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Department of Neuroscience (B.X., S.P.K.), Department of Physiology (P.S.K.), Interdisciplinary Center for Biotechnology Research (W.G.F.), University of Florida College of Medicine, Gainesville, Florida 32610
Address all correspondence and requests for reprints to: Satya P. Kalra, Ph.D., University of Florida College of Medicine, Department of Neuroscience, 100 South Newell Drive Building 59, Box 100244, Gainesville, Florida 32610-0244. E-mail: skalra{at}ufbi.ufl.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-MSH (MSH), and gene expression of GAL in the ARC and
other sites in the basal hypothalamus, also vary in accordance with
changes in energy balance (2, 19, 20). Several attempts have been made
to analyze the daily changes in gene expression and peptide levels of
each of these orexigenic peptides in the hypothalamus (21, 22, 23, 24);
however, the precise temporal relationship between their daily patterns
and nocturnal feeding in rats maintained on a standard light-dark cycle
and ad libitum food intake is not known. Attempts to analyze the anatomical relationships among orexigenic signals in the hypothalamus document interconnections among neurons producing NPY, GAL, and ß-END. NPY-producing neurons in the ARC synapse with GAL and ß-END containing soma and dendrites and GAL-producing neurons were shown to be synaptically linked with ß-END immunopositive soma and dendrites in the ARC (25, 26, 27). That these anatomical links may be functionally relevant was suggested by the demonstrations that both NPY and GAL-induced feeding were partially suppressed by the opioid-receptor antagonist, nalaxone (28, 29), and that NPY stimulated the release of ß-END and GAL in the hypothalamus (26, 29, 30). Because of this communication among the three orexigenic neuropeptides, we tested the hypothesis that a daily pattern of fluctuations in their gene expression in the medial basal hypothalamus (MBH), which contains NPY, POMC, and GAL-producing neurons (31, 32, 33, 34), may manifest in relation to the robust feeding during the dark phase of the daily light-dark cycle.
Leptin, an adipocyte hormone, has recently been shown to inhibit food intake and is thought to play an important role in regulating the daily pattern of food intake in the rat (35, 36, 37, 38). Interestingly, morphological evidence suggests that the leptin receptor (leptin-R) is localized in hypothalamic neurons that produce NPY, GAL, and POMC (39, 40, 41, 42). Because leptin can modulate NPY gene expression and release (43), as well as GAL and POMC gene expression (40, 41, 44), the emerging view is that leptin restrains feeding, in part, by diminishing the output of these orexigenic signals and/or their actions at target sites (1, 2, 3). Further, whereas blood leptin levels and leptin gene expression in adipocytes show a daily pattern (45, 46, 47), the precise temporal relationship between these patterns and nocturnal feeding is not clear. Similarly, whereas coexpression of leptin-R (48) and NPY, GAL, and POMC in hypothalamic neurons is well documented (39, 40, 41, 42), little information is available on the nature of the dynamic changes in leptin-R gene expression, especially in relation to the daily pattern of leptin secretion. Consequently, in addition to the daily pattern of hypothalamic gene expression of the three neuropeptides, we have also analyzed gene expression of leptin-R in the MBH and leptin in adipocytes as well as blood leptin levels.
