Michael F. Wiater

Synergy Between Leptin and Cholecystokinin (CCK) to Control Daily Caloric Intake

Both cholecystokinin (CCK), a short-term meal-related satiety signal, and the ob protein leptin, a postulated long-term adiposity hormone, are thought to be important signals in the multiple interacting systems that control appetite and adiposity. We hypothesized that these hormones may synergistically interact to suppress feeding. Following IP administration of leptin (two doses of 50 micrograms each) and CCK (2, 4, 8, or 16 micrograms) total daily caloric intake was significantly reduced by leptin and CCK compared to leptin alone. These results support the hypothesis that CCK and leptin may synergistically interact to control long-term feeding.

Circadian integration of sleep-wake and feeding requires NPY receptor-expressing neurons in the mediobasal hypothalamus

Sleep and feeding rhythms are highly coordinated across the circadian cycle, but the brain sites responsible for this coordination are unknown. We examined the role of neuropeptide Y (NPY) receptor-expressing neurons in the mediobasal hypothalamus (MBH) in this process by injecting the targeted toxin, NPY-saporin (NPY-SAP), into the arcuate nucleus (Arc). NPY-SAP-lesioned rats were initially hyperphagic, became obese, exhibited sustained disruption of circadian feeding patterns, and had abnormal circadian distribution of sleep-wake patterns. Total amounts of rapid eye movement sleep (REMS) and non-REMS (NREMS) were not altered by NPY-SAP lesions, but a peak amount of REMS was permanently displaced to the dark period, and circadian variation in NREMS was eliminated. The phase reversal of REMS to the dark period by the lesion suggests that REMS timing is independently linked to the function of MBH NPY receptor-expressing neurons and is not dependent on NREMS pattern, which was altered but not phase reversed by the lesion. Sleep-wake patterns were altered in controls by restricting feeding to the light period, but were not altered in NPY-SAP rats by restricting feeding to either the light or dark period, indicating that disturbed sleep-wake patterns in lesioned rats were not secondary to changes in food intake. Sleep abnormalities persisted even after hyperphagia abated during the static phase of the lesion. Results suggest that the MBH is required for the essential task of integrating sleep-wake and feeding rhythms, a function that allows animals to accommodate changeable patterns of food availability. NPY receptor-expressing neurons are key components of this integrative function.

NPAS2 deletion impairs responses to restricted feeding but not to metabolic challenges.

Neuronal PAS domain protein 2 (Npas2) is a clock gene expressed widely in brain and peripheral tissues. NPAS2 is responsive to cellular metabolic state and mutation of this gene impairs adaptation to restricted feeding schedules, suggesting that NPAS2 is required for effective control of a food-entrainable oscillator. However, an alternative possibility, that NPAS2 is required for detection of metabolic cues signaling energy deficiency or for arousal of appropriate behavioral responses to such cues, as not been directly examined. Therefore, we examined the effect of targeted disruption of Npas2 on responses to several acute and chronic metabolic challenges. We found that under normal light-dark and ad libitum feeding conditions, Npas2 knockout (KO) mice did not differ from wild-type (WT) controls with respect to diurnal feeding or blood glucose levels, body weight or size or body composition. Furthermore, feeding responses to overnight food deprivation, insulin- or 2-deoxy-d-glucose (2DG)-induced glucoprivation, mercaptoacetate (MA)-induced blockade of fatty acid oxidation and cold exposure did not differ by genotype. However, KO mice lost more weight than WT during overnight food deprivation and when placed on a 4-h restricted feeding schedule, even though food intake did not differ between groups. Thus, it appears that NPAS2 is not required for detection of or behavioral responses to a variety of acute or chronic metabolic deficits, but is more likely to be involved in effective synchronization of feeding behavior with scheduled food availability.

Leptin does not attenuate the hyperphagia induced by 2-deoxy-D-glucose.

