David Wiggins

A role for PPARα in the control of SREBP activity and lipid synthesis in the liver

Inclusion of the PPARalpha (peroxisome-proliferator-activated receptor alpha) activator WY 14,643 in the diet of normal mice stimulated the hepatic expression of not only genes of the fatty acid oxidation pathway, but also those of the de novo lipid synthetic pathways. Induction of fatty acid synthase mRNA by WY 14,643 was greater during the light phase of the diurnal cycle, when food intake was low and PPARalpha expression was high. Hepatic fatty acid pathway flux in vivo showed a similar pattern of increases. The abundance of mRNAs for genes involved in hepatic cholesterol synthesis was also increased by WY 14,643, but was associated with a decrease in cholesterogenic carbon flux. None of these changes were apparent in PPARalpha-null mice. Mice of both genotypes showed the expected decreases in 3-hydroxy-3-methylglutaryl-CoA reductase mRNA levels and cholesterol synthesis in response to an increase in dietary cholesterol. The increase in fatty acid synthesis due to WY 14,643 was not mediated by increased expression of SREBP-1c (sterol regulatory element binding protein-1c) mRNA, but by an increase in cleavage of the protein to the active form. An accompanying rise in stearoyl-CoA desaturase mRNA expression suggested that the increase in lipogenesis could have resulted from an alteration in membrane fatty acid composition that influenced SREBP activation.

Inhibition of cholesterol absorption associated with a PPARα-dependent increase in ABC binding cassette transporter A1 in mice

Dietary supplementation with the peroxisome proliferator-activated receptor α (PPARα) ligand WY 14,643 gave rise to a 4- to 5-fold increase in the expression of mRNA for the ATP binding cassette transporter A1 (ABCA1) in the intestine of normal mice. There was no effect in the intestine of PPARα-null mice. Consumption of a high-cholesterol diet also increased intestinal ABCA1 expression. The effects of WY 14,643 and the high-cholesterol diet were not additive. WY 14,643 feeding reduced intestinal absorption of cholesterol in the normal mice, irrespective of the dietary cholesterol concentration, and this resulted in lower diet-derived cholesterol and cholesteryl ester concentrations in plasma and liver. At each concentration of dietary cholesterol, there was a similar significant inverse correlation between intestinal ABCA1 mRNA content and the amount of cholesterol absorbed. The fibrate-induced changes in the intestines of the normal mice were accompanied by an increased concentration of the mRNA encoding the sterol-regulatory element binding protein-1c gene (SREBP-1c), a known target gene for the oxysterol receptor liver X receptor α (LXRα). There was a correlation between intestinal ABCA1 mRNA and SREBP-1c mRNA contents, but not between SREBP-1c mRNA content and cholesterol absorption. These results suggest that PPARα influences cholesterol absorption through modulating ABCA1 activity in the intestine by a mechanism involving LXRα.

Intracellular triacylglycerol lipase: Its role in the assembly of hepatic very-low-density lipoprotein (VLDL)

Extracellular fatty acids entering the hepatocyte are either esterified to cytosolic TAG or oxidized to ketone bodies. Very little is esterified and secreted directly in association with VLDL. Thus, even when extracellular fatty acids are available, the major, direct source of VLDL TAG is the cytosolic pool. The recruitment of cytosolic TAG for VLDL assembly involves lipolysis followed by re-esterification. At least 70% of the secreted TAG is derived via this route. Fatty acids released at this lipolytic step are utilized exclusively for VLDL TAG synthesis and are not available for ketogenesis. Substantially more cytosolic TAG undergoes lipolysis than is required to meet the needs of VLDL assembly. The remaining fatty acids are re-esterified and re-cycled to the cell cytosol. From a physiological viewpoint, the presence of this indirect route for VLDL TAG recruitment would provide a means of regulation of VLDL secretion which is independent of the plasma fatty acid concentration. In this respect, several pathophysiological conditions are known in which there is a negative association between plasma fatty acid concentration and the rate of VLDL secretion. These are: (a) insulin-dependent diabetes, (b) starvation, (c) fat-feeding. Lipolysis of cytosolic TAG and transfer of fatty acids into the ER lumen may provide a regulatory focus for the control of hepatic VLDL output.

Peroxisome Proliferator-activated Receptor α Deficiency Abolishes the Response of Lipogenic Gene Expression to Re-feeding RESTORATION OF THE NORMAL RESPONSE BY ACTIVATION OF LIVER X RECEPTOR α

