A Murine Model of Obesity With Accelerated Atherosclerosis

The epidemic of obesity sweeping developed nations is accompanied by an increase in atherosclerotic cardiovascular diseases. Dyslipidemia, diabetes, hypertension, and obesity are risk factors for cardiovascular disease. However, delineating the mechanism of obesity‐accelerated atherosclerosis has been hampered by a paucity of animal models. Similar to humans, apolipoprotein E–deficient (apoE^?/?) mice spontaneously develop atherosclerosis over their lifetime. To determine whether apoE^?/? mice would develop obesity with accelerated atherosclerosis, we fed mice diets containing 10 (low fat (LF)) or 60 (high fat (HF)) kcal % from fat for 17 weeks. Mice fed the HF diet had a marked increase in body weight and atherosclerotic lesion formation compared to mice fed the LF diet. There were no significant differences between groups in serum total cholesterol, triglycerides, or leptin concentrations. Plasma concentrations of the acute‐phase reactant serum amyloid A (SAA) are elevated in both obesity and cardiovascular disease. Accordingly, plasma SAA concentrations were increased fourfold (P < 0.01) in mice fed the HF diet. SAA was associated with both pro‐ and antiatherogenic lipoproteins in mice fed the HF diet compared to those fed the LF diet, in which SAA was primarily associated with the antiatherogenic lipoprotein high‐density lipoprotein (HDL). Moreover, SAA was localized with apoB‐containing lipoproteins and biglycan in the vascular wall. Taken together, these data suggest male apoE‐deficient mice are a model of metabolic syndrome and that chronic low level inflammation associated with increased SAA concentrations may mediate atherosclerotic lesion formation.     

Grape proanthocyanidin-induced intestinal bloom of Akkermansia muciniphila is dependent on its baseline abundance and precedes activation of host genes related to metabolic health

We previously showed that C57BL/6J mice fed high-fat diet (HFD) supplemented with 1% grape polyphenols (GP) for 12 weeks developed a bloom of Akkermansia muciniphila with attenuated metabolic syndrome symptoms. Here we investigated early timing of GP-induced effects and the responsible class of grape polyphenols. Mice were fed HFD, low-fat diet (LFD) or formulations supplemented with GP (HFD-GP, LFD-GP) for 14 days. Mice fed HFD-GP, but not LFD-GP, showed improved oral glucose tolerance compared to controls. A. muciniphila bloom occurred earlier in mice fed LFD-GP than HFD-GP; however, timing was dependent on baseline A. muciniphila levels rather than dietary fat. Mice gavaged for 10 days with GP extract (GPE) or grape proanthocyanidins (PACs), each delivering 360 mg PACs/kg body weight, induced a bloom of fecal and cecal A. muciniphila, the rate of which depended on initial A. muciniphila abundance. Grape PACs were sufficient to induce a bloom of A. muciniphila independent of specific intestinal gene expression changes. Gut microbial community analysis and in vitro inhibition of A. muciniphila by GPE or PACs suggest that the A. muciniphila bloom in vivo occurs via indirect mechanisms.     

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.     

Dietary fat,fatty acid saturation and mitochondrial bioenergetics

Fat intake alters mitochondrial lipid composition which can affect function. We used novel methodology to assess bioenergetics, including simultaneous ATP and reactive oxygen species (ROS) production, in liver and heart mitochondria of C57BL/6 mice fed diets of variant fatty acid content and saturation. Our methodology allowed us to clamp ADP concentration and membrane potential (ΔΨ) at fixed levels. Mice received a control diet for 17–19 weeks, a high-fat (HF) diet (60 % lard) for 17–19 weeks, or HF for 12 weeks followed by 6–7 weeks of HF with 50 % of fat as menhaden oil (MO) which is rich in n-3 fatty acids. ATP production was determined as conversion of 2-deoxyglucose to 2-deoxyglucose phosphate by NMR spectroscopy. Respiration and ATP production were significantly reduced at all levels of ADP and resultant clamped ΔΨ in liver mitochondria from mice fed HF compared to controls. At given ΔΨ, ROS production per mg mitochondrial protein, per unit respiration, or per ATP generated were greater for liver mitochondria of HF-fed mice compared to control or MO-fed mice. Moreover, these ROS metrics began to increase at a lower ΔΨ threshold. Similar, but less marked, changes were observed in heart mitochondria of HF-fed mice compared to controls. No changes in mitochondrial bioenergetics were observed in studies of separate mice fed HF versus control for only 12 weeks. In summary, HF feeding of sufficient duration impairs mitochondrial bioenergetics and is associated with a greater ROS “cost” of ATP production compared to controls. These effects are, in part, mitigated by MO.     