Previous studies showed that animals maintained on a food-restricted (FR) regimen (food availability for 4 h from 1100–1500 h) consume approximately 25–30% less and maintain a steady body weight in contrast to rats maintained on a free-feeding (FF) regimen with progressive weight gain (9, 49). In FR rats, NPY levels and release in the paraventricular nucleus (PVN), a site of leptin action (1, 2, 3, 9), are augmented in anticipation of food availability at 1100 h and then as the animals consume food both NPY content and release are diminished. NPY gene expression in the ARC is also augmented at 1100 h but unlike the content and release responses, it remained elevated regardless of food consumption (9, 49). We have extended these findings to evaluate the impact of similar FR on the daily pattern of gene expression of hypothalamic NPY, POMC, GAL, leptin-R, and adipocyte leptin and blood leptin levels.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Rats were divided randomly into two groups: one group was allowed to feed freely (FF) and the other was food restricted (FR), with food available from 1100–1500 h for 4 weeks, as described earlier (49); water was available ad libitum to all rats. Body weights and 24-h food intake were recorded daily in an additional set of FF and FR rats (three rats/group). Groups of FF and FR rats were weighed and killed by decapitation at 4-h intervals through a 24-h period. The autopsy of the animals started 30 min before the designated hour, and all rats in the group were killed within 30 min. The brain was rapidly removed from each rat, and the medial basal hypothalamus was dissected and frozen on dry ice, then stored at -80 C until messenger RNA (mRNA) extraction. The hypothalamic tissue encompasses the neural tissue 1 mm lateral on both sides of midline extending rostral-caudally from optic chiasm to mamillary body recess. In general, the entire arcuate nucleus with minimal surrounding tissue was taken for analysis. Epididymal fat was similarly frozen. Serum from trunk blood was stored at -20 C until analysis of leptin and corticosterone (CORT). Leptin was measured using the rat leptin RIA kit (Linco Research, Inc., St. Charles, MO), and CORT levels were quantitiated by RIA with antiserum (B3–163) purchased from Endocrine Sciences, Inc. (Tarzana, CA). Levels of preproNPY, GAL, POMC, and leptin-R mRNA in the basal hypothalamus and leptin mRNA in epididymal fat were analyzed by ribonuclease protections assays (RPA; 49–55). This experiment was repeated three times to complete various analyses.
Riboprobes. Plasmid containing the rat NPY complementary DNA (cDNA) fragment was kindly provided by Dr. S. L. Sabol (NIH, Bethesda, MD). The pGem2-rat GAL plasmid containing a 585-bp cDNA fragment was generously provided by Dr. J. F. Hyde (University of Kentucky, Lexington, KY). Construction of the plasmid containing rat leptin cDNA has been described earlier (55).
To construct a rat leptin-R plasmid, a 21-mer upper primer (5'-TTG TAA GAG AGG CTG MYR ARA-3') and a 21-mer lower primer (5'-GAT TGG ATT GTG CTG GKT GAC-3') were designed based on mouse leptin-R gene sequence (GenBank Accession No. U42467; 48). RT-PCR of hypothalamic RNA was performed with these primers and the DNA fragment produced was sequenced and matched with the reported rat leptin-R gene sequence (5' position 907 to 3' position 1395, GenBank Accession No. U52966; 56). The leptin-R plasmid was ligated into pCRII vector (Invitrogen, Carlsbad, CA).
Similarly, the rat POMC plasmid was constructed with ligation of a 478-bp cDNA fragment (5' position 220, 3' position 697, GenBank Accession No. J00759; 57), into pGEM-T vector (Promega Corp., Madison, WI). The DNA fragment was prepared by RT-PCR, as described above, using a 20-mer upper (5'-CCC GAG AAA CAG CAG CAG TG-3') and a 20-mer lower (5'-AGG GGG CCT TGG AGT GAG AA-3') primer.
In vitro transcription templates were generated by linearizing with the restriction enzyme, SpeI, and 32P-labeled antisense riboprobes were synthesized using T7 RNA polymerase for use in RPAs.
Ribonuclease protection assays (RPA). Total RNA isolated from hypothalami with RNA-STAT-60 was divided into aliquots for estimation of various mRNAs by RPAs: NPY (6 µg), POMC (10 µg), leptin-R (20 µg), and GAL (20 µg) mRNA. Five micrograms of total RNA extracted from adipocyte tissue were used for analysis of leptin mRNA. A standardized protocol was used for various RPAs (49, 55). Briefly, the antisense probes and RNA samples were hybridized overnight at 45 C, followed by RNase A/T1 digestion for 1 h at 37 C. For GAL mRNA determination, S1 nuclease digestion was performed for 30 min at 37 C after overnight hybridization. Protected RNA fragments were isolated by ethanol precipitation and resolved on a 6% polyacrylamide denaturing sequencing gel. Each gel contained representative samples from each time point for the two groups of rats to account for gel loading variations. The dried gels were quantitated by a PhosphorImager analyzer (Molecular Dynamics, Inc., Sunnyvale, CA). The NPY, GAL, POMC, and leptin-R mRNA values were normalized to cyclophilin mRNA, and leptin mRNA was standardized relative to ß-actin. Antisense riboprobes for cyclophilin and ß-actin were synthesized using templates purchased from Ambion, Inc. (Austin, TX). Values are presented relative to these internal standards that were hybridized simultaneously with each sample.
