in liver and skeletalmuscle, and can be variable even among differentneurons (Hedo et al., 1981; Heidenreich et al., 1983;Ota et al., 1988). 125I-insulin cross-linking studies byWaldbillig et al. (1987a) demonstrated further simila-rities between brain and retinal IRa in which theligand-receptor complex migrates B10 kDa further onSDS-PAGE than liver IRa: Terminally-linked sialicacid residues comprise most of the IR glycosylationmotifs in adipocytes, as shown by neuramidase sensi-tivity, and the glycosylation is absent in brain IRs;however, the core peptide remains identical followingremoval of all N-linked glycans from 14 potential asubunit sites (Heidenreich and Brandenburg, 1986).Four potential N-linked sites are on the extracellularportion of the IRb; which also contains O-linkedoligosaccharides (Collier and Gorden, 1991). Like theIRa; the IRb subunit in retina and brain also migratesslightly faster on SDS-PAGE compared to liver IR(Reiter et al., 2003). Enzymatically catalyzed IRglycosylation is important for normal IR function asdemonstrated by site-directed mutagenesis of the fourN-glycosylation sites on the IRb (Leconte et al., 1992).The mutant IRb subunit is able to undergo autopho-sphorylation in response to insulin in vitro, but the IRlost all kinase activity towards an exogenous substrate.This alteration prevented any effect of insulin in cellsthat overexpressed the mutant IR on either glucoseuptake or thymidine incorporation into DNA. Althoughglycosylation is required for IR functionality, anexplanation for variability of the glycosylated IR among

tissues currently remains elusive. However, one mightspeculate that the decreased glycosylation of neural IRsinfluences tonic IR activity. Further analysis of IRglycosylation by mass spectroscopy may help to clarifystructure–function relationships.The a and b subunits of the IR are transcribed and

translated as a single peptide from one gene on humanchromosome 19 before assembly into the mature a2b2form. A second difference involves exon 11 expressionon the a subunit, which is the only sequence variance inthe mature peptide (Ebina et al., 1985; Ullrich et al.,1985). This splice variant is differentially expressed invarious tissues. Brain exclusively expresses the �exon 11(type A) form of the IR, but the majority of liver IRs arethe þexon 11 (type B) form (Seino and Bell, 1989). Theexpression of the splice variants in retina has not beenevaluated, nor has the tissue-specific mechanism of itsexpression, making the overall function of IR structuralheterogeneity still unclear.Consistent with the ‘‘housekeeping’’ nature of the IR

promoter, with a GC-rich promoter and the lack of aTATA box, (Araki et al., 1987), the IR has been shownto be expressed constitutively and broadly throughoutthe retina on neuronal, endothelial, and retinal pigmen-ted epithelial (RPE) cells. Using B10 anti-sera, a humananti-IRa sera from a patient with severe insulinresistance, Rodrigues et al. (1988) localized the IR tothe outer segments and retinal nuclear layers of bovine,monkey, and human retina. Interestingly, only rod outersegment disks in monkey, and human M .uller glial cellswere also immunoreactive for the IR with this antibody.IRa structural heterogeneity may have contributed to

Table 1

A summary comparison of b cell, brain, and retina insulin production and IR properties among peripheral/vascular tissues, brain, and retina from

mammalian systems

b Cell Brain Retina

Insulin Synthesized and packaged mRNA and immunoreactive mRNA and IRI detected

into secretory granules insulin (IRI) detected

for secretion as an endocrine Not in granules

hormone in response to Not in granules

circulating nutrients May act as autocrine/

Secreted in vitro paracrine effector

Mode of secretion unknown;

may act as an autocrine/

paracrine effector

Insulin receptor Liver/muscle Brain Retina

Molecular weight IRaB135 kDa and IRaB125 kDa and IRaB125 kDa and

IRbB97 kDa IRbB95 kDa IRbB95 kDa

Greater sialic acid content Less extensive glycosylation Less extensive glycosylation

Variable exon 11 expression Mostly �exon 11 Exon 11 expression undetermined

Effects of fasting Reduces kinase activity No change in kinase activity No change in kinase activity

Effects of systemic Rapid tyrosine Increased tyrosine phospho- Increased tyrosine

insulin injection phosphorylation in skeletal rylation after 3 min in phosphorylation after

muscle and liver ðo90 sÞ cerebellum, but no change 30 min

in forebrain


the variable results with this IRa antibody. Therefore,Naeser (1997) and Gosbell et al. (2002) extended thoseobservations by detecting IR immunoreactivity in allretinal layers of human and bovine retina, respectively,using polyclonal antibodies against intracellular por-tions of the IRb that are not differentially glycosylated.Retinal endothelial cells and pericytes both displayspecific binding for insulin. Treatment of these cells invitro rapidly stimulated thymidine incorporation intoDNA suggesting a strong mitogenic effect (King et al.,1985). Interestingly, it was also observed that the retinalvascular cells were more sensitive to the growthpromoting effect of insulin than aortic endothelial andsmooth muscle cells. The IR in RPE cells has also beeninvestigated, and it was found that its form is mostsimilar to the peripheral form and not the neural form.Compared to liver, it also exhibits neuramidase sensi-tivity, and overall specific binding was on the same orderas liver (Waldbillig et al., 1991). Furthermore, the IR inRPE cells localized exclusively to the basolateral surfaceof the cell, suggesting a possible role in unidirectionalinsulin transport from the choroidal circulation to thephotoreceptors (Sugasawa et al., 1994).Lastly, it has been reported that hybrid receptors

exist, and their expression appears to be widelydistributed and to include neurons (Bailyes et al.,1997; Garcia-de Lacoba et al., 1999). That is, an absegment of the IR is crosslinked to an ab segment of thehomologous IGF-1R, and this heterodimer has thepotential to bind either insulin or IGF-1. The functionof these receptors is not completely understood, buttheir existence may be a mechanism to downregulate asignal from one receptor, or to add greater specificity ofsignaling by either insulin or IGF-1. Hybrid IR/IGF-1Rexpression may also change in disease states as in thosewith hyperinsulinemia (Federici et al., 1998). Our labhas detected this complex in immunoprecipitates of theIR followed by immunoblot analysis for the IGF-1R,and vice versa, in retina (unpublished observations).Further investigation is required to determine thefunctions of IR/IGF-1R hybrids in retina and othertissues alike.In summary, because the retina, and other regions of

the CNS, expresses the IR at levels similar to otherclassically insulin-responsive tissues, it must be consid-ered as a major target of insulin action.

