Amanda Pronk

Detrimental effects of constant light exposure and high-fat diet on circadian energy metabolism and insulin sensitivity

Circadian rhythm disturbances are observed in, e.g., aging and neurodegenerative diseases and are associated with an increased incidence of obesity and diabetes. We subjected male C57Bl/6J mice to constant light [12‐h light‐light (LL) cycle] to examine the effects of a disturbed circadian rhythm on energy metabolism and insulin sensitivity. In vivo electrophysiological recordings in the central pacemaker of the suprachiasmatic nuclei (SCN) revealed an immediate reduction in rhythm amplitude, stabilizing at 44% of normal amplitude values after 4 d LL. Food intake was increased (+26%) and energy expenditure decreased (–13%), and we observed immediate body weight gain (d 4: +2.4%, d 14: +5.0%). Mixed model analysis revealed that weight gain developed more rapidly in response to LL as compared to high fat. After 4 wk in LL, the circadian pattern in feeding and energy expenditure was completely lost, despite continuing low‐amplitude rhythms in the SCN and in behavior, whereas weight gain had stabilized. Hyperinsulinemic‐euglycemic clamp analysis revealed complete abolishment of normal circadian variation in insulin sensitivity in LL. In conclusion, a reduction in amplitude of the SCN, to values previously observed in aged mice, is sufficient to induce a complete loss of circadian rhythms in energy metabolism and insulin sensitivity.—Coomans, C. P., van den Berg, S. A. A., Houben, T., van Klinken, J.‐B., van den Berg, R., Pronk, A. C. M., Havekes, L. M., Romijn, J. A., Willems van Dijk, K., Biermasz, N. R., Meijer, J. H. Detrimental effects of constant light exposure and high‐fat diet on circadian energy metabolism and insulin sensitivity. FASEB J. 27, 1721–1732 (2013). www.fasebj.org

The Suprachiasmatic Nucleus Controls Circadian Energy Metabolism and Hepatic Insulin Sensitivity

Disturbances in the circadian system are associated with the development of type 2 diabetes mellitus. Here, we studied the direct contribution of the suprachiasmatic nucleus (SCN), the central pacemaker in the circadian system, in the development of insulin resistance. Exclusive bilateral SCN lesions in male C57Bl/6J mice, as verified by immunochemistry, showed a small but significant increase in body weight (+17%), which was accounted for by an increase in fat mass. In contrast, mice with collateral damage to the ventromedial hypothalamus and paraventricular nucleus showed severe obesity and insulin resistance. Mice with exclusive SCN ablation revealed a loss of circadian rhythm in activity, oxygen consumption, and food intake. Hyperinsulinemic–euglycemic clamp analysis 8 weeks after lesioning showed that the glucose infusion rate was significantly lower in SCN lesioned mice compared with sham-operated mice (−63%). Although insulin potently inhibited endogenous glucose production (−84%), this was greatly reduced in SCN lesioned mice (−7%), indicating severe hepatic insulin resistance. Our data show that SCN malfunctioning plays an important role in the disturbance of energy balance and suggest that an absence of central clock activity, in a genetically intact animal, may lead to the development of insulin resistance.

Long-term niacin treatment induces insulin resistance and adrenergic responsiveness in adipocytes by adaptive downregulation of phosphodiesterase 3B

The lipid-lowering effect of niacin has been attributed to the inhibition of cAMP production in adipocytes, thereby inhibiting intracellular lipolysis and release of nonesterified fatty acids (NEFA) to the circulation. However, long-term niacin treatment leads to a normalization of plasma NEFA levels and induces insulin resistance, for which the underlying mechanisms are poorly understood. The current study addressed the effects of long-term niacin treatment on insulin-mediated inhibition of adipocyte lipolysis and focused on the regulation of cAMP levels. APOE*3-Leiden.CETP transgenic mice treated with niacin for 15 wk were subjected to an insulin tolerance test and showed whole body insulin resistance. Similarly, adipocytes isolated from niacin-treated mice were insulin resistant and, interestingly, exhibited an increased response to cAMP stimulation by 8Br-cAMP, β1- and β2-adrenergic stimulation. Gene expression analysis of the insulin and β-adrenergic pathways in adipose tissue indicated that all genes were downregulated, including the gene encoding the cAMP-degrading enzyme phosphodiesterase 3B (PDE3B). In line with this, we showed that insulin induced a lower PDE3B response in adipocytes isolated from niacin-treated mice. Inhibiting PDE3B with cilostazol increased lipolytic responsiveness to cAMP stimulation in adipocytes. These data show that long-term niacin treatment leads to a downregulation of PDE3B in adipocytes, which could explain part of the observed insulin resistance and the increased responsiveness to cAMP stimulation.

