Mary M. McGrane

Retinoid regulation of the phosphoenolpyruvate carboxykinase gene in liver

The cytosolic PEPCK gene is a model gene for assessing retinoid regulation of liver-specific genes encoding enzymes of carbohydrate metabolism. In vivo, we have demonstrated that the PEPCK gene is inhibited by vitamin A deficiency. Specifically, under conditions of food deprivation, induction of the PEPCK gene is inhibited in the vitamin A deficient mouse. Inhibition of the PEPCK gene by vitamin A deficiency is reversed by all-trans or 9-cis retinoic acid (RA) treatment. In a transgenic mouse model, a -460 and -355 bp PEPCK promoter fragment confers susceptibility to inhibition by vitamin A deficiency and responsiveness to all-trans RA treatment. However, there is a differential effect of 9-cis RA on the PEPCK promoter; the -460 fragment confers responsiveness to 9-cis RA, but the -355 fragment does not. Taken together, these results indicate that the PEPCK retinoic acid response element (RARE)1 is required for 9-cis RA induction-but not all-trans RA induction-of the PEPCK gene. In order to determine if vitamin A deficiency alters specific localized expression of the PEPCK gene in the periportal cells of the liver, the effect of vitamin A status on PEPCK localization in the liver was also measured. The PEPCK transgenes were expressed specifically in the periportal region of the liver acinus and although vitamin A deficiency caused a decrease in PEPCK transgene mRNA levels in periportal cells, it did not alter the periportal cell-specific pattern of expression. Retinoid treatment induced PEPCK transgene mRNA levels in the same population of cells, however, the -355 bp PEPCK promoter fragment did not respond to 9-cis RA treatment. In order to determine the nuclear transcription factor(s) responsible for retinoid regulation of the PEPCK gene in the liver, we investigated retinoic acid receptor (RAR)alpha and beta and the retinoid X receptor (RXR)alpha-the major retinoid receptors in liver-in terms of expression and the ability of the receptors to bind the PEPCK RAREs. Vitamin A deficiency significantly decreased hepatic RAR beta, but not RAR alpha or RXR alpha mRNA levels. In situ hybridization showed that RAR alpha, RAR beta and RXR alpha mRNAs were localized in the periportal region, however, immunohistochemistry showed that RAR alpha and RXR alpha were distributed evenly across the liver acinus, whereas only RAR beta levels were higher in periportal cells. The binding of nuclear receptors to PEPCK RARE1, RARE2 and RARE3 indicates a complex pattern of retinoid receptor and orphan nuclear receptor binding.

Altered lipid catabolism in the vitamin A deficient liver

The transcriptome pattern of metabolic genes in vitamin A deficient (VAD) liver has been compared to the vitamin A-sufficient (VAS) state using the Mouse 32k oligonucleotide (70mer) array. In VAD liver there was a decrease in expression of genes encoding enzymes of mitochondrial fatty acid (FA) oxidation; these genes included fatty acyl CoA ligase, carnitine o-palmitoyl transferase 1, medium chain acyl-CoA dehydrogenase, 3-ketoacyl CoA thiolase, and citrate synthase. Particularly affected was peroxisome metabolism, as genes encoding enzymes of peroxisomal FA oxidation and transport proteins were differentially expressed. These genes included those encoding acyl-CoA oxidase 1, the peroxisomal bifunctional enzyme, peroxisomal thiolase, and carnitine o-octanoyl transferase, the enzyme involved in shuttling FAs from the peroxisome to the mitochondrion. Most genes that were differentially expressed with chronic vitamin A depletion were responsive to treatment with all-trans retinoic acid (RA). Consistent with the decreased expression of genes involved in FA oxidation, we found an increase in hepatic macrocytic lipid accumulation and triglyceride levels. The relevant nuclear receptor gene that was differentially expressed in the VAD liver was that encoding the peroxisome proliferator-activated receptor (PPAR) alpha, the mRNA levels for which were decreased in VAD liver and increased with all-trans RA treatment. Down regulation of the PPAR alpha gene is the likely cause of the altered expression pattern of the above metabolic genes in VAD liver.

