Lynda M. Williams

Hypothalamic dysfunction in obesity.

A growing number of studies have shown that a diet high in long chain SFA and/or obesity cause profound changes to the energy balance centres of the hypothalamus which results in the loss of central leptin and insulin sensitivity. Insensitivity to these important anorexigenic messengers of nutritional status perpetuates the development of both obesity and peripheral insulin insensitivity. A high-fat diet induces changes in the hypothalamus that include an increase in markers of oxidative stress, inflammation, endoplasmic reticulum (ER) stress, autophagy defect and changes in the rate of apoptosis and neuronal regeneration. In addition, a number of mechanisms have recently come to light that are important in the hypothalamic control of energy balance, which could play a role in perpetuating the effect of a high-fat diet on hypothalamic dysfunction. These include: reactive oxygen species as an important second messenger, lipid metabolism, autophagy and neuronal and synaptic plasticity. The importance of nutritional activation of the Toll-like receptor 4 and the inhibitor of NF-κB kinase subunit β/NK-κB and c-Jun amino-terminal kinase 1 inflammatory pathways in linking a high-fat diet to obesity and insulin insensitivity via the hypothalamus is now widely recognised. All of the hypothalamic changes induced by a high-fat diet appear to be causally linked and inhibitors of inflammation, ER stress and autophagy defect can prevent or reverse the development of obesity pointing to potential drug targets in the prevention of obesity and metabolic dysfunction.

High‐Fat Diet Induces Leptin Resistance in Leptin‐Deficient Mice

The occurrence of type II diabetes is highly correlated with obesity, although the mechanisms linking the two conditions are incompletely understood. Leptin is a potent insulin sensitiser and, in leptin‐deficient, insulin insensitive, Lepob/ob mice, leptin improves glucose tolerance, indicating that leptin resistance may link obesity to insulin insensitivity. Leptin resistance occurs in response to a high‐fat diet (HFD) and both hyperleptinaemia and inflammation have been proposed as causative mechanisms. Scrutinising the role of hyperleptinaemia in this process, central hyperleptinaemia in Lepob/ob mice was induced by chronic i.c.v. infusion of leptin (4.2 μg/day) over 10 days. This treatment led to a dramatic decline in body weight and food intake, as well as an improvement in glucose tolerance. Transfer to HFD for 4 days markedly arrested the beneficial effects of leptin on these parameters. Because Lepob/ob mice are exquisitely sensitive to leptin, the possibility that leptin could reverse HFD‐induced glucose intolerance in these animals was investigated. HFD led to increased body weight and glucose intolerance compared to a low‐fat diet (LFD). Older and heavier Lepob/ob mice were used as body weight‐matched controls. Mice in each group received either i.p. leptin (1.25 mg/kg) or vehicle, and glucose tolerance, food intake and the number of phosphorylated signal transducer and activator of transcription (pSTAT)3 immunoreactive cells in the arcuate nucleus (ARC) and ventromedial hypothalamus (VMH) were analysed. Leptin improved glucose tolerance (P = 0. 019) and reduced food intake in Lepob/ob mice on LFD (P ≤ 0.001) but was ineffective in mice on HFD. Furthermore, when leptin was administered centrally, the glucose tolerance of Lepob/ob mice on HFD was significantly impaired (P = 0.007). Although leptin induced the number of pSTAT3 immunoreactive cells in the ARC and VMH of Lepob/ob mice on LFD, HFD was associated with elevated pSTAT3 immunoreactivity in vehicle‐treated Lepob/ob mice that was unaffected by leptin treatment, suggesting central leptin resistance. Negating central inflammation by co‐administering a c‐Jun n‐terminal kinase (JNK) inhibitor reinstated the glucose‐lowering effects of leptin. These findings demonstrate that Lepob/ob mice develop leptin resistance on a HFD independent of hyperleptinaemia and also indicate that the JNK inflammatory pathway plays a key role in the induction of diet‐induced glucose intolerance.

The Development of Diet-Induced Obesity and Glucose Intolerance in C57Bl/6 Mice on a High-Fat Diet Consists of Distinct Phases

High–fat (HF) diet-induced obesity and insulin insensitivity are associated with inflammation, particularly in white adipose tissue (WAT). However, insulin insensitivity is apparent within days of HF feeding when gains in adiposity and changes in markers of inflammation are relatively minor. To investigate further the effects of HF diet, C57Bl/6J mice were fed either a low (LF) or HF diet for 3 days to 16 weeks, or fed the HF-diet matched to the caloric intake of the LF diet (PF) for 3 days or 1 week, with the time course of glucose tolerance and inflammatory gene expression measured in liver, muscle and WAT. HF fed mice gained adiposity and liver lipid steadily over 16 weeks, but developed glucose intolerance, assessed by intraperitoneal glucose tolerance tests (IPGTT), in two phases. The first phase, after 3 days, resulted in a 50% increase in area under the curve (AUC) for HF and PF mice, which improved to 30% after 1 week and remained stable until 12 weeks. Between 12 and 16 weeks the difference in AUC increased to 60%, when gene markers of inflammation appeared in WAT and muscle but not in liver. Plasma proteomics were used to reveal an acute phase response at day 3. Data from PF mice reveals that glucose intolerance and the acute phase response are the result of the HF composition of the diet and increased caloric intake respectively. Thus, the initial increase in glucose intolerance due to a HF diet occurs concurrently with an acute phase response but these effects are caused by different properties of the diet. The second increase in glucose intolerance occurs between 12 - 16 weeks of HF diet and is correlated with WAT and muscle inflammation. Between these times glucose tolerance remains stable and markers of inflammation are undetectable.

