Correction: Boiten et al. Pathologically Decreased CSF Levels of Synaptic Marker NPTX2 in DLB Are Correlated with Levels of Alpha-Synuclein and VGF. Cells 2021, 10, 38.

The authors want to notify that they have made the following changes to the author list of the paper [...].

TLQP-21 changes in response to a glucose load.

BACKGROUND: The TLQP-21 peptide potentiates glucose-stimulated insulin secretion, hence we investigated its endogenous response to glucose. METHODS: Fasted mice received intraperitoneal glucose (3 g/kg), or saline (controls), and were sacrificed 30 and 120 min later (4 groups, n = 6/group). We investigated TLQP-21 in pancreas and plasma using immunohistochemistry, enzyme-linked immunosorbent assay (ELISA) and high performance liquid chromatography (HPLC), as well as TLQP-21 receptors (gC1q-R and C3a-R1) expression in pancreas by immunohistochemistry. RESULTS: In pancreas, TLQP-immunoreactivity (TLQP-ir.) was shown in insulin-, glucagon- and somatostatin-containing cells. Upon glucose, TLQP-ir. decreased at 30 min (∼40 % vs. controls), while returning to basal values at 120 min. In all groups, C3a-R1 was localized in ∼50 % of TLQP labelled islet cells (mostly central), while gC1q-R was detected in ∼25 % of TLQP cells (mainly peripheral). HPLC fractions of control pancreas extracts, assessed by ELISA, confirmed the presence of a TLQP-21 compatible-form (∼2.5 kDa MW). In plasma, TLQP-ir. increased at 30 min (∼30 %), with highest concentrations at 120 min (both: p<0.05 vs. controls), while HPLC fractions showed an increase in the TLQP-21 compatible form. CONCLUSIONS: Upon hyperglycaemia, TLQP-21 would be released from islets, to enhance insulin secretion but we cannot exclude an autocrine activity which may regulate insulin storage/secretion.

Dynamic of TLQP-peptides upon fasting.

BACKGROUND: The VGF-derived TLQP peptides (TLQPp), a new potential drug target for obesity, are expressed in stomach, pancreas, adrenal gland as well as in adipose tissues, and, when exogenously injected, regulate energy expenditure and food intake. However, it is not clear if these peptides physiologically change in these organs in response to fasting. METHODS: Rats were subdivided into four groups: (A) fed ad libitum, (B) fed with restrictions (once a day) (C) fast for 48 h and (D) fast for 48 h and then fed 1 h before sacrifice. Immunosorbent assay was used to possibly reveal TLQPp changes upon fasting in plasma as well as in pancreas, adrenal gland, stomach and adipose tissues. In the latter organs, we also measured the levels of the VGF precursor protein while immunohistochemistry was used to investigate the presence of the TLQP-21 receptors. RESULTS: During fasting, TLQPp were down-regulated in the stomach (45 %), pancreas (47 %), adrenal gland (51 %) and WAT (45.2 %) in parallel with a significant increase in the blood (36.6 %), all versus ad libitum group. In the same organs where the TLQPp were decreased upon fasting, the VGF precursor levels were not changed. In ad libitum rats, TLQP-21 receptors were well represented within the same cells that expressed TLQPp, suggesting an autocrine activity to be better investigated. CONCLUSIONS: During fasting, TLQPp are probably produced and immediately secreted into the blood circulation, until the hypoglycaemia is counteracted.

Identification of novel cerebrospinal fluid biomarker candidates for dementia with Lewy bodies: a proteomic approach.