Raisins and walking alter appetite hormones and plasma lipids by modifications in lipoprotein metabolism and up-regulation of the low-density lipoprotein receptor

The purpose of this study was to determine the effects of consuming raisins, increasing steps walked, or a combination of these interventions on lipoprotein metabolism and appetite hormones by assessing plasma apolipoprotein concentrations, cholesterol ester transfer protein activity, low-density lipoprotein (LDL) receptor messenger RNA (mRNA) abundance, and plasma ghrelin and leptin concentrations. Thirty-four subjects (17 men and 17 postmenopausal women) were matched for weight and sex and randomly assigned to consume 1 cup raisins per day (RAISIN), increase the amount of steps walked per day (WALK), or a combination of both interventions (RAISIN + WALK). The subjects completed a 2-week run-in period, followed by a 6-week intervention. Ribonucleic acid was extracted from mononuclear cells, and LDL receptor mRNA abundance was quantified by use of reverse transcriptase polymerase chain reaction. Plasma apolipoproteins were measured by Luminex (Austin, TX) technology. Apoproteins A-1, B, C-II, and E and cholesterol ester transfer protein activity were not altered for any of the groups. In contrast, apolipoprotein C-III was significantly decreased by 12.3% only in the WALK group (P < .05). Low-density lipoprotein receptor mRNA abundance was increased for all groups after the intervention (P < .001). There was a significant group effect for plasma leptin (P = .026). Plasma concentrations increased for RAISIN and RAISIN + WALK. Similarly, plasma ghrelin concentrations were elevated postintervention for both groups consuming raisins (P < .05). These data suggest that walking and raisin consumption decrease plasma LDL cholesterol by up-regulating the LDL receptor and that raisin consumption may reduce hunger and affect dietary intake by altering hormones influencing satiety.

Low-carbohydrate diets reduce lipid accumulation and arterial inflammation in guinea pigs fed a high-cholesterol diet.

INTRODUCTIONnLow-carbohydrate diets (LCD) efficiently induce weight loss and favorably affect plasma lipids, however, the effect of LCD on atherosclerosis is still argued.nnnOBJECTIVEnTo evaluate the effect of LCD on the prevention of atherosclerosis.nnnMETHODSnTwenty guinea pigs were fed either a LCD or a low-fat diet (LFD) in combination with high-cholesterol (0.25g/100g) for 12 weeks. The percentage energy of macronutrient distribution was 10:65:25 for carbohydrate:fat:protein for the LCD, and 55:20:25 for the LFD. Plasma lipids were measured using colorimetric assays. Plasma and aortic oxidized (oxLDL) were quantified using ELISA methods. Inflammatory cytokines were measured in aortic homogenates using an immunoassay. H&E stained sections of aortic sinus and Schultz stained sections of carotid arteries were examined.nnnRESULTSnLDL cholesterol was lower in the LCD compared to the LFD group (71.9+/-34.8 vs. 81.7+/-26.9mg/dL; p=0.039). Aortic cholesterol was also lower in the LCD (4.98+/-1.3mg/g) compared to the LFD group (6.68+/-2.0mg/g); p<0.05. The Schultz staining method confirmed less aortic cholesterol accumulation in the LCD group. Plasma oxLDL did not differ between groups, however, aortic oxLDL was 61% lower in the LCD compared to the LFD group (p=0.045). There was a positive correlation (r=0.63, p=0.03) between oxLDL and cholesterol concentration in the aorta of LFD group, which was not observed in LCD group (r=-0.05, p=0.96). Inflammatory markers were reduced in guinea pigs from the LCD group (p<0.05) and they were correlated with the decreases in oxLDL in aorta.nnnCONCLUSIONnThese results suggest that LCD not only decreases lipid deposition, but also prevents the accumulation of oxLDL and reduces inflammatory cytokines within the arterial wall and may prevent atherosclerosis.