3. Insulin in the retina

3.1. Insulin and the blood–retina barrier

The blood–retina barrier (BRB) exquisitely regulatesthe exchange of material between the plasma and theneural retina. This partition gives the retina immuneprivilege as in other regions of the body such as the

brain, testes, and anterior chamber of the eye (Fergusonand Griffith, 1997). The BRB selectivity functions instark contrast to the endothelium of other tissues whichcontain numerous fenestrations, and larger gaps allowfor the free diffusion of molecules between theinterstitial space and endothelial lumen. This is the casefor liver, skeletal muscle, or adipose tissue which mustrapidly metabolize nutrients as they are absorbed fromthe gut, and insulin is released into the body. Therefore,the relatively rapid passage of insulin from the blood tosome tissues is necessary, and it is reasonable tohypothesize that insulin must be transported across theBRB for insulin to act on the neural retina. Somecomparisons can be drawn from studies on insulintransport across the blood–brain barrier (BBB). In thebrain, accumulation of exogenous insulin begins after60 min of insulin infusion and is thought to occur by asaturable, receptor-mediated transport process(Schwartz et al., 1992, 1990). After 60 min; insulinaccumulation in brain proceeds in a linear fashiondespite constant levels of insulin in circulation. Thisresults in relatively constant levels of insulin in thecerebrospinal fluid. The mechanism of transport acrossthe BBB is also thought to be more efficient atphysiological levels of insulin, as rates of insulinaccumulation in brain decreases with infusion ofpharmacological levels of insulin (Schwartz et al., 1992).As stated above, the IR is expressed throughout the

retinal endothelial network. The retinal vasculatureexpresses IRs which resemble those expressed in liver,rather than the smaller neural form of the IR, asassessed by insulin cross-linking and SDS-PAGE auto-radiography (Haskell et al., 1984; Im et al., 1986).Investigations lead by King et al. (King and Johnson,1985) spearheaded our understanding of endothelial celltransport of insulin. Using an aortic endothelial cellmonolayer separating two compartments as their model,they demonstrated that insulin is carried unidirection-ally across the monolayer. This transport of insulin wasmediated specifically by the IR, as pre-treatment withanti-IR antibodies blocked the transport, and B80% ofthe transported insulin remained intact. This findingstrongly suggests that endothelial cells do not degradesignificant amounts of insulin, as compared to othergrowth factors, which may be reflected in a lowerexpression of insulin degrading enzymes in retina andvascular cells (Varandani et al., 1982). These datasuggest that a major function of IRs on endothelialcells is to regulate transport of the hormone to a specifictissue. The specific pathway that endothelial cells utilizeto transport insulin has not been fully described.Incubating endothelial cells with leupeptin, a lysosomalprotease inhibitor, had no effect on insulin transportsuggesting those cellular compartments are not in-volved. Monensin, a proton ionophore which raisesendocytic vesicle pH, reduced insulin transport. This


result suggests that a cellular compartment requires anacidic environment to dissociate insulin from the IR forcomplete insulin transport (Hachiya et al., 1988).Using electron microscopy to detect gold-labeled

insulin, transcellular flux of insulin was also describedin cultures of retinal vascular cells (Stitt et al., 1994).Within 2 min of insulin binding, IRs with bound insulinwere clustered into clatherin-coated pits—the beginningof the well-described receptor-mediated endocytosispathway (Brodsky et al., 2001). After 10 min; theinsulin/IR/clathrin-coated pit complex had invaginatedand localized to the interior of vacuole-like compart-ments, which the authors identified as endosomes.Following 20 min of insulin stimulation, the labeledinsulin was associated with what the investigatorstermed a multivesicular body, and the labeled insulinagain was localized to the interior of this electron-dense,highly membranous structure. After 60 min; the major-ity of labeled insulin resided in secondary lysosomalstructures, while only B23% of the labeled insulin hadbeen transported to the basal plasma membrane orreleased to the extracellular space. These experiments invitro demonstrate an IR-mediated transport of insulinacross retinal endothelial cells, and that circulatinginsulin has the potential to reach the neural retina.Similar transport results have been reported in post-

mortem bovine eyes that were reperfused with bovineblood and 125I-insulin (James and Cotlier, 1983). In theirtime course experiments lasting up to 60 min; insulinwas localized to the basal surface of endothelial cells andalso associated with pericytes. However, no labeledinsulin was observed associated with the neural retina,which may suggest either more time is required forinsulin transport in bovine eyes, or the vascular, neural,and glial functions were compromised in eyes ofexsanguinated animals contributing to reductions ininsulin transport rates. By contrast, Shires et al. (1992)showed that 125I-insulin injected into the superior venacava of anesthetized rats accumulated in the vitreous ofnormal rats within 2 h: This accumulation is alsothought to be due to a saturable transport process.The tight junctions that form in retinal endothelial

cells promote a greater resistance across the barrier andmay restrict retinal insulin permeability compared toother tissues. When cultures of aortic endothelial cellswere directly compared to retinal endothelial cellcultures, the electrical resistance across the monolayerwas B4-fold greater in retinal endothelial cells (Gillieset al., 1995). The permeability of insulin in the retinalendothelial cell cultures was significantly less than theaortic endothelial cell cultures, indicating that expres-sion of specific barrier proteins contribute to thepermeability characteristics of retinal cells.The results presented above and observations from

our lab illustrate the substantial differences between thetighter BRB and the endothelium of other tissues.

