Validity of resting energy expenditure predictive equations before and after an energy-restricted diet intervention in obese women

Background

We investigated the validity of REE predictive equations before and after 12-week energy-restricted diet intervention in Spanish obese (30 kg/m²>BMI<40 kg/m²) women.

Methods

We measured REE (indirect calorimetry), body weight, height, and fat mass (FM) and fat free mass (FFM, dual X-ray absorptiometry) in 86 obese Caucasian premenopausal women aged 36.7±7.2 y, before and after (n = 78 women) the intervention. We investigated the accuracy of ten REE predictive equations using weight, height, age, FFM and FM.

Results

At baseline, the most accurate equation was the Mifflin et al. (Am J Clin Nutr 1990; 51: 241–247) when using weight (bias:−0.2%, P = 0.982), 74% of accurate predictions. This level of accuracy was not reached after the diet intervention (24% accurate prediction). After the intervention, the lowest bias was found with the Owen et al. (Am J Clin Nutr 1986; 44: 1–19) equation when using weight (bias:−1.7%, P = 0.044), 81% accurate prediction, yet it provided 53% accurate predictions at baseline.

Conclusions

There is a wide variation in the accuracy of REE predictive equations before and after weight loss in non-morbid obese women. The results acquire especial relevance in the context of the challenging weight regain phenomenon for the overweight/obese population.     

Gene therapy in a humanized mouse model of familial hypercholesterolemia leads to marked regression of atherosclerosis

Background

Familial hypercholesterolemia (FH) is an autosomal codominant disorder caused by mutations in the low-density lipoprotein receptor (LDLR) gene. Homozygous FH patients (hoFH) have severe hypercholesterolemia leading to life threatening atherosclerosis in childhood and adolescence. Mice with germ line interruptions in the Ldlr and Apobec1 genes (Ldlr^−/−Apobec1^−/−) simulate metabolic and clinical aspects of hoFH, including atherogenesis on a chow diet.

Methods/Principal Findings

In this study, vectors based on adeno-associated virus 8 (AAV8) were used to deliver the gene for mouse Ldlr (mLDLR) to the livers of Ldlr^−/−Apobec1^−/− mice. A single intravenous injection of AAV8.mLDLR was found to significantly reduce plasma cholesterol and non-HDL cholesterol levels in chow-fed animals at doses as low as 3×10⁹ genome copies/mouse. Whereas Ldlr^−/−Apobec1^−/− mice fed a western-type diet and injected with a control AAV8.null vector experienced a further 65% progression in atherosclerosis over 2 months compared with baseline mice, Ldlr^−/−Apobec1^−/− mice treated with AAV8.mLDLR realized an 87% regression of atherosclerotic lesions after 3 months compared to baseline mice. Immunohistochemical analyses revealed a substantial remodeling of atherosclerotic lesions.

Conclusions/Significance

Collectively, the results presented herein suggest that AAV8-based gene therapy for FH may be feasible and support further development of this approach. The pre-clinical data from these studies will enable for the effective translation of gene therapy into the clinic for treatment of FH.     

Glucose-dependent insulinotropic polypeptide prevents the progression of macrophage-driven atherosclerosis in diabetic apolipoprotein E-null mice

Aim

We recently reported that glucose-dependent insulinotropic polypeptide (GIP) prevents the development of atherosclerosis in apolipoprotein E-null (Apoe ^−/−) mice. GIP receptors (GIPRs) are found to be severely down-regulated in diabetic animals. We examined whether GIP can exert anti-atherogenic effects in diabetes.

Methods

Nondiabetic Apoe ^−/− mice, streptozotocin-induced diabetic Apoe ^−/− mice, and db/db mice were administered GIP (25 nmol/kg/day) or saline (vehicle) through osmotic mini-pumps for 4 weeks. The animals were assessed for aortic atherosclerosis and for oxidized low-density lipoprotein-induced foam cell formation in exudate peritoneal macrophages.