BACKGROUND: Diagnosis of dementia with Lewy bodies (DLB) is challenging, largely due to a lack of diagnostic tools. Cerebrospinal fluid (CSF) biomarkers have been proven useful in Alzheimer's disease (AD) diagnosis. Here, we aimed to identify novel CSF biomarkers for DLB using a high-throughput proteomic approach. METHODS: We applied liquid chromatography/tandem mass spectrometry with label-free quantification to identify biomarker candidates to individual CSF samples from a well-characterized cohort comprising patients with DLB (n = 20) and controls (n = 20). Validation was performed using (1) the identical proteomic workflow in an independent cohort (n = 30), (2) proteomic data from patients with related neurodegenerative diseases (n = 149) and (3) orthogonal techniques in an extended cohort consisting of DLB patients and controls (n = 76). Additionally, we utilized random forest analysis to identify the subset of candidate markers that best distinguished DLB from all other groups. RESULTS: In total, we identified 1995 proteins. In the discovery cohort, 69 proteins were differentially expressed in DLB compared to controls (p < 0.05). Independent cohort replication confirmed VGF, SCG2, NPTX2, NPTXR, PDYN and PCSK1N as candidate biomarkers for DLB. The downregulation of the candidate biomarkers was somewhat more pronounced in DLB in comparison with related neurodegenerative diseases. Using random forest analysis, we identified a panel of VGF, SCG2 and PDYN to best differentiate between DLB and other clinical groups (accuracy: 0.82 (95%CI: 0.75-0.89)). Moreover, we confirmed the decrease of VGF and NPTX2 in DLB by ELISA and SRM methods. Low CSF levels of all biomarker candidates, except PCSK1N, were associated with more pronounced cognitive decline (0.37 < r < 0.56, all p < 0.01). CONCLUSION: We identified and validated six novel CSF biomarkers for DLB. These biomarkers, particularly when used as a panel, show promise to improve diagnostic accuracy and strengthen the importance of synaptic dysfunction in the pathophysiology of DLB.

Pathologically Decreased CSF Levels of Synaptic Marker NPTX2 in DLB Are Correlated with Levels of Alpha-Synuclein and VGF.

Background: Dementia with Lewy bodies (DLB) is a neurodegenerative disease where synaptic loss and reduced synaptic integrity are important neuropathological substrates. Neuronal Pentraxin 2(NPTX2) is a synaptic protein that drives the GABAergic inhibitory circuit. Our aim was to examine if NPTX2 cerebral spinal fluid (CSF) levels in DLB patients were altered and how these levels related to other synaptic protein levels and to cognitive function and decline. Methods: NPTX2, VGF, and α-synuclein levels were determined in CSF of cognitive healthy (n = 27), DLB (n = 48), and AD (n = 20) subjects. Multiple cognitive domains were tested, and data were compared using linear models. Results: Decreased NPTX2 levels were observed in DLB (median = 474) and AD (median = 453) compared to cognitive healthy subjects (median = 773). Strong correlations between NPTX2, VGF, and α-synuclein were observed dependent on diagnosis. Combined, these markers had a high differentiating power between DLB and cognitive healthy subjects (AUC = 0.944). Clinically, NPTX2 levels related to global cognitive function and cognitive decline in the visual spatial domain. Conclusion: NPTX2 CSF levels were reduced in DLB and closely correlated to decreased VGF and α-synuclein CSF levels. CSF NPTX2 levels in DLB related to decreased functioning in the visual spatial domain.

VGF peptides as novel biomarkers in Parkinson's disease.

Parkinson's disease (PD) is characterized by a progressive degeneration of dopaminergic neurons in the substantia nigra (SN). At disease onset, a diagnosis is often difficult. VGF peptides are abundant in the SN and peripheral circulation; hence, we investigate whether their plasma profile may reflect the brain dopamine reduction. Using antibodies against the VGF C-terminal portion, we analyzed the rat brain and human plasma, with immunohistochemistry and ELISA. Rats were unilaterally lesioned with 6-hyroxydopamine and sacrificed either 3 or 6 weeks later with or without levodopa treatment. Plasma samples were obtained from PD patients, either at the time of diagnosis (group 1, drug naïve, n = 23) or upon dopamine replacement (group 2, 1-6 years, n = 24; group 3, > 6 years, n = 16), compared with age-matched control subjects (group 4, n = 21). Assessment of the olfactory function was carried out in group 2 using the "Sniffin' Sticks" test. VGF immunoreactivity was present in GABAergic neurons and, on the lesioned side, it was reduced at 3 weeks and abolished at 6 weeks after lesion. Conversely, upon levopoda, VGF labeling was restored. In PD patients, VGF levels were reduced at the time of diagnosis (1504 ± 587 vs. 643 ± 348 pmol/mL, means ± S.E.M: control vs. naïve; p < 0.05) but were comparable with the controls after long-term drug treatment (> 6 years). A linear correlation was demonstrated between VGF immunoreactivity and disease duration, levodopa equivalent dose and olfactory dysfunction. Plasma VGF levels may represent a useful biomarker, especially in the early stages of PD.

