Marijke Schreurs

Regulatory enzymes of mitochondrial B-oxidation as targets for treatment of the metabolic syndrome

Insulin sensitizers like metformin generally act through pathways triggered by adenosine monophosphate‐activated protein kinase. Carnitine palmitoyltransferase 1 (CPT1) controls mitochondrial β‐oxidation and is inhibited by malonyl‐CoA, the product of acetyl‐CoA carboxylase (ACC). The adenosine monophosphate‐activated protein kinase‐ACC‐CPT1 axis tightly regulates mitochondrial long‐chain fatty acid oxidation. Evidence indicates that ACC2, the isoform located in close proximity to CPT1, is the major regulator of CPT1 activity. ACC2 as well as CPT1 are therefore potential targets to treat components of the metabolic syndrome such as obesity and insulin resistance. Reversible inhibitors of the liver isoform of CPT1, developed to prevent ketoacidosis and hyperglycemia, have been found to be associated with side effects like hepatic steatosis. However, stimulation of systemic CPT1 activity may be an attractive means to accelerate peripheral fatty acid oxidation and hence improve insulin sensitivity. Stimulation of CPT1 can be achieved by elimination or inhibition of ACC2 activity and through activating transcription factors like peroxisome proliferator‐activated receptors and their protein partners. The latter leads to enhanced CPT1 gene expression. Recent developments are discussed, including a recently identified CPT1 isoform, i.e. CPT1C. This protein is highly expressed in the brain and may provide a target for new tools to prevent obesity.

Soraphen, an inhibitor of the acetyl‐CoA carboxylase system, improves peripheral insulin sensitivity in mice fed a high‐fat diet

Aim: Inhibition of the acetyl‐CoA carboxylase (ACC) system, consisting of the isozymes ACC1 and ACC2, may be beneficial for treatment of insulin resistance and/or obesity by interfering with de novo lipogenesis and β‐oxidation. We have evaluated effects of pharmacological inhibition of ACC by soraphen (SP) on high fat (HF) diet–induced insulin resistance in mice.

Tumor necrosis factor receptor 1 gain‐of‐function mutation aggravates nonalcoholic fatty liver disease but does not cause insulin resistance in a murine model

Ectodomain shedding of tumor necrosis factor receptor 1 (TNFR1) provides negative feedback to the inflammatory loop induced by TNFα. As the significance of this mechanism in obesity‐associated pathologies is unclear, we aimed to unravel how much TNFR1 ectodomain shedding controls the development of nonalcoholic fatty liver disease (NAFLD), as well as its role in the development of insulin resistance. We used knockin mice expressing a mutated TNFR1 ectodomain (p55Δns), incapable of shedding and dampen the inflammatory response. Our data show that persistent TNFα signaling through this inability of TNFR1 ectodomain shedding contributes to chronic low‐grade inflammation, which is confined to the liver. In spite of this, hepatic lipid levels were not affected by the nonshedding mutation in mice fed a chow diet, nor were they worse off following 12 weeks of high‐fat diet (HFD) than controls (p55+/+) fed an HFD. We detected inflammatory infiltrates, hepatocellular necrosis, and apoptosis in livers of p55Δns/Δns mice fed an HFD, suggesting advanced progression of NAFLD toward nonalcoholic steatohepatitis (NASH). Indeed, fibrosis was present in p55Δns/Δns mice, but absent in wildtype mice, confirming that the p55Δns/Δns mice had a more severe NASH phenotype. Despite low‐grade hepatic inflammation, insulin resistance was not observed in p55Δns/Δns mice fed a chow diet, and HFD‐induced insulin resistance was no worse in p55Δns/Δns mice than p55+/+ mice. Conclusion: TNFR1 ectodomain shedding is not an essential feedback mechanism in preventing the development of hepatic steatosis or insulin resistance. It is, however, pivotal in attenuating the progression from “simple steatosis” towards a more serious phenotype with many NASH features. Targeting TNFR1 could therefore be beneficial in attenuating NASH. (HEPATOLOGY 2013)

Type I diabetes mellitus decreases in vivo macrophage-to-feces reverse cholesterol transport despite increased biliary sterol secretion in mice

Type I diabetes mellitus (T1DM) increases atherosclerotic cardiovascular disease; however, the underlying pathophysiology is still incompletely understood. We investigated whether experimental T1DM impacts HDL-mediated reverse cholesterol transport (RCT). C57BL/6J mice with alloxan-induced T1DM had higher plasma cholesterol levels (P < 0.05), particularly within HDL, and increased hepatic cholesterol content (P < 0.001). T1DM resulted in increased bile flow (2.1-fold; P < 0.05) and biliary secretion of bile acids (BA, 10.5-fold; P < 0.001), phospholipids (4.5-fold; P < 0.001), and cholesterol (5.5-fold; P < 0.05). Hepatic cholesterol synthesis was unaltered, whereas BA synthesis was increased in T1DM (P < 0.001). Mass fecal BA output was significantly higher in T1DM mice (1.5-fold; P < 0.05), fecal neutral sterol excretion did not change due to increased intestinal cholesterol absorption (2.1-fold; P < 0.05). Overall in vivo macrophage-to-feces RCT, using [3H]cholesterol-loaded primary mouse macrophage foam cells, was 20% lower in T1DM (P < 0.05), mainly due to reduced tracer excretion within BA (P < 0.05). In vitro experiments revealed unchanged cholesterol efflux toward T1DM HDL, whereas scavenger receptor class BI-mediated selective uptake from T1DM HDL was lower in vitro and in vivo (HDL kinetic experiments) (P < 0.05), conceivably due to increased glycation of HDL-associated proteins (+65%, P < 0.01). In summary, despite higher mass biliary sterol secretion T1DM impairs macrophage-to-feces RCT, mainly by decreasing hepatic selective uptake, a mechanism conceivably contributing to increased cardiovascular disease in T1DM.