Results

Diabetic Apoe ^−/− mice of 21 weeks of age exhibited more advanced atherosclerosis than nondiabetic Apoe ^−/− mice of the same age. GIP infusion in diabetic Apoe ^−/− mice increased plasma total GIP levels by 4-fold without improving plasma insulin, glucose, or lipid profiles. GIP infusion significantly suppressed macrophage-driven atherosclerotic lesions, but this effect was abolished by co-infusions with [Pro³]GIP, a GIPR antagonist. Foam cell formation was stimulated by 3-fold in diabetic Apoe ^−/− mice compared with their nondiabetic counterparts, but this effect was halved by GIP infusion. GIP infusion also attenuated the foam cell formation in db/db mice. In vitro treatment with GIP (1 nM) reduced foam cell formation by 15% in macrophages from diabetic Apoe ^−/− mice, and this attenuating effect was weaker than that attained by the same treatment of macrophages from nondiabetic counterparts (35%). While GIPR expression was reduced by only about a half in macrophages from diabetic mice, it was reduced much more dramatically in pancreatic islets from the same animals. Incubation with high glucose (500 mg/dl) for 9–10 days markedly reduced GIPR expression in pancreatic islet cells, but not in macrophages.

Conclusions

Long-term infusion of GIP conferred significant anti-atherogenic effects in diabetic mice even though the GIPR expression in macrophages was mildly down-regulated in the diabetic state.     

Beneficial effects of alternate dietary regimen on liver inflammation, atherosclerosis and renal activation

Background

Alternate day calorie restriction (CR) has been shown to be almost as beneficial as daily CR. The question arises whether this concept is also applicable to alternating dietary composition.

Objective

To seek evidence that alternating high cholesterol (HC) - cholesterol-free (CON) Western diet can effectively diminish hepatic and renal inflammation and cardiovascular risk factors as compared with daily HC-supplemented Western diet.

Design

Four groups of ApoE*3Leiden mice, a humanized model for atherosclerosis, were subjected to different feeding treatments for 16 weeks. Mice were fed CON diet; CON diet with 1% w/w cholesterol (HC); alternate (ALT) diet regimen of CON (4 days) and HC (3 days); or CON diet supplemented with 0.43% (w/w) cholesterol (MC), with overall dietary cholesterol intake equal to ALT. Plasma was analyzed for cardiovascular risk factors, aorta for atherosclerotic lesion formation, and liver and kidney for inflammation.

Results

ALT diet but not MC was almost as effective as daily CON feeding in preventing disease development. Compared to HC, the ALT group showed 62% lower hepatic nuclear factor kappa B (NF-κB) activity (P<0.001), a reduction of the circulating inflammatory markers E-selectin (−20%; P<0.05), vascular cell adhesion molecule 1 (VCAM-1; −15%; P<0.05) and Serum Amyloid A (SAA; −31%; P<0.05), smaller atherosclerotic lesion sizes (−51%; 46497±10791 µm² vs. 94664±16470 µm²; P<0.05) and diminished renal expression of specific inflammation and activation markers (VCAM-1, −27%; P<0.05; monocyte chemotactic protein-1 (MCP-1); −37%; P<0.01).

Conclusion

Alternate HC-CON feeding reproduced most of the beneficial effects of daily cholesterol-free diet, including strongly diminished hepatic, vascular and renal activation and inflammation; also atherosclerosis was reduced by half as compared to HC, albeit still higher compared to the CON group.     

CD40L deficiency attenuates diet-induced adipose tissue inflammation by impairing immune cell accumulation and production of pathogenic IgG-antibodies

Background

Adipose tissue inflammation fuels the metabolic syndrome. We recently reported that CD40L – an established marker and mediator of cardiovascular disease – induces inflammatory cytokine production in adipose cells in vitro. Here, we tested the hypothesis that CD40L deficiency modulates adipose tissue inflammation in vivo.

Methodology/Principal Findings

WT or CD40L^−/− mice consumed a high fat diet (HFD) for 20 weeks. Inflammatory cell recruitment was impaired in mice lacking CD40L as shown by a decrease of adipose tissue macrophages, B-cells, and an increase in protective T-regulatory cells. Mechanistically, CD40L-deficient mice expressed significantly lower levels of the pro-inflammatory chemokine MCP-1 both, locally in adipose tissue and systemically in plasma. Moreover, levels of pro-inflammatory IgG-antibodies against oxidized lipids were reduced in CD40L^−/− mice. Also, circulating low-density lipoproteins and insulin levels were lower in CD40L^−/− mice. However, CD40L^−/− mice consuming HFD were not protected from the onset of diet-induced obesity (DIO), insulin resistance, and hepatic steatosis, suggesting that CD40L selectively limits the inflammatory features of diet-induced obesity rather than its metabolic phenotype. Interestingly, CD40L^−/− mice consuming a low fat diet (LFD) showed both, a favorable inflammatory and metabolic phenotype characterized by diminished weight gain, improved insulin tolerance, and attenuated plasma adipokine levels.

Conclusion

We present the novel finding that CD40L deficiency limits adipose tissue inflammation in vivo. These findings identify CD40L as a potential mediator at the interface of cardiovascular and metabolic disease.     

