Posttranslational modifications of nerve cytoskeletal proteins in experimental diabetes

Axonal transport is known to be impaired in peripheral nerve of experimentally diabetic rats. As axonal transport is dependent on the integrity of the neuronal cytoskeleton, we have studied the way in which rat brain and nerve cytoskeletal proteins are altered in experimental diabetes. Rats were made diabetic by injection of streptozotocin (STZ). Up to six weeks later, sciatic nerves, spinal cords, and brains were removed and used to prepare neurofilaments, microtubules, and a crude preparation of cytoskeletal proteins. The extent of nonenzymatic glycation of brain microtubule proteins and peripheral nerve tubulin was assessed by incubation with³H-sodium borohydride followed by separation on two-dimensional polyacrylamide gels and affinity chromatography of the separated proteins. There was no difference in the nonenzymatic glycation of brain microtubule proteins from two-week diabetic and nondiabetic rats. Nor was the assembly of microtubule proteins into microtubules affected by the diabetic state. On the other hand, there was a significant increase in nonenzymatic glycation of sciatic nerve tubulin after 2 weeks of diabetes. We also identified an altered electrophoretic mobility of brain actin from a cytoskeletal protein preparation from brain of 2 week and 6 week diabetic rats. An additional novel polypeptide was demonstrated with a slightly more acidic isoelectric point than actin that could be immunostained with anti-actin antibodies. The same polypeptide could be produced by incubation of purified actin with glucose in vitro, thus identifying it as a product of nonenzymatic glycation. These results are discussed in relation to data from a clinical study of diabetic patients in which we identified increased glycation of platelet actin. STZ-diabetes also led to an increase in the phosphorylation of spinal cord neurofilament proteins in vivo during 6 weeks of diabetes. This hyperphosphorylation along with a reduced activity of a neurofilament-associated protein kinase led to a reduced incorporation of³²P into purified neurofilament proteins when they were incubated with³²P-ATP in vitro. Our combined data show a number of posttranslation modifications of neuronal cytoskeletal proteins that may contribute to the altered axonal transport and subsequent nerve dysfunction in experimental diabetes.     

Changes in Organization and Axonal Transport of Cytoskeletal Proteins During Regeneration

Changes in solubility and transport rate of cytoskeletal proteins during regeneration were studied in the motor fibers of the rat sciatic nerve. Nerves were injured by freezing at the midthigh level either 1-2 weeks before (experiment I) or 1 week after radioactive labeling of the spinal cord with L-[35S]methionine (experiment II). Labeled proteins in 6-mm consecutive segments of the nerve 2 weeks after labeling were analyzed following fractionation into soluble and insoluble populations with 1% Triton at 4 degrees C. When axonal transport of newly synthesized cytoskeleton was examined in the regenerating nerve in experiment I, a new faster component enriched in soluble tubulin and actin was observed that was not present in the control nerve. The rate of the slower main component containing most of the insoluble tubulin and actin together with neurofilament proteins was not affected. A smaller but significant peak of radioactivity enriched in soluble tubulin and actin was also detected ahead of the main peak when the response of the preexisting cytoskeleton was examined in experiment II. It is thus concluded that during regeneration changes in the organization take place in both the newly synthesized and the preexisting axonal cytoskeleton, resulting in a selective acceleration in rate of transport of soluble tubulin and actin.     

Glycation of Brain Actin in Experimental Diabetes

Abstract: Actin is a neuronal protein involved in axonal transport and nerve regeneration, both of which are known to be impaired in experimental diabetes. To determine if actin is subject to glycation, we rendered rats diabetic by injection of streptozotocin. Two or 6 weeks later brains were removed and a preparation of cytoskeletal proteins was analyzed by two-dimensional polyacrylamide gel electrophoresis. Brains from diabetic animals contained an extra polypeptide that migrated close to actin and reacted with monoclonal antibody C4 against actin. It was also found in a preparation of soluble synaptic proteins from diabetic rat brain, indicating that it was at least partly neuronal in origin. This polypeptide could be produced by incubation of cytoskeletal proteins from brains of nondiabetic rats with glucose-6-phosphate in vitro. The appearance of this glycated actin in diabetic animals was prevented by administration of insulin for a period of 6 weeks. We could not detect any effect of glycation in vitro on the ability of muscle G-actin to form F-actin filaments and its significance for the function of actin remains to be determined. The finding that glycation of platelet-derived actin from diabetic patients was significantly increased implies that the abnormality may also occur in clinical diabetes.     

