Glycogen synthesis in the perfused liver of streptozotocin-diabetic rats.

1. Net glycogen accumulation was measured in sequentially removed samples during perfusion of the liver of starved streptozotocin-diabetic rats, and shown to be significantly impaired, compared with rates in normal (starved) rats. 2. In perfusions of normal livers with glucose plus C3 substrates, there was an increase in the proportion of glycogen synthetase 'a', compared with that in the absence of substrates. This response to substrates, followed in sequential synthesis and enzymic sensitivity in the perfused liver of diabetic rats were reversed by pretreatment in vivo with glucose plus fructose, or insulin. Glucose alone did not produce this effect. 4. Glucose, fructose, insulin or cortisol added to e perfusion medium (in the absence of pretreatment in vivo) did not stimulate glycogen synthesis in diabetic rats. 5. In intact diabetic rats, there was a decline in rates of net hepatic glycogen accumulation, and the response of glycogen synthetase to substrates. The most rapid rates of synthesis were obtained after fructose administration. 6. These results demonstrate that there is a marked inherent impairment in hepatic glycogen synthesis in starved diabetic rats, which can be rapidly reversed in vivo but no in perfusion. Thus hepatic glycogen synthesis does not appear to be sensitive to either the short-term direct action of insulin (added alone to perfusions) of to long-term insulin deprivation in vivo. The regulatory roles of substrates, insulin and glycogen synthetase in hepatic glycogen accumulation are discussed.     

Regulation of glycogen metabolism in primary cultures of rat hepatocytes. Restoration of acute effects of insulin and glucose in cells from diabetic rats

Defects in the deposition of glycogen and the regulation of glycogen synthesis in the livers of severely insulin-deficient rats can be reversed, in vivo, within hours of insulin administration. Using primary cultures of hepatocytes isolated from normal and diabetic rats in a serum-free chemically defined medium, the present study addresses the chronic action of insulin to facilitate the direct effects of insulin and glucose on the short term regulation of the enzymes controlling glycogen metabolism. Primary cultures were maintained in the presence of insulin, triiodothyronine, and cortisol for 1-3 days. On day 1 in alloxan diabetic cultures, 10(-7) M insulin did not acutely activate glycogen synthase over a period of 15 min or 1 h, whereas insulin acutely activated synthase in cultures of normal hepatocytes. By day 3 in hepatocytes isolated from alloxan diabetic rats, insulin effected an approximate 30% increase in per cent synthase I within 15 min as was also the case for normal cells. The acute effect of insulin on synthase activation was independent of changes in phosphorylase alpha. Whereas glycogen synthase phosphatase activity could not be shown to be acutely affected by insulin, the total activity in diabetic cells was restored to normal control values over the 3-day culture period. The acute effect of 30 mM glucose to activate glycogen synthase in cultured hepatocytes from normal rats after 1 day of culture was missing in hepatocytes isolated from either alloxan or spontaneously diabetic (BB/W) rats. After 3 days in culture, glucose produced a 50% increase in glycogen synthase activity during a 10-min period under the same conditions. These studies clearly demonstrate that insulin acts in a chronic manner in concert with thyroid hormones and steroids to facilitate acute regulation of hepatic glycogen synthesis by both insulin and glucose.     

Regulation of glycogen metabolism in primary cultures of rat hepatocytes. Restoration of acute effects of glucose in cells from diabetic rats involves protein synthesis

Defective acute regulation of hepatic glycogen synthase by glucose and insulin, caused by severe insulin deficiency, can be corrected in adult rat hepatocytes in primary culture by inclusion of insulin, triiodothyronine, and cortisol in a chemically defined serum-free culture medium over a 3-day period (Miller, T. B., Jr., Garnache, A. K., Cruz, J., McPherson, R. K., and Wolleben, C. (1986) J. Biol. Chem. 261, 785-790). Using primary cultures of hepatocytes isolated from normal and diabetic rats in the same serum-free chemically defined medium, the present study addresses the effects of cycloheximide and actinomycin D on the chronic actions of insulin, triiodothyronine, and cortisol to facilitate the direct effects of glucose on the short-term activation of glycogen synthase. The short-term presence (1 h) of the protein synthesis blockers had no effect on acute activation of glycogen synthase by glucose in primary hepatocyte cultures from normal rats. Normal cells maintained in the presence of cycloheximide or actinomycin D for 2 and 3 days exhibited unimpaired responsiveness to glucose activation of synthase. The protein synthesis inhibitors were effective at blocking the restoration of glucose activation of synthase in diabetic cells in media which restored the activation in their absence. Restoration of glycogen synthase phosphatase activity by insulin, triiodothyronine, and cortisol in primary cultures of diabetic hepatocytes was also blocked by cycloheximide or actinomycin D. These data clearly demonstrate that restoration of acute glycogen synthase activation by glucose and restoration of glycogen synthase phosphatase activity in primary cultures of hepatocytes from adult diabetic rats are dependent upon the synthesis of new protein.     