The mRNA expression of lipogenic genes Scd-1 and Fas is regulated partly by the insulin-sensitive transcription factor SREBP-1c and liver X receptor α (LXRα). Compared with normal mice, the increase in the mRNA expression of hepatic Scd-1, Fas, and Srebp-1c was severely attenuated in peroxisome proliferator-activated receptor α (PPARα)-deficient mice during the transition from the starved to the re-fed states. The concentration of the membrane-bound form of SREBP-1c was also lower in the livers of the PPARα-deficient mice during re-feeding but there was little difference in the concentration of the active, nuclear form, or in the abundance of Insig-2a mRNA. The response of plasma insulin to starvation and re-feeding was normal in the PPARα-deficient mice. Rat hepatocytes transfected with an adenovirus encoding a dominant negative form of PPARα were resistant to the stimulatory effects of insulin on Fas and Scd-1 mRNA expression in vitro. When LXRα was activated in vivo by inclusion of a non-steroidal ligand in the diet, the expression of the mRNA for hepatic Srebp-1c, Fas, and Scd-1 was increased severalfold in mice of both genotypes and resistance associated with PPARα deficiency was abolished during re-feeding. However, although re-feeding the LXRα ligand induced the immature form of SREBP-1c equally in the livers of both genotypes, the concentration of the nuclear form remained relatively low in the livers of the PPARα-deficient mice. We conclude that intact PPARα is required to mediate the response of Scd-1 and Fas gene expression to insulin and that this is normally achieved directly by activation of LXRα.

Glucose Phosphorylation Is Essential for the Turnover of Neutral Lipid and the Second Stage Assembly of Triacylglycerol-Rich ApoB-Containing Lipoproteins in Primary Hepatocyte Cultures

Primary hepatocytes cultured in a medium supplemented with amino acids and lipogenic substrates responded to increased extracellular glucose by increasing the secretion of VLDL apoB. This effect was accompanied by an increased secretion of VLDL triacylglycerol (TAG) derived from endogenous stores. Glucose also stimulated intracellular TAG mobilization via the TAG lipolysis/esterification cycle. All these effects were abolished in the presence of mannoheptulose (MH), an inhibitor of glucose phosphorylation. Glucose also gave rise to a modest (50% to 60%) increase in the incorporation of 35S methionine into newly synthesized apoB (P<0.05) and to a doubling of newly-synthesized apoB secretion as VLDL (P<0. 05). The magnitude of these effects was similar for apoB-48 and for apoB-100. MH inhibited apoB-48 and apoB-100 synthesis and VLDL secretion at all glucose concentrations. The effects of glucose and MH on the secretion of newly-synthesized apoB-48 or apoB-100 as small dense particles were less pronounced. Glucose had no effects on the posttranslational degradation of newly-synthesized apoB-100 or apoB-48. However, this process was significantly enhanced by MH. The results suggest that glucose stimulates TAG synthesis, turnover, and output as VLDL. These effects are associated with an increased VLDL output of apoB mediated mainly by an increase in the net synthesis of both apoB-48 and apoB-100. All these changes are prevented by interference with glucose phosphorylation. Output of small, dense, apoB-containing particles is relatively unaffected by the glucose and MH-induced changes in TAG synthesis and lipolysis, an observation which suggests that only the bulk lipid addition step of VLDL assembly is affected by changes in glucose metabolism.

Inhibition of Hepatocyte Lipogenesis by Nitric Oxide Donor: Could Nitric Oxide Regulate Lipid Synthesis?

Tissue lipogenesis is variably controlled by substrate supply and hormones. The possibility that nitric oxide (NO) might regulate lipogenesis derives from the action of NO on coenzyme A (CoA) to produce metabolically inactive S‐nitrosoCoA. The effect of the nitric oxide donor S‐nitrosoglutathione (GSNO) on long chain fatty acid and cholesterol synthesis was measured in isolated cultured rat hepatocytes. [1‐14C] Butyrate was used as substrate to measure 14C incorporation into lipids as butyrate is twice as effective as acetate in hepatic lipogenesis and is ketogenic via the Lynen cycle. NO very significantly (P < 0.01) impaired long chain fatty acid and cholesterol synthesis an observation dependent upon time of exposure (3 h pre‐incubation or 6 h continuous exposure) and concentration of GSNO (500 μM to 2.0 mM). Decrease in hepatic lipogenesis was paralleled by decrease in ketogenesis. ATP levels remained unchanged following short term exposure to GSNO. Exposure of hepatocytes to GSNO together with 2.0 mM glutathione significantly diminished the inhibition of lipogenesis induced by GSNO alone. Impairment of lipogenesis by GSNO appears not to be limited by energy supply and now adduced, but not proven, to be operative via the degree of inactivation of cytosolic CoA. NO control of lipogenesis could be clinically important where NO production is increased as in demyelinating diseases, chronic arthritis or colitis and in wasting diseases such as AIDS. IUBMB Life, 56: 35‐40, 2004

PYY-Dependent Restoration of Impaired Insulin and Glucagon Secretion in Type 2 Diabetes following Roux-En-Y Gastric Bypass Surgery

Summary Roux-en-Y gastric bypass (RYGB) is a weight-reduction procedure resulting in rapid resolution of type 2 diabetes (T2D). The role of pancreatic islet function in this restoration of normoglycemia has not been fully elucidated. Using the diabetic Goto-Kakizaki (GK) rat model, we demonstrate that RYGB restores normal glucose regulation of glucagon and insulin secretion and normalizes islet morphology. Culture of isolated islets with serum from RYGB animals mimicked these effects, implicating a humoral factor. These latter effects were reversed following neutralization of the gut hormone peptide tyrosine tyrosine (PYY) but persisted in the presence of a glucagon-like peptide-1 (GLP-1) receptor antagonist. The effects of RYGB on secretion were replicated by chronic exposure of diabetic rat islets to PYY in vitro. These findings indicate that the mechanism underlying T2D remission may be mediated by PYY and suggest that drugs promoting PYY release or action may restore pancreatic islet function in T2D.