Effects of taxol on slow and fast axonal transport

Axonal transport of tubulin in the rat sciatic nerve is almost completely inhibited by a single subepineural injection of taxol, without affecting that of neurofilament proteins. Actin and a large number of polypeptides cotransported with actin as minor components are also blocked by taxol, although to a lesser extent. Fast axonal transport is essentially free from the inhibitory effect of this drug. Although previous models have suggested that slow axonal transport involves the bulk movement of cytoskeletal structures, these results suggest that such transport may involve an equilibrium between polymerised and depolymerised forms of the axonal cytoskeleton.     

Altered Axonal Transport of Cytoskeletal Proteins in the Mutant Diabetic Mouse

Polypeptides in the motor axons of the sciatic nerve in 120-day-old normal and diabetic mice C57BL/Ks (db/db) were labeled by injection of [35S]methionine into the ventral horn of the spinal cord. At 8, 15, and 25 days after the injection, the distribution of radiolabeled polypeptides along the sciatic nerve was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Four major radiolabeled polypeptides, tentatively identified as actin, tubulin, and the two lightest subunits of the neurofilament triplet, were studied in both diabetic and control mice. In the diabetic animals, the two polypeptides identified as actin and tubulin showed a reduction of average velocity of migration along the sciatic nerve, resulting in a higher fraction of radioactivity in the proximal part of the sciatic nerve, whereas the front of radioactivity (advancing at maximal velocity) moved at a normal rate. In contrast, both the average and maximal velocities of the two neurofilament subunits were slower in the diabetic mice than in the control mice. These results indicate that the axonal transport of the cytoskeletal proteins is differentially affected in the course of diabetic neuropathy, and may suggest that the impairment concerns mainly the proteins carried by the slowest component of axonal transport.     

Quantitative Analysis of Axonal Transport of Cytoskeletal Proteins in Chicken Oculomotor Nerve

We studied the axonal transport characteristics of major cytoskeletal proteins: tubulin, the 69,000 molecular weight protein of chicken neurofilaments, and actin. After intracerebral injection of [35S]methionine, we monitored the specific radioactivity of these proteins as they passed through a very short nerve segment of the chicken oculomotor nerve. Specific radioactivities were assessed by quantitative sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. The transport patterns obtained for tubulin and the neurofilament protein were very similar, corresponding to transport rate ranges of 1-15 and 1-10 mm/day, respectively. A narrower velocity range of 3 to 4.3 mm/day was found for actin. Tubulin and the neurofilament protein appeared to be largely dispersed during the course of their transit along the nerve. The radioactivity associated with the proteins studied persisted in the nerve segment for a long time after the bulk of the labeled molecules had swept down. Finally, none of these proteins was observed to be transported with the fast axonal transport.     

Changes in neurofilament transport coincide temporally with alterations in the caliber of axons in regenerating motor fibers

The delivery of neurofilaments via axonal transport has been proposed as an important mechanism for regulating axonal caliber. If this hypothesis is correct, alterations in axonal caliber should appear coincident with changes in the delivery of neurofilaments to the axon. The purpose of this study was to determine whether alterations in the caliber of axons in the proximal stumps of transected motor fibers precede, coincide with, or occur substantially later than changes in the delivery of neurofilaments via axonal transport. Between 3 d and 12 wk after crushing the sciatic nerves of 7-wk-old rats, lumbar motor neurons were labeled by the intraspinal injection of [35S]methionine. In neurons labeled between 3 d and 6 wk after axotomy, the relative amount of neurofilament protein in the slow component, as reflected by the ratio of the radioactivities of the 145-kD neurofilament protein to tubulin, was reduced to 30-40% of the control value. Moreover, as determined by immunoreactivity on blots, the amounts of neurofilament protein and tubulin in these nerve fibers were reduced fourfold and twofold, respectively. Thus, changes in the ratio of labeled neurofilament protein to tubulin correlated with comparable changes in the quantities of these proteins in nerve fibers. This decrease in the quantity of neurofilament proteins delivered to axons coincided temporally with reductions in axonal caliber. After regeneration occurred, the delivery of neurofilament proteins returned to pre-axotomy levels (i.e., 8 wk after axotomy), and caliber was restored with resumption of normal age-related radial growth of these axons. Thus, changes in axonal caliber coincided temporally with alterations in the delivery of neurofilament proteins. These results suggest that the majority of neurofilaments in these motor fibers continuously move in the anterograde direction as part of the slow component of axonal transport and that the transport of neurofilaments plays an important role in regulating the caliber of these axons.     

