Silvia Bijland

Perfluoroalkyl sulfonates cause alkyl chain length-dependent hepatic steatosis and hypolipidemia mainly by impairing lipoprotein production in APOE*3-leiden CETP mice

Perfluorobutane sulfonate (PFBS), perfluorohexane sulfonate (PFHxS), and perfluorooctane sulfonate (PFOS) are stable perfluoroalkyl sulfonate (PFAS) surfactants, and PFHxS and PFOS are frequently detected in human biomonitoring studies. Some epidemiological studies have shown modest positive correlations of serum PFOS with non-high-density lipoprotein (HDL)-cholesterol (C). This study investigated the mechanism underlying the effect of PFAS surfactants on lipoprotein metabolism. APOE*3-Leiden.CETP mice were fed a Western-type diet with PFBS, PFHxS, or PFOS (30, 6, and 3 mg/kg/day, respectively) for 4-6 weeks. Whereas PFBS modestly reduced only plasma triglycerides (TG), PFHxS and PFOS markedly reduced TG, non-HDL-C, and HDL-C. The decrease in very low-density lipoprotein (VLDL) was caused by enhanced lipoprotein lipase-mediated VLDL-TG clearance and by decreased production of VLDL-TG and VLDL-apolipoprotein B. Reduced HDL production, related to decreased apolipoprotein AI synthesis, resulted in decreased HDL. PFHxS and PFOS increased liver weight and hepatic TG content. Hepatic gene expression profiling data indicated that these effects were the combined result of peroxisome proliferator-activated receptor alpha and pregnane X receptor activation. In conclusion, the potency of PFAS to affect lipoprotein metabolism increased with increasing alkyl chain length. PFHxS and PFOS reduce plasma TG and total cholesterol mainly by impairing lipoprotein production, implying that the reported positive correlations of serum PFOS and non-HDL-C are associative rather than causal.

High levels of dietary stearate promote adiposity and deteriorate hepatic insulin sensitivity

BackgroundRelatively little is known about the role of specific saturated fatty acids in the development of high fat diet induced obesity and insulin resistance. Here, we have studied the effect of stearate in high fat diets (45% energy as fat) on whole body energy metabolism and tissue specific insulin sensitivity.MethodsC57Bl/6 mice were fed a low stearate diet based on palm oil or one of two stearate rich diets, one diet based on lard and one diet based on palm oil supplemented with tristearin (to the stearate level of the lard based diet), for a period of 5 weeks. Ad libitum fed Oxidative metabolism was assessed by indirect calorimetry at week 5. Changes in body mass and composition was assessed by DEXA scan analysis. Tissue specific insulin sensitivity was assessed by hyperinsulinemic-euglycemic clamp analysis and Western blot at the end of week 5.ResultsIndirect calorimetry analysis revealed that high levels of dietary stearate resulted in lower caloric energy expenditure characterized by lower oxidation of fatty acids. In agreement with this metabolic phenotype, mice on the stearate rich diets gained more adipose tissue mass. Whole body and tissue specific insulin sensitivity was assessed by hyperinsulinemic-euglycemic clamp and analysis of insulin induced PKBser473 phosphorylation. Whole body insulin sensitivity was decreased by all high fat diets. However, while insulin-stimulated glucose uptake by peripheral tissues was impaired by all high fat diets, hepatic insulin sensitivity was affected only by the stearate rich diets. This tissue-specific pattern of reduced insulin sensitivity was confirmed by similar impairment in insulin-induced phosphorylation of PKBser473 in both liver and skeletal muscle.ConclusionIn C57Bl/6 mice, 5 weeks of a high fat diet rich in stearate induces a metabolic state favoring low oxidative metabolism, increased adiposity and whole body insulin resistance characterized by severe hepatic insulin resistance. These results indicate that dietary fatty acid composition per sé rather than dietary fat content determines insulin sensitivity in liver of high fat fed C57Bl/6 mice.

An 8-week high-fat diet induces obesity and insulin resistance with small changes in the muscle transcriptome of C57BL/6J mice.