Insulin regulation of glycogen synthase phosphatase in primary cultures of hepatocytes

Activation of glycogen synthase in the perfused rat liver is defective in severely diabetic rats. In the present study, activation of glycogen synthase by glucose and increased incorporation of [14C]glucose into glycogen by insulin are defective in hepatocytes isolated from alloxan diabetic rats. Acute activation of glycogen synthase in hepatocytes isolated from diabetic rats was restored by treatment of the rats with insulin in vivo. Restoration of synthase activation was not achieved by incubation of hepatocytes in the presence of insulin in vitro for up to 12 h. When isolated hepatocytes from diabetic rats were placed in primary culture in a serum-free defined medium over a 3-day period, glycogen synthesis was partially restored by cortisol and triiodothyronine and dramatically increased by insulin. Concomitant with restoration of [14C]glycogen synthesis was an insulin-mediated increase in glycogen synthase I and synthase phosphatase activity. Restoration of regulation of glycogen synthesis in primary cultures of hepatocytes from diabetic rats by insulin required the presence of cortisol and triiodothyronine. Primary cultures of hepatocytes from normal rats did not require triiodothyronine for insulin to effect glycogenesis over a 3-day period. These data demonstrate that insulin acts in a chronic manner in concert with other hormones to control synthase phosphatase activity, an effect which may be influencing acute control of hepatic glycogen synthesis.     

Stimulation of apolipoprotein secretion in very-low-density and high-density lipoproteins from cultured rat hepatocytes by dexamethasone.

The effects of dexamethasone (a synthetic glucocorticoid) and insulin on the secretion of very-low-density lipoprotein (VLDL) and high-density lipoprotein (HDL) were investigated. Rat hepatocytes in monolayer culture were preincubated for 15 h in the presence or absence of combinations of 100 nM-dexamethasone and 2 nM-, 10 nM- or 50 nM-insulin. Dexamethasone increased [3H]oleate incorporation into secreted triacylglycerol by 2.7-fold and the mass of triacylglycerol secreted by 1.5-fold. Insulin alone decreased these parameters and antagonized the effect of dexamethasone. Dexamethasone increased the secretion of [3H]leucine in apolipoprotein (apo) E, and in the large (BH) and small (BI) forms of apo B in VLDL by about 7.1-, 3.6- and 4.0-fold respectively. Insulin alone decreased the secretion of these 3H-labelled apolipoproteins in VLDL. However, 2 nM-insulin with dexamethasone increased the secretion of 3H-labelled apo BH and apo BL by a further 0.8- and 3.2-fold respectively; 50 nM-insulin decreased the secretions of apo E, apo BH and apo BL in VLDL. Similar effects for dexamethasone or insulin alone were also obtained for the masses of apo E and apo BL + H secreted in VLDL. Albumin secretion was not significantly altered by either dexamethasone or insulin alone, but in combination they stimulated by 2.1-2.6-fold. Insulin or dexamethasone alone had little effect on the secretion of apolipoproteins in the HDL fraction. However, dexamethasone plus 2 nM-insulin increased the incorporation of [3H]leucine into apo AI, apo AH plus apo C, apo AIV and apo E of HDL by about 1.8-, 1.6-, 1.7- and 2.0-fold respectively. The apo E in the bottom fraction represented about 69% of the total 3H-labelled apo E secreted. The responses in the total secretion of apo E from the hepatocytes resembled those seen in HDL. The interactions of insulin and dexamethasone are discussed in relation to the general regulation of lipoprotein metabolism, the development of hyperlipidaemias and the predisposition to premature atherosclerosis.     

Inhibition of apolipoprotein B net synthesis and secretion from cultured rat hepatocytes by the calcium-channel blocker diltiazem.

