Search

Cells respond to external signals like insulin to alter metabolic pathways in response to varying physiological environments. Insulin stimulates the protein kinase C beta (PKCbeta) isozymes and preferentially switches the expression to PKCbetaII isozyme, which is shown to have a crucial role in glucose uptake, cellular proliferation, and differentiation. We have developed an insulin-responsive PKCbetaII heterologous minigene to identify cis-elements in vivo in eukaryotes by cloning the PKCbetaII exon and its flanking intronic sequences into the splicing vector pSPL3. The transfected minigene mimicked the endogenous insulin response of PKCbetaII alternative splicing in five distinct cell types, i.e. L6 skeletal muscle, 3T3-L1 pre-adipocytes, HepG2 human hepatoma cells, A10 vascular smooth muscle cells, and murine embryonic fibroblasts within 30 min of insulin stimulation. Sequential deletions of the flanking introns in the minigene demonstrated that insulin regulated elements within the 5'-intron flanking the PKCbetaII exon. Mutational studies indicated the SRp40 binding site promotes splice site selection. In these cases, splicing appears to be regulated by a phosphatidylinositol 3-kinase signaling pathway because LY294002 and wortmannin, its specific inhibitors, blocked exon inclusion. Cotransfection with constitutively active Akt2 kinase mimicked insulin action. Signal-dependent regulation of splicing by insulin is unique from tissue-specific and developmentally regulated mechanisms previously reported and serves as a prototype for studies of alternative splicing involving protein phosphorylation.

The current study assessed in vivo the effect of insulin on triglyceride-rich lipoprotein (TRL) production by rat liver. Hepatic triglyceride and apolipoprotein B (apoB) production were measured in anesthetized, fasted rats injected intravenously with Triton WR-1339 (400 mg/kg). After intravascular catabolism was blocked by detergent treatment, glucose (500 mg/kg) was injected to elicit insulin secretion, and serum triglyceride and apoB accumulation were monitored over the next 3 h. In glucose-injected rats, triglyceride secretion averaged 22.5 +/- 2.1 microg.ml(-1).min(-1), which was significantly less by 30% than that observed in saline-injected rats, which averaged 32.1 +/- 1.4 microg.ml(-1).min(-1). ApoB secretion was also significantly reduced by 66% in glucose-injected rats. ApoB immunoblotting indicated that both B100 and B48 production were significantly reduced after glucose injection. Results support the conclusion that insulin acts in vivo to suppress hepatic very low density lipoprotein (VLDL) triglyceride and apoB secretion and strengthen the concept of a regulatory role for insulin in VLDL metabolism postprandially.

Lipoprotein and apolipoprotein changes were evaluated in 10-week-old Zucker diabetic fatty (ZDF) male rats following 12 weeks of insulin treatment, which normalized blood glucose and maintained weight gaining characteristic of nondiabetic Zucker fatty rats. Compared with untreated ZDF rats (saline-injected), insulin treatment resulted in increased very-low-density lipoprotein (VLDL; d < 1.006 g/mL) and decreased alpha lipoprotein on agarose gel electrophoresis. These findings were consistent with an observed increase in VLDL triglyceride and cholesterol, and decreased high-density lipoprotein (HDL) cholesterol with insulin treatment in isolated lipoproteins. B100 levels were unchanged by insulin treatment, but B48 levels were significantly increased in the VLDL fraction. Insulin treatment depressed apolipoprotein (apo) A-I levels in HDL, but had little effect on total apo E, apo A-IV, or apo C, although apo C was redistributed to the VLDL fraction. These results suggest that insulin treatment of ZDF rats normalizes hyperglycemia and prevents age-related changes in lipoprotein parameters associated with development of insulinopenic diabetes. Insulin therapy in ZDF rats thereby sustains the hyperlipidemic lipoprotein pattern associated with hyperinsulinemia and obesity.