Decreased sensitivity to the inhibitory effect of insulin on the secretion of very—low-density lipoprotein in cultured hepatocytes from fructose-fed rats

Hepatocytes were prepared from rats fed a chow diet (control-fed) and from rats fed a similar diet in which the drinking water contained 10% (wt/vol) fructose (fructose-fed). Both types of hepatocyte preparations were cultured for < or = 48 hours in supplemented Waymouths medium containing increasing concentrations of bovine insulin (0 to 780 nmol/L). During the first 24 hours of culture, hepatocytes from fructose-fed rats secreted more very-low-density lipoprotein (VLDL) triacylglycerol (TAG) than hepatocytes from control-fed rats. This difference persisted at all concentrations of insulin. There was no difference in the rate of secretion of apolipoprotein B (apo B). In both control-fed and fructose-fed animals, the inhibitory effect of insulin on the secretion of VLDL was greater on the second versus the first day of culture. Under these conditions, hepatocytes from fructose-fed groups were less sensitive to insulin inhibition as compared with those from the control-fed group. This was evidenced by the following: (1) the decreased inhibitory effect of insulin on the secretion of both total and newly synthesized VLDL TAG, (2) the attenuated inhibitory effect of insulin on the secretion of VLDL apo B, (3) the decreased potency of insulin in suppressing the secretion of VLDL TAG in TAG-depleted hepatocytes from fructose-fed as compared with control-fed animals, and (4) the larger proportion of newly synthesized TAG secreted as VLDL in hepatocytes from fructose-fed rats as compared with controls. This difference was exacerbated at higher concentrations of insulin.(ABSTRACT TRUNCATED AT 250 WORDS)

Manipulation of cholesterol and cholesteryl ester synthesis has multiple effects on the metabolism of apolipoprotein B and the secretion of very-low-density lipoprotein by primary hepatocyte cultures.

Inhibition of esterified and non-esterified cholesterol synthesis by lovastatin in primary rat hepatocytes suppressed the net synthesis and very-low-density lipoprotein (VLDL) secretion of apolipoprotein B (apoB)-48 and apoB-100. Lovastatin did not alter the rates of apoB-48 and apoB-100 post-translational degradation. 25-Hydroxycholesterol, which inhibited non-esterified cholesterol synthesis but increased the synthesis of cholesteryl ester, showed differential effects on the metabolism of apoB-48 and apoB-100. Whereas the secretion of apoB-48 VLDL was suppressed there was no effect on the secretion of apoB-100 VLDL. The post-translational degradation of apoB-48, but not of apoB-100, was enhanced by 25-hydroxycholesterol. The net synthesis rates of apoB-48 and apoB-100 were unaffected by 25-hydroxycholesterol. The inhibitory effect of lovastatin alone on the net synthesis of apoB-48 and apoB-100 was reversed by the simultaneous presence of 25-hydroxycholesterol, suggesting a role for newly synthesised cholesteryl ester. Prevention of the reversal effect by the acyl-CoA: cholesterol acyltransferase (ACAT) inhibitor YM 17E supported this interpretation. In the presence of lovastatin, restoration of the net synthesis of apoB by 25-hydroxycholesterol was not accompanied by an increased VLDL output of apoB-48 and apoB-100. However, under these conditions there was an increased post-translational degradation of apoB-48 and apoB-100. These results suggest that interference with intracellular cholesterol and cholesteryl ester metabolism interrupts VLDL assembly at sites of both apoB net synthesis and post-translational degradation.

Metabolic characteristics of a human hepatoma cell line stably transfected with hormone-sensitive lipase.

Clones of HepG2 cells were selected that stably express the cDNA for hormone-sensitive lipase (HSL). When cells were cultured in the presence of labelled extracellular oleate, accumulation of labelled fatty acid as cellular triacylglycerol (TAG) was significantly lower in the transfectants compared with the wild-type cells. There was no change in the net rate of phospholipid (PL) synthesis. Culture of cells containing isotopically prelabelled TAG resulted in a greater net loss of TAG from the transfected cells than from the wild-type cells. The excess loss of labelled TAG was primarily due to an increased TAG fatty acid oxidation. Free fatty acid release into the medium was not increased in the transfectants, nor was the very low rate of lipoprotein lipid secretion. Also, there was no increased net trafficking of fatty acids from TAG into PLs. Changes in the 3H:14C ratio of TAG prelabelled with [3H]glycerol and [14C]oleate suggested that none of excess TAG fatty acid released in the transfected cells underwent intracellular re-esterification to TAG prior to oxidation. The results suggest that fatty acids mobilized by HSL are directed immediately into the oxidative pathway and are not available for biosynthetic processes. It appears likely, therefore, that intracellular TAG-derived fatty acids which enter the oxidative pathway exist in a different compartment from those that are directed towards synthesis.