GIP-dependent expression of hypothalamic genes.

GIP (glucose dependent insulinotrophic polypeptide), originally identified as an incretin peptide synthesized in the gut, has recently been identified, along with its receptors (GIPR), in the brain. Our objective was to investigate the role of GIP in hypothalamic gene expression of biomarkers linked to regulating energy balance and feeding behavior related neurocircuitry. Rats with lateral cerebroventricular cannulas were administered 10 µg GIP or 10 microl artificial cerebrospinal fluid (aCSF) daily for 4 days, after which whole hypothalami were collected. Real time Taqman™ RT-PCR was used to quantitatively compare the mRNA expression levels of a set of genes in the hypothalamus. Administration of GIP resulted in up-regulation of hypothalamic mRNA levels of AVP (46.9±4.5 %), CART (25.9±2.7 %), CREB1 (38.5±4.5 %), GABRD (67.1±11 %), JAK2 (22.1±3.6 %), MAPK1 (33.8±7.8 %), NPY (25.3±5.3 %), OXT (49.1±5.1 %), STAT3 (21.6±3.8 %), and TH (33.9±8.5 %). In a second experiment the same set of genes was evaluated in GIPR(-/-) and GIPR(+/?) mice to determine the effect of lack of GIP stimulation on gene expression. In GIPR(-/-) mice expressions of the following genes were down-regulated: AVP (27.1±7.5 %), CART (28.3±3.7 %), OXT (25.2±5.8 %), PTGES (23.9±4.5 %), and STAT3 (8.8±2.3 %). These results suggest that AVP, CART, OXT and STAT3 may be involved in energy balance-related hypothalamic circuits affected by GIP.

Novel treatments for obesity and osteoporosis: targeting apoptotic pathways in adipocytes.

Obesity and osteoporosis have grave consequences for human health, quality of life, and even the efficiency of the labor force and economy. However, these pathologies share a common cell progenitor, revealing a surprising target for drug research and development. Recent findings show that high adipocyte count in bone marrow is directly related to bone loss, as fat cells replace osteoblasts (or bone-forming cells). The objective of this review is to examine the importance of adipocyte apoptosis in the treatment of obesity and/or osteoporosis, with special emphasis on natural products as promising leads for drug development. We have induced in vivo adipocyte apoptosis, using leptin, ciliary neurotrophic factor (CNTF), beta adrenergic agonists and conjugated linoleic acid (CLA) in rodents. The results of leptin treatments on rats are suppressed food intake, reduced body weight, reduced body fat, adipocyte apoptosis, and elevated energy expenditure. Further, leptin treatment of leptin-deficient (ob/ob) mice increases endosteal bone formation and bone mineral density. Adipocyte apoptosis has also been induced in vitro using tumor necrosis factor-alpha (TNF-alpha), (-)-epigallocatechin gallate (EGCG) from Camellia sinensis and ajoene, from Allium sativum. Natural products have potential for inducing apoptosis of adipose tissue, inhibiting bone marrow adipogenesis and increasing the expression of osteogenic factors in bone, thereby yielding effective treatments for obesity and osteoporosis.

Evaluation of adipocyte apoptosis by laser scanning cytometry.

OBJECTIVE: Adipocyte apoptosis plays an important role in adipose tissue homeostasis and can be altered under a variety of physiological and pathological conditions. This study was carried out to determine whether laser scanning cytometry (LSC) can be used to measure changes in apoptosis of adipocytes over time. DESIGN: LSC was used to investigate adipocyte apoptosis induced by tumor necrosis factor-alpha (TNF-alpha), a cytokine that is associated with obesity and insulin resistance. LSC, a slide-based solid phase cytofluorometer, provides quantitative flow fluorescence data together with morphological information for apoptotic detection. Both 3T3-L1 cells and rat adipocytes from primary cell culture were incubated with 0 or 25 nM TNF-alpha for up to 24 h. Both the FITC-conjugated annexin V/propidium iodide assay and the TUNEL assay were used to distinguish cells with apoptotic characteristics from nonapoptotic cells. RESULTS: Apoptosis did not increase over time in the absence of TNF-alpha for both 3T3-L1 cells and rat primary adipocytes. For both 3T3-L1 cells and rat primary adipocytes, a significant increase in the percentage of apoptotic cells was observed by 3-4 h incubation with TNF-alpha (P<0.05). By 24 h, more than 50% of cells incubated with TNF-alpha were apoptotic (P<0.001). This process was also associated with morphological changes typical of adipocytes undergoing apoptosis. By estimating the percentage of cell subpopulations after different times of incubation with TNF-alpha, we were able to develop grading parameters, based on the adipose apoptotic measurements. CONCLUSION: With morphological information, LSC can be a useful tool to evaluate adipocyte apoptosis.

