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The increased prevalence of obesity and its cardiometabolic implications demonstrates the imperative to identify novel therapeutic targets able to effect meaningful metabolic changes in this population. Antibody-mediated targeting of fibroblast growth factor receptor 1c isoform (FGFR1c) has been shown to ameliorate hyperglycemia and protect from diet- and genetically-induced obesity in rodents and nonhuman primates. However, it is currently unknown which tissue(s) contribute to this glucose-lowering effect. Thus, to elucidate this effect, we treated euglycemic mice with H7, a monoclonal antibody that selectively targets FGFR1c, and used whole-body positron emission computed tomography with a glucose tracer (18F-fluorodeoxyglucose). Treatment with H7 increased basal glucose uptake in white adipose tissue (WAT), brown adipose tissue (BAT), the brain, and liver but reduced it in the quadriceps muscles. Consequentially, blood glucose was significantly reduced in response to treatment. Under insulin-stimulated conditions, the effects of H7 were maintained in WAT, BAT, liver, and muscle. Treatment with H7 decreased triglyceride (TG) content and increased adipose TG lipase content in white adipose tissue, while increasing activation of acetyl coenzyme A carboxylase, suggesting futile cycling of TGs, albeit favoring net hydrolysis. We demonstrated, in vitro, this is a direct effect of treatment in adipose tissue, as basal cellular respiration and glucose uptake were increased in response to treatment. Taken together, these data suggest that antibody-mediated targeting of FGFR1c exerts its powerful glucose-lowering efficacy primarily due to increased glucose uptake in adipose tissue., Targeting the FGFR1c isoform reduces blood glucose via increased uptake in adipose tissue. This is a direct effect of treatment and appears to lead to a futile (re)cycling of triglyceride content.

Siberian hamsters display photoperiodically regulated annual cycles in body weight, appetite, and reproduction. Previous studies have revealed a profound up-regulation of type 3 deiodinase (DIO3) mRNA in the ventral ependyma of the hypothalamus associated with hypophagia and weight loss in short-day photoperiods. DIO3 reduces the local availability of T(3), so the aim of this study was to test the hypothesis that decreased hypothalamic T(3) availability underlies the short-day-induced catabolic state. The experimental approach was to determine whether a local increase in T(3) in the hypothalamus of hamsters exposed to short days could reverse the behavioral and physiological changes induced by this photoperiod. In study 1, microimplants releasing T(3) were placed bilaterally into the hypothalamus. This treatment rapidly induced a long-day phenotype including increased appetite and body weight within 3 wk of treatment and increased fat mass and testis size by the end of the 10-wk study period. In study 2, hypothalamic T(3) implants were placed into hamsters carrying abdominal radiotelemetry implants. Again body weight increased significantly, and the occurrence of winter torpor bouts was dramatically decreased to less than one bout per week, whereas sham-implanted hamsters entered torpor up to six times a week. Our findings demonstrate that increased central T(3) induces a long-day metabolic phenotype, but in neither study was the molt cycle affected, so we infer that we had not disrupted the initial detection of photoperiod. We conclude that hypothalamic thyroid hormone availability plays a key role in seasonal regulation of appetite, body weight, and torpor.

The Siberian hamster survives winter by decreasing food intake and catabolizing abdominal fat reserves, resulting in a sustained, profound loss of body weight. VGF gene expression is photoperiodically regulated in the hypothalamus with significantly higher expression in lean Siberian hamsters. The aim of this study was to investigate the role of VGF in regulating these seasonal cycles by determining the effects of a VGF-derived peptide (TLQP-21) on food intake and body weight. Acute intracerebroventricular administration of TLQP-21 decreased food intake, and chronic treatment caused a sustained reduction in food intake and body weight and decreased abdominal fat depots. Behavioral analysis revealed that TLQP-21 reduced meal size but not the frequency of feeding bouts, suggesting a primary action on satiety. Hamsters treated with TLQP-21 lost a similar amount of weight as a pair-fed group in which food intake was matched to that of the TLQP-21-treated group. Central or peripheral treatment with TLQP-21 did not produce a significant effect on resting metabolic rate. We conclude that the primary action of TLQP-21 is to decrease food intake rather than increase energy expenditure. TLQP-21 treatment caused a decrease in UCP-1 mRNA in brown adipose tissue, but hypothalamic expression of orexigenic and anorexigenic neuropeptide genes remained unchanged after TLQP-21 treatment, although compensatory increases in NPY and AgRP mRNA were observed in the pair-fed hamsters. The effects of TLQP-21 administration are similar to those in hamsters in short days, suggesting that increased VGF activity may contribute to the hypophagia that underlies the seasonal catabolic state.