VGF Peptides in Cerebrospinal Fluid of Patients with Dementia with Lewy Bodies.

In a previous proteomic study, we identified the neurosecretory protein VGF (VGF) as a potential biomarker for dementia with Lewy bodies (DLB). Here, we extended the study of VGF by comparing levels in cerebrospinal fluid (CSF) from 44 DLB patients, 20 Alzheimer's disease (AD) patients, and 22 cognitively normal controls selected from the Amsterdam Dementia Cohort. CSF was analyzed using two orthogonal analytical methods: (1) In-house-developed quantitative ELISA and (2) selected reaction monitoring (SRM). We further addressed associations of VGF with other CSF biomarkers and cognition. VGF levels were lower in CSF from patients with DLB compared to either AD patients or controls. VGF was positively correlated with CSF tau and α-synuclein (0.55 < r < 0.75), but not with Aß1-42. In DLB patients, low VGF levels were related to a more advanced cognitive decline at time of first presentation, whereas high levels of VGF were associated with steeper subsequent longitudinal cognitive decline. Hence, CSF VGF levels were lower in DLB compared to both AD and controls across different analytical methods. The strong associations with cognitive decline further points out VGF as a possible disease stage or prognostic marker for DLB.

Photoperiodic changes in adiposity increase sensitivity of female Siberian hamsters to systemic VGF derived peptide TLQP-21.

TLQP-21, a peptide encoded by the highly conserved vgf gene, is expressed in neuroendocrine cells and has been the most prominent VGF-derived peptide studied in relation to control of energy balance. The recent discovery that TLQP-21 is the natural agonist for the complement 3a receptor 1 (C3aR1) has revived interest in this peptide as a potential drug target for obesity. We have investigated its function in Siberian hamsters (Phodopus sungorus), a rodent that displays natural seasonal changes in body weight and adiposity as an adaptation to survive winter. We have previously shown that intracerebroventricular administration of TLQP-21 reduced food intake and body weight in hamsters in their long-day fat state. The aim of our current study was to determine the systemic actions of TLQP-21 on food intake, energy expenditure and body weight, and to establish whether adiposity affected these responses. Peripheral infusion of TLQP-21 (1mg/kg/day for 7 days) in lean hamsters exposed to short photoperiods (SP) reduced cumulative food intake in the home cage (p<0.05), and intake when measured in metabolic cages (P<0.01). Energy expenditure was significantly increased (p<0.001) by TLQP-21 infusion, this was associated with a significant increase in uncoupling protein 1 mRNA in brown adipose tissue (BAT) (p<0.05), and body weight was significantly reduced (p<0.05). These effects of systemic TLQP-21 treatment were not observed in hamsters exposed to long photoperiod (LP) with a fat phenotype. C3aR1 mRNA and protein were abundantly expressed in the hypothalamus, brown and white adipose tissue in hamsters, but changes in expression cannot explain the differential response to TLQP-21 in lean and fat hamsters.

Reduction of Total Brain and Cerebellum Volumes Associated With Neuronal Autoantibodies in Patients With APECED.

Context: In autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), autoantibodies (AutoAbs) labeling brain neurons were reported; conversely, brain MRI alterations associated with these AutoAbs were never reported. Objectives: To describe brain alterations in APECED and to correlate them with AutoAbs against glutamic acid decarboxylase (GAD), tyrosine hydroxylase (TH), and 5-tryptophan hydroxylase (5-HT) neurons. Design and Participants: Fourteen Sardinian patients with APECED and age-matched control subjects were recruited for MRI analysis and blood sampling to detect AutoAbs to GAD, TH, and 5-HT neurons by using rat brain sections. The majority of patients (n = 12) were investigated for AutoAbs a decade earlier, and 7 of 12 were positive for AutoAbs to GAD and TH neurons. Main Outcomes: Patients with APECED had smaller cerebellum and gray matter volumes, with a ventricular enlargement and a total cerebrospinal fluid (CSF) increase, compared with controls (P < 0.01). In 11 of 14 patients, brain abnormalities were associated with AutoAbs to GAD or TH neurons (titer 1:100 to 15,000) that had persisted for 10 years in 7 of 11 patients. AutoAbs to 5-HT neurons were revealed in all patients with AutoAbs to TH neurons. A decrease in whole brain and cerebellum volumes (P = 0.028) was associated with AutoAbs to GAD neurons, and a CSF increase was associated with AutoAbs to GAD and TH/5-HT neurons (P < 0.05). HLA alleles did not appear to be involved in neuronal autoimmunity. Conclusions: Brain alterations and neuronal AutoAbs were observed in 78.6% of Sardinian patients with APECED, suggesting a brain autoimmune reaction. Prolonged clinical follow-up must be conducted for the possible appearance of clinical neurologic consequences.