1. The effect of the Ca2+-channel blocker diltiazem on hepatic apolipoprotein B (apo B) synthesis and secretion was studied in 12-18 h cultures of collagenase-dispersed rat hepatocytes. 2. The presence of diltiazem in the medium decreased apo B secretion by hepatocytes in a concentration-dependent manner. At 25 microM, diltiazem inhibited apo B secretion by approx. 36%, but there was no evidence of intracellular accumulation of apo B. 3. The inhibition of apo B secretion by hepatocytes was significantly correlated with cell-associated diltiazem (r = 0.72, P less than 0.01). 4. The rate of apo B secretion remained linear over 16 h even in the presence of 50 microM-diltiazem. 5. At diltiazem concentrations in the medium which were inhibitory for apo B secretion, [14C]acetate incorporation into cellular lipids and [35S]methionine incorporation into protein were enhanced. 6. Diltiazem inhibited the secretion of the apo B variants with a preferential inhibition of the higher-molecular-mass form of apo B (apo BH) over the lower-molecular-mass form (apo BL) at diltiazem concentrations in the medium greater than 25 microM. 7. Together, these results suggest that Ca2+ may play an important role in the synthesis and secretion of apo B-containing lipoproteins.     

Insulin suppression of VLDL apo B secretion is not mediated by the LDL receptor

Insulin inhibits hepatic very low density lipoprotein (VLDL) apo B secretion in rats. Current studies test whether the insulin effect is LDL receptor-mediated by examining the effect of insulin on VLDL apo B secretion in hepatocytes derived from Ldlr-/- and control mice. Primary hepatocytes were incubated overnight with media containing 14C-leucine and either 0.1nM (basal) or 200nM insulin. Afterwards, secreted VLDL B100 and B48 were quantitated. Insulin reduced 14C-labeled B100 and B48 comparably in control and Ldlr-/- hepatocytes with a 62+/-12% vs. 59+/-12% decrease in B100, and a 56+/-11% vs. 61+/-9% decrease in B48. Results indicate: (1) mouse hepatocytes respond to insulin by reducing VLDL apo B output; (2) both VLDL B100 and B48 secretion are suppressed; and (3) insulin inhibition of VLDL apo B secretion is retained in Ldlr-/- hepatocytes.     

Effects of hyperthyroidism on synthesis, secretion and metabolism of the VLDL apoproteins by the perfused rat liver

Livers from fed male Sprague-Dawley rats, made hyperthyroid by treatment with triiodothyronine (T3), were isolated and perfused in vitro. T3 (9.6 micrograms/day) was administered by osmotic minipump implanted intraperitoneally. Treatment with T3 for either 7 or 28 days reduced hepatic output of very-low-density lipoprotein (VLDL) and net synthesis of total associated apoproteins. After 7 days treatment, incorporation of [4,5-3H]leucine by livers from hyperthyroid rats into VLDL apo E was reduced while incorporation into apo B100, apo B48, and apo C's did not differ from euthyroid controls. The depressed incorporation of radioactivity into total VLDL protein was accounted for almost entirely on the basis of apo E. Incorporation of leucine into the total lipoprotein apo E isolated in the d less than 1.210 was also diminished by the hyperthyroid state, while that into apo B100, apo B48, and apo C in the total perfusate lipoprotein was similar to that of the euthyroid, as was found for the VLDL. Increased amounts of radioactive apo B100 and apo B48, however, were detected in the HDL fraction isolated from the medium perfusing livers from hyperthyroid rats. Hepatic uptake of VLDL protein and lipid was similar in euthyroid and hyperthyroid rats. Reduction of VLDL lipid and protein in the medium perfusing livers from T3-treated rats, therefore reflects hormonal action on synthesis and secretion, rather than uptake. Since the availability of apo B is thought to be required for secretion of VLDL, our observation suggests that synthesis of apo B is not depressed by treatment with T3 and that apoprotein synthesis is not a significant factor in the decreased output of VLDL by the liver, but that, as reported earlier, the lower output is a consequence of decreased synthesis of TG, the result of a diminished supply of hepatic glycero-3-phosphate in the hyperthyroid. The diminished amount of VLDL protein appears to be accounted for by the decreased quantity of apo E associated with a smaller VLDL particle secreted by livers from T3-treated rats.     

Insulin responsiveness of isolated perfused livers from rats with streptozotocin induced diabetes.