Background: Skeletal muscle is responsible for most of the insulin-stimulated glucose uptake and metabolism. Therefore, it plays an important role in the development of insulin resistance, one of the characteristics of the metabolic syndrome (MS). As the prevalence of the MS is increasing, there is an urgent need for more effective intervention strategies. Methods: C57BL/6J mice were fed an 8-week low-fat diet (10 kcal%; LFD) or high-fat diet (45 kcal%; HFD). Microarray analysis was performed by using two comparisons: (1) 8-week HFD transcriptome versus 8-week LFD transcriptome and (2) transcriptome of mice sacrificed at the start of the intervention versus 8-week LFD transcriptome and 8-week HFD transcriptome, respectively. Results: Although an 8-week HFD induced obesity and impaired insulin sensitivity, HFD-responsive changes in the muscle transcriptome were relatively small (<1.3-fold). In fact, 8-weeks of aging induced more pronounced changes than an HFD. One comparison revealed the transcriptional downregulation of the mito- gen-activated protein kinase cascade, whereas both comparisons showed the upregulation of fatty acid oxidation, demonstrating that the two comparison strategies are confirmative as well as complementary. Conclusion: We suggest using complementary analysis strategies in the genome-wide search for gene expression changes induced by mild interventions, such as an HFD.

High-fat diets rich in medium- versus long-chain fatty acids induce distinct patterns of tissue specific insulin resistance

Excess dietary long-chain fatty acid (LCFA) intake results in ectopic lipid accumulation and insulin resistance. Since medium-chain fatty acids (MCFA) are preferentially oxidized over LCFA, we hypothesized that diets rich in MCFA result in a lower ectopic lipid accumulation and insulin resistance compared to diets rich in LCFA. Feeding mice high-fat (HF) (45% kcal fat) diets for 8 weeks rich in triacylglycerols composed of MCFA (HFMCT) or LCFA (HFLCT) revealed a lower body weight gain in the HFMCT-fed mice. Indirect calorimetry revealed higher fat oxidation on HFMCT compared to HFLCT (0.011.0±0.0007 vs. 0.0096±0.0015 kcal/g body weight per hour, P<.05). In line with this, neutral lipid immunohistochemistry revealed significantly lower lipid storage in skeletal muscle (0.05±0.08 vs. 0.30±0.23 area%, P <.05) and in liver (0.9±0.4 vs. 6.4±0.8 area%, P<.05) after HFMCT vs. HFLCT, while ectopic fat storage in low fat (LF) was very low. Hyperinsulinemic euglycemic clamps revealed that the HFMCT and HFLCT resulted in severe whole body insulin resistance (glucose infusion rate: 53.1±6.8, 50.8±15.3 vs. 124.6±25.4 μmol min(-1) kg(-1), P<.001 in HFMCT, HFLCT and LF-fed mice, respectively). However, under hyperinsulinemic conditions, HFMCT revealed a lower endogenous glucose output (22.6±8.0 vs. 34.7±8.5 μmol min(-1) kg(-1), P<.05) and a lower peripheral glucose disappearance (75.7±7.8 vs. 93.4±12.4 μmol min(-1) kg(-1), P<.03) compared to HFLCT-fed mice. In conclusion, both HF diets induced whole body insulin resistance compared to LF. However, the HFMCT gained less weight, had less ectopic lipid accumulation, while peripheral insulin resistance was more pronounced compared to HFLCT. This suggests that HF-diets rich in medium- versus long-chain triacylglycerols induce insulin resistance via distinct mechanisms.

Fenofibrate increases very low density lipoprotein triglyceride production despite reducing plasma triglyceride levels in APOE*3-Leiden.CETP mice

The peroxisome proliferator-activated receptor alpha (PPARα) activator fenofibrate efficiently decreases plasma triglycerides (TG), which is generally attributed to enhanced very low density lipoprotein (VLDL)-TG clearance and decreased VLDL-TG production. However, because data on the effect of fenofibrate on VLDL production are controversial, we aimed to investigate in (more) detail the mechanism underlying the TG-lowering effect by studying VLDL-TG production and clearance using APOE*3-Leiden.CETP mice, a unique mouse model for human-like lipoprotein metabolism. Male mice were fed a Western-type diet for 4 weeks, followed by the same diet without or with fenofibrate (30 mg/kg bodyweight/day) for 4 weeks. Fenofibrate strongly lowered plasma cholesterol (−38%) and TG (−60%) caused by reduction of VLDL. Fenofibrate markedly accelerated VLDL-TG clearance, as judged from a reduced plasma half-life of glycerol tri[3H]oleate-labeled VLDL-like emulsion particles (−68%). This was associated with an increased post-heparin lipoprotein lipase (LPL) activity (+110%) and an increased uptake of VLDL-derived fatty acids by skeletal muscle, white adipose tissue, and liver. Concomitantly, fenofibrate markedly increased the VLDL-TG production rate (+73%) but not the VLDL-apolipoprotein B (apoB) production rate. Kinetic studies using [3H]palmitic acid showed that fenofibrate increased VLDL-TG production by equally increasing incorporation of re-esterified plasma fatty acids and liver TG into VLDL, which was supported by hepatic gene expression profiling data. We conclude that fenofibrate decreases plasma TG by enhancing LPL-mediated VLDL-TG clearance, which results in a compensatory increase in VLDL-TG production by the liver.