Glucose is the major metabolic substrate for brain energy metabolism. Because glucose is not stored in significant quantities in the brain, the brain is dependent on the continuous delivery of glucose by the blood. Metabolic receptor cells that monitor glucose utilization are located within the brain and stimulate feeding when glucose metabolism is reduced (glucoprivic feeding). Thus, the glucoprivic control of feeding plays a key role in the survival and normal function of the brain and does so by sensing ongoing metabolic conditions within the brain.1–3 In contrast to the glucoprivic control, leptin appears to be related to a control of food intake based on body fat stores.4 Leptin levels are directly proportional to body adiposity, and leptin’s major effect on feeding is inhibitory. Thus leptin and glucoprivation would appear to have distinct roles in the overall regulation of food intake and energy metabolism. In the present experiment, we examined the effect of 2-deoxy-D-glucose (2DG)induced glucoprivation on food intake in chronic leptin-treated rats to determine whether an interaction between these controls of feeding could be detected. Adult male Sprague-Dawley rats (n = 5) adapted to a medium-fat diet were injected subcutaneously with leptin (20 mg/kg/day) for five days. Feeding in response to 2DG (250 mg/kg, s.c.) and saline (0.9%) were tested prior to leptin treatment. 2DG was tested again on days 2 and 6 of leptin treatment. Feeding was measured for 4 h beginning immediately after 2DG or saline injection. Tests were conducted during the light phase of the circadian light-dark cycle. Leptin treatment resulted in a significant reduction in the daily food intake (F(7,32) = 5.25, p < .05). On day 1 of leptin treatment food intake was reduced to 74% of the day zero intake (19.9 ± 0.4 g). On days 2, 3, and 7, food intake was reduced to 68%, 50%, and 50% of the day zero intake, respectively. Body weight was reduced by approximately 1% of the pre-leptin weight per day during the five days of leptin treatment (FIG. 1). Despite the large reduction in the food intake induced by leptin treatment, this treatment did not reduce the amount of food consumed in response to 2DG. 2DG increased food intake significantly above control intake (0.1 ± 0.2 g) in all three tests. Rats ate 3.3 ± 0.4 g in response to 2DG in the pre-leptin test, 2.2 ± 0.6 g in the day 2 test, and 2.6 ± 0.8 g in the day 6 2DG test. Moreover, 2DGinduced food intake on days 2 and 6 of leptin treatment did not differ from the preleptin 2DG response (FIG. 2). The 24-h food intake on 2DG test days during leptin treatment did not differ from the 24-h intake on days 3–5 of leptin treatment when no

Hindbrain Catecholamine Neurons Control Rapid Switching of Metabolic Substrate Use during Glucoprivation in Male Rats

Using the retrogradely transported immunotoxin, antidopamine β-hydroxylase-saporin (DSAP), we showed previously that hindbrain catecholamine neurons innervating corticotropin-releasing hormone neurons in the paraventricular nucleus of the hypothalamus are required for glucoprivation-induced corticosterone secretion. Here, we examine the metabolic consequences of the DSAP lesion in male rats using indirect calorimetry. Rats injected into the paraventricular nucleus of the hypothalamus with DSAP or saporin (SAP) control did not differ in energy expenditure or locomotor activity under any test condition. However, DSAP rats had a persistently higher respiratory exchange ratio (RER) than SAPs under basal conditions. Systemic 2-deoxy-D-glucose did not alter RER in DSAP rats but rapidly decreased RER in SAP controls, indicating that this DSAP lesion impairs the ability to switch rapidly from carbohydrate to fat metabolism in response to glucoprivic challenge. In SAP controls, 2-deoxy-D-glucose-induced decrease in RER was abolished by adrenalectomy but not adrenal denervation. Furthermore, dexamethasone, a synthetic glucocorticoid, decreased RER in both SAP and DSAP rats. Thus, rapid switching of metabolic substrate use during glucoprivation appears to be due to impairment of the catecholamine-mediated increase in corticosterone secretion. Sustained elevation of basal RER in DSAP rats indicates that catecholamine neurons also influence metabolic functions that conserve glucose under basal conditions.

Leptin-sensitive neurons in the arcuate nucleus integrate activity and temperature circadian rhythms and anticipatory responses to food restriction

Previously, we investigated the role of neuropeptide Y and leptin-sensitive networks in the mediobasal hypothalamus in sleep and feeding and found profound homeostatic and circadian deficits with an intact suprachiasmatic nucleus. We propose that the arcuate nuclei (Arc) are required for the integration of homeostatic circadian systems, including temperature and activity. We tested this hypothesis using saporin toxin conjugated to leptin (Lep-SAP) injected into Arc in rats. Lep-SAP rats became obese and hyperphagic and progressed through a dynamic phase to a static phase of growth. Circadian rhythms were examined over 49 days during the static phase. Rats were maintained on a 12:12-h light-dark (LD) schedule for 13 days and, thereafter, maintained in continuous dark (DD). After the first 13 days of DD, food was restricted to 4 h/day for 10 days. We found that the activity of Lep-SAP rats was arrhythmic in DD, but that food anticipatory activity was, nevertheless, entrainable to the restricted feeding schedule, and the entrained rhythm persisted during the subsequent 3-day fast in DD. Thus, for activity, the circuitry for the light-entrainable oscillator, but not for the food-entrainable oscillator, was disabled by the Arc lesion. In contrast, temperature remained rhythmic in DD in the Lep-SAP rats and did not entrain to restricted feeding. We conclude that the leptin-sensitive network that includes the Arc is required for entrainment of activity by photic cues and entrainment of temperature by food, but is not required for entrainment of activity by food or temperature by photic cues.