TLQP Peptides in Amyotrophic Lateral Sclerosis: Possible Blood Biomarkers with a Neuroprotective Role.

While the VGF-derived TLQP peptides have been shown to prevent neuronal apoptosis, and to act on synaptic strengthening, their involvement in Amyotrophic Lateral Sclerosis (ALS) remains unclarified. We studied human ALS patients' plasma (taken at early to late disease stages) and primary fibroblast cultures (patients vs controls), in parallel with SOD1-G93A transgenic mice (taken at pre-, early- and late symptomatic stages) and the mouse motor neuron cell line (NSC-34) treated with Sodium Arsenite (SA) to induce oxidative stress. TLQP peptides were measured by enzyme-linked immunosorbent assay, in parallel with gel chromatography characterization, while their localization was studied by immunohistochemistry. In controls, TLQP peptides, including forms compatible with TLQP-21 and 62, were revealed in plasma and spinal cord motor neurons, as well as in fibroblasts and NSC-34 cells. TLQP peptides were reduced in ALS patients' plasma starting in the early disease stage (14% of controls) and remaining so at the late stage (16% of controls). In mice, a comparable pattern of reduction was shown (vs wild type), in both plasma and spinal cord already in the pre-symptomatic phase (about 26% and 70%, respectively). Similarly, the levels of TLQP peptides were reduced in ALS fibroblasts (31% of controls) and in the NSC-34 treated with Sodium Arsenite (53% of decrease), however, the exogeneous TLQP-21 improved cell viability (SA-treated cells with TLQP-21, vs SA-treated cells only: about 83% vs. 75%). Hence, TLQP peptides, reduced upon oxidative stress, are suggested as blood biomarkers, while TLQP-21 exerts a neuroprotective activity.

The Hypothalamic-Pituitary Axis and Autoantibody Related Disorders.

This review summarized different studies reporting the presence of autoantibodies reacting against cells of the pituitary (APAs) and/or hypothalamus (AHAs). Both APAs and AHAs have been revealed through immunofluorescence using different kinds of substrates. Autoantibodies against gonadotropic cells were mainly found in patients affected by cryptorchidism and hypogonadotropic hypogonadism while those against prolactin cells were found in different kinds of patients, the majority without pituitary abnormalities. APAs to growth hormone (GH) cells have been associated with GH deficiency while those against the adrenocorticotropic cells have distinguished central Cushing's disease patients at risk of incomplete cure after surgical adenoma removal. AHAs to vasopressin cells have identified patients at risk of developing diabetes insipidus. APAs have been also found together with AHAs in patients affected by idiopathic hypopituitarism, but both were also present in different kinds of patients without abnormalities of the hypothalamic-pituitary axis. Despite some data being promising, the clinical use of pituitary and hypothalamus autoantibodies is still limited by the low diagnostic sensitivity, irreproducibility of the results, and the absence of autoantigen/s able to discriminate the autoimmune reaction involving the pituitary or the hypothalamus from the other autoimmune states.

Correction: Hypothalamic over-expression of VGF in the Siberian hamster increases energy expenditure and reduces body weight gain.

[This corrects the article DOI: 10.1371/journal.pone.0172724.].

Profiles of VGF Peptides in the Rat Brain and Their Modulations after Phencyclidine Treatment.