Statistical analysis
Data are presented as mean ± SE. Daily body
weight changes are presented as percent of initial body weight. Data
for different time points within a group were compared by one-way ANOVA
followed by Newman-Keul’s multiple comparison test and also analyzed
using Bonferroni’s comparison test post hoc. Data for the
same time point between the two groups were compared by Student’s
t test. Level of significance was set at P
< 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), in one set of representative rats, FF rats
steadily gained body weight (Fig. 1A) during the 4 weeks of
observation. Food availability, restricted to 4 h from 1100–1500
h, initially resulted in loss of body weight during the first 2 days,
thereafter, rats slowly regained their initial body weight by day 11
and then gained weight at a steady but slower rate than the FF rats.
Their body weight at the end of 4 weeks was 27% below that of FF rats.
The lower rate of body weight gain in FR rats can be attributed to a
lower average daily food consumption of 23.4 ± 0.9 g
compared with 35.2 ± 0.7 g for FF rats (P <
0.05). Similarly, the FR rats (n = 108) used for various analyses
weighed 23% less than the FF rats (n = 76) at the termination of
the experiment (FR:360 ± 3 g vs. FF:466 ±
3 g).
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and 3). In FF rats, hypothalamic NPY
mRNA fluctuated with peak levels at 0700 h (P <
0.05); these elevated levels were sustained until 1100 h. A
decrease in NPY gene expression to a significantly lower range occurred
at 1500 h during the light phase, and interestingly, this low
level was sustained throughout the dark phase. In FR rats, NPY gene
expression did not vary during the 24-h period (Fig. 3). A comparison
of FF and FR rats showed that the level of NPY gene expression was
similar in the two groups at 0700 h and 1100 h
(P > 0.05); however, the decrease thereafter, as seen
in FF rats, was absent in FR rats.
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and 3). As with NPY, a
daily pattern was also evident in hypothalamic POMC and GAL mRNA in FF
rats (Fig. 2; P < 0.05), which was abolished by FR
(Fig. 3). However, there were two significant differences between the
patterns of NPY and the GAL and POMC gene expression. First, the
significant rise of GAL and POMC mRNAs occurred between 1100 and
1500 h, 4 h later than that in NPY mRNA (Fig. 2). Second,
unlike NPY gene expression, in FR rats GAL and POMC gene expression
were maintained in the range of the lowest levels found in FF rats
(Fig. 3).
Daily fluctuations in serum CORT levels: effects of FR (Fig. 4)
Serum CORT levels in FF rats rose late in the evening at 1900
h, near the onset of dark phase and feeding; significantly high levels
were also apparent later at 2300 h. Thereafter, during the later
hours of dark phase and throughout the light phase levels were
maintained at a basal level. Food availability restricted to 1100–1500
h shifted the daily rise in serum CORT levels. CORT levels rose at
1100 h, preceding the feeding phase, and returned to basal range
at the end of the feeding at 1500 h.
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and 3), leptin mRNA remained unchanged during the lights-on phase
(0700–1500 h levels) but increased significantly at the onset of
lights-off phase (1900 h). These levels remained elevated throughout
the lights-off phase and returned to the lower range of expression
during the lights-on phase. In FR rats (Fig. 3), there were no
significant fluctuations in leptin gene expression during the lights-on
and lights-off phases of the light-dark cycle. A small increase evident
at 1100 h and 1500 h was statistically not significant.
The pattern of serum leptin levels during the 24-h period in FF rats
was somewhat different from that of adipocyte mRNA pattern (Fig. 2).
Serum leptin concentrations rose during the dark phase to peak at
2300 h followed by a slow decrease to the low range maintained
during the lights-on phase. It is apparent that serum leptin levels
were low at 1900 h at the onset of the lights-off phase but
increased to peak levels 4 h later at 2300 h. Thus, adipocyte
mRNA increased significantly before, whereas serum leptin levels
increased after initiation of feeding during the lights-off period. In
FR rats, serum leptin levels were in the low range until 1100 h,
the time of availability of rat chow (Fig. 3). Thereafter, during the
4 h of intense feeding leptin levels rose to a peak at 1500 h
and then declined gradually to the basal range.
