Jock S. Hamilton

Uncoupling proteins-2 and 3 influence obesity and inflammation in transgenic mice.

OBJECTIVE: To test the hypothesis that either uncoupling protein-2 UCP2 or UCP3 or both together influence obesity and inflammation in transgenic mice.DESIGN: We generated 12 lines of transgenic mice for both human UCP2 and 3 using native promoters from a human bacterial artificial chromosome (BAC) clone. The BAC expresses no genes other than UCP2 and 3. Mice used for experiments are N4 or higher of backcross to C57BL/6J (B6). Each experiment used transgenic mice and their nontransgenic littermates.RESULTS: Northern blots confirmed expression on human UCP2 in adipose and spleen, while human UCP3 expression was detectable in gastrocnemius muscle. Western blots demonstrated a four-fold increase of UCP2 protein in spleens of Line 32 transgenic animals. Heterozygous mice of four lines showing expression of human UCP2 in spleen were examined for obesity phenotypes. There were no significant differences between Lines 1 and 32, but female transgenics of both lines had significantly smaller femoral fat depots than the control (littermate) mice (P=0.015 and 0.005, respectively). In addition, total fat of transgenic females was significantly less in Line 1 (P=0.05) and almost significantly different in Line 32 (P=0.06). Male Line 1 mice were leaner (P=0.04) while male Line 32 mice were almost significantly leaner (P=0.06). Heterozygous mice of Lines 35 and 44 showed no significant differences from the nontransgenic littermate controls. Effects of the UCP2/UCP3 transgene on obesity in Line 32 mice were confirmed by crossing transgenic mice with the B6.Cg-Ay agouti obese mice. B6.Cg-Ay carrying the UCP2/UCP3 transgene from Line 32 were significantly leaner than nontransgenic B6.Cg-Ay mice.Line 32 UCP2/UCP3 transgenics showed increased hypothalamic Neuropeptide (NPY) levels and food intake, with reduced spontaneous physical activity. Transgenic baseline interleukin4 (IL-4) and interleukin6 (IL-6) levels were low with lower or later increases after endotoxin injection compared to wild-type littermates. Endotoxin-induced fever was also diminished in transgenic male animals. Low-density lipoprotein (LDL) cholesterol levels were significantly higher in both Line 1 and 32 transgenics (P=0.05 and 0.001, respectively) after they had been placed on a moderate fat-defined diet containing 32% of calories from fat for 5 weeks.CONCLUSION: Moderate overexpression of UCP2 and 3 reduced fat mass and increased LDL cholesterol in two independent lines of transgenic mice. Thus, the reduced fat mass cannot be due to insertional mutagenesis since virtually identical fat pad weights and masses were observed with the two independent lines. Line 32 mice also have altered inflammation and mitochondrial function. We conclude that UCP2 and/or 3 have small but significant effects on obesity in mice, and that their mechanism of action may include alterations of metabolic rate.

Meal-induced changes in extracellular 5-HT in medial hypothalamus of lean (Fa/Fa) and obese (fa/fa) Zucker rats.

Hypothalamic serotonin (5-HT) is involved in appetite regulation and sympathetic stimulation of thermogenesis. This study tested the hypothesis that the enhanced energetic efficiency of obese Zucker rats involves blunted serotonergic release within the medial hypothalamus (MH). We used microdialysis and HPLC-EC to measure dynamic changes in extracellular 5-HT levels in the MH of 10-13-week-old male lean (Fa/Fa) and obese (fa/fa) Zucker rats before and after a meal. No differences were noted in basal levels of 5-HT between lean and obese rats. Consistent with the suggestion that hypothalamic 5-HT plays a physiological role in feeding, extracellular 5-HT levels increased significantly in both lean and obese rats given a meal. This increase was observed in the 20 min interval in which they ate the 8.1 kcal meal and remained for an additional 60 min. The net release of 5-HT during the meal interval was comparable in the lean (1.46+/-0.38 fmol/microl) and obese (1.21+/-0.82 fmol/microl) rats. However, the 5-HT levels of the leans (1.80+/-0.29 fmol/microl) plateaued in the next 20 min interval, whereas they continued rising (2.74+/-0.53 fmol/microl) in obese rats and were significantly higher than those in the leans during the 40 and 60 min intervals after the meal was presented. This resulted in a total net release during the meal plus the next three 20 min intervals that was significantly higher in obese (9.83+/-1.16 fmol/microl) than in lean (5.59+/-0.85 fmol/microl) rats. Thus, the enhanced energetic efficiency of the obese Zucker rats may not be associated with attenuated serotonin release in response to a meal. Rather their enhanced release of 5-HT in the MH may reflect compensatory mechanisms for the elevated orexigen NPY, the reduction in meal-induced CCK release, and/or a functional resistance to 5-HT.