As biologists, we are brought up with the concept that mammals are homeothermic, so they tightly regulate body temperature through a complex balance between thermogenesis and heat conservation and dissipation mechanisms. One of the remarkable features of the natural world is that many mammals can also show torpor or hibernation. They can change the parameters of this homeostatic process, and either undergo daily (torpor) or extended periods (hibernation) when body temperature falls towards ambient environmental values. Homeostasis, therefore, becomes rheostasis (1), yet neural and peripheral tissues retain their functional integrity despite marked hypoxia and hypoglcemia. We do not understand fully how this profound change in physiology comes about, but in the current issue of Endocrinology, evidence is provided that up-regulation of thioredoxin-interacting protein (Txnip) in the ependymal cell layer of the hypothalamus is associated with this process (2). Previous studies have suggested that Txnip expressed in the mediobasal hypothalamus plays a role in nutrient sensing (3) and may specifically regulate leptin sensitivity in neuropeptide Y/agouti-related peptide neurons (4). Intriguingly, the current study indicates that enhanced expression is likely to be occurring within tanycyte cells (Figure 1). This is a very timely observation, because there is a growing interest in the role of these cells both in nutrient sensing (5) and as regulators of hypothalamic neurogenesis (6–8) and plasticity (9, 10) that may underlie long-term changes in appetite and energy expenditure. Torpor can occur for a number of reasons in small mammals. Commonly, it is an adaptation to survive winter in temperate or arctic environments, but it can also occur as a response to severely limited food supply or caloric restriction, or to acute cold exposure. Hand et al (2) initially found up-regulation of Txnip in a transgenic mouse lacking the G-protein coupled receptor (GPR50) orphan receptor, which readily enters torpor in response to mild fasting, perhaps reflecting a deficit in hypothalamic nutrient sensing (11). They then found that up-regulation of hypothalamic Txnip occurred in all the various natural types of torpor: seasonal torpor in the Siberian hamster, Phodopus sungorus, torpor in wild-type mice during prolonged fasting and cold exposure, and also in a pharmacological model where mice were treated with 2-deoxyglucose to inhibit glycolysis. Up-regulation of Txnip, therefore, appears to be a universal feature of torpor, although whether it is causal in generating the hypothermic state or is a downstream consequence of torpor has not yet been resolved. The authors note that transgenic mice lacking Txnip fail to enter torpor when fasted for 24 hours, supporting a causal role, and they suggest that its role is to reduce whole-body energy expenditure in order to sustain the hypothermic and hypometabolic state. This concept is supported by their observation that Txnip expression is also elevated in some peripheral tissues during torpor, in particular in brown adipose tissue. Most tellingly, the authors note previous studies showing that transgenic mice lacking Txnip die during a prolonged fast (12), implying that Txnip has a protective function during the extreme hypometabolic state experienced during torpor, which is critical for survival of periods of drastically reduced food availability. Perhaps the most interesting aspect of the current study is that it refocuses attention on tanycytes as a key anatomical substrate for the regulation of energy metabolism. The authors themselves are circumspect as to whether the Txnip gene expression in the ependymal cell layer as identified by in situ hybridization is actually in tanycyte cells.