Hypothalamic leptin-R mRNA levels also varied during the 24-h period in
FF rats with significantly higher levels at 1900 h when adipocyte
leptin mRNA also rose (Fig. 2). Thereafter, a steady diminution in
leptin-R mRNA to nadir at 0700 h was observed. In FR rats,
however, hypothalamic leptin-R expression displayed no significant
changes during the 24-h period (Fig. 3).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Second, an examination of the temporal relationships in peptide gene expression showed that increments in NPY gene expression occurred 4 h earlier than in GAL and POMC mRNA in our study. Several possible explanations can be advanced. It is likely that the gene expression of each of these neuropeptides is regulated by three independent mechanisms or that the daily pattern in NPY gene expression is regulated by mechanisms independently from that which regulate the GAL and POMC gene expression simultaneously. The increase in NPY gene expression manifesting 4 h before GAL and POMC is suggestive of the NPY system driving the expression of the other two. Morphological evidence that NPY neurons are synaptically linked with GAL and POMC producing neurons in the ARC (25, 26, 27) and stimulation of GAL and ß-END efflux by NPY are consistent with this possibility (28, 29, 30). However, additional studies are warranted to unequivocally demonstrate this putative NPYergic regulation of gene expression in GAL and POMC neurons in the hypothalamus.
Third, serum leptin levels remained low when hypothalamic gene expression of neuropeptides first increased and then decreased to low ranges. Leptin-R mRNA has been localized in NPY, GAL, and POMC producing neurons and administration of leptin modulates gene expression of these neuropeptides (39, 40, 41, 42, 43, 44). Further, diminution in serum leptin levels, as those produced by fasting (46, 52), resulted in up-regulation of NPY gene expression and conversely, administration of leptin suppressed the fasting-induced increase in hypothalamic NPY gene expression (52, 59). Daily administration of leptin intraventricularly decreased GAL and POMC mRNA in the hypothalamus (44). On the other hand, Schwartz et al. (60) reported increases in POMC mRNA in the rostral division of the arcuate nucleus by leptin treatment of fasted male rats. Also, leptin administration restored POMC gene expression in ob/ob mice to the range in wild-type controls (41). These lines of evidence imply that NPY, GAL, and POMC producing neurons are target sites of leptin action (39, 40, 41, 42, 43, 44). Although temporal periodicities do not strictly reflect causal relationships, our observation of both increases and decreases in hypothalamic neuropeptide gene expression in the presence of low leptin levels advocates reassessment of the physiological significance of circulating leptin. In fact, the temporal profiles of circulating leptin levels and fluctuations in neuropeptide gene expression are suggestive of an independent regulatory mechanism controlling the daily periodicities. Furthermore, from the FR study it is evident that shifting food availability to the lights-on phase abolished the daily patterns in neuropeptide gene expression but leptin hypersecretion after initiation of food consumption, was unaffected and, as seen in rats maintained on ad libitum food, peak serum levels occurred 4 h after initiation of feeding in these rats. Consequently, the physiological relevance of the well-known effects of exogenous leptin on neuropeptide gene expression remains to be clarified.
Fourth, a temporal dichotomy in the daily rise in adipocyte leptin gene expression and leptin secretion was evident in FF rats. Leptin gene expression rose at 1900 h, at the onset of nighttime feeding but before the rise in serum leptin levels. Saladin et al. (45) also reported a daily rhythm in leptin mRNA in adipose tissue of mice, with lowest gene expression during the entire lights-on phase, and enhanced gene expression at 4 h (2000 h) into the dark phase (lights on 0400–1600 h) followed by a peak at 0400 h. The leptin secretion pattern lagged behind the pattern in leptin gene expression, with serum leptin attaining nadir 2 h into the dark phase followed by a slow rise to peak at 0400 h. It is apparent that temporal differences in daily periodicities in leptin gene expression and circulating levels exist in rats and mice maintained on different light-dark cycles, but it is obvious that leptin gene expression precedes leptin secretion and, at least in rats, leptin gene expression is augmented before the impending ingestive activity. In contrast, increases in the rate of leptin secretion, as evident both in FF and FR rats, followed feeding behavior, an observation complementing that of Ahima et al. (47). Our observation of the rise in leptin gene expression before release implies that some unidentified neural and/or hormonal mechanisms may augment gene expression independently from those that elicit leptin secretion. The possibility that feeding-induced depletion in rat gastric epithelium may contribute toward the rise in serum leptin has recently been raised (61).