Lean (Fa/Fa) but not obese (fa/fa) Zucker rats release cholecystokinin at PVN after a gavaged meal

Neuropeptides play an important role in the integration of dietary signals. Cholecystokinin (CCK) has been implicated in regulating ingestive behavior, particularly satiety. The primary objective of this study was to examine whether the hyperphagia characteristic of obese (fa/fa) rats involves impaired neural CCK secretion. Dynamic release of CCK at the hypothalamic paraventricular nucleus (PVN) of age-matched lean (Fa/Fa) and obese Zucker rats was determined using push-pull perfusion. The gavage of a 10.3-kcal (6 ml) liquid diet during lights off was followed by increased CCK release in lean rats (from 13.6 +/- 1.1 to 22.1 +/- 1.4 fmol in the 1st postprandial period and 18.4 +/- 2.5 fmol in the 2nd postprandial period). An identical meal load resulted in no postprandial increase in CCK release in obese rats, despite the fact that high-K+ artificial cerebrospinal fluid evoked CCK outflow in all animals. Intubation of 6 ml of nonnutritive 1% carboxymethylcellulose had no effect. These results are consistent with the suggestion that hypothalamic CCK plays a physiological role in satiety, and they demonstrate that obese Zucker rats have blunted hypothalamic CCK release in response to dietary cues.Neuropeptides play an important role in the integration of dietary signals. Cholecystokinin (CCK) has been implicated in regulating ingestive behavior, particularly satiety. The primary objective of this study was to examine whether the hyperphagia characteristic of obese ( fa/ fa) rats involves impaired neural CCK secretion. Dynamic release of CCK at the hypothalamic paraventricular nucleus (PVN) of age-matched lean ( Fa/ Fa) and obese Zucker rats was determined using push-pull perfusion. The gavage of a 10.3-kcal (6 ml) liquid diet during lights off was followed by increased CCK release in lean rats (from 13.6 ± 1.1 to 22.1 ± 1.4 fmol in the 1st postprandial period and 18.4 ± 2.5 fmol in the 2nd postprandial period). An identical meal load resulted in no postprandial increase in CCK release in obese rats, despite the fact that high-K+ artificial cerebrospinal fluid evoked CCK outflow in all animals. Intubation of 6 ml of nonnutritive 1% carboxymethylcellulose had no effect. These results are consistent with the suggestion that hypothalamic CCK plays a physiological role in satiety, and they demonstrate that obese Zucker rats have blunted hypothalamic CCK release in response to dietary cues.

Influence of Age and Gender on Brown Adipose Tissue Norepinephrine Turnover

Abstract We hypothesized that the attenuated brown adipose tissue thermogenic capacity observed previously in cold-exposed 27-month-old male versus female Fischer 344 rats might result, in part, from blunted sympathetic signaling to the tissue. As an index of sympathetic activity to brown fat, norepinephrine (NE) turnover in this tissue was evaluated at rest (22-24°C) and during 1.5 hr of cold exposure (6°C) in male and female Fischer 344 rats, aged 6, 12, and 26 months. Resting NE turnover as well as the rate constant for NE efflux from brown fat, expressed as total and as per gram of tissue protein, did not, in general, differ from age or gender. During cold exposure, rate constants and NE turnover rates increased significantly from those at rest in all groups. Brown fat NE turnover in cold-exposed 26-month-old male rats was greater than that observed in age-matched females, suggesting greater, not less, sympathetic signaling in the males versus females. These data indicate that the attenuated brown fat thermogenic capacity as well as the blunted cold-induced thermogenic responses of cold-exposed older male versus female rats reported previously cannot be explained by diminished release of NE from sympathetic nerves innervating brown adipose tissue.

Temporal relationships of blood pressure, heart rate, baroreflex function, and body temperature change over a hibernation bout in Syrian hamsters