Administering superphysiological amounts of insulinintraportally to fasted rats induces significant phosphor-ylation of the IRb; IRS, and IRS-1 associated PI3Kactivity in muscle within 90 s (Folli et al., 1992). Bycontrast, we found no effect on retinal IR phosphoryla-tion until 30 min post injection using a similar model(Reiter et al., 2003). These results are consistent withother studies for brain and retina insulin uptake(Niswender et al., 2003; Schwartz et al., 1990; Shireset al., 1992). Collectively, these studies confirm thedifference between the endothelium of retina anddifferent insulin-sensitive tissues, and that the transportof insulin across the BRB is significantly slower thanother vascular beds. This suggests the neural retina isnot likely a major target for immediate nutrientmetabolism as is skeletal muscle, but the steady-statetransport of insulin to the neural retina may provide astable trophic signal as opposed to insulin’s betterknown rapid metabolic functions.Insulin signaling characteristics in endothelial cells

has been investigated and described as in muscle, liver,adipose, and IR-overexpressing cells, but some of thefinal biological outputs of endothelial cells in responseto insulin are unique. As demonstrated by King andothers (Jialal et al., 1985; King et al., 1985; King andJohnson, 1985), retinal vascular endothelium containsfunctional IRs that promote cell division, and they arerequired for unidirectional insulin transport. In general,the IR signaling characteristics in endothelial cellsresembles what has been described in other systems,and the Akt pathway is activated in response to insulin.In human endothelial cell cultures induced to die withTNF-a treatment, insulin restores Akt phosphorylationand reduces rates of apoptosis (Hermann et al., 2000).This mechanism also appeared to be dependent on Akt’sability to inhibit caspase 9 activity, while Bad may notbe a target of Akt in endothelial cells. Insulin’s ability tostimulate production of other autocrine growth factors,such as VEGF, may also play a role in endothelial cellsurvival. Insulin and VEGF may have combined effectsin promoting the formation of endothelial tubes in vitro(Yamagishi et al., 1999). Glucose uptake in retinalendothelial cells is also not affected acutely by insulin,consistent with a lack of expression of the GLUT4transporter in endothelial cells (Betz et al., 1983; Hariket al., 1990; Kumagai, 1999). Together, insulin promotesboth mitogenic and survival effects on endothelial cells.In terms of diabetic retinopathy, these growth promot-ing effects may produce temporary detrimental con-sequences on the retinal vasculature as discussed below.

3.2. Retinal insulin production

An interesting observation by Havrankova et al.added considerable controversy concerning the role ofinsulin in neuronal cells (Havrankova et al., 1978b).


They detected insulin immunoreactivity in brain tissue,and they put forth the hypothesis that insulin may besynthesized de novo by other mammalian tissues thanpancreatic b cells. It was discovered that insulinconcentrations were at least 10-fold greater in brainthan in plasma, but others have disputed those results(Baskin et al., 1983). Therefore, brain may either havemechanisms in which to concentrate insulin or synthe-size the peptide. Devaskar et al. (1994) furthered thoseobservations by investigating whole brain and culturedprimary neurons and glial cells from rabbits. The insulintranscript was detected using sensitive techniques, suchas nuclear run-on assays and reverse-transcription PCR,but not by Northern blot analysis suggesting insulinmRNA in these tissues is a rare transcript. Whenneonatal rabbit neurons and glial cells were cultured, itwas demonstrated that insulin continued to accumulatein the culture media over 6 days in culture, supportingthe hypothesis that neurons synthesize insulin de novo.Further in vivo evidence of brain-derived insulin camefrom mRNA in situ hybridization which showedsignificant binding to neuronal cells over various regionsof the brain. However, insulin antisera was unable todetect specific insulin immunoreactivity as it had inprevious studies, which may be due to the inability ofinsulin antibodies to react across different species. Thisevidence for de novo synthesis was strengthened withelectron microscopic analysis of the rat CNS demon-strating the presence of both insulin I and II mRNAsand insulin protein in the ER and Golgi apparatus(Schechter et al., 1996). Although the amount of insulinin brain is probably considerably lower than thatexpressed in b cells, it is intriguing to speculate thatneuronal insulin may provide a local trophic ordevelopmental signal, act as a neurotransmitter, orregulate nutrient metabolism in neurons. In fact, a lowerconcentration would be predicted on the basis that bcells concentrate insulin in granules, but the brain doesnot (see Table 1).The evidence for neuronal insulin production has

been extended to retina tissue. Using insulin antisera,insulin immunoreactivity was detected in human retinaltissue and in the optic nerve of mice (Das et al., 1984).Insulin was reported to be located in the ganglion celllayer, the inner nuclear layer, and the inner and outerplexiform layers, but it was completely absent from thephotoreceptors. Glial cells from the optic nerve alsostained positive for insulin. A human retinoblastomacell line, Y79, also was positive for insulin immunor-eactivity, mRNA, and binding sites, although this maybe an artifact of the transformed nature of the cells, asinsulin and IR expression may be altered (Das et al.,1991; Pansky et al., 1986). Using cultured primary ratM .uller glial cells, the Das group showed both insulinprotein and mRNA within this specific subset of retinalcells (Das et al., 1987). Given the supportive nature of

glial cells in general, this suggests the M .uller cells maysecrete the insulin for other neurons as a paracrinehormone. Furthermore, intact retina tissue was shownto express the mRNA for preproinsulin in rats (Buddet al., 1993). In embryonic chickens, the amount ofinsulin expression in retina was considerably less thanpancreatic insulin expression, as a more sensitiveanalysis using RT-PCR was required for insulin mRNAdetection (de la Rosa et al., 1994).Given the extensive evidence that retina and numer-