Characterization of a Novel Intestinal Glycerol-3-phosphate Acyltransferase Pathway and Its Role in Lipid Homeostasis

Dietary triglycerides (TG) are absorbed by the enterocytes of the small intestine after luminal hydrolysis into monacylglycerol and fatty acids. Before secretion on chylomicrons, these lipids are reesterified into TG, primarily through the monoacylglycerol pathway. However, targeted deletion of the primary murine monoacylglycerol acyltransferase does not quantitatively affect lipid absorption, suggesting the existence of alternative pathways. Therefore, we investigated the role of the glycerol 3-phosphate pathway in dietary lipid absorption. The expression of glycerol-3-phosphate acyltransferase (GPAT3) was examined throughout the small intestine. To evaluate the role for GPAT3 in lipid absorption, mice harboring a disrupted GPAT3 gene (Gpat3^−/−) were subjected to an oral lipid challenge and fed a Western-type diet to characterize the role in lipid and cholesterol homeostasis. Additional mechanistic studies were performed in primary enterocytes. GPAT3 was abundantly expressed in the apical surface of enterocytes in the small intestine. After an oral lipid bolus, Gpat3^−/− mice exhibited attenuated plasma TG excursion and accumulated lipid in the enterocytes. Electron microscopy studies revealed a lack of lipids in the lamina propria and intercellular space in Gpat3^−/− mice. Gpat3^−/− enterocytes displayed a compensatory increase in the synthesis of phospholipid and cholesteryl ester. When fed a Western-type diet, hepatic TG and cholesteryl ester accumulation was significantly higher in Gpat3^−/− mice compared with the wild-type mice accompanied by elevated levels of alanine aminotransferase, a marker of liver injury. Dysregulation of bile acid metabolism was also evident in Gpat3-null mice. These studies identify GPAT3 as a novel enzyme involved in intestinal lipid metabolism.     

The inflammatory profile and liver damage of a sucrose-rich diet in mice

It is still unclear if an isoenergetic, sucrose-rich diet leads to health consequences.AimsTo investigate the effects of excessive sucrose within an isoenergetic diet on metabolic parameters in male C57BL/6 mice.MethodsAnimals were fed a control diet (10% fat, 8% sucrose — SC group), a high-sucrose diet (10% fat, 32% sucrose — HSu group), a high-fat diet (42% fat, 8% sucrose — HF group) or a high-fat/high-sucrose diet (42% fat, 32% sucrose — HF/HSu group) for 8 weeks.ResultsMice fed HF and HF/HSu diets gained more body mass (BM) and more body adiposity than SC- or Hsu-fed mice. Despite the unchanged BM and adiposity indices, HSu mice presented adipocyte hypertrophy, which was also observed in the HF and HF/HSu groups (P<.0001). The HF, HSu and HF/HSu mice were glucose intolerant and had elevated serum insulin levels (P<.05). The levels of leptin, resistin and monocyte chemotactic protein-1 increased, while the serum adiponectin decreased in the HF, HSu and HF/HSu groups (P<.05). In the adipose tissue, the HF, HSu and HF/HSu groups showed higher levels of leptin expression and lower levels of adiponectin expression in comparison with the SC group (P<.05). Liver steatosis was higher in the HF, HSu and HF/HSu groups than in the SC group (P<.0001). Hepatic cholesterol was higher in the HF and HF/HSu groups, while hepatic TG was higher in the HSu and HF/HSu groups (P<.05). In hepatic tissue, the sterol receptor element-binding protein-1c expression was increased in the HF, HSu and HF/HSu groups, unlike the peroxisome proliferator-activated receptor-alpha expression that decreased in the HF, HSu and HF/HSu groups in comparison with the SC group (P<.05).ConclusionA sucrose-rich diet does not lead to a state of obesity but has the potential to cause changes in the adipocytes (hypertrophy) as well as glucose intolerance, hyperinsulinemia, hyperlipidemia, hepatic steatosis and increases in the number of inflammatory cytokines. The deleterious effects of a sucrose-rich diet in an animal model, even when the sucrose replaces starch isocalorically in the feed, can have far-reaching consequences for health.     