From the VGF precursor protein originate several low molecular weight peptides, whose distribution in the brain and blood circulation is not entirely known. Among the VGF peptides, those containing the N-terminus portion were altered in the cerebro-spinal fluid (CSF) and hypothalamus of schizophrenia patients. "Hence, we aimed to better investigate the involvement of the VGF peptides in schizophrenia by studying their localization in the brain regions relevant for the disease, and revealing their possible modulations in response to certain neuronal alterations occurring in schizophrenia". We produced antibodies against different VGF peptides encompassing the N-terminus, but also C-terminus-, TLQP-, GGGE- peptide sequences, and the so named NERP-3 and -4. These antibodies were used to carry out specific ELISA and immunolocalization studies while mass spectrometry (MS) analysis was also performed to recognize the intact brain VGF fragments. We used a schizophrenia rat model, in which alterations in the prepulse inhibition (PPI) of the acoustic startle response occurred after PCP treatment. In normal rats, all the VGF peptides studied were distributed in the brain areas examined including hypothalamus, prefrontal cortex, hippocampus, accumbens and amygdaloid nuclei and also in the plasma. By liquid chromatography-high resolution mass, we identified different intact VGF peptide fragments, including those encompassing the N-terminus and the NERPs. PCP treatment caused behavioral changes that closely mimic schizophrenia, estimated by us as a disruption of PPI of the acoustic startle response. The PCP treatment also induced selective changes in the VGF peptide levels within certain brain areas. Indeed, an increase in VGF C-terminus and TLQP peptides was revealed in the prefrontal cortex (p < 0.01) where they were localized within parvoalbumin and tyrosine hydroxylase (TH) containing neurons, respectively. Conversely, in the nucleus accumbens, PCP treatment produced a down-regulation in the levels of VGF C-terminus-, N-terminus- and GGGE- peptides (p < 0.01), expressed in GABAergic- (C-terminus/GGGE) and somatostatin- (N-terminus) neurons. These results confirm that VGF peptides are widely distributed in the brain and modulated in specific areas involved in schizophrenia.

Hypothalamic over-expression of VGF in the Siberian hamster increases energy expenditure and reduces body weight gain.

VGF (non-acronymic) was first highlighted to have a role in energy homeostasis through experiments involving dietary manipulation in mice. Fasting increased VGF mRNA in the Arc and levels were subsequently reduced upon refeeding. This anabolic role for VGF was supported by observations in a VGF null (VGF-/-) mouse and in the diet-induced and gold-thioglucose obese mice. However, this anabolic role for VGF has not been supported by a number of subsequent studies investigating the physiological effects of VGF-derived peptides. Intracerebroventricular (ICV) infusion of TLQP-21 increased resting energy expenditure and rectal temperature in mice and protected against diet-induced obesity. Similarly, ICV infusion of TLQP-21 into Siberian hamsters significantly reduced body weight, but this was due to a decrease in food intake, with no effect on energy expenditure. Subsequently NERP-2 was shown to increase food intake in rats via the orexin system, suggesting opposing roles for these VGF-derived peptides. Thus to further elucidate the role of hypothalamic VGF in the regulation of energy homeostasis we utilised a recombinant adeno-associated viral vector to over-express VGF in adult male Siberian hamsters, thus avoiding any developmental effects or associated functional compensation. Initially, hypothalamic over-expression of VGF in adult Siberian hamsters produced no effect on metabolic parameters, but by 12 weeks post-infusion hamsters had increased oxygen consumption and a tendency to increased carbon dioxide production; this attenuated body weight gain, reduced interscapular white adipose tissue and resulted in a compensatory increase in food intake. These observed changes in energy expenditure and food intake were associated with an increase in the hypothalamic contents of the VGF-derived peptides AQEE, TLQP and NERP-2. The complex phenotype of the VGF-/- mice is a likely consequence of global ablation of the gene and its derived peptides during development, as well as in the adult.

VGF Protein and Its C-Terminal Derived Peptides in Amyotrophic Lateral Sclerosis: Human and Animal Model Studies.