Ciliary neurotrophic factor injected icv induces adipose tissue apoptosis in rats.

Recent findings show that ciliary neurotrophic factor (CNTF) and leptin have similar effects on food intake and body weight, suggesting possible overlapping mechanisms. Intracerebroventricular (icv) injection of leptin results in adipose tissue apoptosis. To determine if CNTF has similar activity, male Sprague Dawley rats implanted with lateral cerebroventricular cannulas were randomly assigned to four treatment groups ( N = 8), including control (aCSF), 10 microg/day leptin, 1 microg/day CNTF, and 5 microg/day CNTF. Rats received daily icv injections for 4 successive days. Both leptin and CNTF (5 microg) decreased BW (8.6% and 11.77%, respectively, p <.05) and cumulative food intake was decreased 43% by leptin ( p <.05). Leptin and CNTF (5 microg) reduced adipose tissue mass in epididymal adipose (Epi) by 30 and 33.5%, ( p <.05), in inguinal adipose (Ing) by 51 and 55% ( p <.05), in retroperitoneal adipose (Rp) by 65 and 64% ( p <.05), and in intrascapular brown adipose (iBAT) by 34 and 25% ( p <.05), respectively. Gastrocnemius muscle was not affected. Leptin and CNTF (5 microg) increased apoptosis in Epi by 84 and 150%, respectively ( p <.05) and in Rp by 121 and 146%, respectively ( p <.05). Loss of adipocytes by apoptosis may provide an explanation for the unexpected delay in return to initial energy status following CNTF treatments.

Leptin-induced adipose apoptosis: Implications for body weight regulation.

Great strides have been made in understanding the genetics of body weight regulation, in part due to the study of rodent models of obesity that are characterized by mutations affecting leptin or its receptors. Leptin, produced in adipose tissue, acts both centrally and peripherally to orchestrate complex metabolic and behavioral changes that increase loss of adipose tissue, including suppressing food intake and increasing thermogenesis. In addition, recent evidence indicates that leptin acts centrally to trigger an apoptotic process resulting in adipocyte deletion. Loss of adipocytes by apoptosis may provide an explanation for the unexpected delay in return to initial energy status following leptin treatments. This review summarizes the major aspects of leptin-induced adipose tissue apoptosis, including some of the newest findings about possible mechanisms of action.

TNFalpha induces and insulin inhibits caspase 3-dependent adipocyte apoptosis.

Regulation of fat cell number by apoptosis is proposed to be part of a normal physiological cycle in adipose growth and development. To investigate this process, cultured rat adipocytes were treated with various concentrations of tumor necrosis factor alpha (TNFalpha) and/or insulin to determine the roles of these factors in adipocyte apoptosis. The cells were analyzed by flow cytometry using a TUNEL assay. TNFalpha increased adipocyte apoptosis in a dose-dependent fashion. TNFalpha-mediated apoptosis was detectable within 6 h of treatment and continued to increase with time. Decreasing media insulin concentration from 8.5 to 0.85 nM resulted in increased adipocyte apoptosis, whereas high doses of insulin protected adipocytes from TNFalpha-induced apoptosis. TNFalpha-activated apoptosis was accompanied by an increase in caspase 3 activity and could be inhibited by a caspase 3-specific inhibitor. These data suggest that adipose tissue cell number is regulated, in part, by an apoptotic signaling pathway that involves TNFalpha, insulin, and caspase 3.