Ablation of TSC2 enhances insulin secretion by increasing the number of mitochondria through activation of mTORC1

Aim

We previously found that chronic tuberous sclerosis protein 2 (TSC2) deletion induces activation of mammalian target of rapamycin Complex 1 (mTORC1) and leads to hypertrophy of pancreatic beta cells from pancreatic beta cell-specific TSC2 knockout (βTSC2^−/−) mice. The present study examines the effects of TSC2 ablation on insulin secretion from pancreatic beta cells.

Methods

Isolated islets from βTSC2^−/− mice and TSC2 knockdown insulin 1 (INS-1) insulinoma cells treated with small interfering ribonucleic acid were used to investigate insulin secretion, ATP content and the expression of mitochondrial genes.

Results

Activation of mTORC1 increased mitochondrial DNA expression, mitochondrial density and ATP production in pancreatic beta cells of βTSC2^−/− mice. In TSC2 knockdown INS-1 cells, mitochondrial DNA expression, mitochondrial density and ATP production were increased compared with those in control INS-1 cells, consistent with the phenotype of βTSC2^−/− mice. TSC2 knockdown INS-1 cells also exhibited augmented insulin secretory response to glucose. Rapamycin inhibited mitochondrial DNA expression and ATP production as well as insulin secretion in response to glucose. Thus, βTSC2^−/− mice exhibit hyperinsulinemia due to an increase in the number of mitochondria as well as enlargement of individual beta cells via activation of mTORC1.

Conclusion

Activation of mTORC1 by TSC2 ablation increases mitochondrial biogenesis and enhances insulin secretion from pancreatic beta cells.     

Hyperlipidemia and atherosclerotic lesion development in Ldlr-deficient mice on a long-term high-fat diet

Background

Mice deficient in the LDL receptor (Ldlr ^−/− mice) have been widely used as a model to mimic human atherosclerosis. However, the time-course of atherosclerotic lesion development and distribution of lesions at specific time-points are yet to be established. The current study sought to determine the progression and distribution of lesions in Ldlr ^−/− mice.

Methodology/Principal Findings

Ldlr-deficient mice fed regular chow or a high-fat (HF) diet for 0.5 to 12 months were analyzed for atherosclerotic lesions with en face and cross-sectional imaging. Mice displayed significant individual differences in lesion development when fed a chow diet, whereas those on a HF diet developed lesions in a time-dependent and site-selective manner. Specifically, mice subjected to the HF diet showed slight atherosclerotic lesions distributed exclusively in the aortic roots or innominate artery before 3 months. Lesions extended to the thoracic aorta at 6 months and abdominal aorta at 9 months. Cross-sectional analysis revealed the presence of advanced lesions in the aortic sinus after 3 months in the group on the HF diet and in the innominate artery at 6 to 9 months. The HF diet additionally resulted in increased total cholesterol, LDL, glucose, and HBA1c levels, along with the complication of obesity.

Conclusions/Significance

Ldlr-deficient mice on the HF diet tend to develop site-selective and size-specific atherosclerotic lesions over time. The current study should provide information on diet induction or drug intervention times and facilitate estimation of the appropriate locations of atherosclerotic lesions in Ldlr ^−/− mice.     

High Beta-Palmitate Fat Controls the Intestinal Inflammatory Response and Limits Intestinal Damage in Mucin Muc2 Deficient Mice

Background

Palmitic-acid esterified to the sn-1,3 positions of the glycerol backbone (alpha, alpha’-palmitate), the predominant palmitate conformation in regular infant formula fat, is poorly absorbed and might cause abdominal discomfort. In contrast, palmitic-acid esterified to the sn-2 position (beta-palmitate), the main palmitate conformation in human milk fat, is well absorbed. The aim of the present study was to examine the influence of high alpha, alpha’-palmitate fat (HAPF) diet and high beta-palmitate fat (HBPF) diet on colitis development in Muc2 deficient (Muc2^−/−) mice, a well-described animal model for spontaneous enterocolitis due to the lack of a protective mucus layer.

Methods

Muc2^−/− mice received AIN-93G reference diet, HAPF diet or HBPF diet for 5 weeks after weaning. Clinical symptoms, intestinal morphology and inflammation in the distal colon were analyzed.

