Glucagonlike Peptide-1 Receptor Imaging in Individuals with Type 2 Diabetes.

The glucagonlike peptide-1 receptor (GLP1R) is a gut hormone receptor, intricately linked to regulation of blood glucose homeostasis via several mechanisms. It is an established and emergent drug target in metabolic disease. The PET radioligand 68Ga-DO3A-VS-exendin4 (68Ga-exendin4) has the potential to enable longitudinal studies of GLP1R in the human pancreas. Methods:68Ga-exendin4 PET/CT examinations were performed on overweight-to-obese individuals with type 2 diabetes (n = 13) as part of a larger target engagement study (NCT03350191). A scanning protocol was developed to optimize reproducibility (target amount of 0.5 MBq/kg [corresponding to peptide amount of <0.2 µg/kg], blood sampling, and tracer stability assessment). The pancreas and abdominal organs were segmented, and binding was correlated with clinical parameters. Results: Uptake of 68Ga-exendin4 in the pancreas, but not in other abdominal tissues, was high but variable between individuals. There was no evidence of self-blocking of GLP1R by the tracer in this protocol, despite the high potency of exendin4. The results showed that a full dynamic scan can be simplified to a short static scan, potentially increasing throughput and reducing patient discomfort. The 68Ga-exendin4 concentration in the pancreas (i.e., GLP1R density) correlated inversely with the age of the individual and tended to correlate positively with body mass index. However, the total GLP1R content in the pancreas did not. Conclusion: In summary, we present an optimized and simplified 68Ga-exendin4 scanning protocol to enable reproducible imaging of GLP1R in the pancreas. 68Ga-exendin4 PET may enable quantification of longitudinal changes in pancreatic GLP1R during the development of type 2 diabetes, as well as target engagement studies of novel glucagonlike peptide-1 agonists.

Drug Occupancy Assessment at the Glucose-Dependent Insulinotropic Polypeptide Receptor by Positron Emission Tomography.

Targeting of the glucose-dependent insulinotropic polypeptide receptor (GIPR) is an emerging strategy in antidiabetic drug development. The aim of this study was to develop a positron emission tomography (PET) radioligand for the GIPR to enable the assessment of target distribution and drug target engagement in vivo. The GIPR-selective peptide S02-GIP was radiolabeled with 68Ga. The resulting PET tracer [68Ga]S02-GIP-T4 was evaluated for affinity and specificity to human GIPR (huGIPR). The in vivo GIPR binding of [68Ga]S02-GIP-T4 as well as the occupancy of a drug candidate with GIPR activity were assessed in nonhuman primates (NHPs) by PET. [68Ga]S02-GIP-T4 bound with nanomolar affinity and high selectivity to huGIPR in overexpressing cells. In vivo, pancreatic binding in NHPs could be dose-dependently inhibited by coinjection of unlabeled S02-GIP-T4. Finally, subcutaneous pretreatment with a high dose of a drug candidate with GIPR activity led to a decreased pancreatic binding of [68Ga]S02-GIP-T4, corresponding to a GIPR drug occupancy of almost 90%. [68Ga]S02-GIP-T4 demonstrated a safe dosimetric profile, allowing for repeated studies in humans. In conclusion, [68Ga]S02-GIP-T4 is a novel PET biomarker for safe, noninvasive, and quantitative assessment of GIPR target distribution and drug occupancy.

Gut Mucosal Gene Expression and Metabolic Changes After Roux-en-Y Gastric Bypass Surgery.

OBJECTIVE: Changes in the secretion of gut-derived peptide hormones have been associated with the metabolic benefits of Roux-en-Y gastric bypass (RYGB) surgery. In this study, the effects of RYGB on anthropometrics, postprandial plasma hormone responses, and mRNA expression in small intestinal mucosa biopsy specimens before and after RYGB were evaluated. METHODS: In a cross-sectional study, 20 individuals with obesity undergoing RYGB underwent mixed meal tests and upper enteroscopy with retrieval of small intestinal mucosa biopsy specimens 3 months before and after surgery. Concentrations of circulating gut and pancreatic hormones during mixed meal tests as well as full mRNA sequencing of biopsy specimens were evaluated. RESULTS: RYGB-induced improvements of body weight and composition, insulin resistance, and circulating cholesterols were accompanied by significant changes in postprandial plasma responses of pancreatic and gut hormones. Global gene expression analysis of biopsy specimens identified 2,437 differentially expressed genes after RYGB, including changes in genes that encode prohormones and G protein-coupled receptors. CONCLUSIONS: RYGB affects the transcription of a wide range of genes, indicating that the observed beneficial metabolic effects of RYGB may rely on a changed expression of several genes in the gut. RYGB-induced changes in the expression of genes encoding signaling peptides and G protein-coupled receptors may disclose new gut-derived treatment targets against obesity and diabetes.

