The permeability of insulin (Ins), nerve growth factor (NGF), albumin (Alb), transferrin (Trf), and IgG across the blood-nerve barrier (BNB) and blood-brain barrier (BBB) in normal adult rats was quantified by measur-ing the (permeability coefficient X surface area) product (PS) with the L.v. bolus-injection technique in the canua brachial vein and artery using radionated proteins. The PS values of the BNB for IgG and Alb were low: 0.079 ± 0.029 x 10-6 and 0.101 ± 0.088 x 10-6 ml g-Ls-, (Xa ± SD, respectively). The PS values for NGF and Trf were 16.1-fold and 25.5-fold higher than for Alb. The PS for Ins across the BNB was 33.190 ± 2.053 X 10-6 mlg-'s-1--a remarkable 329-fold increase compared with Alb. The PS values of the BBB for IgG and Alb in different brain ri were all low, from O.O8± 0.017 to 0.151 ± 0.035 X 10-6 ml g'l-s'1 (X-± SD). NGF Van Bree et al. (4), the uptake values were of the same order of magnitude as that of the vascular marker and were, therefore, considered negligible, which leads to possible erroneous conclusions that the barrier was impermeable to proteins.The vascular-space marker chosen must meet several requirements which have been summarized (1). The closer "a vascular volume indicator approaches the biological, physical, and chemical properties of the test substance, the better the former is able to accurately model the intravascular distribution of the latter." In addition, Fenstermacher et al.(1) emphasized the need to determine a vascular-space correction for each individual animal rather than having a mean adjustment factor for a separate group of animals.In our experiments, the residual brain/endoneurial plasma volume (Vp) Abbreviations: BBB, blood-brain barrier; BNB, blood-nerve barrier; PS, (permeability coefficient x surface area) product; VP, residual brain/endoneurial plasma volume; Alb, albumin; NGF, nerve growth factor; Trf, transferrin; Ins, insulin. *To whom reprint requests should be addressed. 5705The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Insulin's effect on the synthesis of liver proteins remains to be fully defined. Previous studies using various surrogate measures of amino acyl-tRNA have reported variable results of insulin's effect on liver protein synthesis. We determined the effect of insulin with or without amino acid supplementation on the synthesis rates of liver proteins (tissue, albumin, and fibrinogen) using L-[1- 15 N]Phe and that of plasma [ 13 C]ketoisocaproatic acid (KIC) were very similar to that of tRNA, plasma isotopic enrichment of both Leu and Phe were substantially higher (P < 0.01) and VLDL apolipoprotein-B 100 enrichment was lower (P < 0.01) than the respective amino acyl؊tRNA enrichment. Plasma KIC enrichment most accurately predicted leucyl-tRNA enrichment, whereas plasma Leu enrichment was best correlated with that of tRNA. Neither insulin alone nor insulin plus amino acid infusion had an effect on liver tissue protein synthesis. In contrast, insulin alone decreased the albumin synthesis rate, and insulin with amino acids maintained the albumin synthesis rate. Insulin with or without amino acids inhibited the fibrinogen synthesis rate. These results, based on synthetic rates using amino acyl-tRNA, were consistent with those obtained using KIC or tissue fluid Leu or Phe as precursor pools. These studies demonstrated that plasma KIC enrichment is a convenient and reliable surrogate measure of leucyl-tRNA in liver. We also concluded that insulin has differential effects on the synthesis rates of liver proteins. Whereas insulin with or without amino acid supplement has no acute effect on the synthesis of liver tissue protein, insulin has a substantial inhibitory effect on fibrinogen synthesis. In contrast, insulin administration along with amino supplement is necessary to maintain albumin synthesis rate. Diabetes 50:947-954, 2001

Quiescent Schwann cells in the distal segment of the permanently transected nerve produced basal levels of the major myelin glycoprotein, P0, in the absence of myelin assembly. Low levels of Po could be detected at 35 days after transection by autoradiographic analysis of radioiodinated lectin binding after protein separation by high-resolution sodium dodecyl sulfate pore gradient electrophoresis and by fluorographic analysis after electrophoresis of [3H] MATERIALS AND METHODSSciatic nerves from Sprague-Dawley rats (200 g), anesthetized with intraperitoneal sodium pentobarbital, were exposed below the sciatic notch, ligated twice with 4/0 sterile sutures, and transected between the pairs of sutures (11). Each end of the transected nerve was undercut for a distance of 10 mm, repositioned by 180°, and sutured to adjacent muscle. This procedure is designed to inhibit the outgrowth of regenerating axons from the proximal segment and prevent their subsequent entry into the distal segment. After closure of the wound, the animals were maintained in separate cages on shavings for 35 days. At this time, the animals were anesthetized, and the entire lengths of the distal segments of the sciatic, tibial, peroneal, and sural nerves were removed and processed. Morphometric evaluation at the light and electron microscopic level showed no new myelinated fibers at 35 days after transection, although new myelinated fibers (<1.5%, expressed as myelin percent of total area of transverse section of endoneurium) were observed at later times (70 and 105 days), indicating reinnervation (unpublished data). Biochemical studies were performed on the endoneurium, which was removed from the perineurium and epineurium by microdissection (18) and homogenized in deionized distilled water with a ground-glass homogenizer. A uniform suspension was obtained which was then treated with 10% NaDodSO4, sonicated for 1 hr, and centrifuged in the 300 A-100 rotor of the Beckman Airfuge at 197,000 x g at 40C. Aliquots of the NaDodSO4-solubilized supernatant were taken for protein determination (19), and the proteins were separated by NaDodSO4 pore gradient electrophoresis (Na- (20,21). Proteins were detected by Coomassie blue stain, and glycoproteins were evaluated by using radioiodinated lectins applied directly to the slab gel after electrophoresis (22,23). In several experiments, radioactive precursor incorporation studies were performed directly on endoneurial slices at 35 days after transection in modified Krebs mammalian Ringer solution (24). RESULTSAt 35 days after permanent transection, analysis of the detergent-solubilized endoneurium by NaDodSO4-PGE suggests that P0 cannot be detected by Coomassie blue stain (sensitivity -100 ng) (Fig. 1A). In contrast, Po is the major endoneurial protein of the normal sciatic nerve (control). This lack of Abbreviation: PGE, pore gradient electrophoresis. 1864The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement...