Results

Both HBPF diet and AIN-93G diet limited the extent of intestinal erosions and morphological damage in Muc2^−/− mice compared with HAPF diet. In addition, the immunosuppressive regulatory T (Treg) cell response as demonstrated by the up-regulation of Foxp3, Tgfb1 and Ebi3 gene expression levels was enhanced by HBPF diet compared with AIN-93G and HAPF diets. HBPF diet also increased the gene expression of Pparg and enzymatic antioxidants (Sod1, Sod3 and Gpx1), genes all reported to be involved in promoting an immunosuppressive Treg cell response and to protect against colitis.

Conclusions

This study shows for the first time that HBPF diet limits the intestinal mucosal damage and controls the inflammatory response in Muc2^−/− mice by inducing an immunosuppressive Treg cell response.     

Adipocyte hypertrophy, fatty liver and metabolic risk factors in South Asians: the Molecular Study of Health and Risk in Ethnic Groups (mol-SHARE)

Objective

We sought to determine if differences in the distribution and characteristics of adipose tissue between South Asians and white Caucasians account for differences in risk factors for cardiovascular disease.

Research Design and Methods

We recruited 108 healthy South Asians (36.8 years) and white Caucasians (34.2 years) within three BMI strata. Body composition, adipocyte size, abdominal fat area, and hepatic adiposity were assessed and related to fasting glucose, insulin, lipids and adiponectin.

Results

After adjustment for age, sex, and BMI, South Asians compared to white Caucasians had higher ln fasting insulin (mean difference (md): 0.44; 95% CI: 0.20–0.69), lower HDL cholesterol (md: −0.13; 95% CI:−0.26 to −0.01), and lower adiponectin (md: −2.38; 95% CI: −3.59 to −1.17). South Asians also had more body fat (md: 2.69; 95% CI: 0.70 to 4.69), lower lean muscle mass (md: −3.25; 95%CI: −5.35 to −1.14), increased waist to hip ratio (md: 0.03; 95% CI: 0.01–0.05), less superficial subcutaneous abdominal adipose tissue (md: −2.94; 95% CI: −5.56 to−0.32), more deep/visceral to superficial adipose tissue ratio (md 0.34; 95% CI: 0.02 to 0.65), and more liver fat (md: 7.43%; 95% CI: 2.30 to 12.55%). Adipocyte area was increased in South Asians compared to white Caucasians (md: 64.26; 95% CI: 24.3 to 104.1) units². Adjustment for adipocyte area attenuated the ethnic differences in insulin (md: 0.22; 95% CI: −0.07 to 0.51), HDL (md: −0.01; 95% CI: −0.16 to 0.13) and adiponectin (md: −1.11; 95% CI: −2.61 to 0.39). Adjustment for differences in adipocyte area and fat distribution attenuated the ethnic difference in liver fat (md: 5.19; 95% CI: 0.31 to 10.06).

Conclusion

South Asians have an increased adipocyte area compared to white Caucasians. This difference accounts for the ethnic differences in insulin, HDL cholesterol, adiponectin, and ectopic fat deposition in the liver.     

Targeted deletion of neuropeptide Y (NPY) modulates experimental colitis

Background

Neurogenic inflammation plays a major role in the pathogenesis of inflammatory bowel disease (IBD). We examined the role of neuropeptide Y (NPY) and neuronal nitric oxide synthase (nNOS) in modulating colitis.

Methods

Colitis was induced by administration of dextran sodium sulphate (3% DSS) or streptomycin pre-treated Salmonella typhimurium (S.T.) in wild type (WT) and NPY (NPY^−/−) knockout mice. Colitis was assessed by clinical score, histological score and myeloperoxidase activity. NPY and nNOS expression was assessed by immunostaining. Oxidative stress was assessed by measuring catalase activity, glutathione and nitrite levels. Colonic motility was assessed by isometric muscle recording in WT and DSS-treated mice.

Results

DSS/S.T. induced an increase in enteric neuronal NPY and nNOS expression in WT mice. WT mice were more susceptible to inflammation compared to NPY^−/− as indicated by higher clinical & histological scores, and myeloperoxidase (MPO) activity (p<0.01). DSS-WT mice had increased nitrite, decreased glutathione (GSH) levels and increased catalase activity indicating more oxidative stress. The lower histological scores, MPO and chemokine KC in S.T.-treated nNOS^−/− and NPY^−/−/nNOS^−/− mice supported the finding that loss of NPY-induced nNOS attenuated inflammation. The inflammation resulted in chronic impairment of colonic motility in DSS-WT mice. NPY –treated rat enteric neurons in vitro exhibited increased nitrite and TNF-α production.