Diet‐induced Obese Mice Are Leptin Insufficient After Weight Reduction

Behavioral therapies aimed at reducing excess body fat result in limited fat loss after dieting. To understand the causes for maintenance of adiposity, high‐fat (HF) diet–induced obese (DIO) mice were switched to a low‐fat chow diet, and the effects of chow on histological and molecular alterations of adipose tissue and metabolic parameters were examined. DIO mice reduced and stabilized their body weights after being switched to chow (HF‐chow), but retained a greater amount of adiposity than chow‐fed mice. Reduction in adipocyte volume, not number, caused a decrease in fat mass. HF‐chow mice showed normalized circulating insulin and leptin levels, improved glucose tolerance, and reduced inflammatory status in white adipose tissue (WAT). Circulating leptin levels corrected for fat mass were lower in HF‐chow mice. Leptin administration was used to test whether reduced leptin level of HF‐chow mice inhibited further fat loss. Leptin treatment led to an additional reduction in adiposity. Finally, HF‐HF mice had lower mRNA levels of β₃ adrenergic receptor (β₃‐AR) in epididymal WAT (EWAT) compared to chow‐fed mice, and diet change led to an increase in the WAT β₃‐AR mRNA levels that were similar to the levels of chow‐fed mice, suggesting an elevation in sympathetic activation of WAT during diet switch relative to HF‐HF mice leading to the reduced leptin level and proinflammatory cytokine content. In summary, HF‐chow mice were resistant to further fat loss due to leptin insufficiency. Diet alteration from HF to low fat improved metabolic state of DIO mice, although their adiposity was defended at a higher level.     

Suppression of QRFP 43 in the hypothalamic ventromedial nucleus of Long-Evans rats fed a high-fat diet

QRFP 43 is a RFamide peptide present in the ventromedial nucleus (VMN) and lateral hypothalamus. It stimulates food intake in mice and its chronic infusion induces hyperphagia, reduced thermogenesis, and obesity. In this experiment, we measured it in the VMN and lateral hypothalamus of Long-Evans rats fed either a high-fat (HF), control, or low-fat (LF) diet in parallel with plasma leptin, adiposity, and energy intake. After 8 weeks of ad libitum diet intake, energy intake of HF rats was similar to that of control rats. In the VMN, QRFP 43 was completely undetectable in HF rats and its tissue concentration in control rats was significantly lower than in LF rats (p < 0.03). HF rats had higher levels of leptin than control rats (+24%; p < 0.03) and than LF rats (+42%; p < 0.002). The QRFP 43 concentration in the VMN was inversely correlated with plasma leptin (r = −0.34; P < 0.04) and with the adipogenic index of the diet (p < 0.02) but not with insulin. We conclude that the decrease of the orexigenic drive mediated by QRFP 43 could contribute to the normalization of caloric intake in HF diet fed rats. QRFP 43 might play a role downstream of leptin in the regulation of feeding behavior.     