Preclinical Studies on Intestinal Administration of Antisense Oligonucleotides as a Model for Oral Delivery for Treatment of Duchenne Muscular Dystrophy

Antisense oligonucleotides (AONs) used to reframe dystrophin mRNA transcripts for Duchenne muscular dystrophy (DMD) patients are tested in clinical trials. Here, AONs are administered subcutaneously and intravenously, while the less invasive oral route would be preferred. Oral delivery of encapsulated AONs supplemented with a permeation enhancer, sodium caprate, has been successfully used to target tumor necrosis factor (TNF)-α expression in liver. To test the feasibility of orally delivered AONs for DMD, we applied 2′-O-methyl phosphorothioate AONs (with or without sodium caprate supplementation) directly to the intestine of mdx mice and compared pharmacokinetics and -dynamics with intravenous, intraperitoneal, and subcutaneous delivery. Intestinally infused AONs were taken up, but resulted in lower plasma levels compared to other delivery routes, although bioavailability could be largely improved by supplementation of sodium caprate. After intestinal infusion, AON levels in all tissues were lower than for other administration routes, as were the ratios of target versus nontarget organ levels, except for diaphragm and heart where comparable levels and ratios were observed. For each administration route, low levels of exon skipping in triceps was observed 3 hours post-AON administration. These data suggest that oral administration of naked 2′-O-methyl phosphorothioate AONs may be feasible, but only when high AON concentrations are used in combination with sodium caprate.

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

Dietary Mannan Oligosaccharides Modulate Gut Microbiota, Increase Fecal Bile Acid Excretion, and Decrease Plasma Cholesterol and Atherosclerosis Development

Scope Mannan oligosaccharides (MOS) have proven effective at improving growth performance, while also reducing hyperlipidemia and inflammation. As atherosclerosis is accelerated both by hyperlipidemia and inflammation, we aim to determine the effect of dietary MOS on atherosclerosis development in hyperlipidemic ApoE*3‐Leiden.CETP (E3L.CETP) mice, a well‐established model for human‐like lipoprotein metabolism. Methods and results Female E3L.CETP mice were fed a high‐cholesterol diet, with or without 1% MOS for 14 weeks. MOS substantially decreased atherosclerotic lesions up to 54%, as assessed in the valve area of the aortic root. In blood, IL‐1RA, monocyte subtypes, lipids, and bile acids (BAs) were not affected by MOS. Gut microbiota composition was determined using 16S rRNA gene sequencing and MOS increased the abundance of cecal Bacteroides ovatus. MOS did not affect fecal excretion of cholesterol, but increased fecal BAs as well as butyrate in cecum as determined by gas chromatography mass spectrometry. Conclusion MOS decreased the onset of atherosclerosis development via lowering of plasma cholesterol levels. These effects were accompanied by increased cecal butyrate and fecal excretion of BAs, presumably mediated via interactions of MOS with the gut microbiota.

IgG is elevated in obese white adipose tissue but does not induce glucose intolerance via Fc gamma-receptor or complement

Background/Objectives:In obesity, B cells accumulate in white adipose tissue (WAT) and produce IgG, which may contribute to the development of glucose intolerance. IgG signals by binding to Fcγ receptors (FcγR) and by activating the complement system. The aim of our study was to investigate whether activation of FcγR and/or complement C3 mediates the development of high-fat diet-induced glucose intolerance.Methods:We studied mice lacking all four FcγRs (FcγRI/II/III/IV−/−), only the inhibitory FcγRIIb (FcγRIIb−/−), only the central component of the complement system C3 (C3−/−), and mice lacking both FcγRs and C3 (FcγRI/II/III/IV/C3−/−). All mouse models and wild-type controls were fed a high-fat diet (HFD) for 15 weeks to induce obesity. Glucose metabolism was assessed and adipose tissue was characterized for inflammation and adipocyte functionality.Results:In obese WAT of wild-type mice, B cells (+142%, P<0.01) and IgG (+128% P<0.01) were increased compared to lean WAT. Macrophages of FcγRI/II/III/IV−/−mice released lower levels of cytokines compared to wild-type mice upon IgG stimulation. Only C3−/− mice showed reduced HFD-induced weight gain as compared to controls (−18%, P<0.01). Surprisingly, FcγRI/II/III/IV−/− mice had deteriorated glucose tolerance (AUC +125%, P<0.001) despite reduced leukocyte number (−30%, P<0.05) in gonadal WAT (gWAT), whereas glucose tolerance and leukocytes within gWAT in the other models were unaffected compared to controls. Although IgG in gWAT was increased (+44 to +174%, P<0.05) in all mouse models lacking FcγRIIb, only FcγRI/II/III/IV/C3−/− mice exhibited appreciable alterations in immune cells in gWAT, for example, increased macrophages (+36%, P<0.001).Conclusions:Lack of FcγRs reduces the activity of macrophages upon IgG stimulation, but neither FcγR nor C3 deficiency protects against HFD-induced glucose intolerance or reduces adipose tissue inflammation. This indicates that if obesity-induced IgG contributes to the development of glucose intolerance, this is not mediated by FcγR or complement activation.