Conclusions

NPY mediated increase in nNOS is a determinant of oxidative stress and subsequent inflammation. Our study highlights the role of neuronal NPY and nNOS as mediators of inflammatory processes in IBD.     

Fat Oxidation,Fitness and Skeletal Muscle Expression of Oxidative/Lipid Metabolism Genes in South Asians: Implications for Insulin Resistance?

Background

South Asians are more insulin resistant than Europeans, which cannot be fully explained by differences in adiposity. We investigated whether differences in oxidative capacity and capacity for fatty acid utilisation in South Asians might contribute, using a range of whole-body and skeletal muscle measures.

Methodology/Principal Findings

Twenty men of South Asian ethnic origin and 20 age and BMI-matched men of white European descent underwent exercise and metabolic testing and provided a muscle biopsy to determine expression of oxidative and lipid metabolism genes and of insulin signalling proteins. In analyses adjusted for age, BMI, fat mass and physical activity, South Asians, compared to Europeans, exhibited; reduced insulin sensitivity by 26% (p = 0.010); lower VO_2max (40.6±6.6 vs 52.4±5.7 ml.kg⁻¹.min⁻¹, p = 0.001); and reduced fat oxidation during submaximal exercise at the same relative (3.77±2.02 vs 6.55±2.60 mg.kg⁻¹.min⁻¹ at 55% VO_2max, p = 0.013), and absolute (3.46±2.20 vs 6.00±1.93 mg.kg⁻¹.min⁻¹ at 25 ml O₂.kg⁻¹.min⁻¹, p = 0.021), exercise intensities. South Asians exhibited significantly higher skeletal muscle gene expression of CPT1A and FASN and significantly lower skeletal muscle protein expression of PI3K and PKB Ser473 phosphorylation. Fat oxidation during submaximal exercise and VO_2max both correlated significantly with insulin sensitivity index and PKB Ser473 phosphorylation, with VO_2max or fat oxidation during exercise explaining 10–13% of the variance in insulin sensitivity index, independent of age, body composition and physical activity.

Conclusions/Significance

These data indicate that reduced oxidative capacity and capacity for fatty acid utilisation at the whole body level are key features of the insulin resistant phenotype observed in South Asians, but that this is not the consequence of reduced skeletal muscle expression of oxidative and lipid metabolism genes.     

Internalization of modified lipids by CD36 and SR-A leads to hepatic inflammation and lysosomal cholesterol storage in Kupffer cells

Background & Aims

Non-alcoholic steatohepatitis (NASH) is characterized by steatosis and inflammation, which can further progress into fibrosis and cirrhosis. Recently, we demonstrated that combined deletion of the two main scavenger receptors, CD36 and macrophage scavenger receptor 1 (MSR1), which are important for modified cholesterol-rich lipoprotein uptake, reduced NASH. The individual contributions of these receptors to NASH and the intracellular mechanisms by which they contribute to inflammation have not been established. We hypothesize that CD36 and MSR1 contribute independently to the onset of inflammation in NASH, by affecting intracellular cholesterol distribution inside Kupffer cells (KCs).

Methods & Results

Ldlr^−/− mice were transplanted with wild-type (Wt), Cd36^−/− or Msr1^−/− bone marrow and fed a Western diet for 3months. Cd36^−/−- and Msr1^−/−- transplanted (tp) mice showed a similar reduction in hepatic inflammation compared to Wt-tp mice. While the total amount of cholesterol inside KCs was similar in all groups, KCs of Cd36^−/−- and Msr1^−/−-tp mice showed increased cytoplasmic cholesterol accumulation, while Wt-tp mice showed increased lysosomal cholesterol accumulation.

Conclusion

CD36 and MSR1 contribute similarly and independently to the progression of inflammation in NASH. One possible explanation for the inflammatory response related to expression of these receptors could be abnormal cholesterol trafficking in KCs. These data provide a new basis for prevention and treatment of NASH.     

Actigraph accelerometer-defined boundaries for sedentary behaviour and physical activity intensities in 7 year old children

Background

Accurate objective assessment of sedentary and physical activity behaviours during childhood is integral to the understanding of their relation to later health outcomes, as well as to documenting the frequency and distribution of physical activity within a population.

Purpose

To calibrate the Actigraph GT1M accelerometer, using energy expenditure (EE) as the criterion measure, to define thresholds for sedentary behaviour and physical activity categories suitable for use in a large scale epidemiological study in young children.