Fish oil supplementation increases expression of mammary tumor apoptosis mediators and reduces inflammation in an obesity-associated HER-2 breast cancer model

Obesity is associated with inflammation and has been shown to increase breast cancer severity. The objective of this study was to examine the effect of fish oil (FO) supplementation in obesity-associated mammary tumorigenesis in the MMTV-neu(ndl)-YD5 mouse model of human epidermal growth factor receptor-2 positive BC. Female mice were fed one of three diets for 16 weeks: i) high fat diet [HF, % kacl: 41.2% lard, 18.7% corn oil (CO)], ii) an isocaloric HF plus menhaden FO diet (HF+FO, % kcal: 41.2 lard, 13.4% CO, 5.3% FO), iii) low fat diet (LF, % kcal: 4.7% lard, 6% CO). HF mice had increased body weight, visceral adipose weight and serum hormone concentrations (increased leptin and resistin; decreased adiponectin) versus LF, which was attenuated in the HF+FO group versus HF (P<.05). Compared to HF, tumor onset was delayed in HF+FO and LF mice (P<0.05). Compared to HF, HF+FO reduced mammary tumor multiplicity (-27%), tumor weight (-46%) and total tumor volume (-50%) (P<0.05). Additionally, HF+FO reduced mammary tumor multiplicity (-33%), tumor weight (-39%) and total tumor volume (-60%) versus LF. HF+FO improved mammary tumor apoptosis status with increased expression of pro-apoptotic Bad and decreased expression of anti-apoptotic Bcl-xLmediators versus HF (P<0.05). Additionally, HF+FO decreased tumor protein expression of activated Akt, NFκB p65 and STAT3, versus HF (P<0.05). Tumor mRNA expression of inflammatory mediators TNFα, IL-6 and leptin were reduced in HF+FO, whereas IL-10 expression was increased compared to HF (P<0.05). Collectively these results demonstrate the efficacy of FO supplementation for improving obesity-associated breast cancer outcomes.     

Increased maternal fat consumption during pregnancy alters body composition in neonatal mice

Maternal overnutrition prior to and during gestation causes pronounced metabolic dysfunction in the adult offspring. However, less is known about metabolic adaptations in the offspring that occur independently of postnatal growth and nutrition. Therefore, we evaluated the impact of excess maternal dietary lipid intake on the in utero programming of body composition, hepatic function, and hypothalamic development in newborn (P0) offspring. Female mice were fed a low-fat (LF) or high-fat (HF) diet and were mated after 4, 12, and 23 wk. A subset of the obese HF dams was switched to the LF diet during the second (DR2) or third (DR3) pregnancies. The HF offspring accrued more fat mass than the LF pups, regardless of duration of maternal HF diet consumption or prepregnancy maternal adiposity. Increased neonatal adiposity was not observed in the DR3 pups. Liver weights were reduced in the HF offspring but not in the DR2 or DR3 pups. Offspring hepatic triglyceride content was reduced in the HF pups, but hepatic inflammation and expression of lipid metabolism genes were largely unaffected by maternal diet. Maternal diet did not alter the hypothalamic expression of orexigenic and anorexigenic neuropeptides in the offspring. Thus, the intrauterine programming of increased neonatal adiposity and reduced liver size by maternal overnutrition is evident in mice at birth and occurs prior to the development of maternal obesity. These observations demonstrate that dietary intervention during pregnancy minimizes the deleterious effects of maternal obesity on offspring body composition, potentially reducing the offsprings' risk of developing obesity and related diseases later in life.     

Time-restricted feeding mice a high-fat diet induces a unique lipidomic profile

Time-restricted feeding (TRF) can reduce adiposity and lessen the co-morbidities of obesity. Mice consuming obesogenic high-fat (HF) diets develop insulin resistance and hepatic steatosis, but have elevated indices of long-chain polyunsaturated fatty acids (LCPUFA) that may be beneficial. While TRF impacts lipid metabolism, scant data exist regarding the impact of TRF upon lipidomic composition of tissues. We (1) tested the hypothesis that TRF of a HF diet elevates LCPUFA indices while preventing insulin resistance and hepatic steatosis and (2) determined the impact of TRF upon the lipidome in plasma, liver, and adipose tissue. For 12 weeks, male, adult mice were fed a control diet ad libitum, a HF diet ad libitum (HF-AL), or a HF diet with TRF, 12 hours during the dark phase (HF-TRF). HF-TRF prevented insulin resistance and hepatic steatosis resulting from by HF-AL treatment. TRF-blocked plasma increases in LCPUFA induced by HF-AL treatment but elevated concentrations of triacylglycerols and non-esterified saturated fatty acids. Analysis of the hepatic lipidome demonstrated that TRF did not elevate LCPUFA while reducing steatosis. However, TRF created (1) a separate hepatic lipid signature for triacylglycerols, phosphatidylcholine, and phosphatidylethanolamine species and (2) modified gene and protein expression consistent with reduced fatty acid synthesis and restoration of diurnal gene signaling. TRF increased the saturated fatty acid content in visceral adipose tissue. In summary, TRF of a HF diet alters the lipidomic profile of plasma, liver, and adipose tissue, creating a third distinct lipid metabolic state indicative of positive metabolic adaptations following HF intake.     