Lipoprotein and apolipoprotein parameters were studied in the male Zucker diabetic fatty (ZDF) rat at 10 and 20 weeks of age, corresponding to hyperinsulinemic and insulinopenic type 2 diabetes mellitus, respectively. At both ages, ZDF rats had elevated serum triglycerides, free fatty acids, and corticosterone, whereas 20-week ZDF rats had reduced thyroid hormones. At 10 weeks, the hyperlipidemia was confined to elevations in pre-beta triglyceride-rich (d < 1.006 g/mL) lipoproteins. By 20 weeks, all lipoprotein density fractions were increased compared with lean rats, with substantial increases in both low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol. In ZDF rats, there was a progressive increase in apolipoprotein B (apo B) from 1.9 times control at 10 weeks to three times control at 20 weeks. The increase in apo B was accompanied by a shift of apo B, particularly B100, from very-low-density lipoprotein (VLDL) into denser lipoproteins corresponding to intermediate-density lipoproteins plus LDLs (1.006 < d < 1.063 g/mL). In Zucker and 10-week ZDF rats, in the presence of hyperinsulinemia, the increase in serum apo B was predominantly apo B48 present in VLDL. By 20 weeks, when ZDF rats are insulinopenic, the mass ratio of B48:B100 shifted from 2.7 to 0.7. The shift was associated with a decrease in hepatic-edited apo B mRNA. Apo E increased in lean rats between 10 and 20 weeks of age. Although apo E also increased in ZDF rats, the increase by 20 weeks was less than that of lean rats. The molar ratio of apo E to B in VLDL was decreased in ZDF rats. In lean rats, greater than 50% of apo E was present in HDL, in contrast to ZDF rats, where less than 20% of apo E was present in HDL. VLDL apo E shifted to denser fractions by 20 weeks of age, similar to apo B. The apo C level was more than double compared with the level in lean rats and was redistributed from the HDL fraction to lipoprotein fractions containing apo B. Both apo A-I and apo A-IV levels more than doubled between 10 and 20 weeks in ZDF rats. The ZDF rat model may be useful in comparative studies of lipoproteins during diabetic progression from hyperinsulinemia to insulinopenia.

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.

Insulin inhibition of the secretion of apolipoprotein B (apo B) was studied in primary cultures of rat hepatocytes by using brefeldin A (BFA), an inhibitor of protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus, and by using the phosphatidylinositol 3-kinase (PI 3-K) inhibitor wortmannin. Incubation of hepatocytes with BFA (10 micrograms/ml) for 1 h inhibited the subsequent secretion of apo B, albumin and transferrin for up to 3 h. BFA treatment resulted in the time-dependent accumulation in cells of [14C]leucine-labelled proteins and apo B. Under conditions where insulin decreased total apo B (cell plus secreted), BFA blocked the insulin-dependent effect. These results suggest that export of apo B from the ER is a prerequisite for the observed insulin effect. Treatment of hepatocytes with wortmannin for 20 min abolished insulin inhibition of apo B secretion, suggesting that the insulin effect on the apo B pathway involves activation of PI 3-K. Enzyme inhibitor studies indicate that chymostatin and (+)-(2S,3S)-3-[(S)-methyl-1-(3-methylbutylcarbamoyl)-butylcarba moyl]-2- oxiranecarboxylate (E-64-c) partially block insulin effects on apo B compared with leupeptin, which had no discernible effect. The cell-permeable derivative of E-64-c, EST, and N-Ac-Leu-Leu-norleucinal (ALLN) were most effective in blocking insulin effects on apo B. These results suggest that insulin action on apo B in primary rat hepatocytes involves (1) vesicular movement of apo B from the ER; (2) activation of PI 3-K and (3) a cellular protease that is either a cysteine- or calcium-activated neutral protease.