Permanent nerve transection of the adult rat sciatic nerve forces Schwann cells in the distal nerve segment from a myelin-maintaining to a quiescent state. This transition was followed by serial morphometric evaluation of the percentage fascicular area having myelin (myelin percent of area) in transverse sections of the distal nerve segment and revealed a rapid decline from a normal value of 36.6% to 3.2% by 14 days for the sciatic nerve to less than 1.0% throughout the remaining time course (up to 105 days). No evidence of axonal reentry into the distal nerve segment or new myelin formation was observed at times under 70 days. In some of the distal nerve segments at 70, 90, and 105 days, new myelinated fibers were observed that usually consisted of only a few myelinated fibers at the periphery and in the worst case amounted to 1.6% (myelin percent of area). Radioactive precursor incorporation of [3H]mannose into endoneurial slices at 4 and 7 days after transection revealed two species of the major myelin glycoprotein, P0, with Mr of 28,500 and 27,700. By 14 days after nerve transection, only the 27,700 Mr species remained. Incorporation of [3H]mannose into the 27,700 Mr species increased progressively to 35 days after transection and then began to decline at 70 and 105 days. Alterations in the oligosaccharide structure of this down-regulated myelin glycoprotein accounted for the progressive increase in mannose incorporation. Lectin affinity chromatography of pronase-digested P0 glycopeptides on concanavalin A-Sepharose revealed that the 28,500 Mr species of P0 had the complex-type oligosaccharide as the predominant oligosaccharide structure (92%). In contrast, the high mannose-type oligosaccharide was the predominate structure for the 27,700 Mr form, which increased to 70% of the total radioactivity by 35 days after nerve transection. Since the biosynthesis of the complex-type oligosaccharide chains on glycoproteins involves high mannose-type intermediates, the mechanism of down-regulation in the biosynthesis of this major myelin glycoprotein, therefore, results in a biosynthetic switch from the complex-type oligosaccharide structure as an end product to the predominantly high mannose-type oligosaccharide structure as a biosynthetic intermediate. This biosynthetic switch occurs gradually between 7 and 14 days after nerve transection and likely reflects a decreased rate of processing through the Golgi apparatus. It remains to be determined if the high mannose-type oligosaccharide chain on P0 can undergo additional processing steps in this permanent nerve transection model.

The adenylyl cyclase‐cyclic AMP (cAMP) second messenger pathway has been proposed to regulate myelin gene expression; however, a clear correlation between endogenous cAMP levels and myelin‐specific mRNA levels has never been demonstrated during the induction or maintenance of differentiation by the myelinating Schwann cell. Endogenous cAMP levels decreased to 8–10% of normal nerve by 3 days after crush or permanent transection injury of adult rat sciatic nerve. Whereas levels remained low after transection injury, cAMP levels reached only 27% of the normal values by 35 days after crush injury. Because P0 mRNA levels were 60% of normal levels by 14 days and 100% by 21 days after crush injury, cAMP increased only well after P0 gene induction. cAMP, therefore, does not appear to trigger myelin gene induction but may be involved in myelin assembly or maintenance. Forskolin, an activator of adenylyl cyclase, increased endoneurial cAMP levels only in the normal nerve, and in the crushed nerve beginning at 16 days after injury, but at no time in the transected nerve. Only by treating transected nerve with 3‐isobutyl‐1‐methylxanthine (IBMX), an inhibitor of cAMP phosphodiesterases, in combination with forskolin was it possible to increase cAMP levels. No induction of myelin genes, however, was observed with short‐ or long‐term treatment with IBMX and forskolin in the transected nerve. A three‐fold increase in phosphodiesterase activity was observed at 35 days after both injuries, and a nonmyelinated nerve was shown to have even higher activity. These experiments, therefore, suggest an important role for phosphodiesterase in the inactivation of this second messenger‐dependent stimuli when Schwann cells are non‐myelinating, such as after sciatic nerve injury or in the nonmyelinated nerve, which again implies that cAMP may be required for the maintenance of the myelin sheath.