High-Fat Diet: Bacteria Interactions Promote Intestinal Inflammation Which Precedes and Correlates with Obesity and Insulin Resistance in Mouse

Background

Obesity induced by high fat (HF) diet is associated with inflammation which contributes to development of insulin resistance. Most prior studies have focused on adipose tissue as the source of obesity-associated inflammation. Increasing evidence links intestinal bacteria to development of diet-induced obesity (DIO). This study tested the hypothesis that HF western diet and gut bacteria interact to promote intestinal inflammation, which contributes to the progression of obesity and insulin resistance.

Methodology/Principal Findings

Conventionally raised specific-pathogen free (CONV) and germ-free (GF) mice were given HF or low fat (LF) diet for 2–16 weeks. Body weight and adiposity were measured. Intestinal inflammation was assessed by evaluation of TNF-α mRNA and activation of a NF-κB^EGFP reporter gene. In CONV but not GF mice, HF diet induced increases in body weight and adiposity. HF diet induced ileal TNF-α mRNA in CONV but not GF mice and this increase preceded obesity and strongly and significantly correlated with diet induced weight gain, adiposity, plasma insulin and glucose. In CONV mice HF diet also resulted in activation of NF-κB^EGFP in epithelial cells, immune cells and endothelial cells of small intestine. Further experiments demonstrated that fecal slurries from CONV mice fed HF diet are sufficient to activate NF-κB^EGFP in GF NF-κB^EGFP mice.

Conclusions/Significance

Bacteria and HF diet interact to promote proinflammatory changes in the small intestine, which precede weight gain and obesity and show strong and significant associations with progression of obesity and development of insulin resistance. To our knowledge, this is the first evidence that intestinal inflammation is an early consequence of HF diet which may contribute to obesity and associated insulin resistance. Interventions which limit intestinal inflammation induced by HF diet and bacteria may protect against obesity and insulin resistance.     

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.     

Improved immune functions with administration of a low-fat diet in a burn animal model

The purpose of this study was to characterize the impact of a low-fat (LF; 1% fat) diet, a high-fat (HF; 25% fat) diet, and a standard (SD; 5% fat) diet on immune and oxidative parameters in a 20% body surface area burn animal model fed ad libitum for 10 days postinjury. Although the mechanisms are poorly understood, the amount of dietary lipid in nutritional support has been shown to have immunomodulatory effects after burn injury. Burned mice fed the LF diet showed a normal response in activated splenocyte proliferation compared to burned animals that received the SD or HF diet. Animals fed the SD and HF diets presented increased production of nitric oxide and prostaglandin E2 response after burn injury, which is associated with inhibited splenocyte proliferation. The total thiol concentration in spleen cells from burned animals kept on the HF diet was significantly higher than that in unburned animals, while no increase in these oxidative parameters was observed in LF-fed burned animals. Moreover, the LF diet significantly reduced hepatic lipid peroxidation, as measured by malonaldehyde concentration, compared to the other two diets. These results suggest that the administration of a LF diet in mice after a burn injury prevents inhibition of in vitro splenocyte proliferation and reduces the intensity of oxidative stress.     

