ELISA of VGF peptides in mouse.

<p><u>Upper panel</u> (plasma): upon fasting (0’), higher VGF C-terminus, NAPPE, and ERVW immunoreactive peptide/s were shown in slim <i>vs</i>. obese mice, while the glucose load (120’) led to increased VGF C-terminus and TLQP peptide/s in slim mouse, with little response in the obese group. <u>Middle panel</u> (brown adipose tissue, BAT): TLQP, NAPPE and ERVW peptides were higher during fasting (0’) in slim mice, which also showed a distinct increase of VGF C-terminus peptide/s after the glucose load (120’). <u>Lower panel</u> (white adipose tissue, WAT): in basal conditions (0’) ERVW peptide/s levels were higher in slim mice, while the glucose load (120’) resulted in a response of all other VGF peptides in slim mice (only). VGF C-term: VGF C-terminus peptide/s; ERVW refers to the C-terminally directed assay used for QQET-30 like peptide/s (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142333#pone.0142333.t001" target="_blank">Table 1</a>); mean±SEM, * = P<0.03.</p

VGF peptides in human plasma.

<p>VGF peptides were studied at fasting (0’), and upon an oral glucose tolerance test (120’). Euglycemic and newly diagnosed type 2 diabetic subjects (T2D) were subdivided in: normal weight, overweight and obese. Normal weight, fasting euglycemic patients showed significantly higher TLQP and NAPPE immunoreactive peptide/s <i>vs</i>. the corresponding obese subjects (first set of bars in each panel), as well as <i>vs</i>. normal weight diabetics (NAPPE peptide/s only, third <i>vs</i>. first set of bars). Upon the glucose load, VGF C-terminus, TLQP and ERVW peptide/s were stimulated in euglycemic normal weight subjects, but not in the other body weight groups (second <i>vs</i>. first set of bars), nor in diabetic patients (fourth <i>vs</i>. third set of bars). VGF C-term: VGF C-terminus peptide/s, ERVW refers to the C-terminally directed assay used for QQET-30 like peptide/s (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142333#pone.0142333.t001" target="_blank">Table 1</a>); means±SEM, * = P<0.03.</p

VGF antibodies and ELISA characterization.

<p>VGF antibodies and ELISA characterization.</p

HPLC-ESI-MS of human plasma.

<p>The NAPP-19 and QQET-30 peptides were unambiguously identified. <u>Panel A</u>: HPLC-ESI-MS total ion current (TIC) profile, with enlarged view (range 4.05–28.03 min) of the human sample (<30kDa preparation). <u>Panels B-D</u>: extracted Ion Current (XIC) peaks corresponding to NAPP-19 (i.e. VGF_485-503: B), its truncated co-eluting form VGF_487-503 (C), and QQET-30 (D). The same panels indicate m/z values of the multiply charged ions used to extract the ion current peaks. <u>Panel E</u>: deconvoluted ESI-MS spectra of NAPP-19 (monoisotopic [M+H]⁺ at 1915.012 m/z) and VGF_487-503 (monoisotopic [M+H]⁺ at 1729.932 m/z). <u>Panel F:</u> deconvoluted ESI-MS spectrum of QQET-30 (monoisotopic [M+H]⁺ at 3407.697 m/z). Sodium ion adducts are also depicted (ibidem). RT = retention time; NL = normalization level; amino acid numbering is referred to human VGF throughout.</p

Immunolocalization of VGF peptides in mouse tissues.

<p><u>A-C</u>: In celiac ganglia from slim mice, TLQP antiserum (A) stained a large number of cell bodies, which also contained TH (B). <u>D-I</u>: In BAT from slim mice, VGF-immunoreactivity was shown in thin, beaded nerve axons running close to adipocytes, most of which also labelled for TH (E). TLQP-labelled fibres (G) were more numerous than VGF C-terminus reactive ones (D), and appeared to form a major portion of TH-containing axons. <u>J-Q</u>: In the pancreas from fasting, slim mice (J: time 0’) VGF C-terminus peptide/s were shown in fairly numerous islet cells, including virtually all TH containing cells (N). Upon <i>i</i>.<i>p</i>. glucose load, distinct TH, as well as VGF C-terminus immunoreactive axons became apparent, running close to and inside the islets (slim mice: K,O <i>vs</i>. J,K), while endocrine cells showed reduced staining intensity. Such nerve labelling was virtually lost in obese mice (M,Q at time 120’), while a comparable reduction in islet cell labelling was revealed. VGF C-term: rat/mouse VGF C-terminus antibody, TH: tyrosine hydroxylase, merged: simultaneous dual-visualization of red and green labelling; arrows: examples of dual immunolabelled cells: Cy3, green labelling, Cy2; scale bars: A-C = 10 um, D-I: 50 um, J-Q = 10 um.</p

Tissue-selectivity of tumors in familial and sporadic MEN1 probands.

<p>Tissue-selectivity of tumors in familial and sporadic MEN1 probands.</p

Clinical characteristics of the patients (probands and whole cohort) according to <i>MEN1</i> genotype.

<p>Clinical characteristics of the patients (probands and whole cohort) according to <i>MEN1</i> genotype.</p

<i>MEN1</i> gene mutations detected in 54 probands with MEN1 syndrome.

<p><i>MEN1</i> gene mutations detected in 54 probands with MEN1 syndrome.</p

Literature studies investigating <i>MEN1</i> gene large deletions using different analysis methodologies.

<p>Literature studies investigating <i>MEN1</i> gene large deletions using different analysis methodologies.</p

MEN1 manifestations in the groups of F-MEN1, S-MEN1 probands and <i>MEN1</i> mutation-negative and mutation-positive probands.

<p>(<b>A</b>) Association of the main MEN1-related tumors and tumor aggressiveness in probands with F-MEN1 and S-MEN1 syndrome. (<b>B)</b> Association of the main MEN1-related tumors and tumor aggressiveness in patients of the whole cohort with F-MEN1 and S-MEN1 syndrome. (<b>C)</b> Association of the main MEN1-related tumors in <i>MEN1</i> mutation-positive and mutation-negative probands. <b>(D)</b> Detection rate of <i>MEN1</i> gene mutations within each main clinical presentation in <i>MEN1</i> mutation-positive probands with and without family history. Aty-MEN1 refers to atypical MEN1. Statistical significance was determined by Fisher or Chi-square test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.</p