Carbohydrate-response-element-binding protein (ChREBP) and not the liver X receptor α (LXRα) mediates elevated hepatic lipogenic gene expression in a mouse model of glycogen storage disease type 1

GSD-1 (glycogen storage disease type 1) is caused by an inherited defect in glucose-6-phosphatase activity, resulting in a massive accumulation of hepatic glycogen content and an induction of de novo lipogenesis. The chlorogenic acid derivative S4048 is a pharmacological inhibitor of the glucose 6-phosphate transporter, which is part of glucose-6-phosphatase, and allows for mechanistic studies concerning metabolic defects in GSD-1. Treatment of mice with S4048 resulted in an ~60% reduction in blood glucose, increased hepatic glycogen and triacylglycerol (triglyceride) content, and a markedly enhanced hepatic lipogenic gene expression. In mammals, hepatic expression of lipogenic genes is regulated by the co-ordinated action of the transcription factors SREBP (sterol-regulatory-element-binding protein)-1c, LXRα (liver X receptor α) and ChREBP (carbohydrate-response-element-binding protein). Treatment of Lxra-/- mice and Chrebp-/- mice with S4048 demonstrated that ChREBP, but not LXRα, mediates the induction of hepatic lipogenic gene expression in this murine model of GSD-1. Thus ChREBP is an attractive target to alleviate derangements in lipid metabolism observed in patients with GSD-1.

Cholesterol-induced hepatic inflammation does not contribute to the development of insulin resistance in male LDL receptor knockout mice

OBJECTIVE It is generally assumed that hepatic inflammation in obesity is linked to the pathogenesis of insulin resistance. Several recent studies have shed doubt on this view, which questions the causality of this association. This study focuses on Kupffer cell-mediated hepatic inflammation as a possible driver of insulin resistance in the absence and presence of obesity. METHODS We used male mice deficient for the low-density lipoprotein receptor (Ldlr(-/-)) and susceptible to cholesterol-induced hepatic inflammation. Whole body and hepatic insulin resistance was measured in mice fed 4 diets for 2 and 15 weeks, i.e., chow, high-fat (HF), HF-cholesterol (HFC; 0.2% cholesterol) and HF without cholesterol (HFnC). Biochemical parameters in plasma and liver were measured and inflammation was determined using immunohistochemistry and RT-PCR. RESULTS At 2 weeks, we did not find significant metabolic effects in either diet group, except for the mice fed a HFC diet which showed pronounced hepatic inflammation (p < 0.05) but normal insulin sensitivity. At 15 weeks, a significant increase in insulin levels, HOMA-IR, and hepatic insulin resistance was observed in mice fed a HFC, HFnC, and HF diet compared to chow-fed mice (p < 0.05). Regardless of the level of hepatic inflammation (HFC > HF, HFnC; p < 0.05) insulin resistance in mice fed HFC was no worse compared to mice on a HFnC and HF diet. CONCLUSION These data show that cholesterol-induced hepatic inflammation does not contribute to the development of insulin resistance in male Ldlr(-/-) mice. This study suggests that Kupffer cell-driven hepatic inflammation is a consequence, not a cause, of metabolic dysfunction in obesity.

Chronic Prednisolone Treatment Aggravates Hyperglycemia in Mice Fed a High-Fat Diet but Does Not Worsen Dietary Fat-Induced Insulin Resistance