Adipocyte insensitivity to insulin in growth hormone-transgenic mice.

Growth hormone (GH) has an inhibitory effect on adipogenesis, and its effect is associated with insulin action in obesity. In this study, the relationship between GH effect on insulin sensitivity and adipocyte differentiation in vivo was investigated. Transgenic (TG) female mice expressing porcine GH had reduced body weights and weights of retroperitoneal and parametrial fat depots. Insulin treatment increased PPARgamma and GLUT4 expression in adipose tissue of WT mice but had no effect in TG mice. Content of transcription factors, PPARgamma and C/EBPalpha and beta, was higher in adipose tissue of WT mice, and for C/EBPalpha and PPARgamma, the difference occurred primarily in 24-, compared to 12-week-old, mice. Expression of preadipocyte factor-1 was higher in adipose tissue of TG mice, and expression of TNF-alpha and leptin was reduced in adipose tissue of 24-week-old TG mice. Our results suggest that increased expression of GH reduces adipogenesis by inducing adipocyte resistance to the adipogenic effect of insulin.

Regulation of metabolism and body fat mass by leptin.

The relative stability of body weight over the long term and under a variety of environmental conditions that alter short-term energy intake and expenditure provides strong evidence for the regulation of body energy content. The lipostatic theory of energy balance regulation proposed 40 years ago that circulating factors, generated in proportion to body fat stores, acted as signals to the brain, eliciting changes in energy intake and expenditure. The discovery of leptin and its receptors has now provided a molecular basis for this theory. Leptin functions as much more than an adipocyte-derived signal of lipid stores, however. Although suppression of food intake is an important centrally mediated effect of leptin, considerable evidence indicates that leptin also functions both directly and indirectly, via the brain, to orchestrate complex metabolic changes in a number of organs and tissues, altering nutrient flux to favor energy expenditure over energy storage.

Effects of chlorhexidine on human taste perception.

Chlorhexidine gluconate at a dose used to control bacteria in the mouth has a reversible effect on taste perception. Taste-intensity ratings and taste-quality identification for concentration series of sucrose, sodium chloride, citric acid and quinine hydrochloride were obtained from 15 healthy humans. The participants rinsed with 0.12% chlorhexidine for 3 min twice a day. Each individual was tested 3 times: before the 4-day rinse period, 30 min after the final rinse, and 4 days after the rinse period. Chlorhexidine rinses reduced the perceptual intensity of sodium chloride and quinine hydrochloride, not sucrose or citric acid. No effects on taste perception were detected 4 days after the rinse period. The identification of sodium chloride as salty was seriously impaired by chlorhexidine but the identification of quinine hydrochloride as bitter was not affected. Specific sites of action of chlorhexidine on the taste epithelium are not known but its effects on salty taste may be related to its strong positive charge and its effect on bitter taste may be related to its amphiphilicity. Chlorhexidine has promise as a probe of taste transduction, as well as for the management of salty/bitter dysgeusias in humans.

Blockade of satiety factors by central injection of neuropeptide Y in sheep.