Organization and slow axonal transport of cytoskeletal proteins under normal and regenerating conditions

The organization of the axonal cytoskeleton was investigated by analyzing the solubility and transport profile of the major cytoskeletal proteins in motor axons of the rat sciatic nerve under normal and regenerating conditions. When extracted with the Triton-containing buffer at low temperature, 50% of tubulin and 30% of actin were recovered in the insoluble form resistant to further depolymerizing treatments. Most of this cold-insoluble form was transported in slow component a (SCa), the slower of the two subcomponents of slow axonal transport, whereas the cold-soluble form showed a biphasic distribution between SCa and SCb (slow component b). Changes in slow transport during regeneration were studied by injuring the nerve either prior to (experiment I) or after (experiment II) radioactive labeling. In experiment I where the transport of proteins synthesized in response to injury was examined, selective acceleration of SCb was detected together with an increase in the relative proportion of this component. In experiment II where the response of the preexisting cytoskeleton was examined, a shift from SCa to SCb of the cold-soluble form was observed. The differential distribution and response of the two forms of tubulin and actin suggest that the cold-soluble form may be more directly involved in axonal transport.     

Changes in cytoskeletal protein synthesis following axon injury and during axon regeneration

Injury to the axons of facial motoneurons stimulates increases in the synthesis of actin, tubulins, and GAP-43, and decreases in the synthesis of neurofilament proteins: mRNA levels change correspondingly. In contrast to this robust response of peripheral neurons to axotomy, injured central nervous system neurons show either an attenuated response that is subsequently aborted (rubrospinal neurons) or overall decreases in cytoskeletal protein mRNA expression (corticospinal and retinal ganglion neurons). There is evidence that these changes in synthesis are regulated by a variety of factors, including loss of endoneurially or target-derived trophic factors, positive signals arising from the site of injury, changes in the intraaxonal turnover of proteins, and substitution of target-derived trophic support by factors produced by glial cells. It is concluded that there is, as yet, no coherent explanation for the upregulation or downregulation of any of the cytoskeletal proteins following axotomy or during regeneration. In considering the relevance of these changes in cytoskeletal protein synthesis to regeneration, it is emphasized that they are unlikely to be involved in the initial outgrowth of the injured axons, both because transit times between cell body and injury site are too long, and because sprouting can occur in isolated axons. Injuryinduced acceleration of the axonal transport of tubulin and actin in the proximal axon is likely to be more important in providing the cytoskeletal protein required for initial axonal outgrowth. Subsequently, the increased synthesis and transport velocity for actin and tubulin increase the delivery of these proteins to support the increased volume of the maturing regenerating axons. Reduction in neurofilament synthesis and changes in neurofilament phosphorylation may permit the increased transport velocity of the other cytoskeletal proteins. There is little direct evidence that alterations in cytoskeletal protein synthesis are necessary for successful regeneration, nor are they sufficient in the absence of a supportive environment. Nevertheless, the correlation that exists between a robust cell body response and successful regeneration suggests that an understanding of the regulation of cytoskeletal protein synthesis following axon injury must be a part of any successful strategy to improve the regenerative capacity of the central nervous system.     

Temporal Profiles of Cytoskeletal Protein Loss following Traumatic Axonal Injury in Mice

To examine the time course and relative extent of proteolysis of neurofilament and tubulin proteins after traumatic axonal injury (TAI), anesthetized mice were subjected to optic nerve stretch injury. Immunohistochemistry confirmed neurofilament accumulation within axonal swellings at 4, 24, and 72 h postinjury (n = 4 injured and 2 sham per time point). Immunoblotting of optic nerve homogenates (n = 5 injured and 1 sham at 0.5, 4, 24 or 72 h) revealed calpain-mediated spectrin proteolytic fragments after injury. Protein levels for NF68 progressively decreased from 0.5 h to 24 h postinjury, while NF200 and α-tubulin levels decreased acutely (0.5–4 h), with a secondary decline at 72 h postinjury. These data demonstrate that diffusely distributed TAI is associated not only with a localized accumulation of neurofilament proteins, but also significant decreases in total cytoskeletal protein levels which may be mediated, in part, by calpains. Protection of the axonal cytoskeleton represents a potential therapeutic target for axonal damage associated with injury or neurodegenerative diseases. Special issue in honor of Naren Banik.     

