Hiroshi Iwakura

Nutrients differentially regulate nucleobindin-2/nesfatin-1 in vitro in cultured stomach ghrelinoma (MGN3-1) cells and in vivo in male mice.

Nesfatin-1 is secreted, meal-responsive anorexigenic peptide encoded in the precursor nucleobindin-2 [NUCB2]. Circulating nesfatin-1 increases post-prandially, but the dietary components that modulate NUCB2/nesfatin-1 remain unknown. We hypothesized that carbohydrate, fat and protein differentially regulate tissue specific expression of nesfatin-1. NUCB2, prohormone convertases and nesfatin-1 were detected in mouse stomach ghrelinoma [MGN3-1] cells. NUCB2 mRNA and protein were also detected in mouse liver, and small and large intestines. MGN3-1 cells were treated with glucose, fatty acids or amino acids. Male C57BL/6 mice were chronically fed high fat, high carbohydrate and high protein diets for 17 weeks. Quantitative PCR and nesfatin-1 assays were used to determine nesfatin-1 at mRNA and protein levels. Glucose stimulated NUCB2 mRNA expression in MGN3-1 cells. L-Tryptophan also increased NUCB2 mRNA expression and ghrelin mRNA expression, and nesfatin-1 secretion. Oleic acid inhibited NUCB2 mRNA expression, while ghrelin mRNA expression and secretion was enhanced. NUCB2 mRNA expression was significantly lower in the liver of mice fed a high protein diet compared to mice fed other diets. Chronic intake of high fat diet caused a significant reduction in NUCB2 mRNA in the stomach, while high protein and high fat diet caused similar suppression of NUCB2 mRNA in the large intestine. No differences in serum nesfatin-1 levels were found in mice at 7 a.m, at the commencement of the light phase. High carbohydrate diet fed mice showed significantly elevated nesfatin-1 levels at 1 p.m. Serum nesfatin-1 was significantly lower in mice fed high fat, protein or carbohydrate compared to the controls at 7 p.m, just prior to the dark phase. Mice that received a bolus of high fat had significantly elevated nesfatin-1/NUCB2 at all time points tested post-gavage, compared to control mice and mice fed other diets. Our results for the first time indicate that nesfatin-1 is modulated by nutrients.

Comprehensive Profiling of GPCR Expression in Ghrelin-Producing Cells

To determine the comprehensive G protein-coupled receptor (GPCR) expression profile in ghrelin-producing cells and to elucidate the role of GPCR-mediated signaling in the regulation of ghrelin secretion, we determined GPCR expression profiles by RNA sequencing in the ghrelin-producing cell line MGN3-1 and analyzed the effects of ligands for highly expressed receptors on intracellular signaling and ghrelin secretion. Expression of selected GPCRs was confirmed in fluorescence-activated cell-sorted fluorescently tagged ghrelin-producing cells from ghrelin-promoter CreERT2/Rosa-CAG-LSL-ZsGreen1 mice. Expression levels of GPCRs previously suggested to regulate ghrelin secretion including adrenergic-β1 receptor, GPR81, oxytocin receptor, GPR120, and somatostatin receptor 2 were high in MGN3-1 cells. Consistent with previous reports, isoproterenol and oxytocin stimulated the Gs and Gq pathways, respectively, whereas lactate, palmitate, and somatostatin stimulated the Gi pathway, confirming the reliability of current assays. Among other highly expressed GPCRs, prostaglandin E receptor 4 agonist prostaglandin E2 significantly stimulated the Gs pathway and ghrelin secretion. Muscarine, the canonical agonist of cholinergic receptor muscarinic 4, stimulated both the Gq and Gi pathways. Although muscarine treatment alone did not affect ghrelin secretion, it did suppress forskolin-induced ghrelin secretion, suggesting that the cholinergic pathway may play a role in counterbalancing the stimulation of ghrelin by Gs (eg, by adrenaline). In addition, GPR142 ligand tryptophan stimulated ghrelin secretion. In conclusion, we determined the comprehensive expression profile of GPCRs in ghrelin-producing cells and identified two novel ghrelin regulators, prostaglandin E2 and tryptophan. These results will lead to a greater understanding of the physiology of ghrelin and facilitate the development of ghrelin-modulating drugs.