The ability of neuropeptide Y (NPY) to stimulate feed intake was tested in combination with two treatments known to depress feed intake in sheep. Six ewe and three wether lambs (mean BW = 40 kg) fitted with lateral cerebral ventricular guide cannulas and ruminal cannulas had free access to a nutritionally complete, pelleted diet. Balloons placed into the rumen were filled with either 0, 30 or 60 ml of water/kg BW and left in place for 6 h; intake was measured. Based on the decline in feed intake observed with increasing balloon volume in the rumen, Exp. 2 was designed to test effects of NPY injection (0 or 3.0 nmol) into the lateral cerebral ventricle and ruminal distension (0 or 35 ml/kg BW) for 6 h. During the 6-h test period, feed intake was depressed (P less than .05) by intraruminal balloon distension, but feed intake was increased by NPY injection (P less than .05); no interaction between NPY and distension was detected. Ruminal evacuation revealed that digesta occupied only 43% of the rumen's total volume capacity. Balloons occupied 14% of capacity, whereas meal size in control sheep following a 1.5-h fast equaled 7% of capacity. In Exp. 3, intraruminal infusion of 8 mmol/min of propionate depressed (P = .11) feed intake, whereas NPY injection enhanced (P less than .05) intake. There was no interaction between NPY and propionate infusion. In none of these experiments was cumulative feed consumption at 24 h influenced. We conclude that NPY is a versatile feeding stimulant. It promotes feed intake in feed-satiated, ruminally distended and propionate-infused sheep.

Intestinal infusion of a liquid diet alters CCK and NPY concentrations in specific brain areas of rats.

Cholecystokinin (CCK) and neuropeptide Y (NPY) have been implicated in the control of food intake in a number of species. This study was carried out to determine 1) whether nutrient-related stimulation of the upper small intestine could activate central CCK and NPY neuronal systems, resulting in changes in concentration of these peptides in specific brain areas, and 2) the influence of the circadian cycle on nutrient-related effects. Four groups of rats received treatments of either 1.0 ml saline (S) or Ensure liquid diet (E) infused into the duodenum either during the dark (D) or light (L) phase of the circadian cycle. CCK and NPY concentrations in extracts of specific brain areas were measured by RIAs. CCK concentration in the supraoptic n. (SON) was higher in D than in L, regardless of infusion treatment, and in the dorsal parabrachial n. area (DPN), CCK concentration was higher in E than S infused rats, regardless of circadian phase. CCK concentration in the dorsal motor vagal n. area (DMV) was higher in E, but only during L. NPY concentration was higher in DPN and paraventricular n. areas (PVN) and lower in the suprachiasmatic n. area (SC) after E, regardless of circadian phase. The changes in concentration of CCK and NPY in specific brain areas in response to food in the upper intestine suggest that nutrient-related signals from the intestine can activate specific CNS CCK and NPY-containing neural pathways.

Alpha 2-adrenoceptor blockade does not block feeding induced by neuropeptide Y in sheep.

We tested the hypothesis that blockade of central alpha 2-adrenergic receptors would prevent neuropeptide Y (NPY)-induced feeding. Nine young female sheep were fitted with lateral ventricula cannulas. Bolus intracerebroventricular (ICV) injection of 3 nmol of NPY increased feed intake after 30 min between 45 and 153% in three experiments. A bolus ICV injection of 400 or 100 nmol of the alpha 2-antagonist, yohimbine, either 5 or 30 min before NPY injection, did not attenuate this response. Instead, yohimbine increased feed intake over NPY-induced feeding by 52 to 55%. We interpret these data as evidence that the putative NPY feeding pathway in feed-sated sheep is not dependent on the type of alpha 2-adrenergic mechanism which can be blocked by ICV injection of yohimbine.

CNS injection of CCK in rats: effects on real and sham feeding and gastric emptying.

Cholecystokinin (CCK) released from the intestine during feeding may have a physiological role in satiety. There is also evidence that activation of central CCK-containing pathways is involved in the control of feeding behavior. This study was carried out to determine whether CCK-8 administered into the lateral cerebral ventricles (lv) of rats suppresses both sham feeding (SF) and real feeding (RF). Rats with lv guides and gastric cannulas ate a liquid diet with cannulas open (SF) or closed (RF) after lv (0, 0.05, 0.5 micrograms) or intraperitoneal (ip) (0, 4 micrograms/kg) injection of CCK-8. Both RF and SF were significantly decreased by ip CCK-8. RF was also decreased in a dose-related manner after lv CCK-8, but SF was not affected by lv CCK-8. Decreased feeding after ip CCK-8 may be due in part to its suppression of gastric emptying rate (GER). To determine whether central nervous system (CNS) CCK might also be involved in the control of gastric function, GER was measured after lv (0, 0.05, 0.5 micrograms) or ip (0, 4 micrograms/kg) injection of CCK-8. GER was significantly decreased after ip CCK-8, but lv CCK-8 had no effect on GER. Although both CNS and peripheral CCK peptide systems may be involved in satiety, CNS CCK appears to depend on concurrent peripheral nutrient-related stimuli in eliciting satiety.

