Janet D. Sparks

Insulin regulation of triacylglycerol-rich lipoprotein synthesis and secretion

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)

Effect of dietary fat on the development of non-insulin dependent diabetes mellitus in obese Zucker diabetic fatty male and female rats

The obese Zucker diabetic fatty male rat (ZDF/Gmi¿trade mark omitted¿-fa) has become a widely used animal model of NIDDM, in contrast to the obese ZDF females that rarely develop NIDDM. However, preliminary observations suggest that obese ZDF females may become diabetic on high-fat diets. Therefore, we studied the effect of dietary fat on development of NIDDM, dyslipidemia, and alterations in organ-specific serum panels in obese ZDF males and females. Results indicated different effects of dietary fat-content on development of diabetes in males and females. Males, even on low fat-content diets, developed diabetes but the process was accelerated as a function of dietary fat-content, whereas only the highest fat-content diet induced development of NIDDM in obese ZDF females. Additionally, triglyceride/apolipoprotein B ratios demonstrated gender-specific differences in the nature of circulating lipoprotein particles independent of diabetic state with values for females approximately twice those of males indicating more highly triglyceride-enriched lipoprotein particles in females. We conclude that the obese ZDF female rat has the potential to become an important animal model of NIDDM especially in women where few models currently exist.

Insulin and leptin resistance with hyperleptinemia in mice lacking androgen receptor

Epidemiological evidence suggests that sex differences exist in type 2 diabetes. Men seem to be more susceptible than women to the consequences of obesity and sedentary lifestyle, possibly because of differences in insulin sensitivity and regional body fat deposition. Thus, lacking androgen receptor (AR) in male individuals may promote insulin resistance. To determine whether lacking AR in male individuals contributes to in vivo insulin resistance, an AR knockout model (AR(-/y)) was used to study the correlation between AR and insulin resistance. Progressive reduced insulin sensitivity and impaired glucose tolerance were seen in AR(-/y) mice with advancing age. Aging AR(-/y) mice displayed accelerated weight gain, hyperinsulinemia, and hyperglycemia, and loss of AR contributes to increased triglyceride content in skeletal muscle and liver. Leptin is higher in serum of AR(-/y) mice. Treatment with exogenous leptin fails to stimulate weight loss in AR(-/y) mice in advanced age, suggesting leptin resistance in the AR(-/y/) mice. Exogenous dihydrotestosterone replacement fails to reverse the metabolic abnormalities and insulin resistance in AR(-/y) mice. Our in vivo studies demonstrate that androgen-AR plays key roles in the development of insulin and leptin resistance, which may contribute to the development of type 2 diabetes and cardiovascular disease.

Phosphoinositide 3-Kinase Activity Is Necessary for Insulin-dependent Inhibition of Apolipoprotein B Secretion by Rat Hepatocytes and Localizes to the Endoplasmic Reticulum

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.

Increased hepatic steatosis and insulin resistance in mice lacking hepatic androgen receptor

Early studies demonstrated that whole‐body androgen receptor (AR)–knockout mice with hypogonadism exhibit insulin resistance. However, details about the mechanisms underlying how androgen/AR signaling regulates insulin sensitivity in individual organs remain unclear. We therefore generated hepatic AR‐knockout (H‐AR−/y) mice and found that male H‐AR−/y mice, but not female H‐AR−/− mice, fed a high‐fat diet developed hepatic steatosis and insulin resistance, and aging male H‐AR−/y mice fed chow exhibited moderate hepatic steatosis. We hypothesized that increased hepatic steatosis in obese male H‐AR−/y mice resulted from decreased fatty acid β‐oxidation, increased de novo lipid synthesis arising from decreased PPARα, increased sterol regulatory element binding protein 1c, and associated changes in target gene expression. Reduced insulin sensitivity in fat‐fed H‐AR−/y mice was associated with decreased phosphoinositide‐3 kinase activity and increased phosphenolpyruvate carboxykinase expression and correlated with increased protein‐tyrosine phosphatase 1B expression. Conclusion: Together, our results suggest that hepatic AR may play a vital role in preventing the development of insulin resistance and hepatic steatosis. AR agonists that specifically target hepatic AR might be developed to provide a better strategy for treatment of metabolic syndrome in men. (HEPATOLOGY 2008.)

Cyclophilin A is an inflammatory mediator that promotes atherosclerosis in apolipoprotein E–deficient mice

Cyclophilin A promotes atherosclerosis in part by inducing reactive oxygen species and promoting endothelial cell apoptosis and macrophage recruitment into lesions.