Hepatocytes derived from lean Zucker rats have reduced secretion of apo B and lowered cellular apo B in response to a physiologic range of insulin (0.1 nM-10 nM). Effects are attenuated in hepatocytes derived from Zucker obese rats and seen only at higher insulin concentrations (> 100 nM) with a significant shifting of the dose-response curve. Decreased sensitivity and responsiveness of hepatocytes derived from obese rats suggests insulin resistance and dose-response curves are consistent with coexistent binding and post-binding defects. Inability to inhibit hepatic apo B secretion in the presence of short-term high levels of insulin may have important implications to the balance of intestinal and hepatic triglyceride-rich lipoprotein secretion post-prandially.

This review has considered a number of observations obtained from studies of insulin in perfused liver, hepatocytes, transformed liver cells and in vivo and each of the experimental systems offers advantages. The evaluation of insulin effects on component lipid synthesis suggests that overall, lipid synthesis is positively influenced by insulin. Short-term high levels of insulin through stimulation of intracellular degradation of freshly translated apo B and effects on synthesis limit the ability of hepatocytes to form and secrete TRL. The intracellular site of apo B degradation may involve membrane-bound apo B, cytoplasmic apo B and apo B which has entered the ER lumen. How insulin favors intracellular apo B degradation is not known. An area of recent investigation is in insulin-stimulated phosphorylation of intracellular substrates such as IRS-1 which activates insulin specific cellular signaling molecules [245]. Candidate molecules to study insulin action on apo B include IRS-1 and SH2-containing signaling molecules. Insulin dysregulation in carbohydrate metabolism occurs in non-insulin-dependent diabetes mellitus due to an imbalance between insulin sensitivity of tissue and pancreatic insulin secretion (reviewed in Refs. [307,308]). Insulin resistance in the liver results in the inability to suppress hepatic glucose production; in muscle, in impaired glucose uptake and oxidation and in adipose tissue, in the inability to suppress release of free FA. This lack of appropriate sensitivity towards insulin action leads to hyperglycemia which in turn stimulates compensatory insulin secretion by the pancreas leading to hyperinsulinemia. Ultimately, there may be failure of the pancreas to fully compensate, hyperglycemia worsens and diabetes develops. The etiology of insulin resistance is being intensively studied for the primary defect may be over secretion of insulin by the pancreas or tissue insulin resistance and both of these defects may be genetically predetermined. We suggest that, in addition to effects in carbohydrate metabolism, insulin resistance in liver results in the inability of first phase insulin to suppress hepatic TRL production which results in hypertriglyceridemia leading to high levels of plasma FA which accentuate insulin resistance in other target organs. As recently reviewed [17,254] the role of insulin as a stimulator of hepatic lipogenesis and TRL production has been long established. Several lines of evidence support that insulin is stimulatory to the production of hepatic TRL in vivo. First, population based studies support a positive relationship between plasma insulin and total TG and VLDL [253]. Second, there is a strong association between chronic hyperinsulinemia and VLDL overproduction [309].(ABSTRACT TRUNCATED AT 400 WORDS)