Seasonal Siberian hamsters lose fat reserves, decrease body weight and leptin concentrations, and suppress reproduction on short-day photoperiod (SD). Chronic leptin infusion at physiological doses caused body weight and fat loss in SD animals but was ineffective in long-day (LD) hamsters. Using ovariectomized estrogen-treated females, we tested the hypothesis that responsiveness to leptin is regulated by photoperiod. On SD, hypothalamic neuropeptide Y, agouti-related peptide, and cocaine- and amphetamine-regulated transcript gene expression in the arcuate nucleus did not exhibit significant changes, and despite SD-induced fat loss, the catabolic peptide proopiomelanocortin was down-regulated. Food restriction of LD-housed animals caused significant reduction of fat reserves and serum leptin concentrations to SD levels, suppressed serum gonadotropins, and induced increased anabolic (neuropeptide Y, agouti-related peptide) and decreased catabolic (proopiomelanocortin, cocaine- and amphetamine-regulated transcript) gene expression in the arcuate nucleus. Leptin infusion in food-restricted animals had no effect on fat reserves or gonadotropins and did not modulate neuropeptide gene expression. Also, leptin treatment did not blunt the refeeding responses or weight and fat gain in LD-housed food-restricted animals. In conclusion, our results strongly suggest that hypothalamic responses to leptin are regulated primarily by photoperiod, rather than seasonal changes in fat reserves, sex steroids, or leptin concentrations.

Leptin may play a role in appetite regulation and metabolism, but its reproductive role is less clear. In photoperiodic Siberian hamsters, seasonal changes in fatness, leptin gene expression, and metabolism occur synchronously with activation or suppression of reproduction, analogous to puberty. Here, we test the hypothesis that seasonal changes in leptin secretion mediate the photoperiodic regulation of reproduction. Mature male and ovariectomized estrogen-treated female Siberian hamsters were kept in long (LD; 16 h of light, 8 h of darkness) or short days (SD; 8 h of light, 16 h of darkness) for 8 weeks, and recombinant murine leptin (15 μg/day) was infused for 2 weeks via osmotic minipumps. SD hamsters exhibited significant weight and fat losses, reduced serum leptin and food intake, and suppressed pituitary LH concentration. Leptin did not suppress food intake over the 2-week treatment on either photoperiod, but significantly reduced fat reserves in SD hamsters. Leptin had no significant effect on pit...

The distribution of immunoreactive (IR) estrogen receptor (ER)-containing cells was studied in the brains of adult Suffolk ewes using a rat monoclonal antibody (H222) which recognizes the human estrogen receptor. IR cells were characterized by dense nuclear reaction product, and in some instances, cytoplasmic immunostaining which filled dendrite-like processes. The greatest densities of ER-IR cells were found in the medial preoptic area, the mediobasal hypothalamus, and in a number of limbic system structures (amygdala, bed nucleus of the stria terminalis, lateral septum). ER-IR cells were found at lower densities in several other subregions of the hypothalamus and limbic system, and in the periaqueductal gray of the caudal midbrain. Cytoplasmic ER immunoreactivity was most prominent among ER-IR cells in the ventrolateral-ventromedial nucleus, the bed nucleus of the stria terminalis, the midbrain periaqueductal gray, and some ER-IR cells in the substantia innominata. The distribution of ER-containing cells in the sheep brain closely parallels that seen in other mammals. ER-IR cells are found in sites such as the medial preoptic area and ventrolateral-ventromedial hypothalamus which have been implicated as targets in this species and others for the influence of estradiol on sexual behavior and reproductive neuroendocrine function.

Puberty in the female lamb is accompanied by an increased frequency of LH pulses, and during normal development this is preceded by a decline in GH. Conversely, in the growth-retarded lamb, when LH levels are depressed by low nutrition, GH secretion is elevated. Based upon this inverse relationship, we tested the hypothesis that GH may act as a metabolic signal from the brain to inhibit the secretion of LH, and that the decline in GH times puberty. Our approach was to extend high circulating GH levels far beyond the early postnatal period, in a physiological pattern and level, in an attempt to block the pubertal LH rise. To evaluate the pattern of LH as a continuous variable under conditions of constant estradiol negative feedback, the gonadotropin was measured in blood samples collected by jugular venipuncture twice weekly; the lambs were ovariectomized and treated chronically with estradiol (Silastic capsule) beginning at 3 weeks of age. Nine lambs served as untreated controls, and 7 were infused iv with pituitary-derived bovine GH (bGH) between 5 and 28 weeks of age. A programmable backpack infusion pump delivered bGH as hourly pulses, with a total dose of 18 micrograms/kg.24 h, to maintain a physiological pattern and level of GH. At various ages, blood samples were collected at 12-min intervals for 6 h to monitor patterns and levels of peripheral LH and GH. Circulating GH in untreated and treated lambs averaged 7.7 +/- 1.5 ng/ml over a 6-h period at 4 weeks of age and declined to 1.1 +/- 0.2 ng/ml by 19 weeks in the untreated lambs; in contrast, bGH-infused lambs averaged 10.4 +/- 0.9 ng/ml at 19 weeks. Although body weights did not differ, back fat depth and quantity of perirenal fat were reduced in bGH-treated females compared to that in controls. Moreover, insulin-like growth factor-I levels were higher in bGH-treated compared with control lambs, and the bGH-treated lambs exhibited glucose intolerance, thus confirming that infused bGH was biologically active. Neuroendocrine sexual maturity, however, was not different in bGH-treated and control lambs, and it occurred at 21-22 weeks of age. The results do not support our hypothesis that decreasing GH secretion is a requirement for puberty in the sheep. Moreover, unlike in children with delayed puberty, exogenous bGH did not advance normal puberty in the lamb.(ABSTRACT TRUNCATED AT 400 WORDS)