Synthetic glucocorticoids such as prednisolone have potent antiinflammatory actions. Unfortunately, these drugs induce severe adverse effects in patients, many of which resemble features of the metabolic syndrome, such as insulin resistance. In this study, we investigated whether adverse effects of prednisolone on glucose homeostasis are aggravated in mice with compromised insulin sensitivity due to a high-fat diet by applying various methods to analyze changes in insulin sensitivity in mice. C57BL/6J mice were fed a high-fat diet for 6 wk and treated with either prednisolone (10 mg/kg · d) or vehicle for the last 7 d. Insulin sensitivity and blood glucose kinetics were analyzed with state-of-the-art stable isotope procedures in different experimental conditions. Prednisolone treatment aggravated fasting hyperglycemia and hyperinsulinemia caused by high-fat feeding, resulting in a higher homeostatic assessment model of insulin resistance. In addition, prednisolone-treated high-fat diet-fed mice appeared less insulin sensitive by detailed analysis of basal glucose kinetics. Remarkably, using hyperinsulinemic-euglycemic or hyperglycemic clamp techniques, neither hepatic nor peripheral insulin resistance was worsened in the group that was treated with prednisolone. Yet analysis of hepatic glucose metabolism revealed that prednisolone did alter glycogen balance by reducing glycogen synthase flux under hyperinsulinemic as well as hyperglycemic conditions. In addition to elevated insulin levels, prednisolone-treated mice showed a major rise in plasma leptin and fibroblast growth factor 21 levels. Our data indicate that prednisolone-induced adverse effects on glucose metabolism in high-fat diet-fed mice do not reflect impaired insulin sensitivity but may be caused by other changes in the hormonal regulatory network controlling glucose metabolism such as fibroblast growth factor 21 and leptin.

Cholesterol-Induced Hepatic Inflammation Does Not Underlie the Predisposition to Insulin Resistance in Dyslipidemic Female LDL Receptor Knockout Mice

Chronic inflammation is considered a causal risk factor predisposing to insulin resistance. However, evidence is accumulating that inflammation confined to the liver may not be causal to metabolic dysfunction. To investigate this, we assessed if hepatic inflammation explains the predisposition towards insulin resistance in low-density lipoprotein receptor knock-out (Ldlr −/−) mice. For this, wild type (WT) and Ldlr −/− mice were fed a chow diet, a high fat (HF) diet, or a high fat, high cholesterol (HFC) diet for 2 weeks. Plasma lipid levels were elevated in chow-fed Ldlr −/− mice compared to WT mice. Although short-term HF or HFC feeding did not result in body weight gain and adipose tissue inflammation, dyslipidemia was worsened in Ldlr −/− mice compared to WT mice. In addition, dyslipidemic HF-fed Ldlr −/− mice had a higher hepatic glucose production rate than HF-fed WT mice, while peripheral insulin resistance was unaffected. This suggests that HF-fed Ldlr −/− mice suffered from hepatic insulin resistance. While HFC-fed Ldlr −/− mice displayed the anticipated increased hepatic inflammation, this did neither exacerbate systemic nor hepatic insulin resistance. Therefore, our results show that hepatic insulin resistance is unrelated to cholesterol-induced hepatic inflammation in Ldlr −/− mice, indicating that hepatic inflammation may not contribute to metabolic dysfunction per se.

Effects of Anthocyanin and Flavanol Compounds on Lipid Metabolism and Adipose Tissue Associated Systemic Inflammation in Diet-Induced Obesity

Background. Naturally occurring substances from the flavanol and anthocyanin family of polyphenols have been proposed to exert beneficial effects in the course of obesity. We hypothesized that their effects on attenuating obesity-induced dyslipidemia as well as the associated inflammatory sequelae especially have health-promoting potential. Methods. Male C57BL/6J mice (n = 52) received a control low-fat diet (LFD; 10 kcal% fat) for 6 weeks followed by 24 weeks of either LFD (n = 13) or high-fat diet (HFD; 45 kcal% fat; n = 13) or HFD supplemented with 0.1% w/w of the flavanol compound epicatechin (HFD+E; n = 13) or an anthocyanin-rich bilberry extract (HFD+B; n = 13). Energy substrate utilization was determined by indirect calorimetry in a subset of mice following the dietary switch and at the end of the experiment. Blood samples were collected at baseline and at 3 days and 4, 12, and 20 weeks after dietary switch and analyzed for systemic lipids and proinflammatory cytokines. Adipose tissue (AT) histopathology and inflammatory gene expression as well as hepatic lipid content were analyzed after sacrifice. Results. The switch from a LFD to a HFD lowered the respiratory exchange ratio and increased plasma cholesterol and hepatic lipid content. These changes were not attenuated by HFD+E or HFD+B. Furthermore, the polyphenol compounds could not prevent HFD-induced systemic rise of TNF-α levels. Interestingly, a significant reduction in Tnf gene expression in HFD+B mice was observed in the AT. Furthermore, HFD+B, but not HFD+E, significantly prevented the early upregulation of circulating neutrophil chemoattractant mKC. However, no differences in AT histopathology were observed between the HFD types. Conclusion. Supplementation of HFD with an anthocyanin-rich bilberry extract but not with the flavanol epicatechin may exert beneficial effects on the systemic early inflammatory response associated with diet-induced obesity. These systemic effects were transient and not observed after prolongation of HFD-feeding (24 weeks). On the tissue level, long-term treatment with bilberry attenuated TNF-α expression in adipose tissue.

PS19 - 89. Absence of JNK1 protects mice against age-induced obesity and insulin resistance

Both a Western-type diet as well as ageing are risk factors for the development of obesity and type 2 diabetes. Dietinduced obesity is associated with an upregulation of stress kinases like c-Jun amino-terminal kinase 1 (JNK1), and JNK1 knockout mice are protected against diet-induced obesity and insulin resistance.