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.

We have previously reported a positive correlation between the expression of BHMT (betaine-homocysteine S-methyltransferase) and ApoB (apolipoprotein B) in rat hepatoma McA (McArdle RH-7777) cells [Sowden, Collins, Smith, Garrow, Sparks and Sparks (1999) Biochem. J. 341, 639-645]. To examine whether a similar relationship occurs in vivo, hepatic BHMT expression was induced by feeding rats a Met (L-methionine)-restricted betaine-containing diet, and parameters of ApoB metabolism were evaluated. There were no generalized metabolic abnormalities associated with Met restriction for 7 days, as evidenced by control levels of serum glucose, ketones, alanine aminotransferase and L-homocysteine levels. Betaine plus the Met restriction resulted in lower serum insulin and non-esterified fatty acid levels. Betaine plus Met restriction induced hepatic BHMT 4-fold and ApoB mRNA 3-fold compared with Met restriction alone. No changes in percentage of edited ApoB mRNA were observed on the test diets. An increase in liver ApoB mRNA correlated with an 82% and 46% increase in ApoB and triacylglycerol production respectively using in vivo Triton WR 1339. Increased secretion of VLDL (very-low-density lipoprotein) with Met restriction plus betaine was associated with a 45% reduction in liver triacylglycerol compared with control. Nuclear run-off assays established that transcription of both bhmt and apob genes was also increased in Met-restricted plus betaine diets. No change in ApoB mRNA stability was detected in BHMT-transfected McA cells. Hepatic ApoB and BHMT mRNA levels were also increased by 1.8- and 3-fold respectively by betaine supplementation of Met-replete diets. Since dietary betaine increased ApoB mRNA, VLDL ApoB and triacylglycerol production and decreased hepatic triacylglycerol, results suggest that induction of apob transcription may provide a potential mechanism for mobilizing hepatic triacylglycerol by increasing ApoB available for VLDL assembly and secretion.

Insulin regulates hepatic VLDL production by activation of phosphatidylinositide 3-kinase (PI3-kinase) which decreases apo B available for lipid assembly. The current study evaluated the dependence of the VLDL apolipoprotein B (apo B) pathway on PI3-kinase activity in vivo. VLDL production was examined in B100 only, apo B mRNA editing catalytic subunit 1 (apobec-1 Ϫ/Ϫ ) mice, using the Triton WR 1339 method. Glucose injection suppressed VLDL triglyceride production by 28% in male and by 32% in female mice compared with salineinjected controls. When wortmannin was injected to inhibit PI3-kinase, VLDL triglyceride production was increased by 52% in males and by 89% in females, and VLDL B100 levels paralleled triglyceride changes. Pulse-chase experiments in primary mouse hepatocytes showed that wortmannin increased net freshly synthesized B100 availability by Ͼ35%. To test whether physiological insulin resistance produced equivalent effects to wortmannin, we studied male apobec-1 Ϫ/Ϫ mice who became hyperlipidemic on being fed a fructoseenriched diet. Fructose-fed apobec-1 Ϫ/Ϫ mice had significantly higher VLDL triglyceride and B100 production rates compared with chowfed mice, and rates were refractile to glucose or wortmannin. Hepatic VLDL triglyceride and B100 production in wortmannin-injected chow-fed mice equaled that observed in fructose-fed mice. Together, results suggest in vivo and in vitro that wortmannin-sensitive PI3-kinases maintain a basal level of VLDL suppression that is sensitive to changes in activation and that can increase VLDL production when PI3-kinase is inhibited to levels similar to those induced by insulin resistance.very low-density lipoprotein INSULIN RESISTANCE IS A KEY component of metabolic syndrome (20,26). The dyslipidemia associated with insulin resistance is characterized by hyperinsulinemia and hypertriglyceridemia that results from enhanced VLDL production by the liver (15). Apolipoprotein B (apo B) is a structural protein necessary for the assembly of VLDL by the liver and for that of chylomicrons (CM) by the intestine (9). Two forms of apo B are synthesized, B100 and B48, through a process involving apo B mRNA editing and apo B mRNA editing catalytic subunit 1 (apobec-1) (reviewed in Ref.2). Assembly of VLDL particles and VLDL secretion are complex processes involving multiple mechanisms that control apo B stability and degradation (12, 13). Regulation of triglyceride, phospholipid, and apo B components of VLDL particles are asynchronous and appear to involve independent control mechanisms (14). Recent studies from our laboratory (6) suggest that in insulin resistance states, hepatic output of VLDL apo B and triglyceride (TG) is increased, which involves altered posttranscriptional regulation of apo B availability and transcriptional changes mediated through sterol regulatory element binding proteins that regulate lipogenesis (19) and increase TG production.Insulin inhibits the secretion of VLDL apo B by perfused rat liver (34), by primary rat (29, 33), human hepatocytes (27), ...