Central nervous system injection of dynorphin-(1-13) overrides gastric satiety factors in sheep.

Signals from the gastrointestinal (GI) tract, arising during ingestion and digestion of food, are important in the termination of feeding. This study was carried out to determine whether the satiety triggered by specific GI stimuli in sheep could be reversed by central nervous system (CNS) administration of dynorphin, a putative endogenous kappa-opiate receptor ligand, which has been shown to be a potent feed-intake stimulant in many species. Rumen distension and increased intraruminal concentration of propionic acid (an energy substrate produced during fermentation in the rumen) both significantly decreased feed intake in fasted sheep. When either of these stimuli were combined with continuous 60-min lateral cerebroventricular injection of [D-Ala2]dynorphin A-(1-13) (0.32 nmol/min), feed intake returned to control levels. Increasing feed intake in food-producing animals is an important way of improving production efficiency. The findings of these experiments illustrate how manipulation of a CNS opioid system can modify the effects of feed intake-limiting factors generated by ingestion of a meal, thereby promoting greater feed intake.

Lateral cerebroventricular injection of neuropeptide Y stimulates feeding in sheep.

We compared the effects of neuropeptide Y (NPY) injected into the lateral ventricle (LV) and periphery (ip in rats; iv in sheep) on feed and water intake. In sated rats, a bolus injection of 1.18 or 2.35 nmol of NPY administered LV, but not ip, increased (P less than 0.05) feed intake on average of 809% within 30 min of injection. In sated sheep, an LV bolus injection of 2.35 nmol of NPY increased feed intake by 154% within 30 min. Similar doses of human pancreatic polypeptide and peptide YY were less orexigenic than NPY in sheep. After 24 h, cumulative feed intakes were similar among control and peptide treatments. Intravenous injection of 2.35 nmol NPY did not increase feed intake in sheep. Water intake was stimulated (P less than 0.05) by NPY (LV injection) in both the presence and absence of feed. We propose that NPY is involved in the central regulation of consummatory behavior in sheep.

Cholecystokinin concentration in specific brain areas of rats fed during the light or dark phase of the circadian cycle.

Measurement of peptide concentration in specific areas can be used as an initial investigative method for identifying brain sites in which the peptides may be acting. In this study cholecystokinin (CCK) concentration in specific hypothalamic and hindbrain areas of male Sprague-Dawley rats was measured in order to determine whether changes occurred as a result of feeding activity during different portions of the circadian cycle. Three groups of 40 rats each were studied: Group 1 were fasted 16 hr during the dark phase then sacrificed immediately or after a 20 min light phase meal. Group 2 were fasted 16 hr during the light phase then sacrificed immediately after lights out or after a 20 min dark-onset meal. Group 3 were fed ad lib and sacrificed immediately after light out or after a 20 min dark-onset meal. CCK was extracted from dissected areas and concentration was measured by RIA. There was no difference in CCK concentration of any of the 9 brain areas in rats fasted during the dark phase and fed during the light phase. In rats fasted during the light phase CCK concentration of the paraventricular nucleus (PVN) was greater in those that subsequently ate a meal at dark-onset than in those that did not eat (p less than 0.05). In ad lib fed rats CCK concentration was less in the anterior hypothalamus (AH) and greater in the supraoptic nucleus (SON) in rats that ate a dark-onset meal than in rats that did not (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Immunohistochemical distribution of cholecystokinin, dynorphin A and Met-enkephalin neurons in sheep hypothalamus.