VGF mRNA is widely expressed in areas of the nervous system known to degenerate in Amyotrophic Lateral Sclerosis (ALS), including cerebral cortex, brainstem and spinal cord. Despite certain VGF alterations are reported in animal models, little information is available with respect to the ALS patients. We addressed VGF peptide changes in fibroblast cell cultures and in plasma obtained from ALS patients, in parallel with spinal cord and plasma samples from the G93A-SOD1 mouse model. Antisera specific for the C-terminal end of the human and mouse VGF proteins, respectively, were used in immunohistochemistry and enzyme-linked immunosorbent assay (ELISA), while gel chromatography and HPLC/ESI-MS/MS were used to identify the VGF peptides present. Immunoreactive VGF C-terminus peptides were reduced in both fibroblast and plasma samples from ALS patients in an advanced stage of the disease. In the G93A-SOD1 mice, the same VGF peptides were also decreased in plasma in the late-symptomatic stage, while showing an earlier down-regulation in the spinal cord. In immunohistochemistry, a large number of gray matter structures were VGF C-terminus immunoreactive in control mice (including nerve terminals, axons and a few perikarya identified as motoneurons), with a striking reduction already in the pre-symptomatic stage. Through gel chromatography and spectrometry analysis, we identified one form likely to be the VGF precursor as well as peptides containing the NAPP- sequence in all tissues studied, while in the mice and fibroblasts, we revealed also AQEE- and TLQP- peptides. Taken together, selective VGF fragment depletion may participate in disease onset and/or progression of ALS.

Involvement of nigral oxytocin in locomotor activity: A behavioral, immunohistochemical and lesion study in male rats.

Oxytocin is involved in the control of different behaviors, from sexual behavior and food consumption to empathy, social and affective behaviors. An imbalance of central oxytocinergic neurotransmission has been also associated with different mental pathologies, from depression, anxiety and anorexia/bulimia to schizophrenia, autism and drug dependence. This study shows that oxytocin may also play a role in the control of locomotor activity. Accordingly, intraperitoneal oxytocin (0.5-2000µg/kg) reduced locomotor activity of adult male rats. This effect was abolished by d(CH2)5Tyr(Me)(2)-Orn(8)-vasotocin, an oxytocin receptor antagonist, given into the lateral ventricles at the dose of 2µg/rat, which was ineffective on locomotor activity. Oxytocin (50-200ng/site) also reduced and d(CH2)5Tyr(Me)(2)-Orn(8)-vasotocin (2µg/site) increased locomotor activity when injected bilaterally into the substantia nigra, a key area in the control of locomotor activity. Conversely, the destruction of nigral neurons bearing oxytocin receptors by the recently characterized neurotoxin oxytocin-saporin injected into the substantia nigra, increased basal locomotor activity. Since oxytocin-saporin injected into the substantia nigra caused a marked reduction of neurons immunoreactive for tyrosine hydroxylase (e.g., nigrostriatal dopaminergic neurons) and for vesicular glutamate transporters VGluT1, VGluT2 and VGluT3 (e.g., glutamatergic neurons), but not for glutamic acid decarboxylase (e.g., GABAergic neurons), together these findings suggest that oxytocin influences locomotor activity by acting on receptors localized presynaptically in nigral glutamatergic nerve terminals (which control the activity of nigral GABAergic efferent neurons projecting to brain stem nuclei controlling locomotor activity), rather than on receptors localized in the cell bodies/dendrites of nigrostriatal dopaminergic neurons.

VGF Peptide Profiles in Type 2 Diabetic Patients' Plasma and in Obese Mice.

To address the possible involvement of VGF peptides in obesity and diabetes, we studied type 2 diabetes (T2D) and obese patients, and high-fat diet induced obese mice. Two VGF peptides (NAPP-19 and QQET-30) were identified in human plasma by HPLC-ESI-MS. The VGF C-terminus, the above two cleaved peptides, and the TLQP-21 related peptide/s were studied using ELISA and immunohistochemistry. In euglycemic patients, plasma NAPPE and TLQP like peptides were significantly reduced with obesity (74±10 vs. 167±28, and 92±10 vs. 191±19 pmol/ml, mean+SEM, n = 10 and 6, obese vs. normal BMI, respectively, p<0.03). Upon a standard glucose load, a distinct response was shown for VGF C-terminus, TLQP and QQET-like (ERVW immunoreactive) peptides in euglycemic normal BMI patients, but was virtually abolished in euglycemic obese, and in T2D patients independently of BMI. High-fat diet induced obese mice showed reduced plasma VGF C-terminus, NAPPE and QQET-like (ERVW) peptide/s (3±0.2 vs. 4.6±0.3, 22±3.5 vs. 34±1.3, and 48±7 vs. 100±7 pmol/ml, mean+SEM, n = 8/group, obese vs. slim, respectively, p<0.03), with a loss of the response to glucose for all VGF peptides studied. In immunohistochemistry, TLQP and/or VGF C-terminus antibodies labelled VGF containing perikarya in mouse celiac ganglia, pancreatic islet cells and thin beaded nerve fibres in brown adipose tissues, with fewer in white adipose tissue. Upon the glucose load, tyrosine hydroxylase and VGF C-terminus immunoreactive axons became apparent in pancreatic islets of slim animals, but not in obese animals. Alltogether, a significant loss of VGF peptide immunoreactivity and/or their response to glucose was demonstrated in obese patients, with or without T2D, in parallel with a similar loss in high-fat diet induced obese mice. An involvement of VGF in metabolic regulations, including those of brown and/or white adipose tissues is underlined, and may point out specific VGF peptides as potential targets for diagnosis and/or treatment.