The ability of insulin to inhibit efflux of potassium (K) and amino acid nitrogen (AAN) from perfused livers of normal and insulin deficient rats was studied. Two groups of rats with different degrees of insulin deficiency were produced by injecting varying amounts of streptozotocin. One group, classified as being moderately diabetic (MD), had fasting plasma glucose levels between 235--425 mg%, while the other group, whose plasma glucose levels greater than 425 mg%, were considered to have severe diabetes (SD). Two other groups of rats were food restricted in order to attain body weights comparable to the two groups of diabetic rats, and livers from these animals were used for control perfusions. The results indicated that the ability of insulin to suppress efflux of K and AAN from perfused livers of rats with MD was comparable to that seen in control perfusions. On the other hand, insulin could not suppress the efflux of either K or AAN from perfused livers of rats with SD. These results indicate that normal hepatic responsiveness to insulin can be lost secondary to the production of insulin deficiency.     

Regulation of very-low-density-lipoprotein lipid secretion in hepatocyte cultures derived from diabetic animals.

Hepatocytes were derived from 2-3-day streptozotocin-diabetic rats and maintained in culture for up to 3 days. Compared with similar cultures from normal animals, these hepatocytes secreted less very-low-density-lipoprotein (VLDL) triacylglycerol, but the decrease in the secretion of VLDL non-esterified and esterified cholesterol was not so pronounced. This resulted in the secretion of relatively cholesterol-rich VLDL particles by the diabetic hepatocytes. Addition of insulin for a relatively short period (24 h) further decreased the low rates of VLDL triacylglycerol secretion from the diabetic hepatocytes. The secretion of VLDL esterified and non-esterified cholesterol also declined. These changes occurred irrespective of whether or not exogenous fatty acids were present in the culture medium. Little or no inhibitory effect of insulin was observed after longer-term (24-48 h) exposure to the hormone. Both dexamethasone and a mixture of lipogenic precursors (lactate plus pyruvate) stimulated VLDL triacylglycerol and cholesterol secretion, but not to the levels observed in hepatocytes from normal animals. The low rate of hepatic VLDL secretion in diabetes contrasts with the increase in whole-body VLDL production rate. This suggests that the intestine is a major source of plasma VLDL in insulin-deficient diabetes.     

Insulin stimulates triacylglycerol secretion by perfused livers from fed rats but inhibits it in livers from fasted or insulin-deficient rats implications for the relationship between hyperinsulinaemia and hypertriglyceridaemia.

We determined whether the direction of the acute effect of insulin on hepatic triacylglycerol secretion is dependent on the prior physiological state or on the in vitro experimental system used. The effect of insulin on triacylglycerol secretion was studied using perfused livers isolated from rats under three metabolic conditions: fed normo-insulinaemic, 24-h fasted and fed, streptozotocin-diabetic (insulin-deficient). Insulin acutely activated triacylglycerol secretion (by 43%) in organs from fed, normo-insulinaemic animals, whereas it inhibited triacylglycerol secretion in livers isolated from fasted or insulin-deficient rats (by 30 and 33%, respectively). By contrast, in 24-h-cultured hepatocytes insulin invariably acutely inhibited triacylglycerol secretion irrespective of the metabolic state of the donor animals. It is concluded that the use of perfused livers enables the observation of a switch in the direction of insulin action on hepatic triacylglycerol secretion from stimulatory, in the normo-insulinaemic state, to inhibitory in the fasting or insulin-deficient state. The possible implications of this switch for the relationship between hyperinsulinaemia, increased hepatic very-low-density lipoprotein-triacylglycerol secretion and hypertriglyceridaemia observed in vivo are discussed.     

Effects of epidermal growth factor on apo B mRNA levels and apo B accumulation in the media of primate hepatocytes in culture.

EGF has been shown to augment albumin and apolipoprotein A-I secretion by cynomolgus monkey hepatocytes in primary culture without stimulating cell division. This study was undertaken to determine what effect EGF had on apo B secretion by those hepatocytes. The results indicate that EGF (3 nM final concentration) severely inhibits the rate at which apo B accumulates in the culture medium of primate hepatocytes. That effect was evident within 48 hours of treatment, and by 72 hours the rate that apo B accumulated was less than half that of cells treated with a hormone-free medium. However, the apo B mRNA levels in the EGF-treated cells were more than double those of hepatocytes given the hormone-free medium. These data indicate that EGF has a potent effect on the rate at which apo B accumulates in the culture medium of primate hepatocytes and that the effect is independent of apo B gene expression.     

