Insulin inhibits apolipoprotein B (apoB) secretion by primary rat hepatocytes through activation of phosphoinositide 3-kinase (PI 3-K). Current studies demonstrate that the PI 3-K inhibitor wortmannin inhibits both basal and insulin-stimulated PI 3-K activities. Wortmannin and LY 294002, two structurally distinct PI 3-K inhibitors, prevent insulin-dependent inhibition of apoB secretion in a dose-dependent manner.To link PI 3-K activation to insulin action on apoB, we investigated whether insulin induced localization of activated PI 3-K to the endoplasmic reticulum (ER), where apoB biogenesis is initiated. Insulin action results in a significant redistribution of PI 3-K to a low density microsome (LDM) fraction containing apoB protein and apoB mRNA. Insulin stimulates a significant increase in PI 3-K activity associated with insulin receptor substrate-1 as well as an increase in insulin receptor substrate-1/PI 3-K mass in LDM. Subfractionation of LDM on sucrose density gradients shows that insulin significantly increases the amount of PI 3-K present in an ER fraction containing apoB. Insulin stimulates PI 3-K activity in smooth and rough microsomes isolated from rat hepatocytes, the latter of which contain rough ER as demonstrated by electron microscopy. Studies indicate that 1) PI 3-K activity is necessary for insulin-dependent inhibition of apoB secretion by rat hepatocytes; 2) insulin action leads to the activation and localization of PI 3-K in an ER fraction containing apoB; and 3) insulin stimulates PI 3-K activity in the rough ER. Apolipoprotein B (apoB)1 is a major structural protein component of chylomicrons, very low density lipoproteins (VLDL), and low density lipoproteins and is required for the assembly and secretion of triglyceride-rich lipoproteins. ApoB synthesis and secretion are regulated by insulin (1). Insulin suppresses VLDL triglyceride and apoB secretion by rat hepatocytes (2-4), human hepatocytes (5), human hepatoma cell line HepG2 (6), and human fetal intestine (7). Studies in primary rat hepatocytes indicate that the inhibition of apoB secretion by insulin is a result of enhanced intracellular degradation of newly synthesized apoB (8) and that the effect of insulin is mediated by the insulin receptor (9).It is not known how insulin receptor signaling leads to the inhibition of apoB secretion. Insulin binding to its receptor activates the intrinsic tyrosine kinase activity of the receptor, which tyrosine phosphorylates insulin receptor substrates 1 and 2 (IRS-1 and IRS-2) (10). These modified proteins function as adaptor molecules to activate several downstream effector proteins, among which is phosphoinositide 3-kinase (PI 3-K). Growth factor-activated PI 3-K consists of an 85-kDa (p85) regulatory subunit and a 110-kDa (p110) catalytic subunit (11)(12)(13)(14). PI 3-K phosphorylates phosphatidylinositol (PtdIns), PtdIns-4-P, and PtdIns-4,5-P 2 in the 3Ј-position of the inositol ring (15, 16). Activation of PI 3-K is necessary for vesicular transport of lysosomal proteins (17, 18) and for ...

The effects of hypoinsulinemic nonketotic streptozotocin diabetes on hepatic apo B synthesis and secretion was studied in primary cultures of rat hepatocytes. Diabetic rats were characterized by their significantly elevated serum glucose, apo B, and triglyceride levels, while serum insulin levels were less than a third of normal. Serum transaminase activities of diabetic rats were significantly elevated when compared with control rats, which was attributed to an increase in liver transaminase activity in diabetic rats. The pattern of enzyme activities of hepatocytes reflected that observed in livers of donor rats and the pattern was retained by primary cultures of hepatocytes over the culture period. Hepatocytes from diabetic rats secreted only one third of the apo B secreted by hepatocytes from control rats, which was determined by monoclonal immunoassay of rat total apo B. Decreases in secretion were confirmed by measurement of secretory [35S~methionine-labeled lipoprotein apo B radioactivity. The decreased apo B content of media of hepatocytes from diabetic rats was not due to increased apo B catabolism since hepatocytes from diabetic rats were shown to degrade less lipoprotein-apo B than hepatocytes from normal rats in control experiments. In addition, the apo B content of detergent-solubilized hepatocytes from diabetic rats was significantly less than that of hepatocytes from control rats. These results suggest that insulin is necessary for normal hepatic apo B synthesis and secretion and that the hyperlipidemia associated with hypoinsulinemia in vivo is primarily of intestinal origin.

The patterns of disposition of intravenously injected doses of 131 I proinsulin and I31 I insulin were compared in the rat with respect to plasma clearance, uptake, and degradation in selected organs and tissues, and rates of accumulation of degraded products in plasma and urine. Small doses of 131 I insulin (4 mptg. per 100 gm. rat body weight) were cleared from plasma approximately twice as rapidly as large doses (4 fig. per 100 gm. rat body weight) and three times as rapidly as equi molar small doses (6 m/u,g. per 100 gm. rat body weight) or large doses (6/xg.per 100 gm. rat body weight) of l31 I proinsulin. Conversely, the relative rate of accumulation of l 3 l Ilabeled degradation products in plasma after injection of low doses of l 3 l I insulin was about twice as rapid as the rate of accumulation after large doses and about three times as rapid as the rate of accumulation after either small or large doses of l3 'I proinsulin. Peak uptake and degradation in the liver at one minute was greatest after injection of low doses of l 3 l I insulin (28.8 ±'2.5 per cent of injected radioactivity), less after injection of large doses (15.1 ± 1 . 5 per cent), and least after injection of low doses (5.5 ± 0.9 per cent) or high doses (4.6 ± 0 . 6 per cent) of I31 I proinsulin. In contrast, peak uptake and degradation in the kidneys at seven to eleven minutes was greatest after injection of low doses (24.7 ± 2 . 7 per cent) or high doses (27.5 ± 1 . 2 per cent of l 3 l I proinsulin), least after injection of low doses (9.6 ± 0 . 6 per cent) of l 3 l I insulin, and intermediate after injection of high doses (17.5 ± 2 . 6 per cent). No large differences were noted in patterns of uptake of radioactivity in muscle, fat, or skin compartments. Excretion of degraded products in urine was more delayed after l 3 l I proinsulin injections. An inverse relationship was noted between initial concentration of hormone in plasma and uptake by the liver. Compared to | 3 | I insulin, the hypoglycemic effect of l 3 l I proinsulin was weaker (58 per cent) and more delayed in onset. No evidence of conversion of l 3 l I proinsulin to l 3 l I insulin was noted.The studies indicate that the differences in dose dependency in plasma clearance and degradation of 13I I insulin and | 3 | I proinsulin can be attributed to differences in relative uptakes and degradation by the liver and kidneys. The liver appeared to be the major organ involved in the removal and degradation of insulin, whereas the kidney appeared to be the major organ involved in the case of proinsulin. The hepatic process was rapid but relatively saturable, whereas the kidney process was slower but relatively unsaturable. The inverse relationship between initial concentration of hormone in plasma and uptake by liver suggests that the ratio may provide a sensitive index of the role of the liver in plasma hormone clearance. DIABETES 27:400-10, April, 1978.In 1967 Steiner and his co-workers first demonstrated the existence of a precursor in the biosynthesis of insulin in the pancreatic...

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