High levels of whole-body energy expenditure are associated with a lower coupling of skeletal muscle mitochondria in C57Bl/6 mice

Considerable variation in energy expenditure is observed in C57Bl/6 mice on a high-fat diet. Because muscle tissue is a major determinant of whole-body energy expenditure, we set out to determine the variation in energy expenditure and its possible association with skeletal muscle mitochondrial function upon high-fat diet intervention. Metabolic cages using indirect calorimetry were used to assess whole-body energy metabolism in C57Bl/6 male mice during the first 3 days of high-fat diet intervention. Mice were grouped in a negative or positive residual nocturnal energy expenditure group after correction of total nocturnal energy expenditure for body mass by residual analysis. The positive residual energy expenditure group was characterized by higher uncorrected total nocturnal energy expenditure and food intake. On day 7, mitochondria were isolated from the skeletal muscle of the hind limb. Mitochondrial density was determined by mitochondrial protein content and did not differ between the positive and negative residual energy expenditure groups. Using high-resolution respirometry, mitochondrial oxidative function was assessed using various substrates. Mitochondria from the positive residual energy expenditure group were characterized by a lower adenosine diphosphate-stimulated respiration and lower respiratory control rates using palmitoyl-coenzyme A as substrate. These results indicate that reduced mitochondrial coupling is associated with positive residual energy expenditure and high rates of total energy expenditure in vivo.

CETP does not affect triglyceride production or clearance in APOE*3-Leiden mice

The cholesteryl ester transfer protein (CETP) facilitates the bidirectional transfer of cholesteryl esters and triglycerides (TG) between HDL and (V)LDL. By shifting cholesterol in plasma from HDL to (V)LDL in exchange for VLDL-TG, CETP aggravates atherosclerosis in hyperlipidemic APOE*3-Leiden (E3L) mice. The aim of this study was to investigate the role of CETP in TG metabolism and high-fat diet-induced obesity by using E3L mice with and without the expression of the human CETP gene. On chow, plasma lipid levels were comparable between both male and female E3L and E3L.CETP mice. Further mechanistic studies were performed using male mice. CETP expression increased the level of TG in HDL. CETP did not affect the postprandial plasma TG response or the hepatic VLDL-TG and VLDL-apolipoprotein B production rate. Moreover, CETP did not affect the plasma TG clearance rate or organ-specific TG uptake after infusion of VLDL-like emulsion particles. In line with the absence of an effect of CETP on tissue-specific TG uptake, CETP also did not affect weight gain in response to a high-fat diet. In conclusion, the CETP-induced increase of TG in the HDL fraction of E3L mice is not associated with changes in the production of TG or with tissue-specific clearance of TG from the plasma.

Apolipoprotein A5 deficiency aggravates high-fat diet-induced obesity due to impaired central regulation of food intake