Leptin receptor gene expression and number in the brain are regulated by leptin level and nutritional status

Hormone potency depends on receptor availability, regulated via gene expression and receptor trafficking. To ascertain how central leptin receptors are regulated, the effects of leptin challenge, high‐fat diet, fasting and refeeding were measured on leptin receptor number and gene expression. These were measured using quantitative 125I‐labelled leptin in vitro autoradiography and in situ hybridisation, respectively. Ob‐R (all forms of leptin receptor) expression in the choroid plexus (CP) was unchanged by high‐fat diet or leptin challenge, whereas fasting increased but refeeding failed to decrease expression. 125I‐labelled leptin binding to the CP was increased by fasting and returned to basal levels on refeeding. 125I‐Labelled leptin was reduced by leptin challenge and increased by high‐fat feeding. Ob‐Rb (signalling form) in the arcuate (ARC) and ventromedial (VMH) nuclei was increased after fasting and decreased by refeeding. Leptin challenge increased Ob‐Rb expression in the ARC, but not after high‐fat feeding. In general, changes in gene expression in the ARC and VMH appeared to be largely due to changes in area rather than density of labelling, indicating that the number of cells expressing Ob‐Rb was the parameter that contributed most to these changes. Leptin stimulation of suppressor of cytokine signalling 3 (SOCS3), a marker of stimulation of the Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) pathway, was unchanged after high‐fat diet. Thus, early loss of leptin sensitivity after high‐fat feeding is unrelated to down‐regulation of leptin receptor expression or number and does not involve the JAK/STAT pathway. The effect of leptin to decrease 125I‐labelled leptin binding and the loss of ability of leptin to up‐regulate Ob‐Rb expression in the ARC after high‐fat feeding offer potential mechanisms for the development of leptin insensitivity in response to both hyperleptinaemia and high‐fat diet.

Central Inhibition of IKKβ/NF-κB Signaling Attenuates High-Fat Diet–Induced Obesity and Glucose Intolerance

Metabolic inflammation in the central nervous system might be causative for the development of overnutrition-induced metabolic syndrome and related disorders, such as obesity, leptin and insulin resistance, and type 2 diabetes. Here we investigated whether nutritive and genetic inhibition of the central IκB kinase β (IKKβ)/nuclear factor-κB (NF-κB) pathway in diet-induced obese (DIO) and leptin-deficient mice improves these metabolic impairments. A known prominent inhibitor of IKKβ/NF-κB signaling is the dietary flavonoid butein. We initially determined that oral, intraperitoneal, and intracerebroventricular administration of this flavonoid improved glucose tolerance and hypothalamic insulin signaling. The dose-dependent glucose-lowering capacity was profound regardless of whether obesity was caused by leptin deficiency or high-fat diet (HFD). To confirm the apparent central role of IKKβ/NF-κB signaling in the control of glucose and energy homeostasis, we genetically inhibited this pathway in neurons of the arcuate nucleus, one key center for control of energy homeostasis, via specific adeno-associated virus serotype 2–mediated overexpression of IκBα, which inhibits NF-κB nuclear translocation. This treatment attenuated HFD-induced body weight gain, body fat mass accumulation, increased energy expenditure, and reduced arcuate suppressor of cytokine signaling 3 expression, indicative for enhanced leptin signaling. These results reinforce a specific role of central proinflammatory IKKβ/NF-κB signaling in the development and potential treatment of DIO-induced comorbidities.

