. Abbreviations used: HFD, high fat diet; HFL, high fat lard diet; HO-1, heme oxygenase-1; NQO-1, NAD(P)H:quinone oxidoreductase 1; Nrf2, NF-E2-related factor 2; WD, western diet. AbstractLong term consumption of a high fat diet (HFD) contributes to increased morbidity and mortality. Yet the specific effects of HFD consumption on brain aging are poorly understood. In the present study 20-month old male C57Bl/6 mice were fed either 'western diet' (41% fat), very high fat lard diet (60% fat), or corresponding control diets for 16 weeks and then assessed for changes in metabolism and brain homeostasis. Although both HFDs increased adiposity and fasting blood glucose, only the high fat lard diet increased age-related oxidative damage (protein carbonyls) and impaired retention in the behavioral test. This selective increase in oxidative damage and cognitive decline was also associated with a decline in NF-E2-related factor 2 (Nrf2) levels and Nrf2 activity, suggesting a potential role for decreased antioxidant response. Taken together, these data suggest that while adiposity and insulin resistance following HFD consumption are linked to increased morbidity, the relationship between these factors and brain homeostasis during aging is not a linear relationship. More specifically, these data implicate impaired Nrf2 signaling and increased cerebral oxidative stress as mechanisms underlying HFDinduced declines in cognitive performance in the aged brain.

This study was undertaken to investigate the effects of prenatal and postnatal exposure to high fat diet on the brain. Female rats were divided into high fat diet (HFD) and control diet (CD) groups 4 weeks prior to breeding and throughout gestation and lactation. After weaning, male progeny were placed on a chow diet until 8 weeks old, and then segregated into HFD or CD groups. At 20 weeks old, rats were evaluated in the Morris water maze, and markers of oxidative stress and inflammation were documented in brain. In comparison to rats fed CD, cognitive decline in HFD progeny from HFD dams manifested as a decline in retention, but not acquisition, in the water maze. HFD was also associated with significant increases in 3-nitrotyrosine, inducible nitric oxide synthase, IL-6, and glial markers Iba-1 and GFAP, with the largest increases frequently observed in HFD animals born to HFD dams. Thus, these data collectively suggest that HFD increases oxidative and inflammatory signaling in brain, and further indicate that maternal HFD consumption might sensitize offspring to the detrimental effects of HFD.

We tested the hypothesis that maternal consumption of dietary fat, independent from obesity, increases serum leptin in neonatal pups and predisposes them to adult obesity. Female rats either were fed a high-fat (HF) diet or a low-fat (LF) diet or were fed the HF diet but pair fed (PF) to the caloric intake of the LF group for 4 wk before breeding and throughout gestation and lactation. Dams consuming the HF diet had increased adiposity and were hyperphagic. At weaning, pups born to obese dams had significantly higher body fat and serum leptin levels and reduced insulin tolerance compared with offspring of LF-fed dams. Pups were weaned onto a chow diet until 8 wk of age, when they were then fed either HF or LF diet. At 18 wk of age, offspring from obese HF dams weighed more than offspring from nonobese LF or PF dams, and offspring eating HF diet weighed significantly more than those eating LF diet. Consequently, HF-fed offspring of obese HF dams weighed the most and LF-fed offspring from obese HF dams were similar in weight to HF-fed offspring from nonobese LF dams. These data suggest that maternal obesity exerts an independent effect on offspring body weight that is of similar magnitude as the effect of the offspring's adult diet. Furthermore, there was no difference in body weight between the nonobese LF and PF offspring on either diet. Together, these data suggest that maternal adiposity, and not dietary fat per se, induces hyperleptinemia and insulin resistance in offspring, as well as an increased body weight that persists into adulthood.

After a period of forced overfeeding, many individuals actively compensate for this weight gain by reducing food intake and maintaining this state of hypophagia well into the post-overfeeding period. Our central goal is to define the mechanism underlying this adaptive reduction in food intake. When male Long Evans rats were implanted with indwelling gastric cannula and overfed a liquid low-fat (10% fat) diet for 17 days, overfed rats exhibited increased weight gain (P < 0.01) but decreased food intake, and this hypophagia persisted for 4-6 days post-overfeeding (P < 0.05). Leptin levels were increased 8-fold by overfeeding (P < 0.01), yet returned to baseline within 2 days post-overfeeding, despite the persistent hypophagia. Energy expenditure and oxygen consumption (VO2) were increased on the first day post-overfeeding (P < 0.05), but subsequently normalized prior to the normalization of food intake. Lastly, in leptin receptor deficient Obese Zucker (fa/fa) rats, overfeeding produced a significant decrease in food intake during active overfeeding. However, food intake returned to near baseline levels within one day post-overfeeding. Contrastingly, food intake remained suppressed in lean controls for 6 days post-overfeeding. Thus intact leptin signaling is not required for the decrease in food intake that occurs during overfeeding, but the ability to maintain this hypophagia is substantially impaired in the absence of leptin signaling. In addition, this post-overfeeding leptin effect appears to occur despite the fact that leptin levels normalize relatively rapidly post-overfeeding.