Methods

Accelerometer-based assessments of physical activity (counts per minute) were calibrated against EE measures (kcal.kg⁻¹.hr⁻¹) obtained over a range of exercise intensities using a COSMED K4b² portable metabolic unit in 53 seven-year-old children. Children performed seven activities: lying down viewing television, sitting upright playing a computer game, slow walking, brisk walking, jogging, hopscotch and basketball. Threshold count values were established to identify sedentary behaviour and light, moderate and vigorous physical activity using linear discriminant analysis (LDA) and evaluated using receiver operating characteristic (ROC) curve analysis.

Results

EE was significantly associated with counts for all non-sedentary activities with the exception of jogging. Threshold values for accelerometer counts (counts.minute⁻¹) were <100 for sedentary behaviour and ≤2240, ≤3840 and ≥3841 for light, moderate and vigorous physical activity respectively. The area under the ROC curves for discrimination of sedentary behaviour and vigorous activity were 0.98. Boundaries for light and moderate physical activity were less well defined (0.61 and 0.60 respectively). Sensitivity and specificity were higher for sedentary (99% and 97%) and vigorous (95% and 91%) than for light (60% and 83%) and moderate (61% and 76%) thresholds.

Conclusion

The accelerometer cut points established in this study can be used to classify sedentary behaviour and to distinguish between light, moderate and vigorous physical activity in children of this age.     

LDL receptor knock-out mice are a physiological model particularly vulnerable to study the onset of inflammation in non-alcoholic fatty liver disease

Background & Aims

Non-alcoholic steatohepatitis (NASH) involves steatosis combined with inflammation, which can progress into fibrosis and cirrhosis. Exploring the molecular mechanisms of NASH is highly dependent on the availability of animal models. Currently, the most commonly used animal models for NASH imitate particularly late stages of human disease. Thus, there is a need for an animal model that can be used for investigating the factors that potentiate the inflammatory response within NASH. We have previously shown that 7-day high-fat-high-cholesterol (HFC) feeding induces steatosis and inflammation in both APOE2ki and Ldlr^−/− mice. However, it is not known whether the early inflammatory response observed in these mice will sustain over time and lead to liver damage. We hypothesized that the inflammatory response in both models is sufficient to induce liver damage over time.

Methods

APOE2ki and Ldlr^−/− mice were fed a chow or HFC diet for 3 months. C57Bl6/J mice were used as control.

Results

Surprisingly, hepatic inflammation was abolished in APOE2ki mice, while it was sustained in Ldlr^−/− mice. In addition, increased apoptosis and hepatic fibrosis was only demonstrated in Ldlr^−/− mice. Finally, bone-marrow-derived-macrophages of Ldlr^−/− mice showed an increased inflammatory response after oxidized LDL (oxLDL) loading compared to APOE2ki mice.

Conclusion

Ldlr^−/− mice, but not APOE2ki mice, developed sustained hepatic inflammation and liver damage upon long term HFC feeding due to increased sensitivity for oxLDL uptake. Therefore, the Ldlr^−/− mice are a promising physiological model particularly vulnerable for investigating the onset of hepatic inflammation in non-alcoholic steatohepatitis.     

Peripheral effects of FAAH deficiency on fuel and energy homeostasis: role of dysregulated lysine acetylation

Background

FAAH (fatty acid amide hydrolase), primarily expressed in the liver, hydrolyzes the endocannabinoids fatty acid ethanolamides (FAA). Human FAAH gene mutations are associated with increased body weight and obesity. In our present study, using targeted metabolite and lipid profiling, and new global acetylome profiling methodologies, we examined the role of the liver on fuel and energy homeostasis in whole body FAAH^−/− mice.

Methodology/Principal Findings

FAAH^−/− mice exhibit altered energy homeostasis demonstrated by decreased oxygen consumption (Indirect calorimetry). FAAH^−/− mice are hyperinsulinemic and have adipose, skeletal and hepatic insulin resistance as indicated by stable isotope phenotyping (SIPHEN). Fed state skeletal muscle and liver triglyceride levels was increased 2–3 fold, while glycogen was decreased 42% and 57% respectively. Hepatic cholesterol synthesis was decreased 22% in FAAH^−/− mice. Dysregulated hepatic FAAH^−/− lysine acetylation was consistent with their metabolite profiling. Fasted to fed increases in hepatic FAAH^−/− acetyl-CoA (85%, p<0.01) corresponded to similar increases in citrate levels (45%). Altered FAAH^−/− mitochondrial malate dehydrogenase (MDH2) acetylation, which can affect the malate aspartate shuttle, was consistent with our observation of a 25% decrease in fed malate and aspartate levels. Decreased fasted but not fed dihydroxyacetone-P and glycerol-3-P levels in FAAH^−/− mice was consistent with a compensating contribution from decreased acetylation of fed FAAH^−/− aldolase B. Fed FAAH^−/− alcohol dehydrogenase (ADH) acetylation was also decreased.