Assessment of glucagon receptor occupancy by Positron Emission Tomography in non-human primates.

The glucagon receptor (GCGR) is an emerging target in anti-diabetic therapy. Reliable biomarkers for in vivo activity on the GCGR, in the setting of dual glucagon-like peptide 1/glucagon (GLP-1/GCG) receptor agonism, are currently unavailable. Here, we investigated [68Ga]Ga-DO3A-S01-GCG as a biomarker for GCGR occupancy in liver, the tissue with highest GCGR expression, in non-human primates (NHP) by PET. [68Ga]Ga-DO3A-S01-GCG was evaluated by dynamic PET in NHPs by a dose escalation study design, where up to 67 µg/kg DO3A-S01-GCG peptide mass was co-injected. The test-retest reproducibility of [68Ga]Ga-DO3A-S01-GCG binding in liver was evaluated. Furthermore, we investigated the effect of pre-treatment with acylated glucagon agonist 1-GCG on [68Ga]Ga-DO3A-S01-GCG binding in liver. [68Ga]Ga-DO3A-S01-GCG bound to liver in vivo in a dose-dependent manner. Negligible peptide mass effect was observed for DO3A-S01-GCG doses <0.2 µg/kg. In vivo Kd for [68Ga]Ga-DO3A-S01-GCG corresponded to 0.7 µg/kg, which indicates high potency. The test-retest reproducibility for [68Ga]Ga-DO3A-S01-GCG binding in liver was 5.7 ± 7.9%. Pre-treatment with 1-GCG, an acylated glucagon agonist, resulted in a GCGR occupancy of 61.5 ± 9.1% in liver. Predicted human radiation dosimetry would allow for repeated annual [68Ga]Ga-DO3A-S01-GCG PET examinations. In summary, PET radioligand [68Ga]Ga-DO3A-S01-GCG is a quantitative biomarker of in vivo GCGR occupancy.

Investigating Intestinal Glucagon After Roux-en-Y Gastric Bypass Surgery.

CONTEXT: After Roux-en-Y gastric bypass (RYGB) surgery, postprandial plasma glucagon concentrations have been reported to increase. This occurs despite concomitant improved glucose tolerance and increased circulating plasma concentrations of insulin and the glucagon-inhibiting hormone glucagon-like peptide 1 (GLP-1). OBJECTIVE: To investigate whether RYGB-induced hyperglucagonemia may be derived from the gut. DESIGN AND SETTING: Substudy of a prospective cross-sectional study at a university hospital in Copenhagen, Denmark. PARTICIPANTS: Morbidly obese individuals undergoing RYGB (n = 8) with or without type 2 diabetes. INTERVENTIONS: Three months before and after RYGB, participants underwent upper enteroscopy with retrieval of gastrointestinal mucosal biopsy specimens. Mixed-meal tests were performed 1 week and 3 months before and after RYGB. MAIN OUTCOME MEASURES: The 29-amino acid glucagon concentrations in plasma and in mucosal gastrointestinal biopsy specimens were assessed using mass spectrometry-validated immunoassays, and a new monoclonal antibody reacting with immunoreactive glucagon was used for immunohistochemistry. RESULTS: Postprandial plasma concentrations of glucagon after RYGB were increased. Expression of the glucagon gene in the small intestine increased after surgery. Glucagon was identified in the small-intestine biopsy specimens obtained after, but not before, RYGB. Immunohistochemically, mucosal biopsy specimens from the small intestine harbored cells costained for GLP-1 and immunoreactive glucagon. CONCLUSION: Increased concentrations of glucagon were observed in small-intestine biopsy specimens and postprandially in plasma after RYGB. The small intestine harbored cells immunohistochemically costaining for GLP-1 and glucagon-like immunoreactivity after RYGB. Glucagon derived from small-intestine enteroendocrine l cells may contribute to postprandial plasma concentrations of glucagon after RYGB.

A novel dual glucagon-like peptide and glucagon receptor agonist SAR425899: Results of randomized, placebo-controlled first-in-human and first-in-patient trials.

