Alterations in the Serotonin and Dopamine Pathways by Cystathionine Beta Synthase Overexpression in Murine Brain.

Cystathionine beta synthase (CBS) is one of the 225 genes on chromosome 21 (HSA 21) that are triplicated in persons with trisomy 21 (Down syndrome). Although most triplicate HSA21 genes have their orthologous genes on murine chromosome 16, the murine ortholog of hCBS is on murine chromosome 17 and thus is not present in the well-studied Ts65Dn mouse model of trisomy 21. Persons with trisomy 21 (T21) present deficits in neurotransmission and exhibit early brain aging that can partially be explained by monoamine neurotransmitter alterations. We used transgenic mice for the hCBS gene, which overexpress the CBS protein in various brain regions, to study if CBS overexpression induces modifications in the monoamine neurotransmitters in the hypothalamus, thalamus, hippocampus, and striatum from transgenic and control female and male mice aged 3-4 months and 11-12 months. Sex, age, and brain area each influenced neurotransmitter levels. Briefly, the serotonin pathway was modified by CBS overexpression in various brain areas in female mice but not in male mice. The dopamine pathway was modified in brain regions according to sex and age. These results may allow us to better understand the role of the transsulfuration pathway and especially CBS overexpression in the metabolism of biogenic amines and the catecholamine catabolism in persons with trisomy 21.

A novel, double intra-carotid cannulation technique to study the effect of central nutrient sensing on glucose metabolism in the rat.

BACKGROUND: The hypothalamus plays a key role in central nutrient sensing and glucose homeostasis. Due to its position next to the third ventricle, intracerebroventricular (ICV) injections or osmotic minipumps are widely applied techniques in studying effects of hormones and other molecules on the hypothalamus and glucose metabolism. NEW METHODS: The intracarotid catheter technique in which a catheter is placed in the carotid artery, pointing towards the brain, provides a physiological route to centrally infuse blood-borne molecules in an undisturbed animal. To measure effects of central interventions on peripheral glucose metabolism, endogenous glucose production (EGP) and insulin sensitivity can be measured using a stable isotope technique. To combine both techniques, it is necessary to combine different catheters. We here describe a novel cannulation technique for the carotid artery, enabling stress-free infusions towards the brain and blood sampling from the carotid artery concomitantly, and infuse a stable isotope via the jugular vein. RESULTS: We showed accurate EGP measurements when intracarotically infusing saline towards the brain. The stress-hormone corticosterone, as well as energy expenditure, did not alter upon central infusion. COMPARISON EXISTING METHOD(S): ICV infusions bypass the blood-brain-barrier (BBB) and are thus a less physiological approach when studying central effects of blood-borne factors. Furthermore, ICV injections can elicit a stress response which can interfere with outcomes of glucose metabolism. We described a stress-free, physiological method to study effects of central infusions on peripheral parameters. CONCLUSIONS: This technique provides new opportunities for studying central effects of, for instance, hormones and nutrients, on glucose metabolism.

High fat induces acute and chronic inflammation in the hypothalamus: effect of high-fat diet, palmitate and TNF-α on appetite-regulating NPY neurons.