diabetic fatty (ZDF) rats, a model of insulin-resistant type 2 diabetes progression. TRL production was measured in vivo by blocking catabolism with Triton WR-1339. Ten-week ZDF rats are hyperinsulinemic with increased TRL production [both triglyceride and apolipoprotein B (apoB)]. Twenty-week ZDF rats are insulinopenic, and TRL production is similar to lean controls. Insulin infusion suppresses glucose and free fatty acids in 10-and 20-wk ZDF rats. Increased TRL production is not reduced by insulin in 10-wk rats; however, at 20 wk, TRL production is suppressed by insulin. In vitro studies with hepatocytes derived from 10-wk ZDF rats showed minimal insulin dose effects on apoB secretion compared with the response and sensitivity of hepatocytes derived from 20-wk ZDF and control lean rats. Hepatic sterol regulatory-binding protein (SREBP)-1c mRNA levels are increased at 10 wk but return to control levels at 20 wk. ApoB mRNA levels are similar to lean controls at 10 and 20 wk. The following two mechanisms for hypertriglyceridemia associated with hyperinsulinemia are suggested: increased TRL synthesis and loss of TRL suppression. Increased triglyceride production in hyperinsulinemic rats likely relates to increased expression of SREBP-1c, whereas increased apoB production involves posttranscriptional processes. hyperinsulinemic diabetes; insulinopenic diabetes; apolipoprotein b metabolism; triglyceride-rich lipoprotein production HYPERTRIGLYCERIDEMIA IS A key component of metabolic syndrome and type 2 diabetes and is strongly correlated with increased risk of cardiovascular disease (12). Insulin increases the intracellular availability of triglyceride (TG) for very low density lipoprotein (VLDL) production (1), possibly through enhanced lipogenesis (23). Pancreatic availability of insulin is diminished as disease progresses, with late-stage diabetes being characterized by insulinopenic hyperglycemia. Previous in vitro studies performed in cultured hepatocytes isolated from fructose-fed, insulin-resistant rats demonstrate blunting of the acute suppressive effects of insulin on VLDL secretion (37). Similar results are observed in hepatocytes from insulin-resistant obese rats (31). Furthermore, insulin fails to suppress hepatic VLDL apolipoprotein B (apoB) production in diabetic patients (16) and in hyperinsulinemic individuals during euglycemic clamp experiments (13,21). It is known that, during late-stage diabetes, pancreatic insulin release is progressively diminished; however, the effect of insulin on liver VLDL production during this transition from hyperinsulinemia to insulinopenia is not known.Hepatic expression of key enzymes involved in lipogenesis and cholesterogenesis is regulated by a family of transcription factors known as sterol regulatory element-binding proteins (SREBP; see Ref. 9). SREBP-1c is mainly involved in regulation of TG-synthesizing genes, whereas SREBP-2 has its main effect on cholesterol-synthesizing genes. SREBP-1c has been implicated as a mediator of insulin action in hepatic lipogenesis...

Both in humans and animal models, an acute increase in plasma insulin levels, typically following meals, leads to transient depression of hepatic secretion of very low density lipoproteins (VLDL). One contributing mechanism for the decrease in VLDL secretion is enhanced degradation of apolipoprotein B100 (apoB100), which is required for VLDL formation. Unlike the degradation of nascent apoB100, which occurs in the endoplasmic reticulum (ER), insulin-stimulated apoB100 degradation occurs post-ER and is inhibited by pan-phosphatidylinositol (PI)3-kinase inhibitors. It is unclear, however, which of the three classes of PI3-kinases is required for insulin-stimulated apoB100 degradation, as well as the proteolytic machinery underlying this response. Class III PI3-kinase is not activated by insulin, but the other two classes are. By using a class I-specific inhibitor and siRNA to the major class II isoform in liver, we now show that it is class II PI3-kinase that is required for insulin-stimulated apoB100 degradation in primary mouse hepatocytes. Because the insulin-stimulated process resembles other examples of apoB100 post-ER proteolysis mediated by autophagy, we hypothesized that the effects of insulin in autophagy-deficient mouse primary hepatocytes would be attenuated. Indeed, apoB100 degradation in response to insulin was significantly impaired in two types of autophagy-deficient hepatocytes. Together, our data demonstrate that insulin-stimulated apoB100 degradation in the liver requires both class II PI3-kinase activity and autophagy.