AIMS: To evaluate the safety, pharmacokinetics and pharmacodynamics of SAR425899, a novel polypeptide, active as an agonist at both the glucagon-like peptide-1 receptor (GLP-1R) and the glucagon receptor (GCR), in healthy volunteers and in overweight/obese patients with type 2 diabetes (T2D). METHODS: Subcutaneous administrations of SAR425899 were tested in two randomized, placebo-controlled, double-blind clinical trials. In the first trial, healthy overweight volunteers (body mass index [BMI] 25-30 kg/m2 ; n = 32) received single-ascending doses (0.01-0.1 mg) of SAR425899 or placebo. In the second, a multiple-ascending-dose trial (NCT02411825), healthy normal- to overweight volunteers (BMI 20-30 kg/m2 ; n = 40) and overweight/obese patients with T2D (BMI 28-42 kg/m2 ; n = 36) received daily doses of SAR425899 or placebo over 21 or 28 days, respectively. RESULTS: The most frequently reported adverse events were gastrointestinal; gastrointestinal side effects were less pronounced in patients with T2D compared with healthy volunteers. SAR425899 significantly reduced levels of fasting plasma glucose (P < 0.05 vs. placebo) and glycated haemoglobin (P < 0.001 versus placebo) in patients with T2D. Additionally, SAR425899 led to reductions in body weight, with a maximal reduction of 5.32 kg in healthy volunteers and 5.46 kg in patients with T2D (P < 0.001 vs. placebo) at end of treatment. CONCLUSIONS: SAR425899 was well tolerated and led to favourable glycaemic effects in patients with T2D and weight reduction in both healthy volunteers and patients. Whether dual GLP-1R/GCR agonism represents a treatment method that is superior to pure GLP-1R agonists for obesity and diabetes treatment remains to be confirmed.

Team Players or Opponents: Coadministration of Selective Glucagon and GLP-1 Receptor Agonists in Obese Diabetic Monkeys.

We assessed the therapeutic contribution of the individual components of glucagon-like peptide-1 receptor (GLP-1R) and glucagon receptor (GCGR) agonists alone and in combination upon energy homeostasis and glycemic control in diet-induced obese, diabetic nonhuman primates. The pharmacological active dose ranges of selective agonists were established through a dose-finding study, followed by a 6-week chronic study. Repeated subcutaneous administration of a selective GCGR agonist (30 µg/kg once daily) did not affect food intake or body weight, whereas the selective GLP-1R agonist (3 µg/kg once daily) alone decreased energy intake by 18% and body weight by 3.8% ± 0.9%. Combination of both agonists reduced significantly cumulative food intake by 27% and body weight by 6.6% ± 0.9%. Fasting plasma glucose (FPG) was improved by GLP-1R agonist (baseline vs end of study, 176.7 ± 34.0 vs 115.9 ± 16.1 mg/dL). In contrast, groups exposed to GCGR agonist experienced nonsignificant elevations of FPG. More accurate assessment of therapeutic interventions on glucose homeostasis was tested by an IV glucose tolerance test. Glucose excursion was significantly elevated by chronic GCGR agonist administration, whereas it was significantly decreased in GLP-1R agonist-treated monkeys. In the combination group, a nonsignificant increase of glucose excursion was seen, concomitantly with significantly increased insulin secretion. We conclude that chronic glucagon agonism does not affect energy homeostasis in nonhuman primates. In combination with GLP-1R agonism, glucagon agonism synergistically enhances negative energy balance with resulting larger body weight loss. However, adding GCGR to GLP-1R agonism diminishes glycemic control in diabetic monkeys. Therefore, long-term therapeutic implications of using GLP-1R/GCGR coagonists for weight management in diabetes warrants further scrutiny.

The imprinted gene neuronatin is regulated by metabolic status and associated with obesity.

Using restriction fragment differential display (RFDD) technology, we have identified the imprinted gene neuronatin (Nnat) as a hypothalamic target under the influence of leptin. Nnat mRNA expression is decreased in several key appetite regulatory hypothalamic nuclei in rodents with impaired leptin signaling and during fasting conditions. Furthermore, peripheral administration of leptin to ob/ob mice normalizes hypothalamic Nnat expression. Comparative immunohistochemical analysis of human and rat hypothalami demonstrates that NNAT protein is present in anatomically equivalent nuclei, suggesting human physiological relevance of the gene product(s). A putative role of Nnat in human energy homeostasis is further emphasized by a consistent association between single nucleotide polymorphisms (SNPs) in the human Nnat gene and severe childhood and adult obesity.