Human TP53 polymorphism (rs1042522) modelled in mouse does not affect glucose metabolism and body composition

Variation in TP53 has been associated with cancer. The pro-allele of a TP53 polymorphism in codon 72 (rs1042522) has been associated with longevity. Recently, we showed that the same allele might be involved in preservation of glucose metabolism, body composition and blood pressure during ageing. Here, we assessed glucose tolerance and body composition in mice carrying the human polymorphism. Our data do not support the previous findings in humans, suggesting that this polymorphism does not play a major role in development of glucose metabolism and body composition during ageing. Alternatively, the mouse model may not be suitable to validate these rs1042522-associated traits up to the age tested.

Dietary yeast-derived mannan oligosaccharides have immune-modulatory properties but do not improve high fat diet-induced obesity and glucose intolerance

The indigestible mannan oligosaccharides (MOS) derived from the outer cell wall of yeast Saccharomyces cerevisiae have shown potential to reduce inflammation. Since inflammation is one of the underlying mechanisms involved in the development of obesity-associated metabolic dysfunctions, we aimed to determine the effect of dietary supplementation with MOS on inflammation and metabolic homeostasis in lean and diet-induced obese mice. Male C57BL/6 mice were fed either a low fat diet (LFD) or a high fat diet (HFD) with, respectively, 10% or 45% energy derived from lard fat, with or without 1% MOS for 17 weeks. Body weight and composition were measured throughout the study. After 12 weeks of intervention, whole-body glucose tolerance was assessed and in week 17 immune cell composition was determined in mesenteric white adipose tissue (mWAT) and liver by flow cytometry and RT-qPCR. In LFD-fed mice, MOS supplementation induced a significant increase in the abundance of macrophages and eosinophils in mWAT. A similar trend was observed in hepatic macrophages. Although HFD feeding induced a classical shift from the anti-inflammatory M2-like macrophages towards the pro-inflammatory M1-like macrophages in both mWAT and liver from control mice, MOS supplementation had no effect on this obesity-driven immune response. Finally, MOS supplementation did not improve whole-body glucose homeostasis in both lean and obese mice.Altogether, our data showed that MOS had extra-intestinal immune modulatory properties in mWAT and liver. However these effects were not substantial enough to significantly ameliorate HFD-induced glucose intolerance or inflammation.

The Prebiotic Inulin Aggravates Accelerated Atherosclerosis in Hypercholesterolemic APOE*3-Leiden Mice

The prebiotic inulin has proven effective at lowering inflammation and plasma lipid levels. As atherosclerosis is provoked by both inflammation and hyperlipidemia, we aimed to determine the effect of inulin supplementation on atherosclerosis development in hypercholesterolemic APOE*3-Leiden (E3L) mice. Male E3L mice were fed a high-cholesterol (1%) diet, supplemented with or without 10% inulin for 5 weeks. At week 3, a non-constrictive cuff was placed around the right femoral artery to induce accelerated atherosclerosis. At week 5, vascular pathology was determined by lesion thickness, vascular remodeling, and lesion composition. Throughout the study, plasma lipids were measured and in week 5, blood monocyte subtypes were determined using flow cytometry analysis. In contrast to our hypothesis, inulin exacerbated atherosclerosis development, characterized by increased lesion formation and outward vascular remodeling. The lesions showed increased number of macrophages, smooth muscle cells, and collagen content. No effects on blood monocyte composition were found. Inulin significantly increased plasma total cholesterol levels and total cholesterol exposure. In conclusion, inulin aggravated accelerated atherosclerosis development in hypercholesterolemic E3L mice, accompanied by adverse lesion composition and outward remodeling. This process was not accompanied by differences in blood monocyte composition, suggesting that the aggravated atherosclerosis development was driven by increased plasma cholesterol.