The Lipoprivic Control of Feeding Is Governed by Fat Metabolism, Not by Leptin or Adipose Depletion

A lipoprivic control of feeding has been proposed based on the finding that appetite is stimulated by drugs such as beta-mercaptoacetate (MA) that reduce fatty acid oxidation. The adipose-derived hormone, leptin, has effects on feeding and fat oxidation that are opposite those produced by MA. However, effects of this hormone on MA-induced feeding are not known. Here we examined the effects of endogenous leptin levels and of acute central and peripheral leptin administration on MA-induced feeding. We also examined leptin-induced changes in feeding, body weight, and plasma fuels after capsaicin-induced deletion of the lipoprivic control. MA-induced feeding was not altered under any of these conditions, and leptins effects were not altered by capsaicin. We then examined MA-induced feeding during chronic leptin treatment. Because chronic leptin produces several distinct metabolic states as body adiposity is reduced, we tested MA before, during, and after leptin treatment at times that coincided with these states. MA-induced feeding was unchanged on d 3 of leptin treatment when rats were in a lipolytic state and rapidly metabolizing body fat stores but reduced on d 10 when they were adipose deplete and their level of fat oxidation was reduced. Together results suggest that the lipoprivic control is normally less active in the fat deplete state than during states associated with fat availability. If so, its insensitivity to leptin would enable the lipoprivic control to operate when dietary fat, adiposity, and leptin levels are elevated. The role played by the lipoprivic control under such conditions remains uncertain.

Deletion of GPR40 fatty acid receptor gene in mice blocks mercaptoacetate-induced feeding

Both increased and decreased fatty acid (FA) availability contribute to control of food intake. For example, it is well documented that intestinal FA reduces feeding by triggering enterondocrine secretion of satietogenic peptides, such as cholecystokinin (CCK) and glucagon-like peptide-1 (GLP-1). In contrast, mechanisms by which decreased FA availability increase feeding are not well understood. Over the past three decades substantial research related to FA availability and increased feeding has involved use of the orexigenic compound mercaptoacetate (MA). Because MA reportedly inhibits FA oxidation, it has been assumed that reduced FA oxidation accounts for the orexigenic action of MA. Recently, however, we demonstrated that MA antagonizes G protein-coupled receptor 40 (GPR40), a membrane receptor for long and medium chain FA. We also demonstrated that, by antagonizing GPR40, MA inhibits GLP-1 secretion and attenuates vagal afferent activation by FA. Because both vagal afferent activation and GLP-1 inhibit food intake, we postulated that inhibition of GPR40 by MA might underlie the orexigenic action of MA. We tested this hypothesis using male and female GPR40 knockout (KO) and wild-type (WT) mice. Using several testing protocols, we found that MA increased feeding in WT, but not GPR40 KO mice, and that GPR40 KO mice gained more weight than WT on a high-fat diet. Metabolic monitoring after MA or saline injection in the absence of food did not reveal significant differences in respiratory quotient or energy expenditure between treatment groups or genotypes. These results support the hypothesis that MA stimulates food intake by blocking FA effects on GPR40.

Hindbrain catecholamine neurons contribute to control of daily food intake during chronic leptin treatment.

Chronic leptin treatment sufficient to cause profound reduction of food intake and body weight does not suppress glucoprivic feeding, a control of feeding that requires hindbrain catecholamine neurons. Therefore, we speculated that hindbrain catecholamine neurons may function during leptin treatment to avert brain glucose deficit. If so, elimination of these neurons would potentiate reduction of food intake during chronic leptin treatment. To test this hypothesis, we microinjected the immunotoxin, anti-dopamine beta-hydroxylase saporin (DSAP) or IgG-saporin control (SAP), into the PVH to retrogradely destroy catecholamine neurons with projections to the hypothalamus. This DSAP lesion selectively abolishes the glucoprivic control of feeding, but does not cause anorexia or loss of body weight during ad libitum access to food. Beginning 2 weeks after injections, we administered leptin into the lateral cerebroventricle daily and measured food (standard chow) intake, body weight and plasma metabolic fuels. We found that leptin treatment caused clear changes in plasma NEFA, glycerol and triglyceride levels, but these did not differ between DSAP and SAP rats. However, DSAP lesions significantly reduced food intake during leptin treatment and potentiated leptin-induced weight loss. DSAP-lesioned rats also had lower plasma glucose levels than SAP rats beginning on day 8 of leptin treatment. Results reveal a contribution of hindbrain catecholamine neurons to control of feeding and energy homeostasis during chronic leptin. Catecholamine neurons and/or the glucoprivic control may play a more prominent role in control of daily food intake in the absence of body fat stores.

Energy Homeostasis and the Tumor/Host Interaction: The role of the Brain

The defensive regulation of energy homeostasis by neural and endocrine systems is examined to evaluate the role of the brain in macroenvironmental metabolic control systems that help counterattack the aggressive tumor. Brain homeostatic mechanisms (neural and hormonal) discussed are those linked to metabolic rhythms, food intake and adiposity. Homeostasis is discussed in terms of rheostasis, the low probability of dysregulation, and the potential risks for the defense. The perspective of this review is that many of the metabolic alterations observed in tumor progression may be due to appropriate central homeostatic regulation. Clear deficits in homeostatic regulation during tumor growth have not been unequivocally demonstrated. Yet the brain of the host is clearly under duress due to the tumor. Clarification of homeostatic macroenvironmental regulatory responses may be useful in developing strategies that collaborate with these brain mechanisms. Further analysis of these regulatory systems may identify key changes that ultimately subserve the lethal failure of all host responses. Strategies that protect the brain from the pathological consequences of tumor growth (glucoprivation, oxidative stress, ketogenic diets) and thereby enable a stronger defense are discussed.