[Central fat deposits contribute to atherosclerosis and insulin resistance and peripheral fat deposits protect against atherosclerosis and insulin resistance in postmenopausal women--secondary publication]. / Centralt fedt fremmer og perifert fedt beskytter postmenopausale kvinder mod aterosklerose og insulinresistens--secondary publication.

An analysis of 290 postmenopausal women, subdivided into four distinct body fat distribution groups, showed that peripheral fat mass (PFM) confers insulin-sensitizing and anti-atherogenic effects. PFM contributes to circulating adiponectin, an adipocyte-derived hormone with insulin-sensitizing, anti-inflammatory, and anti-atherogenic effects. In generally obese women, the secretion of adiponectin by PFM provides effective anti-atherogenic protection. In postmenopausal women, body fat distribution has more critical implications for metabolic and cardiovascular risk than overall obesity per se.

Enlarged waist combined with elevated triglycerides is a strong predictor of accelerated atherogenesis and related cardiovascular mortality in postmenopausal women.

BACKGROUND: Upward trends of obesity urge more effective identification of those at cardiovascular risk. A simple dichotomous indicator, enlarged waist (> or =88 cm) combined with elevated triglycerides (> or =1.45 mmol/L) (EWET), was shown to offer advantages in identifying individuals with atherogenic "lipid overaccumulation" compared with other indicators, including the metabolic syndrome defined by the National Cholesterol Education Program (MS-NCEP). Whether EWET offers superior disease and event prediction in postmenopausal women, however, remains unknown. METHODS AND RESULTS: A community-based sample of 557 women (48 to 76 years of age) were followed up for 8.5+/-0.3 years to assess the utility of EWET and MS-NCEP in estimating the risk of all-cause and cardiovascular mortality and the annual progression rate of aortic calcification. At baseline, 15.8% of women had EWET and 17.6% had MS-NCEP. All-cause mortality and cardiovascular mortality were increased in carriers of the dichotomous indicators (P<0.001). After adjustment for age, smoking, and LDL cholesterol, presence of EWET was associated with a 4.7-fold (95% CI, 2.2 to 9.8; P<0.001) increased risk and presence of MS-NCEP was associated with a 3.2-fold (95% CI, 1.5 to 6.5; P<0.001) increased risk for fatal cardiovascular events. Exclusion of women with prevalent diabetes did not change these trends; respective hazard ratios were 4.2 (95% CI, 1.9 to 9.3; P<0.001) and 2.5 (95% CI, 1.1 to 5.5; P<0.05). Among those who were discordant for EWET and MS-NCEP at baseline, those who had EWET alone (n=21) had a higher annual progression rate of aortic calcification compared with those who had MS-NCEP alone (n=31; P<0.05). CONCLUSIONS: The combined presence of EWET may be the best indicator of cardiovascular risk in postmenopausal women. Other components of the MS-NCEP add little medical value to screening in general practices.

Novel associations between bioavailable estradiol and adipokines in elderly women with different phenotypes of obesity: implications for atherogenesis.

BACKGROUND: Peripheral adiposity confers protection against diabetes and atherosclerosis in elderly women. The underlying mechanisms, however, remain to be elucidated. METHODS AND RESULTS: On the basis on dual-energy X-ray absorptiometry measurements of central fat mass (CFM) and peripheral fat mass (PFM), we identified 290 elderly women with distinct forms of body fat distribution (lean, peripheral obesity, central obesity, or general obesity). Study parameters were plasma tumor necrosis factor-alpha, interleukin (IL)-6, adiponectin, estradiol, sex hormone-binding globulin, insulin resistance, and aortic calcification, graded on lateral radiography. In peripherally and generally obese women, plasma estradiol and insulin resistance were significantly lower, whereas sex hormone-binding globulin and adiponectin were significantly higher compared with centrally obese women independent of age, body mass index, total fat mass, and smoking habits (all P<0.05). After adjustment for these confounders, IL-6 in centrally obese women was comparable with that seen in generally obese (similar high CFM%) but significantly higher than in peripherally obese women and lean women (low CFM%). Atherosclerosis was less severe in generally obese (2.5+/-0.3) compared with centrally obese women (5.0+/-0.7, P=0.001). In multiple regression analysis, total fat mass, body fat distribution, insulin resistance, estradiol, current smoking, treated hyperlipidemia, and treated hypertension contributed independently to the variation of aortic calcification (R=0.55, SEE=3.60, P<0.001). CONCLUSIONS: Abundant presence of PFM in generally obese women is associated with increased plasma adiponectin and higher insulin sensitivity, which could explain the apparent protection against the atherogenic effects of IL-6 derived from CFM. Low peripheral exposure to estradiol appears to be a sine qua non of maintained adiponectin secretion from PFM.