Hibernating mammals undergo torpor during which blood pressure (BP), heart rate (HR), metabolic rate, and core temperature (TC) dramatically decrease, conserving energy. While the cardiovascular system remains functional, temporal changes in BP, HR, and baroreceptor-HR reflex sensitivity (BRS) over complete hibernation bouts and their relation to TC are unknown. We implanted BP/temperature telemetry transmitters into Syrian hamsters to test three hypotheses: H-1) BP, HR, and BRS decrease concurrently during entry into hibernation and increase concurrently during arousal; H-2) these changes occur before changes in TC; and H-3) the pattern of changes is consistent over successive bouts. We found: 1) upon hibernation entry, BP and HR declined before TC and BRS, suggesting baroreflex control of HR continues to regulate BP as the BP set point decreases; 2) during the later phase of entry, BRS decreased rapidly whereas BP and TC fell gradually, suggesting the importance of TC in further BP declines; 3) during torpor, BP slowly increased (but remained relatively low) without changes in HR or BRS or increased TC, suggesting minimal baroreflex or temperature influence; 4) during arousal, increased TC and BRS significantly lagged increases in BP and HR, consistent with establishment of tissue perfusion before increased TC/metabolism; and 5) the temporal pattern of these changes was similar over successive bouts in all hamsters. These results negate H-1, support H-2 with respect to BP and HR, support H-3, and indicate that the baroreflex contributes to cardiovascular regulation over a hibernation bout, albeit operating in a fundamentally different manner during entry vs. arousal.

Extracellular hypothalamic serotonin levels after dorsal raphe nuclei stimulation of lean (Fa/Fa) and obese (fa/fa) Zucker rats

Serotonin (5-HT), acting in the medial hypothalamus (MH), is involved in appetite/satiety and sympathetic stimulation of thermogenesis. This study tested the hypothesis that the enhanced energetic efficiency of obese Zucker rats is associated with a reduced capacity of activated dorsal raphe (DR) neurons to release 5-HT in the MH. We used microdialysis and HPLC-EC to measure dynamic changes in extracellular 5-HT levels in the MH of urethane-anesthetized, 10-14 week old male lean and obese Zucker rats. These concentrations did not differ significantly between the two genotypes prior to stimulation (mean+/-S.E.M.=3.8+/-0.5 fmol/microl, lean; 3.6+/-1.0 fmol/microl, obese) or following DR stimulation at 25 Hz (200 microA). The latter elicited initial net increases of 0.54+/-0.15 fmol/microl in lean and 0.58+/-0.20 fmol/microl in obese rats; and 20 min post-stimulus, 5-HT values were still elevated and comparable in the two genotypes. Although a 50-Hz (200 microA) stimulus evoked initial increases that were similar in lean (1.37+/-0.23 fmol/microl) and obese (0.95+/-0.24 fmol/microl,) rats, the net increase in 5-HT concentration during the next 20-40 min period was higher in the lean (2.03+/-0.55 fmol/microl vs. 1.18+/-0.24 fmol/microl in the obese animals). Also, in the lean, but not obese rats, extracellular 5-HT levels were significantly greater at 50 vs. 25 Hz. These results support the hypothesis that the capacity of midbrain serotonergic neurons to release 5-HT at the MH is reduced in obese Zucker rats, consistent with their blunted responsiveness to dietary stimuli and greater energetic efficiency.

Neuroprotection supports signal processing in the hippocampus of Syrian hamsters, a facultative hibernator

Studies on several species of mammalian seasonal hibernators (those hibernating only in winter) show that their neurons are more tolerant to hypoxia than those in non-hibernating species. Such tolerance has not been studied in facultative hibernators [e.g., Syrian hamsters (Mesocricetus auratus)], which can hibernate at any time of year. We tested the hypotheses that, when exposed to hypoxia, hamster hippocampal pyramidal cells more effectively support signal processing than do rat hippocampal neurons and this protection is enhanced in slices from hibernating versus non-hibernating hamsters and as temperature decreases. Population spike amplitudes (PSAs) were recorded from CA1 pyramidal cells. Slices were perfused in oxygenated artificial cerebral spinal fluid (O(2)ACSF) to establish a baseline. Oxygen was then replaced by nitrogen (N(2)ACSF) for 15 min, followed by a 30-min recovery period in O(2)ACSF. Three minutes after slices were returned to O(2)ACSF, PSAs recovered to 62.4 ± 6.8% of baseline in 15 slices from 8 non-hibernating hamsters but only to 22.7 ± 5.6% in 17 slices from 5 rats. Additionally, PSA recovery was greater in slices from hibernating than non-hibernating hamsters and recovery increased as temperature decreased. These significant differences (P ≤ 0.05) suggest Syrian hamsters are a useful model for studying naturally occurring neuroprotective mechanisms.