Insulin inhibition of apolipoprotein B (apoB) secretion by primary cultures of rat hepatocytes was investigated in pulse-chase experiments using [35S]methionine as label. Radioactivity incorporation into apoBH and apoBL, the higher and lower molecular weight forms, was assessed after immunoprecipitation of detergent-solubilized cells and media and separation of the apoB forms using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Hepatocyte monolayers were incubated for 12-14 h in medium with and without an inhibitory concentration of insulin. Cells were then incubated for 10 min with label, and, after differing periods of chase with unlabeled methionine, cellular medium and media labeled apoB were analyzed; greater than 90% of labeled apoB was present in cells at 10 and 20 min after pulse, and labeled apoB did not appear in the medium until 40 min of chase. Insulin treatment inhibited the incorporation of label into total apoB by 48%, into apoBH by 62%, and into apoBL by 40% relative to other cellular proteins. Insulin treatment favored the more rapid disappearance of labeled cellular apoBH with an intra-cellular retention half-time of 50 min (initial half-life of decay, t1/2 = 25 min) compared with 85 min in control (t1/2 = 60 min). Intracellular retention half-times of labeled apoBL were similar in control and insulin-treated hepatocytes and ranged from 80 to 100 min. After 180 min of chase, 44% of labeled apoBL in control and 32% in insulin-treated hepatocytes remained cell associated. Recovery studies indicated that insulin stimulated the degradation of 45 and 27% of newly synthesized apoBH and apoBL, respectively. When hepatocyte monolayers were continuously labeled with [35S]methionine and then incubated in chase medium with and without insulin, labeled apoBH was secreted rapidly, reaching a plateau by 1 h of chase, whereas labeled apoBL was secreted linearly over 3-5 h of chase. Insulin inhibited the secretion of immunoassayable apoB but not labeled apoB. Results demonstrate that 1) insulin inhibits synthesis of apoB from [35S]methionine, 2) insulin stimulates degradation of freshly translated apoB favoring apoBH over apoBL, and 3) an intracellular pool of apoB, primarily apoBL, exists that is largely unaffected by insulin. Overall, insulin action in primary hepatocyte cultures reduces the secretion of freshly synthesized apoB and favors secretion of preformed apoB enriched in apoBL.

Lipoprotein synthesis and secretion were examined in primary cultures of rat hepatocytes cultured on collagen-coated plates and incubated with pharmacologic and physiologic concentrations of insulin. Media insulin concentration declined rapidly over the course of incubation indicating that hepatocytes rapidly degrade insulin. When insulin was present in the media, cellular triglyceride accumulated while lipid secretion declined. Insulin inhibited the incorporation of labeled amino acids into total secretory lipoprotein apoproteins and apolipoprotein B (apo B) as well as apo B mass as measured by monoclonal radioimmunoassay. The effect of insulin on apo B secretion occurred as early as three hours after the addition of insulin to the culture media and both apo B of higher molecular weight (apo BH) and apo B of lower molecular weight (apo BL) were affected. Cellular apo B did not accumulate within cells. The majority of secretory lipid radioactivity synthesized from acetate was in VLDL density lipoproteins. The composition of newly synthesized lipids as assessed by thin layer chromatography was not significantly altered with insulin. These studies support the finding that insulin inhibits VLDL secretion by hepatocytes while at the same time stimulating overall triglyceride synthesis. A suggested mechanism is that insulin uncouples triglyceride and apo B synthesis, which influences subsequent lipoprotein assembly and secretory pathways. These results are consistent with the concept that postprandial insulin release inhibits hepatic lipoprotein secretion while intestinal lipoprotein metabolic pathways are most active.

Previous studies have demonstrated the presence of insulin receptors on the maternal surface of the placenta in several species and the specific binding of insulin to the placenta in sheep. However, both in-vitro and in-vivo studies have produced conflicting evidence concerning the effect of insulin on placental glucose uptake. To clarify this problem, we measured maternal hindlimb, uterine and fetal glucose and oxygen extractions and glucose/oxygen quotients in chronically catheterized, non-stressed, late-gestation pregnant sheep over 1 h at a constant concentration of arterial plasma glucose, and again during the next 2 h at the same glucose level but at a higher insulin concentration using glucose 'clamp' methodology. Insulin produced a 4.9-fold increase in glucose extraction and a 3.5-fold increase in glucose/oxygen quotient across the hindlimb; in contrast, insulin did not significantly affect uterine or fetal glucose extraction or glucose/oxygen quotient. We conclude that in contrast to other tissues of the pregnant ewe, placental glucose uptake and transfer are insensitive to variations in maternal insulin concentration.