Central and peripheral fat mass have contrasting effect on the progression of aortic calcification in postmenopausal women.

AIM: To investigate the long-term effects of central fat mass (CFM) and peripheral fat mass (PFM) on atherogenic risk profile and the progression of aortic calcification (AC) in postmenopausal women. METHODS AND RESULTS: Participants were 316 women aged 50-76 years, who were followed for 7.7 years. CFM and PFM were measured at baseline by DXA and related to follow-up measures of atherogenic metabolites, blood pressure, and the progression of AC assessed on lateral radiographs. CFM and PFM independently of each other exhibited contrasting influence on follow-up measures of atherogenic risk factors and the progression of AC. In a multiple regression model, the negative contribution of PFM (P<0.05), but not the adverse contribution of CFM, was independent of confounders. When comparing different extreme forms of obesity, women with central obesity showed the greatest (2.36+/-0.60, n=11), whereas those with peripheral obesity the smallest changes in AC (0.50+/-0.34, n=10) over the study period. Women with general obesity also tended to show less progression of AC compared with women with central obesity (1.23+/-0.42, n=21). CONCLUSIONS: This study provides direct support for the independent anti-atherogenic influence of PFM and calls on further research to define the adipocyte-derived factors involved in this favourable effect.

Central pre-proglucagon derived peptides: opportunities for treatment of obesity.

Modern societies have moved from famine to feast and obesity and its co-morbidities now sweep the world as a global epidemic. Numerous scientific laboratories and pharmaceutical companies have taken the challenge and are now exploiting novel molecular targets for treatment of obesity. The pre-proglucagon system constitutes interesting candidates as potential targets for new anti-obesity drugs. In the periphery, pre-proglucagon derived peptides, Glucagon-Like Peptide-1 (GLP-1), Glucagon-Like Peptide-2 (GLP-2) and oxyntomodulin (OXM) are involved in a wide variety of physiological functions, including glucose homeostasis, gastric emptying, intestinal growth, insulin secretion as well as the regulation of food intake. Peripheral administration of GLP-1 derivatives and analogues to both rodents and man have shown promising effects on food intake and body weight suggesting that such therapies constitute potential anti-obesity treatment. In the central nervous system, pre-proglucagon and hence GLP-1, GLP-2 and OXM are exclusively found in a small population of nerve cells in the nucleus of the solitary tract. These constitute a neural pathway linking the "viscero-sensory" brainstem to hypothalamic nuclei involved in energy homeostasis. Intracerebroventricular administration of all of the three derived peptides robustly decrease food intake. It is evident that central GLP-1 agonism probably in combination with GLP-2 and/or OXM agonism constitute a potential pharmacological tool to reduce food intake and maybe also enhance energy expenditure. This and other aspects of the current state of the role of central pre-proglucagon in energy homeostasis are reviewed.

Peripheral adiposity exhibits an independent dominant antiatherogenic effect in elderly women.

BACKGROUND: Although several lines of evidence point to an atherogenic role of central fat mass (CFM), few data are available to address the specific role played by peripheral fat mass (PFM). METHODS AND RESULTS: This study was a cross-sectional analysis of 1356 women aged 60 to 85 years. Study variables were physical measures, CFM and PFM measured by DEXA, aortic calcification (AC) graded on lateral radiographs, lipid and glucose metabolites, blood pressure, and information on lifestyle factors and coronary disease. Peripheral fat mass showed independent negative correlation with both atherogenic metabolic risk factors and AC (P<0.001). The most severe insulin resistance-dyslipidemic syndrome and AC (score 5.10+/-0.76) was found in women with high central fat percentage (CF%, 21.7+/-0.2%) and low peripheral fat percentage (PF%, 18.3+/-0.2%, n=48). The least severe AC (score 2.45+/-0.31) was found in obese women with high CF% (21.6+/-0.1%) and high PF% (27.3+/-0.14%, n=112). The insulin resistance-dyslipidemic syndrome was also less severe compared with those with the same CF% but low PF%. The most favorable metabolic profile characterized women with low CF% (11.56+/-0.16%) and high PF% (26.86+/-0.33%, n=44). In women with a history of myocardial infarct (18.41+/-0.55%, n=45), CF% was significantly higher compared with women with no manifest coronary disease (16.48+/-0.12%, n=1210) without differences in PF%. CONCLUSIONS: In elderly women, localization of fat mass is apparently more important for atherosclerosis than obesity per se; although CFM is associated with atherogenic tendencies, PFM seems to exhibit an independent dominant antiatherogenic effect.