ous other extra-pancreatic tissues express insulin-likemolecules (LeRoith et al., 1988), we must now recognizethe potential of insulin to modulate neuronal functions.The observations in chick retina, as observed in otherspecies, suggests that retinal (or neuronal) expression ofinsulin is not purely a mammalian phenomenon, andthat insulin, or other insulin-related peptides in otherspecies, may have an evolutionarily conserved role ingeneral neuronal development. Retinal development issculpted by coordinated differentiation and apoptosis,and Diaz et al. have established that retinal cell survivalin chicks depends on insulin produced by the retina(Diaz et al., 2000). Using antibodies to inhibit insulinaction in developing chick retina, they observed asignificant elevation in apoptosis; ganglion cells wereparticularly sensitive to the induction of apoptosis byblocking insulin signaling. Lastly, consistent with ourobservations in mammalian retina, chick retina re-sponds to insulin in vitro with significant elevations inAkt phosphorylation, while the Ras/Raf/MEK/ERKpathway is relatively insensitive to insulin stimulation.This supports the hypothesis that retinal insulin servesas a trophic factor in either an autocrine or paracrinemanner. However, developing chick retina stimulatedwith insulin exhibits increased 3H-thymidine-DNAincorporation and protein synthesis, suggesting insulinmay play a role in mitogenesis and differentiationduring embryonic stages (Hernandez-Sanchez et al.,1995). These results suggest insulin signaling maybeimportant in the adult retina as well, and it also plays amajor role in development. In fact, insulin binding in theretina is greater in fetal versus adult chick retina(Waldbillig et al., 1991).

3.3. Insulin signaling in Caenorhabditis elegans and

Drosophila melanogaster

Recent studies of the insulin-like signaling in wormsand flies have provided important insights into insulinphysiology of mice and men. A particularly interestingline of study in the nematode worm, Caenorhabditis

elegans, has revealed a neuron-specific insulin-likesignaling system that controls the worm’s life span(Kimura et al., 1997). This signaling system closelymirrors what has been established in mammalian cellsand described above. The IR homolog in C. elegans,


DAF-2, signals to AGE-1 (homolog to mammalianPI3K), AKT-1 and -2, and DAF-16 (homolog tomammalian Forkhead transcription factors, Fig. 1)(Paradis and Ruvkun, 1998). In wild-type C. elegans,when nutrients are abundant, the worms’ life span isB12 days, but in the absence of nutrients, their growthbecomes arrested and they enter a dauer stage inresponse to a dauer-inducing pheromone. This is a formof ‘‘hibernation’’ that allows the worm to prolong its lifespan until its environment becomes more favorable forreproduction. Experiments by Ruvkun and co-workers(Paradis and Ruvkun, 1998) have established the IR-likesignaling of DAF-2 in C. elegans to be intimately linkedto regulation of life span.Genetic sequencing of the DAF-2 gene revealed that it

is an IR homolog in C. elegans (Kimura et al., 1997). Itis 36% identical to the human IR within its ligandbinding domain (35% identical to the IGF-1R), and50% identical with 70% amino acid similarity within thetyrosine kinase domain. The genetic organization is alsoquite similar as it has a proform, a proteolytic site, and atransmembrane domain. C. elegans carrying a mutationin DAF-2 displayed arrested growth and a rise in fataccumulation that is associated with dauer formation,despite a favorable environment for survival andreproduction. This evidence suggests a strong evolu-tionary link in IR function in which the insulin signalingpathway regulates metabolism.Since that seminal report established a significant role

of insulin-like signaling in C. elegans, an entire cascadehomolog has been described (Paradis and Ruvkun,1998). Comprehensive genetic studies of C. elegans

mutants have established a pathway from DAF-2 toDAF-16, the Forkhead homolog, that regulates life spanin the worm. For example, essentially all viable wormswith DAF-2 mutations alone enter the dauer stage andhave a prolonged life span, as discussed above. Acomplementary mutation in DAF-16 will reestablish anormal phenotype (Ogg et al., 1997). Therefore, activityof the Forkhead transcription factor, DAF-16, isrequired for dauer formation, and signaling fromDAF-2 directly antagonizes such activity, as the IRaffects forkhead activity in mammalian cells. Using thatstrategy, other mediators of the DAF-2 signalingpathway were linked. C. elegans with AGE-1 mutations,like DAF-2 mutations, enter the dauer stage, andnormal life span is restored when an activating mutantof AKT-1 is also expressed. Furthermore, the geneproducts of AKT-1 and AKT-2 in C. elegans directlyantagonize DAF-16 as is the case in mammalian cells(Paradis and Ruvkun, 1998).Now the question becomes, again as in mammalian

systems, what is the role of the IR-like DAF-2 signalingsystem in neurons? Using tissue specific promoters,Wolkow et al. (2000) elegantly demonstrated in C.

elegans that restoring the insulin-like signaling system to

DAF-2 and AGE-1 mutants in neuronal cells regulateslongevity. This was in contrast to restoring DAF-2signaling to muscle and gut cells of C. elegans, which didnot restore normal life span. However, a functionaldivergence for insulin-like signaling emerged from theirstudy. It was discovered that restoring DAF-2 to themuscle and intestinal cells reduced fat accumulation andrestored normal metabolic processes, but did not alterlife span. This signaling dichotomy in lower organismssuggests it may also be conserved in mammals.Furthermore, 37 insulin peptide-like genes have beenidentified in C. elegans, and deletion of one of them doesnot effect life span, so there is probably functionalredundancy (Pierce et al., 2001). Interestingly, the INSgenes were expressed by neurons suggesting a possibleneuroendocrine role for insulin conserved throughoutevolution.The fruit fly, Drosophila melanogaster, also possesses

