Impaired branched chain amino acid metabolism alters feeding behavior and increases orexigenic neuropeptide expression in the hypothalamus.

Elevation of dietary or brain leucine appears to suppress food intake via a mechanism involving mechanistic target of rapamycin, AMPK, and/or branched chain amino acid (BCAA) metabolism. Mice bearing a deletion of mitochondrial branched chain aminotransferase (BCATm), which is expressed in peripheral tissues (muscle) and brain glia, exhibit marked increases in circulating BCAAs. Here, we test whether this increase alters feeding behavior and brain neuropeptide expression. Circulating and brain levels of BCAAs were increased two- to four-fold in BCATm-deficient mice (KO). KO mice weighed less than controls (25·9 vs 20·4 g, P<0·01), but absolute food intake was relatively unchanged. In contrast to wild-type 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 (HP) 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, avoidance of dietary protein, and reduction in neuropeptide expression in BCATm-KO mice. Instead, these mice exhibit an increased expression of orexigenic neuropeptides and an avoidance of BCAAs but not HP. These data thus suggest that either BCAAs do not act as physiological signals of protein status or the loss of BCAA metabolism within brain glia impairs the detection of protein balance.

High fat diet increases hippocampal oxidative stress and cognitive impairment in aged mice: implications for decreased Nrf2 signaling.

Long 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 HFD-induced declines in cognitive performance in the aged brain.

Innervation of skeletal muscle by leptin receptor-containing neurons.

In addition to suppressing food intake, leptin reduces body adiposity by altering metabolism within peripheral tissues such as adipose tissue and muscle. Recent work indicates that leptin action within the brain is sufficient to promote glucose uptake and increase fat oxidation within skeletal muscle, and that these effects are dependent on the sympathetic nervous system. To identify neuronal circuits through which leptin impacts skeletal muscle metabolism, we used LepRb-GFP reporter mice in combination with muscle-specific injection of an mRFP-expressing pseudorabies virus (PRV), which acts as a transsynaptic retrograde tracer. Consistent with previous observations in the rat, muscle-specific PRV injection lead to labeling within multiple areas of the hypothalamus and brainstem. However, the only areas in which PRV and LepRb colocalization was detected were within the brainstem nucleus of the solitary tract (NTS) and the hypothalamic retrochiasmatic area. Within the NTS 28.5+/-9.4% of PRV-positive neurons contained LepRb-GFP, while in the RCH 37+/-1.7% of PRV neurons also contained LepRb. In summary, these data clearly implicate the NTS and RCH as key sites through which brain leptin impacts skeletal muscle, and as such provide an anatomical framework within which to interpret physiological data indicating that leptin acts in the brain to influence metabolism within skeletal muscle.

Decreased food intake following overfeeding involves leptin-dependent and leptin-independent mechanisms.

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.

Inhibition of DNA methyltransferase 1 expression in bovine fibroblast cells used for nuclear transfer.

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.

Effects of high fat diet on Morris maze performance, oxidative stress, and inflammation in rats: contributions of maternal diet.

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 the 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 the brain, and further indicate that maternal HFD consumption might sensitize offspring to the detrimental effects of HFD.

Maternal obesity is necessary for programming effect of high-fat diet on offspring.

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.

Effect of epigenetic modifications of donor somatic cells on the subsequent chromatin remodeling of cloned bovine embryos.

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.

DNA methylation and histone acetylation patterns in cultured bovine fibroblasts for nuclear transfer.

Evidence indicates that failure of nuclear transfer (NT) embryos to develop normally can be attributed, at least partially, to the use of a differentiated cell nucleus as the donor karyoplast. It has been hypothesized that blastocyst production and development to term of cloned embryos may differ between population doublings (PDs) of the same cell line as a consequence of changes in DNA methylation and histone acetylation patterns during in vitro culture. The objective of this study was to determine gene expression patterns of the chromatin remodeling proteins DNA methyltransferase-1 (Dnmt1), methyl CpG binding protein-2 (MeCP2), and histone deacetyltransferse-1 (HDAC1), in addition, to measuring levels of DNA methylation and histone acetylation of bovine fibroblast cells at different PDs. Bovine fibroblast cell lines were established from four 50-day fetuses. Relative levels of Dnmt1, MeCP2, HDAC1, methylated DNA, and acetylated histone were analyzed at PDs 2, 7, 15, 30, 45, and 70. RNA levels of Dnmt1, HDAC1, and MeCP2 were examined using Q-PCR. Global levels of methylated DNA and acetylated histone were determined by incubation of fixed cells with an anti-5-methylcytidine and anti-acetyl-histone H3 antibody, respectively. Cells were labeled with a second antibody, counter-stained with propidium iodide and analyzed by flow cytometry. These data demonstrate that chromatin remodeling protein mRNAs involved in epigenetic modifications are altered during in vitro culture. Methylated DNA and acetylated histone patterns of in vitro cells change with time in culture. Subsequent use of these cells for NT will provide insight as to how these epigenetic modifications affect reprogramming.