Hypothalamic inflammation and food intake regulation during chronic illness.

Anorexia is a common symptom in chronic illness. It contributes to malnutrition and strongly affects survival and quality of life. A common denominator of many chronic diseases is an elevated inflammatory status, which is considered to play a pivotal role in the failure of food-intake regulating systems in the hypothalamus. In this review, we summarize findings on the role of hypothalamic inflammation on food intake regulation involving hypothalamic neuropeptide Y (NPY) and pro-opiomelanocortin (POMC). Furthermore, we outline the role of serotonin in the inability of these peptide based food-intake regulating systems to respond and adapt to changes in energy metabolism during chronic disease.

A role for orexin in cytotoxic chemotherapy-induced fatigue.

Fatigue is the most common symptom related to cytotoxic chemotherapeutic treatment of cancer. Peripheral inflammation associated with cytotoxic chemotherapy is likely a causal factor of fatigue. The neural mechanisms by which cytotoxic chemotherapy associated inflammation induces fatigue behavior are not known. This lack of knowledge hinders development of interventions to reduce or prevent this disabling symptom. Infection induced fatigue/lethargy in rodents is mediated by suppression of hypothalamic orexin activity. Orexin is critical for maintaining wakefulness and motivated behavior. Though there are differences between infection and cytotoxic chemotherapy in some symptoms, both induce peripheral inflammation and fatigue. Based on these similarities we hypothesized that cytotoxic chemotherapy induces fatigue by disrupting orexin neuron activity. We found that a single dose of a cytotoxic chemotherapy cocktail (cyclophosphamide, adriamycin, 5-fluorouracil - CAF) induced fatigue/lethargy in mice and rats as evidenced by a significant decline in voluntary locomotor activity measured by telemetry. CAF induced inflammatory gene expression - IL-1R1 (p<0.001), IL-6 (p<0.01), TNFα (p<0.01), and MCP-1 (p<0.05) - in the rodent hypothalamus 6-24h after treatment during maximum fatigue/lethargy. CAF decreased orexin neuron activity as reflected by decreased nuclear cFos localization in orexin neurons 24h after treatment (p<0.05) and by decreased orexin-A in cerebrospinal fluid 16 h after treatment (p<0.001). Most importantly, we found that central administration of 1 µg orexin-A restored activity in CAF-treated rats (p<0.05). These results demonstrate that cytotoxic chemotherapy induces hypothalamic inflammation and that suppression of hypothalamic orexin neuron activity has a causal role in cytotoxic chemotherapy-induced fatigue in rodents.

Changes in melanocortin expression and inflammatory pathways in fetal offspring of nonhuman primates fed a high-fat diet.

The hypothalamic melanocortin system, which controls appetite and energy expenditure, develops during the third trimester in primates. Thus, maternal nutrition and health may have a profound influence on the development of this system. To study the effects of chronic maternal high-fat diet (HFD) on the development of the melanocortin system in the fetal nonhuman primate, we placed adult female macaques on either a control (CTR) diet or a HFD for up to 4 yr. A subgroup of adult female HFD animals was also switched to CTR diet during the fifth year of the study (diet reversal). Third-trimester fetuses from mothers on HFD showed increases in proopiomelanocortin mRNA expression, whereas agouti-related protein mRNA and peptide levels were decreased in comparison with CTR fetuses. Proinflammatory cytokines, including IL-1beta and IL-1 type 1 receptor, and markers of activated microglia were elevated in the hypothalamus, suggesting an activation of the local inflammatory response. Fetuses of diet-reversal mothers had normal melanocortin levels. These results raise the concern that chronic consumption of a HFD during pregnancy, independent of maternal obesity and diabetes, can lead to widespread activation of proinflammatory cytokines that may alter the development of the melanocortin system. The abnormalities in the fetal POMC system, if maintained into the postnatal period, could impact several systems, including body weight homeostasis, stress responses, and cardiovascular function. Indeed, the HFD offspring develop early-onset excess weight gain. These abnormalities may be prevented by healthful nutrient consumption during pregnancy even in obese and severely insulin-resistant individuals.

Activation-dependent regulation of galanin gene expression in gonadotropin-releasing hormone neurons in the female rat.