Binding of γ-Aminobutyric AcidA Receptors to Tubulin

Abstract: The rate of axonal transport of tubulin, actin, and the neurofilament proteins was measured in the peripheral and central projections of the rat L5 dorsal root ganglion (DRG). [³⁵S]Methionine was injected into the DRG, and the "front" of the radiolabeled protein was located 7, 14, and 20 days postinjection. Transport rates calculated for the neurofilament triplet proteins, tubulin, and actin in the peripheral nerve were ∼ 1.5-fold faster than those in the dorsal root. A progressive decrease in the rate of transport was observed from 7 to 20 days after radiolabeling in both the central and peripheral directions (neurofilaments, ∼ 1.7-fold; tubulin/actin, 2.1-fold). A surgical preparation, leaving the peripheral sciatic nerve with predominantly sensory fibers, was the basis for ELISAs for phosphorylation-dependent immunoreactivity of the high-molecular-weight neurofilament protein. In both dorsal roots and peripheral sensory axons the degree of phosphorylation was greater in nerve segments further away from the cell bodies. The degree of phosphorylation-related immunoreactivity correlates with the slowing of transport of radiolabeled cytoskeletal protein.     

Intracellular protein glycation in cultured retinal capillary pericytes and endothelial cells exposed to high-glucose concentration.

There is now increasing evidence suggesting that non-enzymatic glycation (NEG) of proteins is involved in the pathogenesis of chronic diabetic complication. In this study we demonstrate that chronic exposure to high-glucose concentration leads to intracellular protein glycation in cultured bovine retinal capillary pericytes and endothelial cells. The level of intracellular protein glycation, as measured using a competitive enzyme-linked immunoabsorbant assay (ELISA), was found to increase in both pericytes and endothelial cells as function of time. As expected products of NEG were only detected when the Schiff base and the Amadori products were chemically reduced to glucitollysine by sodium borohydride. Despite the accumulation of early glycation products on cellular proteins there was no further rearrangement reaction into advanced glycation endproducts (AGEs), even after 12 days of incubation in high-glucose medium. Immunofluorescence microscopy demonstrated that the monoclonal antibody reacting with glucitollysine stains the cytoplasm of both pericytes and endothelial cells in a finely punctate pattern. Further studies using Western blot analysis suggested that a number of cellular proteins, including smooth muscle actin in pericytes, become rapidly glycated. The results from this in vitro study suggest that excessive accumulation of early products of non-enzymatic glycation in pericytes and endothelial cells may play an important role in the pathogenesis of diabetic retinopathy.     

Transport complexes associated with slow axonal flow

Cytoskeletal proteins-neurofilament polypeptides, tubulin and actin-are transported along axons by slow transport. How or in what form they are transported is not known. One hypothesis is that they are assembled into the cytoskeleton at the cell body and transported as intact polymers down the axon. However, recent radiolabeling and photobleaching studies have shown that tubulin and actin exist in both a mobile phase and a stationary phase in the axon. Consequently, it is more likely that cytoskeletal proteins move along the axon in some form of transport complex and are assembled into a cytoskeleton which is stationary. In this overview we discuss these topics and consider the evidence for the existence of transport complexes associated with slow axonal flow. Such evidence includes the slow transport of particulate complexes containing tubulin and neurofilament polypeptides along reconstituted microtubules in vitro, and the coordinate slow transport of actin with actin-binding in vivo.Special issue dedicated to Dr. Lawrence Austin.     

Non-enzymatic glycation of proteins: From diabetes to cancer

Incubation of proteins with glucose leads to their non-enzymatic glycation and formation of Amadori products known as an early glycation product. Oxidative cleavage of Amadori products is considered as a major route to advanced glycation endproducts (AGEs) formation in vivo. Non-enzymatic glycation of proteins or Maillard reaction is increased in diabetes mellitus due to hyperglycemia and leads to several complications such as blindness, heart disease, nerve damage, and kidney failure. The early and advanced glycation products are accumulated in plasma and tissues of diabetic patients and cause production of autoantibodies against corresponding products. The advanced glycation products are also associated with other diseases like cancer. This review summarizes current knowledge of these stage specific glycated products as common and early diagnostic biomarkers for the associated diseases and the complications with the aim of a novel therapeutic target for the diseases.     