Mutations in apolipoprotein A5 (APOA5) have been associated with hypertriglyceridemia in humans and mice. This has been attributed to a stimulating role for APOA5 in lipoprotein lipase‐mediated triglyceride hydrolysis and hepatic clearance of lipoprotein remnant particles. However, because of the low APOA5 plasma abundance, we investigated an additional signaling role for APOA5 in high‐fat diet (HFD)‐induced obesity. Wild‐type (WT) and Apoa5–/– mice fed a chow diet showed no difference in body weight or 24‐h food intake (Apoa5–/–, 4.5±0.6 g; WT, 4.2±0.5 g), while Apoa5–/– mice fed an HFD ate more in 24 h (Apoa5–/–, 2.8±0.4 g; WT, 2.5±0.3 g, P<0.05) and became more obese than WT mice. Also, intravenous injection of APOA5‐loaded VLDL‐like particles lowered food intake (VLDL control, 0.26±0.04 g; VLDL+APOA5, 0.11±0.07 g, P<0.01). In addition, the HFD‐induced hyperphagia of Apoa5–/– mice was prevented by adenovirus‐mediated hepatic overexpression of APOA5. Finally, intracerebroventricular injection of APOA5 reduced food intake compared to injection of the same mouse with artificial cerebral spinal fluid (0.40±0.11 g; APOA5, 0.23±0.08 g, P<0.01). These data indicate that the increased HFD‐induced obesity of Apoa5–/– mice as compared to WT mice is at least partly explained by hyperphagia and that APOA5 plays a role in the central regulation of food intake.—Van den Berg, S. A. A., Heemskerk, M. M., Geerling, J. J., van Klinken, J.‐B., Schaap, F. G., Bijland, S., Berbée, J. F. P., van Harmelen, V. J. A., Pronk, A. C. M., Schreurs, M., Havekes, L. M., Rensen, P. C. N., van Dijk, K. W., Apolipoprotein A5 deficiency aggravates high‐fat diet‐induced obesity due to impaired central regulation of food intake. FASEB J. 27, 3354–3362 (2013). www.fasebj.org

Treatment of genetically obese mice with the iminosugar N-(5-adamantane-1-yl-methoxy-pentyl)-deoxynojirimycin reduces body weight by decreasing food intake and increasing fat oxidation

Obesity and its associated conditions such as type 2 diabetes mellitus are major causes of morbidity and mortality. The iminosugar N-(5-adamantane-1-yl-methoxy-pentyl)-deoxynojirimycin (AMP-DNM) improves insulin sensitivity in rodent models of insulin resistance and type 2 diabetes mellitus. In the current study, we characterized the impact of AMP-DNM on substrate oxidation patterns, food intake, and body weight gain in obese mice. Eight ob/ob mice treated with 100 mg/(kg d) AMP-DNM mixed in the food and 8 control ob/ob mice were placed in metabolic cages during the first, third, and fifth week of the experiment for measurement of substrate oxidation rates, energy expenditure, activity, and food intake. Mice were killed after 6 weeks of treatment. Initiation of treatment with AMP-DNM resulted in a rapid increase in fat oxidation by 129% (P = .05), a decrease in carbohydrate oxidation by 35% (P = .01), and a reduction in food intake by approximately 26% (P < .01) compared with control mice. Treatment with AMP-DNM decreased hepatic triglyceride content by 66% (P < .01) and, in line with the elevated fat oxidation rates, increased hepatic carnitine palmitoyl transferase 1a expression. Treatment with AMP-DNM increased plasma levels of the appetite-regulating peptide YY compared with control mice. Treatment with AMP-DNM rapidly reduces food intake and increases fat oxidation, resulting in improvement of the obese phenotype. These features of AMP-DNM, together with its insulin-sensitizing capacity, make it an attractive candidate drug for the treatment of obesity and its associated metabolic derangements.

Supplementary Material for: An 8-Week High-Fat Diet Induces Obesity and Insulin Resistance with Small Changes in the Muscle Transcriptome of C57BL/6J Mice

Background: Skeletal muscle is responsible for most of the insulin-stimulated glucose uptake and metabolism. Therefore, it plays an important role in the development of insulin resistance, one of the characteristics of the metabolic syndrome (MS). As the prevalence of the MS is increasing, there is an urgent need for more effective intervention strategies. Methods: C57BL/6J mice were fed an 8-week low-fat diet (10 kcal%; LFD) or high-fat diet (45 kcal%; HFD). Microarray analysis was performed by using two comparisons: (1) 8-week HFD transcriptome versus 8-week LFD transcriptome and (2) transcriptome of mice sacrificed at the start of the intervention versus 8-week LFD transcriptome and 8-week HFD transcriptome, respectively. Results: Although an 8-week HFD induced obesity and impaired insulin sensitivity, HFD-responsive changes in the muscle transcriptome were relatively small ( Conclusion: We suggest using complementary analysis strategies in the genome-wide search for gene expression changes induced by mild interventions, such as an HFD.