Glycerol-3-phosphate Acyltransferase Isoform-4 (GPAT4) Limits Oxidation of Exogenous Fatty Acids in Brown Adipocytes

Glycerol-3-phosphate acyltransferase-4 (GPAT4) null pups grew poorly during the suckling period and, as adults, were protected from high fat diet-induced obesity. To determine why Gpat4^−/− mice failed to gain weight during these two periods of high fat feeding, we examined energy metabolism. Compared with controls, the metabolic rate of Gpat4^−/− mice fed a 45% fat diet was 12% higher. Core body temperature was 1 ºC higher after high fat feeding. Food intake, fat absorption, and activity were similar in both genotypes. Impaired weight gain in Gpat4^−/− mice did not result from increased heat loss, because both cold tolerance and response to a β3-adrenergic agonist were similar in both genotypes. Because GPAT4 comprises 65% of the total GPAT activity in brown adipose tissue (BAT), we characterized BAT function. A 45% fat diet increased the Gpat4^−/− BAT expression of peroxisome proliferator-activated receptor α (PPAR) target genes, Cpt1α, Pgc1α, and Ucp1, and BAT mitochondria oxidized oleate and pyruvate at higher rates than controls, suggesting that fatty acid signaling and flux through the TCA cycle were enhanced. To assess the role of GPAT4 directly, neonatal BAT preadipocytes were differentiated to adipocytes. Compared with controls, Gpat4^−/− brown adipocytes incorporated 33% less fatty acid into triacylglycerol and 46% more into the pathway of β-oxidation. The increased oxidation rate was due solely to an increase in the oxidation of exogenous fatty acids. These data suggest that in the absence of cold exposure, GPAT4 limits excessive fatty acid oxidation and the detrimental induction of a hypermetabolic state.     

Colonic inflammation accompanies an increase of β-catenin signaling and Lachnospiraceae/Streptococcaceae bacteria in the hind gut of high-fat diet-fed mice

Consumption of an obesigenic/high-fat diet (HFD) is associated with a high colon cancer risk and may alter the gut microbiota. To test the hypothesis that long-term high-fat (HF) feeding accelerates inflammatory process and changes gut microbiome composition, C57BL/6 mice were fed HFD (45% energy) or a low-fat (LF) diet (10% energy) for 36 weeks. At the end of the study, body weights in the HF group were 35% greater than those in the LF group. These changes were associated with dramatic increases in body fat composition, inflammatory cell infiltration, inducible nitric oxide synthase protein concentration and cell proliferation marker (Ki67) in ileum and colon. Similarly, β-catenin expression was increased in colon (but not ileum). Consistent with gut inflammation phenotype, we also found that plasma leptin, interleukin 6 and tumor necrosis factor α concentrations were also elevated in mice fed the HFD, indicative of chronic inflammation. Fecal DNA was extracted and the V1–V3 hypervariable region of the microbial 16S rRNA gene was amplified using primers suitable for 454 pyrosequencing. Compared to the LF group, the HF group had high proportions of bacteria from the family Lachnospiraceae/Streptococcaceae, which is known to be involved in the development of metabolic disorders, diabetes and colon cancer. Taken together, our data demonstrate, for the first time, that long-term HF consumption not only increases inflammatory status but also accompanies an increase of colonic β-catenin signaling and Lachnospiraceae/Streptococcaceae bacteria in the hind gut of C57BL/6 mice.     

Long-term high-fat feeding induces greater fat storage in mice lacking UCP3

Uncoupling protein-3 (UCP3) is a mitochondrial inner-membrane protein highly expressed in skeletal muscle. While UCP3's function is still unknown, it has been hypothesized to act as a fatty acid (FA) anion exporter, protecting mitochondria against lipid peroxidation and/or facilitating FA oxidation. The aim of this study was to determine the effects of long-term feeding of a 45% fat diet on whole body indicators of muscle metabolism in congenic C57BL/6 mice that were either lacking UCP3 (Ucp3(-/-)) or had a transgenically induced approximately twofold increase in UCP3 levels (UCP3tg). Mice were fed the high-fat (HF) diet for a period of either 4 or 8 mo immediately following weaning. After long-term HF feeding, UCP3tg mice weighed an average of 15% less than wild-type mice (P < 0.05) and were 20% less metabolically efficient than both wild-type and Ucp3(-/-) mice (P < 0.01). Additionally, wild-type mice had 21% lower, whereas UCP3tg mice had 36% lower, levels of adiposity compared with Ucp3(-/-) mice (P < 0.05 and P < 0.001, respectively), indicating a protective effect of UCP3 against fat gain. No differences in whole body oxygen consumption were detected following long-term HF feeding. Glucose and insulin tolerance tests revealed that both the UCP3tg and Ucp3(-/-) mice were more glucose tolerant and insulin sensitive compared with wild-type mice after short-term HF feeding, but this protection was not maintained in the long term. Findings indicate that UCP3 is involved in protection from fat gain induced by long-term HF feeding, but not in protection from insulin resistance.     