Slow transport of the cytoskeleton after axonal injury

The delivery of cytoskeletal proteins to the axon occurs by slow axonal transport. We examined how the rate of slow transport was altered after axonal injury. When retinal ganglion cell (RGC) axons regenerated through peripheral nerve grafts, an increase in the rate of slow transport occurred during regrowth of the injured axons. We compared these results to axonal injury in the optic nerve where no substantial regrowth occurs and found a completely different response. Slow transport was decreased approximately tenfold in rate in the proximal segment of crushed optic nerves. This decreased rate of slow transport was not induced immediately, but occurred about 1 week after injury. To explore whether a decrease in the rate of slow transport was induced when the regeneration of peripheral nerves was physically blocked, we examined slow transport in motor neurons after the sciatic nerve was transected and ligated. In this case, no change in the rate of the comigrating tubulin and neurofilament (NF) radioactive peaks were observed. We discuss how the changes in the rate of slow transport may reflect different neuronal responses to injury and speculate about the possible molecular changes in the expression of tubulin which may contribute to the observed changes. © 1992 John Wiley & Sons, Inc.     

Slow transport of the cytoskeleton after axonal injury.

The delivery of cytoskeletal proteins to the axon occurs by slow axonal transport. We examined how the rate of slow transport was altered after axonal injury. When retinal ganglion cell (RGC) axons regenerated through peripheral nerve grafts, an increase in the rate of slow transport occurred during regrowth of the injured axons. We compared these results to axonal injury in the optic nerve where no substantial regrowth occurs and found a completely different response. Slow transport was decreased approximately tenfold in rate in the proximal segment of crushed optic nerves. This decreased rate of slow transport was not induced immediately, but occurred about 1 week after injury. To explore whether a decrease in the rate of slow transport was induced when the regeneration of peripheral nerves was physically blocked, we examined slow transport in motor neurons after the sciatic nerve was transected and ligated. In this case, no change in the rate of the comigrating tubulin and neurofilament (NF) radioactive peaks were observed. We discuss how the changes in the rate of slow transport may reflect different neuronal responses to injury and speculate about the possible molecular changes in the expression of tubulin which may contribute to the observed changes.     

Electrical Stimulation Accelerates and Enhances Expression of Regeneration-Associated Genes in Regenerating Rat Femoral Motoneurons

1. In this study we investigated whether electrical stimulation accelerates the upregulation of Talpha1-tubulin and GAP-43 (regeneration-associated genes; RAGs) and the downregulation of the medium-molecular-weight neurofilament (NFM), in concert with stimulation-induced acceleration of BDNF and trkB gene expression and axonal regeneration. 2. Two weeks prior to unilateral femoral nerve transection and suture, fluorogold (Fluorochrome Inc., Denver) or fluororuby (Dextran tetramethylrhodamine, Mol. Probes, D-1817, Eugene, OR) was injected into quadriceps muscles of the left and right hindlimbs to label the femoral motoneuron pools as previously described. Over a period of 7 days, fresh spinal cords were processed for semiquantitation of mRNA by using in situ hybridization. 3. There was an increase in Talpha1-tubulin and GAP-43 mRNA and a decline in the NFM mRNA at 7 days after nerve suture and sham stimulation but not in intact nerves. In contrast, 1-h stimulation of sutured but not intact nerves dramatically accelerated the changes in gene expression: mRNA levels of Talpha1-tubulin and GAP-43 were significantly elevated above control levels by 2 days while NFM mRNA was significantly reduced by 2 days in the sutured nerves. Thereby, the neurofilament/tubulin expression ratio was reduced at 2 days after suture and stimulation, possibly allowing more tubulin to be transported faster into the growing axons to accelerate the elongation rate following stimulation. Importantly, the changes in RAGs and NFM gene expression were delayed relative to the accelerated upregulation of BDNF and trkB mRNA by electrical stimulation. 4. The temporal sequence of upregulation of BDNF and trkB, altered gene expression of RAGs and NFM, and accelerated axonal outgrowth from the proximal nerve stump are consistent with a key role of BDNF and trkB in mediating the altered expression of RAGs and, in turn, the promotion of axonal outgrowth after electrical stimulation.     