The regulation of circulating ghrelin — with recent updates from cell-based assays [Review]

Ghrelin is a stomach-derived orexigenic hormone with a wide range of physiological functions. Elucidation of the regulation of the circulating ghrelin level would lead to a better understanding of appetite control in body energy homeostasis. Earlier studies revealed that circulating ghrelin levels are under the control of both acute and chronic energy status: at the acute scale, ghrelin levels are increased by fasting and decreased by feeding, whereas at the chronic scale, they are high in obese subjects and low in lean subjects. Subsequent studies revealed that nutrients, hormones, or neural activities can influence circulating ghrelin levels in vivo. Recently developed in vitro assay systems for ghrelin secretion can assess whether and how individual factors affect ghrelin secretion from cells. In this review, on the basis of numerous human, animal, and cell-based studies, we summarize current knowledge on the regulation of circulating ghrelin levels and enumerate the factors that influence ghrelin levels.

Overexpression of intraislet ghrelin enhances β-cell proliferation after streptozotocin-induced β-cell injury in mice

Previously, we reported that exogenous administration of ghrelin ameliorates glucose metabolism in a neonate streptozotocin (STZ)-induced diabetic rat model through enhancement of β-cell proliferation. However, it was not clear whether the observed β-cell proliferation was a direct or indirect effect (e.g., via orexigenic or growth hormone-stimulated pathways) of ghrelin activity. Here, we aimed to investigate whether ghrelin directly impacts β-cell proliferation after STZ-induced injury in mice. Seven-week-old male rat insulin II promoter-ghrelin internal ribosomal sequence ghrelin O-acyltransferase transgenic (RIP-GG Tg) mice, which have elevated pancreatic ghrelin levels, but only minor changes in plasma ghrelin levels when fed a medium-chain triglyceride-rich diet, were treated with STZ. Then, serum insulin, pancreatic insulin mRNA expression, and islet histology were evaluated. We found that the serum insulin levels, but not blood glucose levels, of RIP-GG Tg mice were significantly ameliorated 14 days post-STZ treatment. Pancreatic insulin mRNA expression was significantly elevated in RIP-GG Tg mice, and β-cell numbers in islets were increased. Furthermore, the number of phospho-histone H3⁺ or Ki67⁺ proliferating β-cells was significantly elevated in RIP-GG Tg mice, whereas the apoptotic indexes within the islets, as determined by TUNEL assay, were not changed. These results indicate that ghrelin can directly stimulate β-cell proliferation in vivo after β-cell injury even without its orexigenic or GH-stimulating activities, although it did not have enough impact to normalize the glucose tolerance in adult mice.

Soy‐ghretropin, a novel ghrelin‐releasing peptide derived from soy protein

We investigated exogenous secretagogues of ghrelin, which is an orexigenic hormone isolated from the stomach. We found that the tryptic digest of soy β‐conglycinin stimulated ghrelin secretion by the ghrelin‐producing cell line, MGN3‐1. We then identified a 22‐amino acid peptide corresponding to the β‐conglycinin α‐subunit(192–213) [βCGα(192–213)] from an active fraction separated by HPLC. The N‐terminal undecapeptide of βCGα(192–213), NKNPFLFGSNR, exhibited ghrelin‐releasing activity at a lower dose than that of βCGα(192–213). We named NKNPFLFGSNR ‘soy‐ghretropin’, which corresponds to βCGα(192–202). Neither [des‐N1K2]‐soy‐ghretropin nor [des‐R11]‐soy‐ghretropin stimulated ghrelin secretion; hence, both the N‐ and C‐terminal structures of soy‐ghretropin were indispensable. Orally administered soy‐ghretropin increased plasma ghrelin levels and food intake in vivo. Soy‐ghretropin is the first exogenous ghrelin‐releasing peptide derived from food protein.

High incorporation of long‐chain fatty acids contributes to the efficient production of acylated ghrelin in ghrelin‐producing cells

The mechanisms for supplying octanoic acid for ghrelin acylation in X/A‐like cells are incompletely understood. We found that long‐chain fatty acids were incorporated at a higher rate in the ghrelin‐producing cell line MGN3‐1 than in MIN6 cells, in part due to higher expression level of long‐chain fatty acyl‐CoA synthetase family member 1 (Acsl1). Inhibition of ACSLs by triacsin C profoundly suppressed acylated ghrelin production. These results suggest that high incorporation of long‐chain fatty acids into the ghrelin‐producing cells plays a role in the supply of octanoic acid for ghrelin acylation.