Conclusions/Significance

Whole body FAAH deletion contributes to a pre-diabetic phenotype by mechanisms resulting in impairment of hepatic glucose and lipid metabolism. FAAH^−/− mice had altered hepatic lysine acetylation, the pattern sharing similarities with acetylation changes reported with chronic alcohol treatment. Dysregulated hepatic lysine acetylation seen with impaired FAA hydrolysis could support the liver''s role in fostering the pre-diabetic state, and may reflect part of the mechanism underlying the hepatic effects of endocannabinoids in alcoholic liver disease mouse models.     

High fat diet-induced changes in mouse muscle mitochondrial phospholipids do not impair mitochondrial respiration despite insulin resistance

Background

Type 2 diabetes mellitus and muscle insulin resistance have been associated with reduced capacity of skeletal muscle mitochondria, possibly as a result of increased intake of dietary fat. Here, we examined the hypothesis that a prolonged high-fat diet consumption (HFD) increases the saturation of muscle mitochondrial membrane phospholipids causing impaired mitochondrial oxidative capacity and possibly insulin resistance.

Methodology

C57BL/6J mice were fed an 8-week or 20-week low fat diet (10 kcal%; LFD) or HFD (45 kcal%). Skeletal muscle mitochondria were isolated and fatty acid (FA) composition of skeletal muscle mitochondrial phospholipids was analyzed by thin-layer chromatography followed by GC. High-resolution respirometry was used to assess oxidation of pyruvate and fatty acids by mitochondria. Insulin sensitivity was estimated by HOMA-IR.

Principal Findings

At 8 weeks, mono-unsaturated FA (16∶1n7, 18∶1n7 and 18∶1n9) were decreased (−4.0%, p<0.001), whereas saturated FA (16∶0) were increased (+3.2%, p<0.001) in phospholipids of HFD vs. LFD mitochondria. Interestingly, 20 weeks of HFD descreased mono-unsaturated FA while n-6 poly-unsaturated FA (18∶2n6, 20∶4n6, 22∶5n6) showed a pronounced increase (+4.0%, p<0.001). Despite increased saturation of muscle mitochondrial phospholipids after the 8-week HFD, mitochondrial oxidation of both pyruvate and fatty acids were similar between LFD and HFD mice. After 20 weeks of HFD, the increase in n-6 poly-unsaturated FA was accompanied by enhanced maximal capacity of the electron transport chain (+49%, p = 0.002) and a tendency for increased ADP-stimulated respiration, but only when fuelled by a lipid-derived substrate. Insulin sensitivity in HFD mice was reduced at both 8 and 20 weeks.

Conclusions/Interpretation

Our findings do not support the concept that prolonged HF feeding leads to increased saturation of skeletal muscle mitochondrial phospholipids resulting in a decrease in mitochondrial fat oxidative capacity and (muscle) insulin resistance.     

The bile acid receptor GPBAR-1 (TGR5) modulates integrity of intestinal barrier and immune response to experimental colitis

Background

GP-BAR1, a member G protein coupled receptor superfamily, is a cell surface bile acid-activated receptor highly expressed in the ileum and colon. In monocytes, ligation of GP-BAR1 by secondary bile acids results in a cAMP-dependent attenuation of cytokine generation.

Aims

To investigate the role GP-BAR1 in regulating intestinal homeostasis and inflammation-driven immune dysfunction in rodent models of colitis.

Methods

Colitis was induced in wild type and GP-BAR1^−/− mice by DSS and TNBS administration. Potential GP-BAR1 agonists were identified by in silico screening and computational docking studies.

Results

GP-BAR1^−/− mice develop an abnormal morphology of colonic mucous cells and an altered molecular architecture of epithelial tight junctions with increased expression and abnormal subcellular distribution of zonulin 1 resulting in increased intestinal permeability and susceptibility to develop severe colitis in response to DSS at early stage of life. By in silico screening and docking studies we identified ciprofloxacin as a GP-BAR1 ligand. In monocytes, ciprofloxacin increases cAMP concentrations and attenuates TNFα release induced by TLR4 ligation in a GP-BAR1 dependent manner. Treating mice rendered colitic by TNBS with ciprofloxacin and oleanolic acid, a well characterized GP-BAR1 ligand, abrogates signs and symptoms of colitis. Colonic expression of GP-BAR1 mRNA increases in rodent models of colitis and tissues from Crohn''s disease patients. Flow cytometry analysis demonstrates that ≈90% of CD14+ cells isolated from the lamina propria of TNBS-treated mice stained positively for GP-BAR1.