Evidence indicates that failure of nuclear transfer (NT) embryos to develop normally can be attributed, at least partially, to the use of differentiated cells as the donor karyoplast. Blastocyst production and development to term of cloned embryos has been hypothesized to differ between population doublings of the same cell line as a consequence of changes in the levels of DNA methyltransferase 1 (DNMT1) and methylated DNA during in vitro culture. The objective of this study was to determine embryo production, developmental potential, and gene expression patterns of prehatched and posthatched embryos generated using donor cells with different levels of DNMT1 transcript. Day 7 embryos generated using donor cells with high and low levels of DNMT1 mRNA were transferred to recipient cows. Embryos recovered on Day 13 were morphologically characterized or used for gene expression analysis of DNMT, INFT, and MHC1. A higher proportion of 8- to 16-cell embryos developed to the blastocyst stage when cells with low levels of DNMT1 mRNA were used as donor nuclei. Day 13 NT embryos generated using donor cells with decreased levels of DNMT1 mRNA and capable of developing beyond the 8- to 16-cell stage produced a larger number of apparently developing embryos, larger conceptuses, and a higher expression of DNMT3A transcript than NT embryos reconstructed using cells with high levels of DNMT1 mRNA. However, abnormal gene expression of DNMT, INFT, and MHC1 was noted in the majority of cloned embryos, indicating inefficient nuclear reprogramming and retarded embryo development. Furthermore, aberrant DNMT1 expression may partially contribute to the inefficient nuclear reprogramming observed in cloned embryos.

Elevation of dietary or brain leucine appears to suppress food intake via a mechanism involving mTOR, AMPK and/or branched chain amino acid (BCAA) metabolism. Mice bearing a deletion of mitochondrial branched chain amino transferase (BCATm), which is expressed in peripheral tissues (muscle) and brain glia, exhibit marked increases in circulating BCAAs. Here we test whether this increase in circulating BCAAs alters feeding behavior and brain neuropeptide expression. Circulating and brain levels of BCAAs were increased 2-4 fold in BCATm-deficient mice (KO). KO mice weighed less than controls (25.9 vs. 20.4g, P < 0.01), but absolute food intake was relatively unchanged. In contrast to wildtype mice, KO mice preferred a low BCAA diet to a control diet (P < 0.05), but exhibited no change in preference for low vs. high protein diets. KO mice also exhibited low leptin levels and increased hypothalamic NPY and AgRP mRNA. Normalization of circulating leptin levels had no effect on either food preference or the increased NPY and AgRP mRNA expression. If BCAAs act as signals of protein status, one would expect reduced food intake, an avoidance of dietary protein, and a reduction in neuropeptide expression in BCATm-KO mice. Instead, these mice exhibit increased expression of orexigenic neuropeptides and an avoidance of BCAAs but not high protein. These data thus suggest either that BCAAs do not act as physiological signals of protein status, or that the loss of BCAA metabolism within brain glia impairs the detection of protein balance.

Numerous studies have reported aberrant gene expression levels attributed to suboptimal in vitro culture conditions. This study investigated the effects of different culture systems and protein sources on the developmental competence of in vitro production (IVP) embryos measured by cleavage and blastocyst rates, cell number, and relative abundance of POU5F1 (OCT4), nanog, GJA1 (connexin 43), and SLC2A1 (GLUT1) transcripts when compared to in vivo embryos. Experiment 1 compared IVP embryos cultured in either synthetic oviductal fluid (SOFaa) or potassium simplex optimized medium supplemented with amino acids (KSOMaa). Experiment 2 compared the same two culture systems with and without the addition of calf serum (CS). Results from both experiments indicated that despite similar developmental rates, significant differences were observed at the mRNA level. In Experiment 1, OCT4 was the only transcript to have a mean abundance level significantly higher in KSOMaa blastocysts when compared with both SOFaa and in vivo embryos. The same pattern of upregulation of OCT4 mRNA was noted in Experiment 2. There were no significant alterations of the ICM specific transcript nanog in either experiment. In contrast to reports by others, connexin 43 mRNA was not expressed at detectable levels in in vivo embryos analyzed in our studies. Blastocysts cultured in SOFaa with CS or KSOMaa had a significant upregulation of GLUT1 mRNA when compared with other treatments and in vivo embryos. Until differences between IVP and in vivo embryos are minimized, aberrations in IVP will continue to arise.

The aberrant expression of DNA methyltransferase 1 (DNMT1) in cloned embryos has been implicated as a possible factor in the improper donor genome reprogramming during nuclear transfer. DNMT1 is responsible for maintaining DNA methylation and the subsequent differentiation status of somatic cells. The presence of DNMT1 transcript in the donor cell may contribute to perpetuation of the highly methylated status of the somatic nuclei in cloned embryos. The objective of the present study was to determine the methylation pattern of cloned embryos reconstructed with cells treated with DNMT1-specific small interfering RNA (siRNA). Bovine fibroblasts were transfected with a DNMT1-specific siRNA under optimised conditions. The expression patterns of DNMT1 were characterised by Q-PCR using the DeltaDeltaC(T) method. The level of DNMT1 was successfully decreased in bovine fibroblast cells using a DNMT1-specific siRNA. Additionally, reduction in the expression of DNMT1 mRNA and DNMT1 protein led to a moderate hypomethylation pattern in the siRNA-treated cells. The use of siRNA-treated cells as donor nuclei during nuclear transplantation induced a reduction in methylation levels compared with controls but did not reduce methylation levels to that of IVF embryos. Further studies are required to determine if this level of reduced methylation is sufficient to improve subsequent development.