an insulin-like signaling cascade with many similaritiesto C. elegans and mammalian systems with mutationsthat effect fly growth. Comparisons among the con-served insulin signaling pathways can be seen in Fig. 1(Garofalo, 2002). In D. melanogaster, evidence suggeststhat the primary role for the insulin-like signaling is tocontrol growth by regulating cell size and number, andmutations within the Dinr gene result in abnormaldevelopment (Brogiolo et al., 2001; Fernandez et al.,1995). Like C. elegans, D. melanogaster express multipleinsulin-like peptides, three of which localize to neuro-secretory cells in the brain. When one of these peptides isoverexpressed, it results in increases in both cell size andnumber. When the Dinr gene is also overexpressed, theeye (comprised mostly of photoreceptor cells) displaysan overgrowth, while Dinr mutations result in reducedbody weight and fewer ommatidia, the fly photoreceptorunits. Genetic analysis of these mutants suggest that theinsulin-like peptides are ligands for the DINR as the eyephenotypes are corrected in insulin-like peptide over-expressing mutants with Dinr mutations (Brogiolo et al.,2001). The Dinr peptide is unique in that it expresses aB60 kDa C-terminal extension that is proteolyticallycleaved in some cells, and this extension is hypothesizedto give the receptor different functions in different cells.A similar function of the IR may occur in mammaliancells; that is, some functions of the IR may depend onthe cell type expressing the IR rather than a proteolyticevent which modifies the receptor. Given the conservednature of the IR throughout evolution, the function ofthe IR remains very similar in promoting anaboliceffects, yet other modifications or effector moleculesremain to be discovered. Glycosylation and exon 11expression are two other modifications of the IR thatalso may be contributing to differential IR activity andregulation in mammalian cells. Taken together, theconservation of insulin-like peptide expression inneurons or neurosecretory cells from lower species to


mammals suggest a specialized function of insulin andthe IR, and that neuronal cells have evolved to utilizeinsulin signaling differently. In terms of the retina, thisfunction may be in place to regulate proper embryonicor adult development and cell growth, maintain cellularsurvival, metabolism, or regulate phototransduction, allof which require further investigation. Indeed, aninsulin-secreting, gut-associated b cell does not ariseuntil after neurons in evolution, so the b cells may bemodified neural cells. In fact, they express proteinspreviously described as only neuron-specific, such asneurofilament (Escurat et al., 1991). Such properties ofcells blur the line between a pure neuron and ahormone-secreting endocrine cell in complex mamma-lian systems. Therefore, some of the properties attrib-uted solely to pancreatic b cells may be in common withretinal and other neurons, and vice versa.

4. Retinal insulin signaling

The physiological outcomes of IR activation in retinaand other neurons are beginning to be elucidated. It hasbeen established that IRs are expressed on differentneurons and glia, and co-localization studies havedetermined that the IR, IRS-1, PI3K, and phosphotyr-osine motifs topographically overlap in neurons (Baskinet al., 1993; Folli et al., 1994). Hence, an insulinsignaling pathway could be functional in those cellssince it resembles IR signaling in other systems, but thephysiological role has received little attention.Our observations extended previous observations and

have begun to establish a possible function of insulinsignaling in retina (Reiter et al., 2003). As stated above,we observed slow in vivo phosphorylation of the IR andAkt in retina with systemic insulin administration. Invitro, we saw no significant change in IRS-1 tyrosinephosphorylation, unlike IRS-2, which was rapidlyphosphorylated and dephosphorylated. Retina expressesmRNA for all three Akt isoforms, but insulin stimulatesonly Akt-1 activity. This suggests tissue specific signal-ing in the retina tissue. Consistent with the findings ofDiaz et al. (2000), the ERK 1/2 pathway stimulation wasrelatively unaffected compared to the Akt pathway withinsulin. It cannot be ruled out that other proteinsmediate the insulin signal in retina, or that subsets of theIR expressed on different parts of the retina may signaldifferently. In brain, the IR is a component of thesynapse, and signals arising there may be mediated byp58/53, a substrate of the IR which is expressed in brainand absent in liver and skeletal muscle (Abbott et al.,1999; Yeh et al., 1996). The IR is expressed broadly onsingle neurons, while IR and p58/53 co-localize in thesynapse, suggesting a specialized function for insulinsignaling within the same neuron. Indeed, novelmediators of insulin signaling may exist in retina and

other neurons that have not been described in other celltypes. Our hypothesis in retina is that the IR-Aktpathway in retina is tonically active and does notfluctuate in response to the organism’s nutritional state,and that this signal is required for proper developmentand cell survival.Similar experiments analyzing systemic insulin in rats

have produced interesting results suggesting differentparts of brain may be more insulin sensitive. Within3 min of systemic injection, cerebellar IRs were phos-phorylated, but there was no effect on the frontal cortex(de L.A. Fernandes et al., 1999). Therefore, regions ofthe brain with low permeability to insulin behavesimilarly to retina in which transport is slower, resultingin delayed activation of the IR. A recent report byNiswender et al. (2003) characterized signaling inhippocampus in response to intraperitoneally adminis-tered insulin. In this model, PI3K activity was notsignificantly elevated until 15 min post injection. Theseresults suggest that, like retina, the brain can respond tocirculating insulin, and it does so by signaling to knownmediators of the signaling pathway. However, thecomplete physiologic outcome of neuronal insulinsignaling in vivo remains to be seen.We have also identified some of the molecular

mechanisms of how insulin may reduce apoptosis bystudying retinal neurons in culture (Barber et al., 2001).Serum starvation-induced apoptosis of retinal neuronswas partially inhibited with insulin treatment, andinhibitors of the PI3K pathway inhibited insulin’seffects. Furthermore, insulin reduced cleavage andactivation of caspase 3, suggesting it may also be atarget of Akt. Interestingly, the mitotic rates of theseretinal neurons were unaffected with insulin treatment,in direct contrast to retinal endothelial cells whichproliferate in response to insulin, as reported by Kinget al. (1985). Together with our results in vivo, thesedata strongly point to the Akt-mediated survival path-way as a mediator of insulin-induced retinal cellsurvival. This, however, does not rule out effects fromother growth factors that are present in the retinalmilieu. For instance, to promote retinal ganglion cellsurvival in vitro, combinations of growth factors arerequired in addition to membrane depolarization, cell–cell contact, and the second-messenger cAMP (Gold-berg et al., 2002; Meyer-Franke et al., 1995).Heidenreich et al. previously showed reduced rates of