LOXL null mice demonstrate selective dentate structural changes but maintain dentate granule cell and CA1 pyramidal cell potentiation in the hippocampus

Lysyl oxidase-like protein (LOXL), part of the lysyl oxidase copper-dependent amine oxidase family, is expressed in the extracellular matrix and in the nucleus. It likely plays a role in cross-linking collagen and elastin, possibly modulating cellular functions. Immunohistochemical studies show the presence of LOXL in the pyramidal cell layer of the hippocampus; and in this study, we report that cells in the granule cell layer have significantly smaller somas in LOXL -/- compared to LOXL +/+ mice. In addition we tested the hypothesis that these structural alterations in the dentate granule layer were associated with synaptic efficacy and thus muted long-term potentiation in mice lacking the protein. Electrical recordings were obtained in 300-mum hippocampal slices in dentate and CA1 pyramidal cell layers in age-matched wild type and LOXL null mice. Potentiation in the CA1 cell layer of 10 LOXL -/- and 8 LOXL +/+ mice was 191.0+/-9.3% and 181.6+/-9.1%, respectively (mean+/-S.E.M.). Dentate potentiation was 120.8+/-7.0% and 121.0+/-3.4% in 11 LOXL -/- and 11 LOXL +/+ mice, respectively. No phenotypic difference in potentiation of population spike amplitude (or in EPSP slope) in either layer was observed. Thus, contrary to expectation, structural changes in the hippocampus of LOXL -/- mice did not affect synaptic remodeling in a manner that impaired the establishment of LTP.

Altered α1-adrenoceptor binding in intact and adrenalectomized obese Zucker rats (fa/fa)

Abstract While many autonomic and metabolic defects associated with genetic obesity in the Zucker rat are corrected by adrenalectomy (Adx), brain adrenoceptor function has not been examined in this context. Here, 3 weeks after Adx or sham surgery, brains of 11 weeks old lean ( Fa/Fa ) and obese ( fa/fa ) male Zucker rats were assayed for α 1 -([ 3 H]prazosin; [ 3 H]PRZ) and α 2 -adrenoceptor ([ 3 H]paraminoclonidine; [ 3 H]PAC) binding by autoradiography. By genotype, obese rats had 19–256% higher [ 3 H]PRZ binding than lean rats in the amygdala (central [ACN], basolateral [ABL], basomedial [ABM] and medial [MAN] nuclei [n.]), hypothalamus (dorsomedial n. [DMN] and lateral [LH]) and somatosensory cortex. In the ABL and ACN, increased maximal binding ( B max ) in obese rats was associated with decreased affinity (increased K d ). Three weeks after surgery, sham-operated obese rats gained 27% more weight than lean rats but lean and obese Adx rats gained the same amount of weight. Adx reduced [ 3 H]PRZ binding in both lean and obese rats by 37–70% in the amygdala (ABM, ACN, MAN) compared to sham-operated rats. But, Adx selectively reduced [ 3 H]PRZ binding only in lean rats in the ABL, DMN, ventromedial hypothalamic n. (VMN) and ventroposteromedial thalamic n. In most areas, decreases in maximal binding ( B max ) associated with Adx were accompanied by decreases in K d . Unlike [ 3 H]PRZ binding, there was no consistent genotype difference in [ 3 H]PAC binding although Adx was followed by increased binding in obese and decreased binding in lean rats in the ABL. In only the VMN, obese rats had a 21% higher α 2 -toα 1 -adrenoceptor ratio than lean White widespread differences in brain α 1 -adrenoceptor binding between lean and obese rats may be important, the selectively higher VMN α 2 -/α 1 -ratio may be critical to the pathogenesis of obesity.

Decreasing temperature shifts hippocampal function from memory formation to modulation of hibernation bout duration in Syrian hamsters

Previous studies in hibernating species have characterized two forms of neural plasticity in the hippocampus, long-term potentiation (LTP) and its reversal, depotentiation, but not de novo long-term depression (LTD), which is also associated with memory formation. Studies have also shown that histamine injected into the hippocampus prolonged hibernation bout duration. However, spillover into the ventricles may have affected brain stem regions, not the hippocampus. Here, we tested the hypothesis that decreased brain temperature shifts the major function of the hippocampus in the Syrian hamster (Mesocricetus auratus) from one of memory formation (via LTP, depotentiation, and de novo LTD) to increasing hibernation bout duration. We found reduced evoked responses in hippocampal CA1 pyramidal neurons following low-frequency stimulation in young (<30 days old) and adult (>60 days old) hamsters, indicating that de novo LTD was generated in hippocampal slices from both pups and adults at temperatures >20°C. However, at temperatures below 20°C, synchronization of neural assemblies (a requirement for LTD generation) was markedly degraded, implying that de novo LTD cannot be generated in hibernating hamsters. Nonetheless, even at temperatures below 16°C, pyramidal neurons could still generate action potentials that may traverse a neural pathway, suppressing the ascending arousal system (ARS). In addition, histamine increased the excitability of these pyramidal cells. Taken together, these findings are consistent with the hypothesis that hippocampal circuits remain operational at low brain temperatures in Syrian hamsters and suppress the ARS to prolong bout duration, even though memory formation is muted at these low temperatures.