Acute suppression of apo B secretion by insulin occurs independently of MTP

Secretion of apolipoprotein (apo) B-containing lipoproteins by the liver depends mainly upon apo B availability and microsomal triglyceride transfer protein (MTP) activity and is subject to insulin regulation. Hepatic MTP mRNA expression is negatively regulated by insulin which correlates with inhibition of apo B secretion suggesting that insulin might suppress apo B secretion through an MTP-dependent mechanism. To investigate this possibility, we examined the acute effect of insulin on hepatic MTP expression and activity levels in vivo utilizing apobec-1^−/− mice. Insulin did not significantly alter hepatic MTP mRNA levels or lipid transfer activity 2 h following injection, but suppressed expression of genes important in gluconeogenesis. To study the specific role of MTP, we expressed human MTP (hMTP) in primary rat hepatocytes using adenoviral gene transfer. Increased expression of hMTP resulted in a 47.6 ± 17.9% increase in total apo B secreted. Incubation of hepatocytes with insulin suppressed apo B secretion by 50.1 ± 10.8% in cells over-expressing hMTP and by 53.0 ± 12.4% in control transfected hepatocytes. Results indicate that even under conditions of increased hepatic apo B secretion mediated by MTP, responsiveness of hepatocytes to insulin to suppress apo B secretion is maintained.     

Combined administration of sodium chloro-2-propionate and insulin on the secretion of glucagon by the pancreas in the diabetic rat

This work was designed to study the effects of sodium 2-chloropropionate (2CP) alone or combined with insulin, in vitro, on glucagon secretion from pancreas isolated from rats, made diabetic by streptozotocin (66 mg/kg i.p.). The pancreata were perfused with a physiological solution containing 2.8 mM glucose (0.5 g/l) and glucagon secretion was stimulated by an arginine infusion (5 mM) for 30 min. When 2CP (1 mM) and/or insulin (4 IU/l) were applied, they were infused from the start of the organ perfusion. In the presence of glucose alone, a marked decrease in glucagon output was observed in diabetic rat pancreas. The arginine perfusion induced a biphasic glucagon secretion both in normal and diabetic rat pancreas; this response was however clearly reduced in diabetic rat pancreas. In diabetic rat pancreas, the infusion of either 2CP or insulin had no effect on glucagon output in presence of glucose alone, nor did it modify the response to arginine. In contrast, the combined infusion of insulin and 2CP induced different effects depending on the conditions: whereas in presence of glucose alone it restored a glucagon output close to that recorded in normal rat pancreas, it did not modify the response to arginine.     

Secretion of triglyceride lipase from rat hepatocytes in culture: modulation by insulin and phenobarbital

Hepatic triglyceride lipase (HTGL) was measured in primary rat hepatocytes maintained for 3 days under three different culture conditions: basal medium, basal medium plus insulin, and basal medium plus insulin and phenobarbital. The activity of HTGL secreted by these cells was measured by treating intact cells with heparin; intracellular enzyme was subsequently measured in cell homogenates. Insulin stimulated intracellular triglyceride lipase activity by 48% and extracellular lipase by 30%. Phenobarbital, an enzyme-inducing drug, caused a further 15% increase in extracellular hepatic triglyceride lipase; whereas, the intracellular activity was reduced. The presence of insulin greatly stimulated the rate of enzyme secretion, and this rate was not notably affected by the presence of phenobarbital. After 3 days in culture, the short term (2-8 h) synthesis and secretion of enzyme from cultures treated with insulin or insulin plus phenobarbital were equally inhibited by cycloheximide. Monensin also inhibited enzyme secretion in both cultures and caused a similar increase in intracellular lipase activities. Insulin did not significantly affect the proportion of intracellular enzyme (17.7% basal vs. 15.8% insulin). On the other hand phenobarbital produced a 20-30% reduction in the proportion of intracellular enzyme (12.5 vs. 17.7% basal or 15.8% insulin). These findings suggest a drug-induced redistribution of triglyceride lipase.     

Taurolithocholic acid-3 sulfate impairs insulin signaling in cultured rat hepatocytes and perfused rat liver.