Selective Hepatic Insulin Resistance, VLDL Overproduction, and Hypertriglyceridemia

Insulin plays a central role in regulating energy metabolism, including hepatic transport of very low-density lipoprotein (VLDL)–associated triglyceride. Hepatic hypersecretion of VLDL and consequent hypertriglyceridemia leads to lower circulating high-density lipoprotein levels and generation of small dense low-density lipoproteins characteristic of the dyslipidemia commonly observed in metabolic syndrome and type 2 diabetes mellitus. Physiological fluctuations of insulin modulate VLDL secretion, and insulin inhibition of VLDL secretion upon feeding may be the first pathway to become resistant in obesity that leads to VLDL hypersecretion. This review summarizes the role of insulin–related signaling pathways that determine hepatic VLDL production. Disruption in signaling pathways that reduce generation of the second messenger phosphatidylinositide (3,4,5) triphosphate downstream of activated phosphatidylinositide 3-kinase underlies the development of VLDL hypersecretion. As insulin resistance progresses, a number of pathways are altered that further augment VLDL hypersecretion, including hepatic inflammatory pathways. Insulin plays a complex role in regulating glucose metabolism, and it is not surprising that the role of insulin in VLDL and lipid metabolism will prove equally complex.

Insulin effects on apolipoprotein B lipoprotein synthesis and secretion by primary cultures of rat hepatocytes

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.

Loss of TR4 Orphan Nuclear Receptor Reduces Phosphoenolpyruvate Carboxykinase-Mediated Gluconeogenesis

OBJECTIVE—Regulation of phosphoenolpyruvate carboxykinase (PEPCK), the key gene in gluconeogenesis, is critical for glucose homeostasis in response to quick nutritional depletion and/or hormonal alteration. RESEARCH DESIGN/METHODS AND RESULTS— Here, we identified the testicular orphan nuclear receptor 4 (TR4) as a key PEPCK regulator modulating PEPCK gene via a transcriptional mechanism. TR4 transactivates the 490-bp PEPCK promoter-containing luciferase reporter gene activity by direct binding to the TR4 responsive element (TR4RE) located at −451 to −439 in the promoter region. Binding to TR4RE was confirmed by electrophoretic mobility shift and chromatin immunoprecipitation assays. Eliminating TR4 via knockout and RNA interference (RNAi) in hepatocytes significantly reduced the PEPCK gene expression and glucose production in response to glucose depletion. In contrast, ectopic expression of TR4 increased PEPCK gene expression and hepatic glucose production in human and mouse hepatoma cells. Mice lacking TR4 also display reduction of PEPCK expression with impaired gluconeogenesis. CONCLUSIONS—Together, both in vitro and in vivo data demonstrate the identification of a new pathway, TR4 → PEPCK → gluconeogenesis → blood glucose, which may allow us to modulate metabolic programs via the control of a new key player, TR4, a member of the nuclear receptor superfamily.

Impaired hepatic apolipoprotein B and E translation in streptozotocin diabetic rats.

Studies of streptozotocin-induced diabetes in rats have demonstrated that hepatic apo B and apo E production are reduced. To determine if reductions are related to decreases in hepatic mRNAs, we performed blotting analysis of total liver RNA with rat apo B, apo E, and albumin cDNA probes. The expected reduction in albumin mRNA levels to 48% of control livers occurred in diabetic rat liver, while apo B and apo E mRNA levels were unchanged. The proportion of translational stop codon (BSTOP) mRNA averaged 43% of total in diabetic rats similar to control levels. Long-term labeling experiments using [35S]methionine in primary cultures of rat hepatocytes and specific immunoprecipitations demonstrated production of apo B and apo E, and albumin by hepatocytes from diabetic rats was reduced to 37%, 53%, and 23% of controls. Pulse-chase studies, together with mRNA analyses, suggest that reduced hepatic secretion of apo B and apo E in diabetics is primarily a result of impaired translation and not intracellular degradation. Ribosome transit studies directly confirmed the prolonged elongation rates for apo B and apo E mRNAs in hepatocytes derived from diabetic rats. This effect was more pronounced on apo BH (higher molecular weight) than on apo BL (lower molecular weight). Treatment of diabetic rats with insulin for 7 d led to normalization of hepatic albumin mRNA levels with no substantial change in apo E mRNA levels. In contrast, insulin treatment resulted in significant increases in hepatic apo B mRNA over control levels. Results suggest hepatic albumin and apo B mRNA levels are responsive to insulin in the diabetic state.