In this context, it is noteworthy that the temporal patterns of gene expression of adipocyte leptin and hypothalamic leptin-R were quite similar in terms of low levels during the lights-on phase and abrupt increase at the onset of ingestive behavior. However, whereas leptin-R gene expression decreased steadily thereafter, leptin gene expression remained elevated throughout the dark phase and decreased only during the lights-on phase. The causal factors responsible for this short-lived increase in hypothalamic leptin-R gene expression concomitant with adipocyte leptin gene expression remains to be ascertained.
Fifth, these results also demonstrated that changing the time of food availability to a 4-h period and limiting it to about 25% below the FF daily intake during this period, produced profound effects on hypothalamic neuropeptide and adipocyte gene expression and leptin secretion. A major finding is that FR abolished the daily patterns of fluctuations in gene expression of all three neuropeptides. Levels of gene expression were maintained either in the high range (NPY) or low range (GAL and POMC). In an earlier study (49), rats maintained on a similar FR regimen exhibited elevated NPY gene expression when examined during the lights-on phase. Thus, the current study extends these findings to show that despite an unchanged light-dark cycle, gene expression of orexigenic peptides is profoundly affected by FR and shift in time of food availability limited to the lights-on period. Whether shifting the FR regimen to other times during the light/dark cycle will produce similar impact is under investigation. The current results indicate that ad libitum food availability may be necessary to generate the daily patterns in gene expression of hypothalamic orexigenic peptides and adipocyte leptin but not leptin secretion.
As reported previously (62, 63), increase in serum CORT levels reflecting augmented hypothalamo-pituitary-adrenal activity, shifted with the time of food availability. Interestingly, serum CORT levels rose at the onset of feeding and decreased to a basal range at the end of 4 h of feeding, thereby affirming a tight relationship with the onset of daily corticosterone hypersecretion and onset of feeding behavior (62). Extending these studies, an analysis of the temporal relationship between CORT and leptin secretion and leptin adipocyte gene expression demonstrate that peak in serum CORT and adipocyte leptin gene expression coincide in FF rats and this coincidence was not apparent in FR rats due to absence of a daily pattern in adipocyte leptin gene expression. However, in FF and FR rats, the serum CORT levels rose before the rise in serum leptin levels. The physiological relevance of the contemporaneous rise in serum CORT and adipocyte leptin gene expression in FF rats, on one hand, and lag period between the rise in serum CORT and leptin levels, on the other, remains to be ascertained.
Finally, with respect to leptin biology, these studies showed also that FR eliminates the daily rise in adipocyte leptin and hypothalamic leptin-R gene expression. In contrast, increased leptin secretion during the period of feeding was unaffected by FR. Clearly, this association with feeding behavior of leptin secretion in FF and FR rats implies that the trigger for increased secretion is closely tied to ingestive behavior, a finding corroborating several reports in rodents (2, 45, 46, 47).
In conclusion, these findings reveal the occurrence of daily increases and decreases in hypothalamic gene expression of orexigenic neuropeptides in conjunction with low circulating leptin levels during the lights-on phase when rats display little feeding. A dichotomy in the daily rise in adipocyte leptin and hypothalamic leptin-R gene expression and serum leptin concentration was also evident, thereby suggesting disparate mechanisms regulating these daily responses. Whereas food availability restricted to 4 h during the lights-on phase abolished the daily pattern of gene expression of hypothalamic orexigenic peptides and leptin-R and adipocyte leptin, leptin secretion was found to be linked to feeding behavior. Thus, for elucidation of mechanisms underlying hypothalamic integration of energy balance under normal and altered conditions of food availability, it may be important to consider the daily patterns of gene expression and availability of hypothalamic orexigenic peptides and their relationship to adipocyte leptin feedback signaling.
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Acknowledgments |
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Footnotes |
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Received September 16, 1998.
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