glucose uptake in response to insulin in neurons in vitro(Heidenreich et al., 1988), and insulin can stimulateamino acid uptake in brain neurons (Yang and Fellows,1980). The question of insulin-stimulated substrateuptake has also been addressed in human Y79retinoblastoma cells (Yorek et al., 1987). Of the aminoacids assayed, only glycine uptake was stimulated byboth insulin and IGF-1. In this way, insulin couldpotentially regulate neurotransmission as glycine is a


putative neurotransmitter. However, uptake of otherneurotransmitters such as GABA and dopamine wereunaltered in this cell line.Other aspects of retinal function have been investi-

gated in response to insulin. Insulin has been reported tomediate differentiation and synaptogenesis (Puro andAgardh, 1984), and to regulate acetyltransferase pro-teins (Holdengreber et al., 1998; Kyriakis et al., 1987).Embryonic chick retinal cell in vitro increased theircholinergic acetyltransferase activity over time in aninsulin dose-dependent manner. Holdengreber et al. alsoshowed an insulin-mediated increase in choline acetyl-transferase protein with short-term exposure to insulin,and that this increase was found in the ganglion andinner nuclear cell layers. Insulin, known to modulate theactivity of various transcription factors, may regulatecholine acetyltransferase protein by regulating jun (Renet al., 1997), but other transcription factors may also beinvolved. Insulin may also synergize with other trophicfactors in retina to promote differentiation. In thepostnatal chicken retina, insulin and FGF2 wererequired to induce waves of proliferation and expressionof progenitor markers, such as Pax6, in M .uller glia cells(Fischer et al., 2002). Together, insulin appears topromote some aspects of neurogenesis in retina (Her-nandez-Sanchez et al., 1995).Acute effects of insulin on retinal electrophysiology

have also been studied. For instance, the a- and b-wavesof the electroretinogram (ERG) were reduced wheninsulin was applied to an in vitro system (Gosbell et al.,1996). Such an effect on the photoreceptors is consistentwith our and other’s observations of IR expression onphotoreceptors (Gosbell et al., 2002; Naeser, 1997;Rodrigues et al., 1988). A mechanism by which insulinmay alter the ERG was provided by Stella et al. (2001)when they showed an insulin-dependent reduction ofCa2þ influx in photoreceptors. However, these resultsare in contrast to those of Lansel and Niemeyer (1997)in cat retina where, under normoglycemic conditions,the b-wave amplitude of the ERG was unaffected. Onlywhen the eye was perfused with hypoglycemic solutions,which reduced the b-wave, did insulin recover retinalnerve ERG activity. The physiological relevance of theseresults is questionable because under hypoglycemicconditions, circulating insulin is also usually reduced,but their results also suggest insulin crosses the BRB.So, if the BRB were more permeable to insulin, one mayexpect vision to change briefly with fluctuations incirculating insulin. Therefore, IRb phosphorylation inretina may be maintained stably for proper phototrans-duction and synapse function.Since the retina is the only neural tissue exposed to

light, some questions concerning light-mediated IRactivity are beginning to be answered. Rajala et al.,found that the IRb interacts with the regulatory subunitof PI3K, p85, in bovine rod outer segments. In pull

down assays, the interaction between the IR and PI3Kwas dependent on tyrosine phosphorylation of the IRb;and treatment of the tissue with insulin in vitroincreased PI3K association and activity associated withthe IRb (Rajala and Anderson, 2001). This suggestssome aspects of insulin signaling in retina may occurindependent of the IRS proteins in activating PI3K, orthis IRb-PI3K interaction may be specific to the rodouter segments. In a second report, the same group(Rajala et al., 2002) showed that light increases IRbphosphorylation and PI3K activity in rod outersegments associated with the IR in mice and rats. Theypropose that this phenomenon may be a mechanism toprotect the retina from light induced damage. Thissuggests Akt would also be activated by light since it isimmediately downstream of PI3K. Our observations inretina treated in vitro with insulin suggest this would bethe case as all nuclear layers of the retina, includingphotoreceptors, had increases in immunoreactivity forthe active, phosphorylated form of Akt, and Akt-1 and -3 mRNA localized to this region of the retina (Reiteret al., 2003). Even more interesting is their suggestionthat IR activation could be modulated by other ligandsand that the intracellular portion of the IRb may bemodulated by novel ligand-independent mechanisms toincrease its phosphorylation, or that the IR may playsome role in phototransduction.

5. Insulin and insulin receptors in retinal diseases

Theories about the pathogenesis of diabetic retino-pathy have been plentiful and include increased polyolpathway flux, nonenzymatic glycosylation and receptorfor advanced glycation endproducts (RAGE) activation,increased oxidative stress, protein kinase C activation,altered growth factor expression, and hypoxia (Aielloand Gardner, 2000; Brownlee et al., 1988; King et al.,1996; Kowluru et al., 2001). As of this writing nocomprehensive mechanism has yet been established thatprovides a causal link between the fundamental meta-bolic abnormalities of diabetes and the initiation andprogression of retinopathy that includes an explanationto account for loss of vision. During this period therehave been two implicit assumptions underlying mostdiabetes complications research: first, that retinopathy issolely or primarily a blood vessel disorder; and second,that excess glucose is necessary and sufficient to accountfor the phenotype (Gardner et al., 2002). Until recentlythese appeared to be reasonable operative assump-tions. The clinical signs of retinopathy are determinedby the appearance of hemorrhages, microaneurysms,cotton–wool spots, macular edema, lipid exudates, andneovascularization. These features are visible withophthalmoscopy because they contain pigment (hemo-globin) or reduce retinal transparency (cotton–wool