Advanced liver steatosis accompanies an increase in hepatic inflammation,colonic, secondary bile acids and Lactobacillaceae/Lachnospiraceae bacteria in C57BL/6 mice fed a high-fat diet

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in Western countries, and the gut-liver axis is implicated in liver disease pathogenesis. We hypothesize that advanced liver steatosis accompanies an increase in hepatic inflammation, colonic secondary bile acids (BAs) and secondary BA-producing bacteria in mice fed a high-fat (HF) diet model of obesity. Four-week old male C57BL/6 mice were fed an HF (45% energy) or a low-fat (LF) (10% energy) diet for 21 weeks. At the end of the study, body weight and body fat percentage in the HF group were 0.23- and 0.41-fold greater than those in the LF group, respectively. Similarly, the HF group exhibited an increase in hepatic lipid droplets, inflammatory cell infiltration, inducible nitric oxide synthase, and hepatocellular ballooning (but without hepatic Mallory bodies) which are key histological features of advanced hepatic steatosis. Furthermore, RNA sequencing, qPCR and immunohistological methods found that nicotinamide n-methyltransferase and selenoprotein P, two inflammation-related hepatic genes, were upregulated in the HF group. Consistent with the hepatic inflammation, the levels of proinflammatory plasma-cytokines (TNF-α and IL6), colonic secondary BAs (LCA, DCA) and secondary BA producing bacteria (e.g., lactobacillaceae/Lachnospiraceae) were at least 0.5-fold greater in the HF group compared with the LF group. Taken together, the data demonstrate that advanced liver-steatosis is concurrent with an elevated level of hepatic inflammation, colonic secondary bile acids and their associated bacteria in mice fed an HF diet. These data suggest a potential gut-liver crosstalk at the stage of advanced liver-steatosis.     

A dairy‐based high calcium diet improves glucose homeostasis and reduces steatosis in the context of preexisting obesity

Objective:

High dietary calcium (Ca) in the context of a dairy food matrix has been shown to reduce obesity development and associated inflammation in diet‐induced obese (DIO) rodents. The influence of Ca and dairy on these phenotypes in the context of preexisting obesity is not known. Furthermore, interpretations have been confounded historically by differences in body weight gain among DIO animals fed dairy‐based protein or high Ca.

Design and Methods:

Adiposity along with associated metabolic and inflammatory outcomes were measured in DIO mice previously fattened for 12 week on a soy protein‐based obesogenic high fat diet (45% energy, 0.5% adequate Ca), then fed one of three high fat diets (n = 29‐30/group) for an additional 8 week: control (same as lead‐in diet), high‐Ca (1.5% Ca), or high‐Ca + nonfat dry milk (NFDM).

Results and Conclusion:

Mice fed high‐Ca + NFDM had modestly, but significantly, attenuated weight gain compared to mice fed high‐Ca or versus controls (P < 0.001), whereas mice fed high‐Ca alone had increased weight gain compared to controls (P < 0.001). Total measured adipose depot weights between groups were similar, as were white adipose tissue inflammation and macrophage infiltration markers (e.g. TNFα, IL‐6, CD68 mRNAs). Mice fed high‐Ca + NFDM had significantly improved glucose tolerance following a glucose tolerance test, and markedly lower liver triglycerides compared to high‐Ca and control groups. Improved metabolic phenotypes in prefattened DIO mice following provision of a diet enriched with dairy‐based protein and carbohydrates appeared to be driven by non‐Ca components of dairy and were observed despite minimal differences in body weight or adiposity.