Conclusions

GP-BAR1 regulates intestinal barrier structure. Its expression increases in rodent models of colitis and Crohn''s disease. Ciprofloxacin is a GP-BAR1 ligand.     

The D299G/T399I Toll-like receptor 4 variant associates with body and liver fat: results from the TULIP and METSIM Studies

Background

Toll-like-receptor 4 (TLR) is discussed to provide a molecular link between obesity, inflammation and insulin resistance. Genetic studies with replications in non-diabetic individuals in regard to their fat distribution or insulin resistance according to their carrier status of a common toll-like receptor 4 (TLR4) variant (TLR4^D299G/T399I) are still lacking.

Methodology/Principal Findings

We performed a cross-sectional analysis in individuals phenotyped for prediabetic traits as body fat composition (including magnetic resonance imaging), blood glucose levels and insulin resistance (oral glucose tolerance testing, euglycemic hyperinsulinemic clamp), according to TLR4 genotype determined by candidate SNP analyses (rs4986790). We analyzed N = 1482 non-diabetic individuals from the TÜF/TULIP cohort (South Germany, aged 39±13 y, BMI 28.5±7.9, mean±SD) and N = 5327 non-diabetic participants of the METSIM study (Finland, males aged 58±6 y, BMI 26.8±3.8) for replication purposes. German TLR4^D299G/T399I carriers had a significantly increased body fat (XG in rs4986790: +6.98%, p = 0.03, dominant model, adjusted for age, gender) and decreased insulin sensitivity (XG: −15.3%, Matsuda model, p = 0.04; XG: −20.6%, p = 0.016, clamp; both dominant models adjusted for age, gender, body fat). In addition, both liver fat (AG: +49.7%; p = 0.002) and visceral adipose tissue (AG: +8.2%; p = 0.047, both adjusted for age, gender, body fat) were significantly increased in rs4986790 minor allele carriers, and the effect on liver fat remained significant also after additional adjustment for visceral fat (p = 0.014). The analysis in METSIM confirmed increased body fat content in association with the rare G allele in rs4986790 (AG: +1.26%, GG: +11.0%; p = 0.010, additive model, adjusted for age) and showed a non-significant trend towards decreased insulin sensitivity (AG: −0.99%, GG: −10.62%).

Conclusions/Significance

TLR4^D299G/T399I associates with increased total body fat, visceral fat, liver fat and decreased insulin sensitivity in non-diabetic Caucasians and may contribute to diabetes risk. This finding supports the role of TLR4 as a molecular link between obesity and insulin resistance.     

MFGE8 does not influence chorio-retinal homeostasis or choroidal neovascularization in vivo

Purpose

Milk fat globule-epidermal growth factor-factor VIII (MFGE8) is necessary for diurnal outer segment phagocytosis and promotes VEGF-dependent neovascularization. The prevalence of two single nucleotide polymorphisms (SNP) in MFGE8 was studied in two exsudative or “wet” Age-related Macular Degeneration (AMD) groups and two corresponding control groups. We studied the effect of MFGE8 deficiency on retinal homeostasis with age and on choroidal neovascularization (CNV) in mice.

Methods

The distribution of the SNP (rs4945 and rs1878326) of MFGE8 was analyzed in two groups of patients with “wet” AMD and their age-matched controls from Germany and France. MFGE8-expressing cells were identified in Mfge8 ^+/− mice expressing ß-galactosidase. Aged Mfge8 ^+/− and Mfge8 ^−/− mice were studied by funduscopy, histology, electron microscopy, scanning electron microscopy of vascular corrosion casts of the choroid, and after laser-induced CNV.

Results

rs1878326 was associated with AMD in the French and German group. The Mfge8 promoter is highly active in photoreceptors but not in retinal pigment epithelium cells. Mfge8^−/− mice did not differ from controls in terms of fundus appearance, photoreceptor cell layers, choroidal architecture or laser-induced CNV. In contrast, the Bruch''s membrane (BM) was slightly but significantly thicker in Mfge8^−/− mice as compared to controls.

Conclusions

Despite a reproducible minor increase of rs1878326 in AMD patients and a very modest increase in BM in Mfge8^−/− mice, our data suggests that MFGE8 dysfunction does not play a critical role in the pathogenesis of AMD.