In rats, galanin is colocalized in GnRH neurons, and galanin mRNA in GnRH neurons is increased coincidentally with the preovulatory gonadotropin surge. Whether the induction of galanin mRNA in GnRH neurons at proestrus reflects the action of sex steroids is unknown. We tested this hypothesis by challenging ovariectomized rats (n = 7) with estrogen and progesterone (E/P) to induce a LH surge and measuring galanin mRNA in GnRH neurons to determine whether there was an associated induction of galanin message in these cells. We used single and double label in situ hybridization and image analysis to compare among groups the levels of both galanin mRNA and GnRH mRNA in GnRH neurons. We found that steroid-primed animals showed an approximately 400% induction of galanin mRNA signal in GnRH neurons over that in vehicle-treated animals. Second, we hypothesized that steroid-dependent events which induce the expression of galanin mRNA in GnRH neurons depend on transsynaptic input to GnRH neurons. We tested this hypothesis by examining the effect of a pharmacological blockade of the steroid-induced activation of GnRH neurons on levels of galanin mRNA in these cells. We killed groups of ovariectomized adult female rats at the peak of a E/P-primed LH surge (n = 7) and after steroid priming followed by blockade of the LH surge with either the general anesthetic pentobarbital (n = 7) or the specific alpha-adrenergic receptor blocker phenoxybenzamine (n = 7). When we examined signal levels representing galanin mRNA content in GnRH neurons, we observed a 4-fold increase in signal for galanin mRNA in the GnRH neurons of steroid-primed (E/P surge) animals compared with that in oil-treated controls (P < 0.0004). This increase in galanin mRNA was prevented when the LH surge was blocked by treatment with either pentobarbital or phenoxybenzamine (P < 0.03 and P < 0.0001 vs. E/P surge controls, respectively). Cellular levels of GnRH mRNA were not different among control, E/P, and E/P plus pentobarbital groups (P > 0.2). These observations suggest that an increase in galanin mRNA levels in GnRH neurons is tightly coupled to the occurrence of a LH surge. By inference, induction of galanin mRNA in GnRH neurons reflects their activation, possibly via afferent neurons that transduce the steroid signal to GnRH neurons.

Regulation of gonadotropin-releasing hormone (GnRH) and galanin gene expression in GnRH neurons during lactation in the rat.

Galanin is colocalized with GnRH, and its expression in these neurons is enhanced at proestrus, a time of activation of GnRH neurons. We tested the hypothesis that the expression of both the GnRH and galanin mRNAs in GnRH neurons decrease during lactation in the rat, a reproductive state characterized by reduced gonadotropin secretion. For double label in situ hybridization, GnRH mRNA was detected with an antisense cRNA probe labeled with the hapten digoxigenin, whereas galanin mRNA was detected with a cRNA probe labeled with 35S. The number of silver grains deposited over a digoxigenin-labeled cell body provided an index of galanin mRNA levels in GnRH cells. We observed a 60% reduction in signal (grains per cell) for galanin mRNA in GnRH neurons of lactating animals compared with those of diestrus animals (P < 0.004), with no difference in the number of GnRH neurons between groups. To compare cellular GnRH mRNA content between groups, we used single label in situ hybridization and image analysis. Signal levels (grains per cell) for GnRH mRNA were not different between diestrus and lactating animals in either an initial (diestrus, 121.4 +/- 5.9; lactation, 117.3 +/- 8.0; P > 0.7) or in a subsequent trial (diestrus, 184.0 +/- 10.4; lactation, 197.5 +/- 13.0; P > 0.7). To confirm and extend these findings, we used a RNAse protection assay to measure and compare the content of GnRH mRNA in hypothalamic fragments between diestrus and lactating animals. The concentration of GnRH mRNA (picograms of mRNA per 25 micrograms total RNA) was not different between the two groups (diestrus, 1.21 +/- 0.25; lactation, 1.25 +/- 0.13; P > 0.7). A determination of the total GnRH peptide content by RIA in a separate set of hypothalamic dissections revealed no difference between groups in the level of GnRH content (nanograms) per hypothalamus (diestrus, 6.0 +/- 0.6; lactation, 5.7 +/- 0.4; P > 0.4). We conclude that galanin mRNA expression in GnRH neurons of the rat is diminished during lactation, whereas GnRH expression continues unabated. This decrease in galanin gene expression associated with lactation may lead to decreased synthesis and secretion of galanin, which, in turn, could diminish the pulsatile secretion of GnRH or reduce its activity at the pituitary.

Measurement of total protein in plant samples in the presence of tannins.

A method for measuring total protein in situ in plant samples has been developed using the determination of amino acids released by acid hydrolysis of dried plant material. Standard proteins and plant samples were hydrolyzed with 3% sulfuric acid at 100 degrees C for 24 h and the amino acids released were measured with ninhydrin. Unhydrolyzed plant extracts were also analyzed for free amino acids with ninhydrin. Total amino acid equivalents (protein plus free amino acids) of a diverse set of plant samples was significantly correlated with total protein as estimated by elemental analysis (N X 6.25). The Lowry method as modified by precipitation of proteins with trichloroacetic acid was found to be unsatisfactory for dried plant samples due to the incomplete extractability of proteins. Although some alkaloids caused increased absorbance with ninhydrin, interference with quantification of protein is likely to be minimal. Tannins interfered with the Lowry and Bradford methods but not the ninhydrin method.