spots and lipids). However, at least 95% of the retinalvolume and mass is comprised of neurons and glial cellsthat lack pigment and are thus indistinguishable bybiomicroscopy, fluorescein angiography, or ultrasound,and only marginally detectable by current opticalcoherence tomography. Recent work by several inves-tigators now provides evidence that challenges this firstassumption. There is now clear evidence for loss ofretinal neurons by apoptosis (Barber et al., 1998; Zenget al., 2000), activation of glial cells (Barber et al., 2000;Rungger-Brandle et al., 2000), and microglial cells(Rungger-Brandle et al., 2000; Zeng et al., 2000) byaltered expression of glial fibrillary acidic protein(GFAP). These changes develop early in the course ofexperimental diabetes and precede detectable structuralmicrovascular lesions as assessed by fluorescein angio-graphy or trypsin digest preparations. In fact, the loss ofretinal neurons was first described 40 years ago (Blood-worth, 1962; Wolter, 1961), and close histologicexamination of eyes with macular edema revealsextensive loss of ganglion cells and inner nuclear layerneurons (Yanoff and Fine, 1989). These anatomicchanges support the well-recognized functional changesin the ERG, color vision, and contrast sensitivity thatprecede vascular changes (Ghirlanda et al., 1997).Collectively these data strongly support a major if notdirect effect of diabetes on retinal neural function thatmay or not depend on alterations in vascular function(Lieth et al., 2000a).The second assumption regarding the singular causa-

tive role of glucose appears to be based on theobservation that insulin does not increase glucoseuptake in retinal endothelium via the GLUT1 glucosetransporter. Moreover, the results of the DiabetesControl and Complications Trial (DCCT) (DCCTResearch Group, 1993) have been misinterpreted as‘‘proving’’ that hyperglycemia alone accounts for retino-pathy and other complications (Davis and Granner,1996).The major indices of metabolic control in the DCCT

were hemoglobin A1c and blood glucose values; thesetests reflect the net carbohydrate metabolism (intakeminus utilization) and the degree of insulin action.Deficient insulin action (qualitative or quantitative)—rather than hyperglycemia—is the cardinal metabolicfeature of diabetes, and the improved control achievedin the DCCT was accomplished via significantly higherinsulin doses (DCCT, 1995). Thus, the major questionrelevant to retinopathy research remains: what is themechanism of the effects observed in the DCCT? Is itfrom obvious changes such as lowering the glucoseburden or higher insulin levels and greater insulinaction, or from effects on amino acid or lipidmetabolism that are also central to diabetes? At thispoint the answer remains elusive but it is clearlypremature to exclude factors other than glucose. In

fact, no single parameter may account for vascular andneural lesions.With these points in mind the question may be asked

whether retinal insulin signaling changes in diabetes andif these changes are relevant to the genesis of retino-pathy. The published work in this area is remarkablylimited. Zetterstrom et al. (1992) found that retinas fromrats with 4 weeks of experimental diabetes expressedhigher IR levels than controls, and this increase reflecteda doubling of the neuronal and a 20% decrease in theperipheral (vascular) IR subtypes. Neuronal insulinreceptors in wheat-germ agglutinin-purified corticalsynaptosomal membranes were also increased, and thereceptors exhibited normal autophosphorylation prop-erties. The authors concluded that retinal and corticalIR are sensitive to circulating insulin and glucose levels.Wheat-germ agglutinin binds to sialic acid residues sothe method employed to isolate IRs in this study selectedfor vascular over non-vascular IR. Studies of retinal IRkinase activity are now ongoing in our lab.It is not possible to investigate retinal IR in intact

human eyes, and the only one study has examinedvitreous insulin levels in patients (Feman et al., 1978).However, details of the subjects’ medical history,medications, diabetes type, or duration were notincluded, making interpretation of the data difficult.Type I diabetic patients may have reduced vitreousinsulin, whereas hyperinsulinemic Type II patients mayhave normal or elevated vitreous insulin concentrations.Insulin levels have been reported to be reduced byapproximately half in the vitreous of rats with 4 weeksof streptozotocin-induced diabetes (Shires et al., 1992).Thus, it appears that circulating insulin readily gainsaccess to the vitreous cavity in normal and diabetic rats,and that at least part of the vitreous insulin contentderives from the pancreas. Unfortunately it is difficult toinvestigate this point in humans.While long-term intensive insulin therapy clearly

reduces the risk of retinopathy progression, a minorityof patients may develop exacerbation of their retino-pathy after institution of tight control. In the DCCT,13% of intensively treated patients versus 7.6% ofconventionally treated patients who had retinopathy atbaseline developed worsening within 6 months oftreatment (Anonymous, 1998). However, only two of1441 patients developed proliferative retinopathy and 3developed clinically significant macular edema requiringlaser treatment. Thus, ‘‘early worsening’’ is uncommonand seldom associated with vision loss. Recently,Poulaki et al. (2002) found that subcutaneous insulinimplants delivering low-dose (2 units) of insulin dailyincreased blood–retinal barrier permeability and VEGFexpression in rats with short-term streptozotocin dia-betes. These short term responses may reflect impairedability of diabetic rat retinas to respond to insulintherapy, but are in contrast to our observations that 3


days of insulin treatment substantially improves tightjunction protein expression and glial reactivity andglutamine synthetase activity without complete restora-tion of euglycemia (Barber et al., 2000; Lieth et al.,2000b), or that chronic insulin therapy reduces apopto-sis (Barber et al., 1998). The long-term benefits ofintensive insulin therapy are unequivocal, and one mightreasonably interpret the DCCT data to represent aninsulin dose–response trial (Fig. 2). If so, systemicallyadministered insulin may be a direct pharmacologicintervention for the retina and other complications-prone tissues, including the kidneys and peripheralnerves. Indeed, a direct role for insulin on retinal cellviability is strongly suggested by the observations that indeveloping chicken retinas, insulin reduces apoptotic celldeath (Diaz et al., 1999), and insulin antibodies induceganglion cell apoptosis (Diaz et al., 2000). Theseobservations have yet to be confirmed in human retinasbut the high degree of homology in insulin signalingand retinal development across species suggests animportant role for insulin in the maintenance of retinalcell survival as the insulin-signaling pathway is akey determinant of neuronal survival in species fromC. elegans (Lin et al., 2001), Drosophila melanogaster