BACKGROUND: Consumption of dietary fat is one of the key factors leading to obesity. High-fat diet (HFD)-induced obesity is characterized by induction of inflammation in the hypothalamus; however, the temporal regulation of proinflammatory markers and their impact on hypothalamic appetite-regulating neuropeptide Y/agouti-related peptide (NPY/AgRP) neurons remains undefined. METHODS: Mice were injected with an acute lipid infusion for 24 h or fed a HFD over 8-20 weeks. Characterized mouse NPY/AgRP hypothalamic cell lines were used for in vitro experimentation. Immunohistochemistry in brain slices or quantitative real-time PCR in cell lines, was performed to determine changes in the expression of key inflammatory markers and neuropeptides. RESULTS: Hypothalamic inflammation, indicated by tumor necrosis factor (TNF)-α expression and astrocytosis in the arcuate nucleus, was evident following acute lipid infusion. HFD for 8 weeks suppressed TNF-α, while significantly increasing heat-shock protein 70 and ciliary neurotrophic factor, both neuroprotective components. HFD for 20 weeks induced TNF-α expression in NPY/AgRP neurons, suggesting a detrimental temporal regulatory mechanism. Using NPY/AgRP hypothalamic cell lines, we found that palmitate provoked a mixed inflammatory response on a panel of inflammatory and endoplasmic reticulum (ER) stress genes, whereas TNF-α significantly upregulated IκBα, nuclear factor (NF)-κB and interleukin-6 mRNA levels. Palmitate and TNF-α exposure predominantly induced NPY mRNA levels. Utilizing an I kappa B kinase ß (IKKß) inhibitor, we demonstrated that these effects potentially occur via the inflammatory IKKß/NF-κB pathway. CONCLUSIONS: These findings indicate that acute lipid and chronic HFD feeding in vivo, as well as acute palmitate and TNF-α exposure in vitro, induce markers of inflammation or ER stress in the hypothalamic appetite-stimulating NPY/AgRP neurons over time, which may contribute to a dramatic alteration in NPY/AgRP content or expression. Acute and chronic HFD feeding in vivo temporally regulates arcuate TNF-α expression with reactive astrocytosis, which suggests a time-dependent neurotrophic or neurotoxic role of lipids.

Physiological and pathophysiological implications of lipid sensing in the brain.

Fatty acid (FA)-sensitive neurons are present in the brain, especially the hypothalamus, and play a key role in the neural control of energy homeostasis. Through neuronal output, FA may modulate feeding behaviour as well as insulin secretion and action. Subpopulations of neurons in the ventromedial and arcuate hypothalamic nuclei are selectively either inhibited or activated by FA. Molecular effectors of these FA effects probably include chloride or potassium ion channels. While intracellular metabolism and activation of the ATP-sensitive K⁺ channel appear to be necessary for some of the signalling effects of FA, at least half of the FA responses in ventromedial hypothalamic neurons are mediated by interaction with FAT/CD36, an FA transporter/receptor that does not require intracellular metabolism to activate downstream signalling. Thus, FA or their metabolites can modulate neuronal activity as a means of directly monitoring ongoing fuel availability by brain nutrient-sensing neurons involved in the regulation of energy and glucose homeostasis. Recently, the role of lipoprotein lipase in FA sensing has also been shown in animal models not only in hypothalamus, but also in hippocampus and striatum. Finally, FA overload might impair neural control of energy homeostasis through enhanced ceramide synthesis and may contribute to obesity and/or type 2 diabetes pathogenesis in predisposed subjects.

Dietary triglycerides act on mesolimbic structures to regulate the rewarding and motivational aspects of feeding.

Circulating triglycerides (TGs) normally increase after a meal but are altered in pathophysiological conditions, such as obesity. Although TG metabolism in the brain remains poorly understood, several brain structures express enzymes that process TG-enriched particles, including mesolimbic structures. For this reason, and because consumption of high-fat diet alters dopamine signaling, we tested the hypothesis that TG might directly target mesolimbic reward circuits to control reward-seeking behaviors. We found that the delivery of small amounts of TG to the brain through the carotid artery rapidly reduced both spontaneous and amphetamine-induced locomotion, abolished preference for palatable food and reduced the motivation to engage in food-seeking behavior. Conversely, targeted disruption of the TG-hydrolyzing enzyme lipoprotein lipase specifically in the nucleus accumbens increased palatable food preference and food-seeking behavior. Finally, prolonged TG perfusion resulted in a return to normal palatable food preference despite continued locomotor suppression, suggesting that adaptive mechanisms occur. These findings reveal new mechanisms by which dietary fat may alter mesolimbic circuit function and reward seeking.

Central orchestration of peripheral nutrient partitioning and substrate utilization: implications for the metabolic syndrome.