Growth retardation induced by dietary restriction in the lamb results in a low frequency of episodic LH secretion and, thus, delayed puberty. Such lambs respond normally to physiological doses of GnRH, indicating that the pituitary gland can function adequately during diet-induced hypogonadotropism. The current studies investigated central mechanisms underlying diet-induced hypogonadotropism. The first aim was to determine whether the hypothalamic GnRH secretory system is capable of normal function. The initial approach was to compare hypothalamic GnRH content between lambs on a restricted diet with low LH pulse frequency (less than 1 pulse/4 h; n = 5) and lambs on an ad libitum diet with high LH pulse frequency (4.5 +/- 0.4 pulses/4 h; n = 5). RIA of extracts of preoptic area and mediobasal hypothalamus/median eminence tissue blocks revealed no differences in GnRH content between lambs on a restricted diet and those on an ad libitum diet. The second approach was to determine if LH secretion could be induced by chemical stimulation of neuronal function with N-methyl-D,L-aspartate (NMA), an excitatory amino acid agonist. Initially, a single iv bolus of NMA was given to hypogonadotropic lambs on a restricted diet. There was a dose-dependent immediate rise in serum LH concentrations. All lambs responded to the highest dose (5.0 mg/kg BW; n = 6), and four of five lambs responded to the intermediate dose (1.0 mg/kg). No lambs responded to the lowest dose (0.2 ng/kg), despite a normal response to GnRH (2.5 ng/kg BW, iv). In a second experiment, hypogonadotropic lambs on a restricted diet were treated with repeated injections of NMA (5 mg/kg BW, iv) at either hourly intervals (n = 6) or every 3 h (n = 6). Each NMA injection induced a LH pulse in both treatment regimens over the entire 7-h experimental period. Thus, the nutritionally growth-limited lamb is capable of sustained production of LH pulses, which, we presume, reflect GnRH secretion. The second aim was to test the hypothesis that endogenous opioid mechanisms inhibit LH secretion during nutritionally induced hypogonadotropism, because opioid pathways are a poor inhibitory regulator of LH secretion in the normally developing sheep, even in the absence of ovarian steroids. We were unable to detect any effects of the opiate antagonist naloxone on LH secretion in the nutritionally growth-limited lamb. We conclude that central mechanisms controlling the release, rather than synthesis, of GnRH are limiting LH secretion when sexual maturation is delayed by growth retardation. Moreover, opioid inhibition is not the primary reason for hypogonadotropism during dietary restriction.

Previous studies show that limited nutrition in the ovariectomized lamb results in an impairment of LH and FSH secretion, a phenomenon that is rapidly reversible by increasing the level of nutrition and independent of ovarian steroid feedback. The present study characterizes the biosynthesis of pituitary hormones in the nutritionally growth-limited female lamb by measuring steady state mRNA concentrations. These changes were examined relatively to pituitary and serum hormone concentrations to establish the relationship(s) of synthesis and secretion in response to nutritional manipulation. Ad libitum feeding of nutritionally growth-restricted ovariectomized lambs for 14 days resulted in an increase in the frequency of episodic LH release, thereby increasing mean serum LH concentrations (P less than 0.001). Similarly, mean circulating concentrations of FSH were increased (P less than 0.001). By contrast, serum GH concentrations were lowered significantly as a result of ad libitum feeding (P less than 0.05). Serum PRL concentrations remained unchanged. Although pituitary LH and PRL concentrations were also unchanged in response to increased nutrition feeding, FSH and GH concentrations increased (P less than 0.05). Short-term ad libitum feeding of the chronically food-restricted lamb resulted in significant changes in mRNA concentrations for all hormones except PRL. The concentrations of gonadotropin subunit mRNAs (i.e. alpha, LH beta, and FSH beta) were all significantly higher in response to increased nutrition (P less than 0.001). GH mRNA was also affected; however, feeding decreased concentrations (P less than 0.001). The results demonstrate that an increase in the level of nutrition in the restricted diet lamb produces profound changes in the synthesis, storage, and secretion of LH, FSH, and GH. These changes appear to be coordinated, although the response differs depending upon the hormone.