(Brogiolo et al., 2001; Chen et al., 1996), and mice(White, 2002b). We have also found that insulin is apotent inhibitor of cell death in rat retinal neurons inculture in response to serum deprivation (Barber et al.,2001). Thus, the cumulative data support a direct andbeneficial role of insulin on retinal cell function andsurvival during embryologic development and stressfulconditions such as nutritional deprivation and hypoxia.In clinical medicine, exceptional cases often provide

opportunities to gain new insights into the specificetiologic factors in disease pathogenesis. Although

hyperglycemia is most often considered to be a strongpredictor of diabetes complications, more than 15 well-documented cases of diabetic retinopathy and nephro-pathy have been described in patients without overthyperglycemia or glucose intolerance (Barnes et al.,1985; Chan et al., 1985; Collens et al., 1959; Harrowerand Clark, 1976; Johansen, 1969). One of us (TWG) hasalso evaluated two obese adult males with classicalproliferative diabetic retinopathy who did not haveblood glucose values greater than 120 mg=dl (unpub-lished data). Together, these cases suggest that hyper-glycemia may not be essential for the development orprogression of retinopathy. How could impaired insulinaction, and thus diabetes, not alter glucose levels and yetresult in retinopathy? Insulin has protean consequencesfrom tissue to tissue and cell to cell that are modified bysubstrate levels and interactions with other hormonesand growth factors. The signaling pathways for insulinare highly complex and divergent, and many probablyremain undiscovered. It is conceivable that somepatients may have selective impairments in insulinaction that affect glucose uptake and metabolism lessthan other aspects of diabetes such as lipid metabolismas suggested by Chaturvedi (2002). That is, specificmutations in the complex signaling pathways of insulincould lead to a phenotype in which lipid or amino acidmetabolism is defective with minimal changes in glucosemetabolism, so patients may be ‘‘diabetic’’ in terms ofoverall insulin action without frank hyperglycemia. Thisconcept requires additional investigation.Further evidence for a role of insulin action derives

from observations that insulin sensitivity is an importantdeterminant for the progression of retinopathy in TypesI and II diabetes. The European Diabetes ProspectiveDiabetes Complications Study (EURODIAB-PCS)

Fig. 2. In individuals with IDDM, the maintainance of retinal function depends on insulin. Adapted from the DCCT trial (N. Engl. J. Med. 329,

977–986), the percentage of patients developing new retinopathy (y-axis), is significantly reduced in individuals receiving greater amounts of insulin

per day (intensive insulin therapy). Similar results were also found in patients with pre-existing mild diabetic retinopathy. Therefore, insulin per se

may have therapeutic implications in preventing diabetic complications.


found that insulin resistance (as measured by body massindex and triglyceride levels) is nearly as an importantpredictor of retinopathy as is HBA1c (Chaturvedi et al.,2001). Similar results have also been found in the DCCTcohort (Zhang et al., 2001), and these findings confirmearlier studies showing that acute insulin sensitivity is astrong predictor of retinopathy and other vascularcomplications in Type I diabetes (Martin and Hopper,1987), and that patients with retinopathy are moreinsulin resistant than those without retinopathy (Man-eschi et al., 1983). Insulin sensitivity was also found tobe a major risk factor for the presence of retinopathy atthe time of Type II diabetes diagnosis in the UnitedKingdom Prospective Diabetes Study (UKPDS) (Koh-ner et al., 1998).Taken together, these laboratory, epidemiologic, and

clinical trial data strongly suggest that altered insulinaction, at least at the systemic level, plays an importantrole in the pathogenesis of diabetic retinopathy. It isnow important to determine if retina-specific changes ininsulin action has causal relationship to cell death,vascular permeability, and vision loss. Better under-standing of the importance of insulin sensitivity andaction will provide the opportunity to develop newdrugs that enhance the general or specific actions ofinsulin and the insulin receptor.

6. Conclusions and future directions

Genes and their protein, lipid, nucleic acid, andcarbohydrate products are assigned names based ontheir initial context of tissue origin or predictedfunction. Inevitably the scope of understanding of theexpression and function of genes expands over time. Forexample, presumed gastric hormones such as gastrin andcholecystokinin, are also expressed in the CNS. Thus,classical concepts of gene function and disease patho-genesis is often limited by assumptions. Three suchinstances are been pointed out in this review. First, thatinsulin is solely of pancreatic origin and functions onlyto control glucose levels; second, that diabetic retino-pathy is purely a vascular disease; and third, thatdiabetic retinopathy development is related purely toplasma glucose levels. By examining these assumptionsit is possible to develop new concepts about the role ofinsulin in normal retinal physiology and in diseasepathogenesis.Several additional questions can now be asked based

on these concepts:

1. Does retinal insulin content change in diabetes?2. Does retinal insulin serve a metabolic or trophic role

in adult mammalian retinas, and, if so, what is themechanism?

3. Does retinal insulin receptor signaling change indiabetes or other disease states, and is it causative forthe pathology?

Insulin undoubtedly functions as but one factor in acomplex system to regulate retinal processes. However,as the primary anabolic peptide hormone it undoubtedlymakes a major contribution to normal vision. Furtherstudies will be required to answer key questions that willprovide the information needed to better prevent andtreat retinal diseases.

Acknowledgements

This work is supported by the ADA, JDRF, NIHEY12021, The Pennsylvania Lions Sight Conservation& Eye Research Foundation, Fight for Sight, and Mr.and Mrs. Jack Turner, Athens, GA.

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