BACKGROUND/AIMS: The role of bile acids for insulin resistance in cholestatic liver disease is unknown. METHODS: The effect of taurolithocholic acid-3 sulfate (TLCS) on insulin signaling was studied in cultured rat hepatocytes and perfused rat liver. RESULTS: TLCS induced insulin resistance at the level of insulin receptor (IR) beta Tyr(1158) phosphorylation, phosphoinositide (PI) 3-kinase activity and protein kinase (PK)B Ser(473) phosphorylation in cultured hepatocytes. Consistently, the insulin stimulation of the PI 3-kinase-dependent K(+) uptake, hepatocyte swelling and proteolysis inhibition was blunted by TLCS in perfused rat liver. The PKC inhibitor Go6850 and tauroursodeoxycholate (TUDC) counteracted the suppression of insulin-induced IRbeta and PKB phosphorylation by TLCS. Rapamycin and dibutyryl-cAMP, which inhibited basal signaling via mammalian target of rapamycin (mTOR), restored insulin-induced PKB- but not IRbeta phosphorylation. In livers from 7 day bile duct-ligated rats PKB Ser(473) phosphorylation was decreased by about 50%. CONCLUSION: TLCS induces insulin resistance by a PKC-dependent suppression of insulin-induced IRbeta phosphorylation and the PI 3-kinase/PKB path. This can in part be compensated by a decrease of mTOR activity, which may release insulin-sensitive components downstream of the insulin receptor from tonic inhibition. The data suggest that retention of hydrophobic bile acids confers insulin resistance on the cholestatic liver.     

Secretion of alpha-tocopherol from cultured rat hepatocytes

Primary cultures of rat hepatocytes and rat liver perfusions were used to study hepatic secretion of alpha-tocopherol. The secretion of alpha-tocopherol from hepatocytes in culture was linear with time for 4 h. Ultracentrifugation of the medium revealed that 89.4 +/- 2.1% of alpha-tocopherol secreted during 4 h incubation was associated with the very-low density lipoprotein fraction (VLDL, d less than 1.006 g/ml). Oleic acid had no significant effect on the secretory rate of alpha-tocopherol, whereas eicosapentaenoic acid reduced the amount of alpha-tocopherol secreted to 48.4 +/- 12.7% of the control value after 20 h incubation (P less than 0.01). Monensin, a known inhibitor of VLDL secretion, reduced the secretion of alpha-tocopherol to 14.1 +/- 4.3% of the control value (P less than 0.02). Colchicine and chloroquine inhibited the secretion of alpha-tocopherol in the same order of magnitude as monensin. Hepatic perfusion after intravenous injection of in vivo labeled alpha-[3H]tocopherol lymph, showed that about 75% of the secreted radioactivity was in the VLDL fraction. From these results we conclude that most alpha-tocopherol is secreted from the liver associated with nascent VLDL in rats.     

Secretion of high and low molecular weight phosphorylated apolipoprotein B by hepatocytes from control and diabetic rats. Phosphorylation of APO BH and APO BL

Apolipoprotein B (apo B) phosphorylation was examined in primary cultures of hepatocytes from control and nonketotic streptozotocin diabetic rats. Following a 5-h incubation with ortho[32P]phosphate, media lipoproteins (d less than 1.063 g/ml) were isolated, and delipidated apolipoproteins were separated by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis (SDS-PAGGE), and gels were heat fixed. Autoradiographic bands corresponding to high (apo BH) and low molecular weight apo B (apo BL) were observed in media lipoproteins isolated from control rats, and these bands were more prominent in media lipoproteins isolated from diabetic rats. Apo B-specific activity was estimated from aqueous alcohol-precipitated radioactivity and apo B monoclonal immunoassay of isolated media lipoproteins. In lipoproteins secreted by hepatocytes of diabetic rats, the calculated apo B specific activity was between 18- and 31-fold greater than that secreted by hepatocytes of control rats, consistent with the SDS-PAGGE gel data. The increase in secretory 32P-labeled apo B from hepatocytes of diabetic rats was due, at least in part, to an increase in labeled phospho-tyrosine as determined by phosphoamino acid analysis. These data demonstrate that apo BH may be secreted as a phosphorylated protein and that apo B phosphorylation occurs on tyrosine as well as serine residues.     

Enhanced synthesis and secretion of apolipoprotein E from sciatic nerves of streptozotocin-induced diabetic rats after injury

To elucidate the pathogenesis of diabetic neuropathy, synthesis and secretion of apolipoprotein E (apo E) from sciatic nerves after injury was studied in normal and streptozotocin-induced diabetic rats. Seven, 14, 28, 45 and 59 days after making crush injury on sciatic nerves with concomitant administration of streptozotocin (50 mg/kg body weight), the nerves were taken out and incubated with [35S]methionine. The [35S]labeled apo E was precipitated with specific antiserum. The amounts of apo E secreted into medium by nerves of diabetic rats were 7 times greater than those of non-diabetic rats 7 days after injury. This enhanced secretion of apo E was relatively selective for this protein, since the ratio of the immunoprecipitable apo E to the TCA preciptitable protein in the medium increased in diabetic rats. Intriguing possibility deduced from these results is that the secretion of apo E is involved in the development of diabetic neuropathy.