The nutritionally growth-retarded ovariectomized lamb provides a model system to investigate the regulation of immunoreactive and bioactive FSH release in the absence of feedback effects of ovarian products. We examined the acute effects of nutritional repletion on FSH release in this model. Five March-born lambs were weaned at 7 weeks of age and placed on a limited diet. All were ovariectomized at 31 weeks. At 37 weeks, they were fed ad libitum for 14 days. Blood samples were collected at 12 min intervals for 4 h on day -1 (restricted) and days 2, 7, and 14 of ad libitum feeding, and serum FSH concentrations were measured by both RIA and a Sertoli cell aromatase in vitro bioassay. Mean concentrations of serum immunoreactive FSH (I-FSH) and the bioactive FSH (B-FSH) increased 2- and 3-fold, respectively, after ad libitum feeding for 14 days. Pulse analyses by two objective computerized programs, DETECT and CLUSTER, revealed the presence of FSH pulses (predominantly B-FSH) during the restricted phase, as well as ad libitum fed phases, at times where I-LH pulses were not detected. In contrast to the increases in I-LH pulse frequency during ad libitum fed states, FSH pulse frequency remained stable whereas pulse amplitude increased. In addition, the overall B-FSH pulse amplitudes were 57 +/- 7% (mean +/- SE of three different pulse analyses) greater than I-FSH pulse amplitudes, implying preferential enrichment of FSH bioactivity. The results suggest that the mechanisms governing both the quantitative and qualitative changes in circulating FSH concentrations are extremely sensitive to changes in level of nutrition. The asynchronous occurrence of FSH pulses in the absence of I-LH pulses suggests that FSH may be more sensitive to small changes in GnRH secretion than I-LH, or that a hypothalamic releasing factor specific for FSH may be released.

This study tested the hypothesis that in the female sheep, a short day (SD) breeder, puberty can occur normally in the absence of ambient short days. More specifically, the photoperiod cue timing the transition into adulthood is exposure to and then termination of a long day melatonin rhythm. Control lambs born in the spring were exposed to 5 weeks of long days (LD; 16 h of light, 8 h of darkness; 18-23 weeks of age) and were raised in SD (8 h of light, 16 h of darkness) at other times. As expected from previous studies, this alternating photoperiod sequence (SD-LD-SD) induced puberty at the normal age in autumn [33 +/- 2 weeks (mean +/- SEM); n = 6]. The other three groups were exposed only to LD from birth; the superior cervical ganglia were removed bilaterally at different ages to denervate the pineal gland in order to block transduction of subsequent LD cues. Puberty occurred normally (31 +/- 1 weeks; n = 7) after ganglionectomy at 23 weeks of age, indicating that ambient short days are not required to initiate reproductive cycles. LD are necessary, as evidenced by the results for the other two groups ganglionectomized neonatally at 4 weeks of age. With no further treatment, puberty was either delayed (n = 1) or did not occur during the first year of life (n = 5), after which the study ended. This delay was prevented in the other group of ganglionectomized lambs by a 5-week (18-23 weeks of age) exposure to LD melatonin patterns by means of 8-h melatonin infusions nightly; 12 weeks after melatonin replacement therapy, puberty occurred at the normal time (34 +/- 1 weeks; n = 6). The inference is that for puberty to occur in the female lamb the animal must be exposed to relatively limited periods of LD, followed by the blockade or absence of further LD cues (pineal denervation, termination of LD melatonin infusion, or presence of SD). This supports the concept that the LD of summer, followed by their disappearance in autumn, time puberty in the female sheep.