Energy homoeostasis is maintained through a complex interplay of nutrient intake and energy expenditure. The central nervous system is an essential component of this regulation, as it integrates circulating signals of hunger and satiety to develop adaptive responses at the behavioural and metabolic levels, while the hypothalamus is regarded as a particularly crucial structure in the brain in terms of energy homoeostasis. The arcuate nucleus (ARC) of the hypothalamus contains at least two intermingled neuronal populations: the neurons that produce neuropeptide Y (NPY); and the Agouti-related protein (AgRP) produced by AgRP/NPY neurons situated below the third ventricle in close proximity to proopiomelanocortin (POMC)-producing neurons. POMC neurons exert their catabolic and anorectic actions by releasing α-melanocyte-stimulating hormone (α-MSH), while AgRP neurons oppose this action by exerting tonic GABAergic inhibition of POMC neurons and releasing the melanocortin receptor inverse agonist AgRP. The release of neurotransmitters and neuropeptides by second-order AgRP neurons appears to take place on a multiple time scale, thereby allowing neuromodulation of preganglionic neuronal activity and subsequent control of nutrient partitioning - in other words, the coordinated regulation of conversion, storage and utilization of carbohydrates vs. lipids. This suggests that the function of AgRP neurons extends beyond the strict regulation of feeding to the regulation of efferent organ activity, such that AgRP neurons may now be viewed as an important bridge between central detection of nutrient availability and peripheral nutrient partitioning, thus providing a mechanistic link between obesity and obesity-related disorders.

Alterations of peroxisome proliferator-activated receptor delta activity affect fatty acid-controlled adipose differentiation.

Fatty acids have been postulated to regulate adaptation of adipose mass to nutritional changes by controlling expression of genes implicated in lipid metabolism via activation of nuclear receptors. Ectopic expression of the nuclear receptors PPARgamma or PPARdelta promotes adipogenesis in fibroblastic cells exposed to thiazolidinediones or long-chain fatty acids. To investigate the role of PPARdelta in fatty acid regulation of gene expression and adipogenesis in a preadipose cellular context, we studied the effects of overexpressing the native receptor or the dominant-negative PPARdelta mutant in Ob1771 and 3T3-F442A cells. Overexpression of PPARdelta enhanced fatty acid induction of the adipose-related genes for fatty acid translocase, adipocyte lipid binding protein, and PPARgamma and fatty acid effects on terminal differentiation. A transactivation-deficient form of PPARdelta mutated in the AF2 domain severely reduced these effects. Findings are similar in Ob1771 or 3T3-F442A preadipose cells. These data demonstrate that PPARdelta plays a central role in fatty acid-controlled differentiation of preadipose cells. Furthermore, they suggest that modulation of PPARdelta expression or activity could affect adaptive responses of white adipose tissue to nutritional changes.

Peroxisome-proliferator-activated receptor delta mediates the effects of long-chain fatty acids on post-confluent cell proliferation.

Nutritional long-chain fatty acids control adipose tissue mass by regulating the number and the size of adipocytes. It is now established that peroxisome-proliferator-activated receptors (PPARs) play crucial functions in the control of gene expression and the level of cell differentiation. PPARgamma, which is activated by specific prostanoids, is a key factor in activating terminal differentiation and adipogenesis. We have recently demonstrated that PPARdelta, once activated by fatty acids, drives the expression of a limited set of genes, including that encoding PPARgamma, thereby inducing adipose differentiation. Thus far, the mechanism of action of fatty acids in the control of preadipocyte proliferation has remained unknown. We show here that PPARdelta is directly implicated in fatty acid-induced cell proliferation. Ectopic expression of PPARdelta renders 3T3C2 cells capable of responding to treatment with long-chain fatty acids by a resumption of mitosis, and this effect is limited to a few days after confluence. This response is restricted to PPARdelta activators and, for fatty acids, takes place within the range of concentrations found to trigger differentiation of preadipocytes both in vitro and in vivo. Furthermore, the use of a mutated inactive PPARdelta demonstrated that transcriptional activity of the nuclear receptor is required to mediate fatty acid-induced proliferation. These data demonstrate that PPARdelta, as a transcription factor, is directly implicated in fatty acid-induced proliferation, and this could explain the hyperplastic development of adipose tissue that occurs in high-fat-fed animals.