Tubulin and Neurofilament Proteins Are Transported Differently in Axons of Chicken Motoneurons

SUMMARY 1. We previously showed that actin is transported in an unassembled form with its associated proteins actin depolymerizing factor, cofilin, and profilin. Here we examine the specific activities of radioactively labeled tubulin and neurofilament proteins in subcellular fractions of the chicken sciatic nerve following injection of L-[³⁵S]methionine into the lumbar spinal cord.2. At intervals of 12 and 20 days after injection, nerves were cut into 1-cm segments and separated into Triton X-100-soluble and particulate fractions. Analysis of the fractions by high-resolution two-dimensional gel electrophoresis, immunoblotting, fluorography, and computer densitometry showed that tubulin was transported as a unimodal wave at a slower average rate (2–2.5 mm/day) than actin (4–5 mm/day). Moreover, the specific activity of soluble tubulin was five times that of its particulate form, indicating that tubulin is transported in a dimeric or small oligomeric form and is assembled into stationary microtubules.3. Neurofilament triplet proteins were detected only in the particulate fractions and transported at a slower average rate (1 mm/day) than either actin or tubulin.4. Our results indicate that the tubulin was transported in an unpolymerized form and that the neurofilament proteins were transported in an insoluble, presumably polymerized form.     

Wallerian degeneration and axonal regeneration after sciatic nerve crush are altered in ICAM-1-deficient mice

The intercellular cell adhesion molecule-1 (ICAM-1) has been implicated in the recruitment of immune cells during inflammatory processes. Previous studies investigating its involvement in the process of Wallerian degeneration and focusing on its potential role in macrophage recruitement have come to controversial conclusions. To examine whether Wallerian degeneration is altered in the absence of ICAM-1, we have analyzed changes in the expression of axonal and Schwann cell markers following sciatic nerve crush in wildtype and ICAM-1-deficient mice. We report that the lack of ICAM-1 leads to impaired axonal degeneration and regeneration and to alterations in Schwann cell responses following sciatic nerve crush. Degradation of neurofilament protein, the collapse of axonal profiles, and the re-expression of neurofilament proteins are substantially delayed in the distal nerve segment of ICAM-1^-/- mice. In contrast, the degradation of myelin, as determined by immunostaining for myelin protein zero, is unaltered in the mutants. Upregulation of GAP-43 and p75 neurotrophin receptor (p75^NTR) expression, characteristic for Schwann cells dedifferentiating in response to nerve injury, is differentially altered in the mutant animals. These results indicate that ICAM-1 is essential for the normal progression of axonal degeneration and regeneration in distal segments of injured peripheral nerves.     

Beneficial role of amino acids in mitigating cytoskeletal actin glycation and improving F-actin content: In vitro

Aims: The actin filaments present in circulating leukocytes facilitate their passage through microvenules and capillaries by helping in their deformability. Decreased deformability of granulocytes is now known to cause occlusion of the retinal microcapillaries leading to hypoxia and the subsequent development of diabetic retinopathy. Structural and functional loss of proteins, due to non-enzymatic glycation and glycoxidation, has been reported to cause diabetic pathogenesis. As amino acids have been earlier reported to have antidiabetic properties, the present study involves the investigation of the susceptibility of the cytoskeletal actin to glycation and its mitigation by free amino acids. This study also involves quantifying F-actin in cultured mononuclear cells obtained from diabetic and normal healthy volunteers and on the effect of glucose and free amino acids on F-actin content. Methods: Commercial non-muscle actin and actin immuno-pre-cipitated from granulocytes obtained from (a) normal healthy human volunteers and (b) patients with type 2 diabetes mellitus were subjected to glycation studies using [U] ¹⁴C glucose. The effect of free amino acids, as antiglycating agents, was determined using various concentrations of lysine, arginine, alanine, aspartic acid and glutamic acid. F-actin content in cultured mononuclear cells was estimated by flow cytometry using fluorescein isothiocynate (FITC)-Phalloidin. Results: Commercial actin at physiological conditions of pH and temperature was found to undergo non-enzymatic glycation. The extent of in vitro glycation was significantly low (P<0.001) in actin isolated from patients with type2 diabetes when compared to the non-diabetic group, suggesting an increased in vitro structural modification of actin in patients with diabetes. All the free amino acids tested were found to have varying degrees of antiglycating effect. The F-actin content in the intact mononuclear cells obtained from diabetic patients was found to be low when compared with normal healthy volunteers (P<0.001). Similarly the F-actin content was significantly low when the normal mononuclear cells were incubated with glucose. This effect was reversed upon the addition of free amino acids to the incubation mixture. Conclusions: Free amino acids can play a positive role in improving leukocyte deformability by mitigating cytoskeletal actin glycation and improving F-actin content.