To evaluate possible mechanisms by which insulin inhibits hepatic apolipoprotein B (apoB) secretion, we incubated primary cultures of rat hepatocytes with sodium orthovanadate, a phosphotyrosine phosphatase inhibitor and insulin-mimetic agent. Vanadate (10 microM) and insulin (10 nM) inhibited the medium accumulation of apoB (secretion) by 21 and 37%, respectively, without increasing intracellular apoB. The effects of insulin and vanadate together were not additive. Both insulin and vanadate enhanced intracellular glycogen accumulation by 82 and 37%, respectively. Unlike insulin, vanadate, at a concentration that inhibited apoB secretion (10 microM), had no effect on intracellular lipogenesis, inhibited the secretion of newly synthesized hepatic proteins, and had a delayed onset and termination of action on inhibition of apoB secretion. At higher concentrations (40 and 80 microM), vanadate stimulated intracellular lipogenesis. In conclusion, our data indicate that vanadate mimics insulin action in hepatocytes with regard to the inhibition of medium accumulation of apoB. These data are consistent with the hypothesis that inhibition of apoB secretion may be secondary to an increase in phosphotyrosine content at its site of synthesis. The kinases responsible for this effect have not been identified. Several effects of vanadate, however, are different from those of insulin, suggesting a differential sensitivity to vanadate, a divergence of the signal transfer by insulin and vanadate at the insulin-receptor or postreceptor level, or both.

Glucose clamp experiments were performed in 27 chronically catheterized, late-gestation fetal lambs in order to measure the effect of fetal insulin concentration on fetal glucose uptake at a constant glucose concentration. Fetal arterial blood glucose concentration was measured over a 30-min control period and then maintained at the control value by a variable glucose infusion into the fetus while insulin was infused at a constant rate into the fetus. Plasma insulin concentration increased from 21 +/- 10 (SD) to 294 +/- 179 (SD) microU X ml-1. The exogenous glucose infusion rate necessary to maintain constant glycemia during the plateau hyperinsulinemia averaged 4.3 +/- 1.6 (SD) mg X min-1 X kg-1. In a subset of 13 animals, total fetal exogenous glucose uptake (FGU; sum of glucose uptake from the placenta via the umbilical circulation plus the steady-state exogenous glucose infusion rate) was measured during the control and hyperinsulinemia period. FGU was directly related to insulin concentration (y = 4.24 + 0.07x) at insulin levels less than 100 microU/ml and increased 132% above control at insulin levels above 100 microU/ml. Hyperinsulinemia did not affect fetal glucose uptake from the placenta via the umbilical circulation. These studies demonstrate that insulin concentration is a major factor controlling glucose uptake in the near-term fetal lamb, and that an increase of fetal insulin does not affect the transport of glucose to the fetus from the placenta.

1. The effect of insulin on apolipoprotein (apo B) secretion was studied in 24 h recirculating liver perfusions of isolated normal, diabetic and insulin-treated diabetic rats. In single perfusions from each group apo B accumulated in the media in a linear fashion. 2 In perfusions of normal rat livers, when the medium contained insulin plus cortisol, apo B production was significantly inhibited (by 35.8%), demonstrating a hormone effect on apo B secretion. 3. In perfusions of diabetic-rat livers, apo B production was decreased to 11.8% of normal when the medium contained no hormones, and was not significantly changed by the addition of insulin plus cortisol to the medium, suggesting that the hormone effect on apo B secretion is missing in long-term hypoinsulinaemic states. 4. Treatment of diabetic rats with daily insulin injection restored apo B production and restored the effect of insulin plus cortisol in the medium to inhibit apo B secretion during perfusion. 5. Parallel studies of apo B secretion with insulin alone, cortisol alone and insulin plus cortisol in the medium were performed in primary cultures of hepatocytes to compare results from liver perfusions. 6. Apo B secretion by hepatocytes from normal, diabetic and treated-diabetic rats was inhibited (by 36.8%, 57.1% and 57.9% respectively) when insulin alone was added to the medium. 7. Insulin plus cortisol inhibited apo B secretion by hepatocytes from normal and treated diabetic rats (by 30.2% and 47.2% respectively), but failed to inhibit apo B secretion by hepatocytes from diabetic rats.