Cocaine-amphetamine regulated transcript (CART) expression is not regulated by amphetamine.

Cocaine-amphetamine-regulated transcript (CART) is one of the most abundantly expressed mRNAs in the rat hypothalamus. Nevertheless, CART was identified from striatal extracts as a transcript induced acutely by cocaine or amphetamine treatment. In the hypothalamus, CART mRNA expression has been shown to be regulated by leptin, and CART peptides have been implicated in feeding behavior and in the regulation of the HPA-axis. In the present experiments we have re-examined the effects of amphetamine on CART expression in the forebrain and in the hypothalamus by the use of in situ hybridization. Although we used the same amphetamine treatment paradigm as that originally reported we failed to demonstrate any regulation of CART mRNA levels by amphetamine in either the forebrain or in the hypothalamus. The present results question the acronym chosen for this predominantly hyopthalamic neuropeptide.

Ups and downs for neuropeptides in body weight homeostasis: pharmacological potential of cocaine amphetamine regulated transcript and pre-proglucagon-derived peptides.

Although most humans experience an underlying upwards drift of the body-weight set-point, body weight appears tightly regulated throughout life. The present review describes the structural basis of the adipostat and hypothesise, which components may constitute available targets for pharmacotherapy of excess body weight. Hypothalamic neurones constitute the major components of the body weight homeostasis maintaining device. Together with neurones of the nucleus of the solitary tract, neurones of the hypothalamic arcuate nucleus constitute the sensory components of the adipostat. The arcuate nucleus neurones respond to circulating levels of leptin and insulin, both of which reflect the levels of energy stored as triacylglycerol in adipocytes. The arcuate nucleus projects heavily to the hypothalamic paraventricular nucleus. Neurones of the hypothalamic paraventricular nucleus are hypothesised to constitute, at least partly, the adipostat motor pattern generator, which upon stimulation activates either net anabolic or catabolic physiological responses. The overall sensitivity of the adipostat is influenced by gain setting neurones hypothesised to be located in the dorsomedial hypothalamic nucleus and lateral hypothalamic area. Cocaine amphetamine regulated transcript (CART) peptides and pre-proglucagon derived peptides, glucagon-like peptide-1 (GLP-1) and glucagon-like peptide-2 (GLP-2) are catabolic neurotransmitters synthesised in neurones of the arcuate nucleus and the nucleus of the solitary tract, respectively. The present review summarises the available evidence that both families of peptides constitute endogenous transmitters mediating satiety and touch upon potential pharmacological exploitation of this knowledge.

Light-induced c-Fos expression in suprachiasmatic nuclei neurons targeting the paraventricular nucleus of the hamster hypothalamus: phase dependence and immunochemical identification.

The suprachiasmatic nuclei (SCN) contain a master clock driving the majority of circadian rhythms in mammals. It is believed that the SCN confers circadian rhythmicity as well as light responsiveness to pineal melatonin secretion via a direct projection to the paraventricular nucleus of the hypothalamus (PVN). Neurons in the SCN respond to light during subjective night with an expression of the immediate early gene c-fos. The number and distribution of c-Fos protein-containing neurons depend on the zeitgeber time (ZT) at which the light stimulus is presented. To investigate whether this phase-dependent activity is present in the SCN output neurons targeting the PVN, we combined retrograde cholera toxin subunit B (ChB) tracing from the PVN with c-Fos immunohistochemistry. Male golden hamsters were injected iontophoretically with ChB into the PVN area and 7 days later given a 1.5-hr light stimulus at either ZT 14 or ZT 19 followed by vascular fixation. Light stimulation at ZT 19 gave rise to more c-Fos containing neurons in the SCN than light presented at ZT 14. Double immunostaining for ChB and c-Fos revealed that light stimulation at ZT 14 induced c-Fos expression in 26.6% +/- 2.8% of the retrogradely filled perikarya, whereas light-stimulation at ZT 19 increased this fraction to 40.7% +/- 1.9%. This demonstrates the presence of a phase-dependent c-Fos induction in the suprachiasmatic-paraventricular projection system. Triple immunohistochemistry showed that light-activated output neurons contained both gastrin-releasing peptide and vasoactive intestinal polypeptide and to a lesser extent vasopressin. The present findings provide functional evidence of light activation of central pathways involved in the regulation of circadian output rhythms.