Photoperiod Regulates vgf-Derived Peptide Processing in Siberian Hamsters.

VGF mRNA is induced in specific hypothalamic areas of the Siberian hamster upon exposure to short photoperiods, which is associated with a seasonal decrease in appetite and weight loss. Processing of VGF generates multiple bioactive peptides, so the objective of this study was to determine the profile of the VGF-derived peptides in the brain, pituitary and plasma from Siberian hamsters, and to establish whether differential processing might occur in the short day lean state versus long day fat. Antisera against short sequences at the C- or N- termini of proVGF, as well as against NERP-1, TPGH and TLQP peptides, were used for analyses of tissues, and both immunohistochemistry and enzyme linked immunosorbent assay (ELISA) coupled with high-performance liquid (HPLC) or gel chromatography were carried out. VGF peptide immunoreactivity was found within cortex cholinergic perikarya, in multiple hypothalamic nuclei, including those containing vasopressin, and in pituitary gonadotrophs. ELISA revealed that exposure to short day photoperiod led to a down-regulation of VGF immunoreactivity in the cortex, and a less pronounced decrease in the hypothalamus and pituitary, while the plasma VGF levels were not affected by the photoperiod. HPLC and gel chromatography both confirmed the presence of multiple VGF-derived peptides in these tissues, while gel chromatography showed the presence of the VGF precursor in all tissues tested except for the cortex. These observations are consistent with the view that VGF-derived peptides have pleiotropic actions related to changing photoperiod, possibly by regulating cholinergic systems in the cortex, vasopressin hypothalamic pathways, and the reproductive axis.

VGF changes during the estrous cycle: a novel endocrine role for TLQP peptides?

Although the VGF derived peptide TLQP-21 stimulates gonadotropin-releasing hormone (GnRH) and gonadotropin secretion, available data on VGF peptides and reproduction are limited. We used antibodies specific for the two ends of the VGF precursor, and for two VGF derived peptides namely TLQP and PGH, to be used in immunohistochemistry and enzyme-linked immunosorbent assay complemented with gel chromatography. In cycling female rats, VGF C-/N-terminus and PGH peptide antibodies selectively labelled neurones containing either GnRH, or kisspeptin (VGF N-terminus only), pituitary gonadotrophs and lactotrophs, or oocytes (PGH peptides only). Conversely, TLQP peptides were restricted to somatostatin neurones, gonadotrophs, and ovarian granulosa, interstitial and theca cells. TLQP levels were highest, especially in plasma and ovary, with several molecular forms shown in chromatography including one compatible with TLQP-21. Among the cycle phases, TLQP levels were higher during metestrus-diestrus in median eminence and pituitary, while increased in the ovary and decreased in plasma during proestrus. VGF N- and C-terminus peptides also showed modulations over the estrous cycle, in median eminence, pituitary and plasma, while PGH peptides did not. In ovariectomised rats, plasmatic TLQP peptide levels showed distinct reduction suggestive of a major origin from the ovary, while the estrogen-progesterone treatment modulated VGF C-terminus and TLQP peptides in the hypothalamus-pituitary complex. In in vitro hypothalamus, TLQP-21 stimulated release of growth hormone releasing hormone but not of somatostatin. In conclusion, various VGF peptides may regulate the hypothalamus-pituitary complex via specific neuroendocrine mechanisms while TLQP peptides may act at further, multiple levels via endocrine mechanisms involving the ovary.