High-Fat Diet Alters Gene Expression in the Liver and Colon: Links to Increased Development of Aberrant Crypt Foci

BackgroundObesity is associated with an increased risk of colon cancer. High-fat diets that lead to obesity may be a contributing factor, but the mechanisms are unknown.AimsThis study examines susceptibility to azoxymethane (AOM)-induced precancerous lesions in mice in response to consumption of either a low or a high-fat diet and associated molecular changes in the liver and colon.MethodsGene markers of xenobiotic metabolism, leptin-regulated inflammatory cytokines and proliferation were assessed in liver and colon in response to high-fat feeding to determine links with increased sensitivity to AOM.ResultsHigh-fat feeding increased development of AOM-induced precancerous lesions and was associated with increased CYP2E1 gene expression in the liver, but not the colon. Leptin receptors and the colon stem cell marker (Lgr5) were down-regulated in the proximal colon, with a corresponding up-regulation of the inflammatory cytokine (IL6) in response to high-fat feeding. Notably in the distal colon, where aberrant crypt foci develop in response to AOM, the proliferative stem cell marker, Lgr5, was significantly up-regulated with high-fat feeding.ConclusionsThe current study provides evidence that high-fat diets can alter regulation of molecular markers of xenobiotic metabolism that may expose the colon to carcinogens, in parallel with activation of β-catenin-regulated targets regulating colon epithelial cells. High-fat diets associated with obesity may alter multiple molecular factors that act synergistically to increase the risk of colon cancer associated with obesity.

Leptin up‐regulates pro‐inflammatory cytokines in discrete cells within mouse colon

Dysregulation of leptin associated with obesity is implicated in obesity‐related colon cancer, but mechanisms are elusive. Increased adiposity and elevated plasma leptin are associated with perturbed metabolism in colon and leptin receptors are expressed on colon epithelium. We hypothesise that obesity increases the sensitivity of the colon to cancer by disrupting leptin‐regulated gene targets within colon tissues. PCR arrays were used to firstly identify leptin responsive genes and secondly to identify responses to leptin challenge in wild‐type mice, or those lacking leptin (ob/ob). Leptin‐regulated genes were localised in the colon using in situ hybridisation. IL6, IL1β and CXCL1 were up‐regulated by leptin and localised to discrete cells in gut epithelium, lamina propria, muscularis and at the peritoneal serosal surface. Leptin regulates pro‐inflammatory genes such as IL6, IL1β and CXCL1, and might increase the risk of colon cancer among obese individuals. J. Cell. Physiol. 226: 2123–2130, 2011.

Ghrelin: New Molecular Pathways Modulating Appetite and Adiposity

Ghrelin is a unique endogenous peptidic hormone regulating both hunger and adiposity. Many of the actions of ghrelin are modulated specifically by the central nervous system. A number of molecular events triggered via the activation of the ghrelin receptor (GHS-R1a), leading to increased levels of neuropeptide Y (NPY) and agoutirelated peptide (AgRP) and ultimately responsible for the orexigenic effect of ghrelin have been characterized. Moreover, the discovery of ghrelin O-acyltransferase (GOAT), the enzyme responsible for the octanoylation of ghrelin, provides a mechanism allowing specific targeting of the ghrelin/GHS-R1a system without affecting the role of des-acyl-ghrelin in other pathways involved in the regulation of energy balance. This review aims to summarize novel roles of ghrelin in energy balance, focusing particularly on both the newly identified neuronal pathways mediating the effects of ghrelin and on peripheral mechanisms leading to increased adiposity.

The NuGO proof of principle study package: a collaborative research effort of the European Nutrigenomics Organisation

Acknowledgments This project is funded by the Nutrigenomics Organisation, EC funded Network of Excellence, grant nr.FOOD- 2004-506360.

Effects of conjugated linoleic acid plus n -3 polyunsaturated fatty acids on insulin secretion and estimated insulin sensitivity in men

Background/Objectives:Dietary addition of either conjugated linoleic acid (CLA) or n-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFAs) has been shown to alter adiposity and circulating lipids, risk markers of cardiovascular diseases. However, CLA may decrease insulin sensitivity, an effect that may be reversed by n-3 LC-PUFA. Thus, the potential of CLA plus n-3 LC-PUFA to affect insulin secretion and sensitivity in non-diabetic young and old, lean and obese subjects was tested.Subjects/Methods:CLA (3u2009g daily) plus n-3 LC-PUFA (3u2009g daily) or control oil (6u2009g daily) was given to lean (n=12; BMI 20–26u2009kg/m2) or obese (n=10; BMI 29–35u2009kg/m2) young (20–37 years old) or lean (n=16) or obese (n=11) older men (50–65 years) for 12 weeks. The study had a double-blind, placebo-controlled randomized crossover design, and primary end points were insulin secretion and sensitivity during a standardized meal test, evaluated by modeling glucose, insulin and C-peptide data.Results:The combination was well tolerated. There was no significant difference in fasting levels of glucose, insulin or C-peptide after CLA/n-3 LC-PUFA treatment compared with control oil. Neither insulin secretion nor estimated sensitivity was affected by CLA/n-3 LC-PUFA in lean or obese young subjects or in older lean subjects. However, in older obese subjects, estimated insulin sensitivity was reduced with CLA/n-3 LC-PUFA compared with control (P=0.024).Conclusions:The results do not support beneficial effects of CLA/n-3 LC-PUFA for β-cell dysfunction or insulin resistance in humans but suggest that insulin sensitivity in older obese subjects is reduced.