The distribution of cholecystokinin (CCK)-, Met-enkephalin (M-ENK)- and dynorphin (DYN)-like immunoreactive perikarya were examined in the sheep hypothalamus using the peroxidase-anti-peroxidase technique. CCK- and DYN-containing neurons were found primarily in the suprachiasmatic nucleus (SCH) and supraoptic nucleus (SO). No CCK- or DYN-containing neurons were found in the paraventricular nucleus (PVN). M-ENK-containing neurons were found mainly in the PVN of the hypothalamus. In addition, M-ENK neurons were found in the dorsomedial (DMH), lateral (LH), anterior (AH) and periventricular hypothalamic areas. The distribution of these neuropeptides may provide a basis for understanding differences in responsiveness to centrally administered peptides.

Effects of somatostatin immunoneutralization on growth and endocrine parameters in chickens.

The effects of somatostatin immunoneutralization on growth rate, growth hormone (GH) secretion and circulating insulin-like growth factor I (IGF-I) concentrations were investigated in chickens through the use of passive and active immunization techniques. Intravenous bolus injection of goat-antisomatostatin stimulated a significant (P less than .05) increase in plasma GH levels for one hour post-injection in four and six week old male broiler chickens. The GH response to an intravenous bolus injection of hGRF44NH2 was similar in the antisomatostatin treated chicks and normal goat serum treated controls. Despite the presence of circulating somatostatin antisera after 28 hours, plasma GH levels were not different between control and antisomatostatin-treated chicks at that time. Continuous administration of somatostatin antisera by Alzet pump over a two-week period resulted in significant (P less than .05) elevations in plasma GH levels at one week post-implantation and in circulating IGF-I concentrations after two weeks of administration. Chicks which developed antibodies against somatostatin following active immunization exhibited a 7.1% increase in growth rate which was associated with a significant decrease in abdominal fat. However, neither GH nor IGF-I concentrations were elevated in the chicks which developed somatostatin antibodies. Thus, the benefits gained from somatostatin immunoneutralization may be exerted through mechanisms other than GH.

Differential distributions of cholecystokinin in hamster and rat forebrain.

Rats and golden hamsters show a differential feeding response to intracranial injections of cholecystokinin (CCK). Rats, but not hamsters reduce food intake after CCK injections into the hypothalamic paraventricular nucleus. In view of this species difference, we undertook an immunohistochemical study of the distribution of CCK-immunoreactivity in the hamster hypothalamus and remaining forebrain. CCK-immunoreactive perikarya were abundant in the neocortex, claustrum, hippocampal formation, amygdaloid complex, bed nucleus of the stria terminalis, nucleus of the lateral olfactory tract and in the magnocellular basal nucleus. CCK-immunoreactive neurons had a more restricted distribution in the diencephalon and were relatively rare in the preoptic area-hypothalamus. The only exception was the suprachiasmatic nucleus and adjacent medial anterior hypothalamus, in which CCK-immunoreactive neurons were numerous. CCK-containing perikarya were not observed in the hamster hypothalamic paraventricular and supraoptic nuclei, where they have been reported to occur in the rat. Groups of CCK-positive perikarya were also noted in the hamster thalamic paratenial and parafascicular nuclei. CCK-immunoreactive fibers/terminals were localized in the caudate and putamen, periventricular zones, dorsolateral geniculate, thalamic reticular nucleus and the superficial layer of the optic tectum. Fiber/terminal labeling was also present in those regions associated with CCK-immunoreactive perikarya. Our results indicate that the telencephalic distribution of CCK-containing neurons in the hamster appears to be similar to that reported in the rat. However, several differences occur in the diencephalon. Perhaps the most striking is that the hamster differs from the rat in having a large group of CCK-containing neurons in the suprachiasmatic nucleus, and in lacking the CCK-containing perikarya observed in the rat paraventricular and supraoptic nuclei. These differences may underly species differences in feeding responses to intracranial CCK injections and gonadal responses to short photoperiods. Our data further suggest that the distribution of neuropeptides and other neuroactive substances may not always be conserved during evolution.