Carbohydrate restriction and dietary cholesterol modulate the expression of HMG-CoA reductase and the LDL receptor in mononuclear cells from adult men

The liver is responsible for controlling cholesterol homeostasis in the body. HMG-CoA reductase and the LDL receptor (LDL-r) are involved in this regulation and are also ubiquitously expressed in all major tissues. We have previously shown in guinea pigs that there is a correlation in gene expression of HMG-CoA reductase and the LDL-r between liver and mononuclear cells. The present study evaluated human mononuclear cells as a surrogate for hepatic expression of these genes. The purpose was to evaluate the effect of dietary carbohydrate restriction with low and high cholesterol content on HMG-CoA reductase and LDL-r mRNA expression in mononuclear cells. All subjects were counseled to consume a carbohydrate restricted diet with 10–15% energy from carbohydrate, 30–35% energy from protein and 55–60% energy from fat. Subjects were randomly assigned to either EGG (640 mg/d additional dietary cholesterol) or SUB groups [equivalent amount of egg substitute (0 dietary cholesterol contributions) per day] for 12 weeks. At the end of the intervention, there were no changes in plasma total or LDL cholesterol (LDL-C) compared to baseline (P > 0.10) or differences in plasma total or LDL-C between groups. The mRNA abundance for HMG-CoA reductase and LDL-r were measured in mononuclear cells using real time PCR. The EGG group showed a significant decrease in HMG-CoA reductase mRNA (1.98 ± 1.26 to 1.32 ± 0.92 arbitrary units P < 0.05) while an increase was observed for the SUB group (1.13 ± 0.52 to 1.69 ± 1.61 arbitrary units P < 0.05). Additionally, the LDL-r mRNA abundance was decreased in the EGG group (1.72 ± 0.69 to 1.24 ± 0.55 arbitrary units P < 0.05) and significantly increased in the SUB group (1.00 ± 0.60 to 1.67 ± 1.94 arbitrary units P < 0.05). The findings indicate that dietary cholesterol during a weight loss intervention alters the expression of genes regulating cholesterol homeostasis.

Low-carbohydrate diet disrupts the association between insulin resistance and weight gain

The cornerstone to treat metabolic syndrome and insulin resistance is dietary intervention. Both low-carbohydrate diet (LCD) and low-fat diet (LFD) have been reported to induce weight loss and improve these conditions. One of the factors associated with a subjects adherence to the diet is satiety. The aim of this study was to evaluate the effects of LCD and LFD on body weight, appetite hormones, and insulin resistance. Twenty guinea pigs were randomly assigned to LCD or LFD (60%:10%:30% or 20%:55%:25% of energy from fat/carbohydrate/protein, respectively) for 12 weeks. Weight and food intake were recorded every week. After this period, animals were killed and plasma was obtained to measure plasma glucose and insulin, appetite hormones, and ketone bodies. Guinea pigs fed LCD gained more weight than those fed LFD. The daily amount of food intake in grams was not different between groups, suggesting that food density and gastric distension played a role in satiety. There was no difference in leptin levels, which excludes the hypothesis of leptin resistance in the LCD group. However, plasma glucagon-like peptide-1 was 47.1% lower in animals fed LCD (P < .05). Plasma glucose, plasma insulin, and insulin sensitivity were not different between groups. However, the heavier animals that were fed LFD had impairment in insulin sensitivity, which was not observed in those fed LCD. These findings suggest that satiety was dependent on the amount of food ingested. The weight gain in animals fed LCD may be related to their greater caloric intake, lower levels of glucagon-like peptide-1, and higher protein consumption. The adoption of LCD promotes a unique metabolic state that prevents insulin resistance, even in guinea pigs that gained more weight. The association between weight gain and insulin resistance seems to be dependent on high carbohydrate intake.