Differential role of GPR142 in tryptophan-mediated enhancement of insulin secretion in obese and lean mice

Tryptophan is reportedly the most potent agonist for GPR142. Glucose-stimulated insulin secretion (GSIS) from pancreatic beta cells are enhanced by GPR142-mediated signal. It is not clear, however, if GPR142-mediated signals is solely attributable to GSIS enhancement after tryptophan load in various pathophysiological settings. This study aims to reveal the significance of GPR142 signaling in tryptophan-mediated GSIS enhancement in normal and obese mice. Tryptophan significantly improved glucose tolerance in both lean and DIO mice, but the extent of improvement was bigger in DIO mice with augmented glucose-stimulated insulin secretion (GSIS) enhancement. The same results were obtained in ob/ob mice. GPR142 deletion almost completely blocked tryptophan actions in lean mice, suggesting that GPR142 signaling was solely responsible for the GSIS enhancement. In obese GPR142KO mice, however, a significant amount of tryptophan effects were still observed. Calcium-sensing receptors (CaSR) are also known to recognize tryptophan as ligand. Expression levels of CaSR were significantly elevated in the pancreas of DIO mice, and CaSR antagonist further blocked tryptophan’s actions in DIO mice with GPR142 deletion. Although GPR142 signaling had a major role in tryptophan recognition for the enhancement of GSIS in lean mice, other pathways including CaSR signaling also had a significant role in obese mice, which seemed to contribute to the augmented enhancement of GSIS by tryptophan in these animals.

Endogenous peptide profile for elucidating biosynthetic processing of the ghrelin precursor

Ghrelin is an orexigenic peptide primarily produced by gastric endocrine cells. The biosynthetic cleavage site of ghrelin has been well documented, but how its downstream region undergoes proteolytic processing remains poorly explored. Here, we provide the first snapshot of endogenous peptides from the ghrelin precursor by profiling the secretopeptidome of cultured mouse ghrelin-producing cells during exocytosis. Mapping of MS/MS sequenced peptides to the precursor highlighted three atypical monobasic processing sites, including the established C-terminus of ghrelin and the N-terminal cleavage site for obestatin, a putative 23-amino-acid C-terminally amidated peptide. However, we found that mouse obestatin does not occur in the form originally reported, but that a different amidation site is used to generate a shorter peptide. These data can be extended to study and characterize the precursor-derived peptides located downstream of ghrelin in different biological contexts.

Establishment of Leptin-Responsive Cell Lines from Adult Mouse Hypothalamus

Leptin resistance is considered to be the primary cause of obesity. However, the cause of leptin resistance remains incompletely understood, and there is currently no cure for the leptin-resistant state. In order to identify novel drug-target molecules that could overcome leptin resistance, it would be useful to develop in vitro assay systems for evaluating leptin resistance. In this study, we established immortalized adult mouse hypothalamus—derived cell lines, termed adult mouse hypothalamus (AMH) cells, by developing transgenic mice in which SV40 Tag was overexpressed in chromogranin A—positive cells in a tamoxifen-dependent manner. In order to obtain leptin-responsive clones, we selected clones based on the phosphorylation levels of STAT3 induced by leptin. The selected clones were fairly responsive to leptin in terms of STAT3, ERK, and Akt phosphorylation and induction of c-Fos mRNA induction. Pretreatment with leptin, insulin, and palmitate attenuated the c-Fos mRNA response to leptin, suggesting that certain aspects of leptin resistance might be reconstituted in this cellular model. These cell lines are useful tools for understanding the molecular nature of the signal disturbance in the leptin-resistant state and for identifying potential target molecules for drugs that relieve leptin resistance, although they have drawbacks including de-differentiated nature and lack of long-time stability.

Fulminant Type 1 Diabetes Mellitus Accompanied by Positive Conversion of Anti-insulin Antibody after the Administration of Anti-CTLA-4 Antibody Following the Discontinuation of Anti-PD-1 Antibody

An 80-year-old woman with malignant melanoma received 20 cycles of anti-programmed death 1 (PD-1) antibody (nivolumab) treatment and showed normal glucose tolerance. Three weeks after switching to anti-cytotoxic T-lymphocyte associated antigen 4 (CTLA-4) antibody (ipilimumab), her plasma glucose level was elevated to 639 mg/dL, her HbA1c was 7.7%, and her fastening serum C-peptide immunoreactivity was undetectable. Anti-glutamic acid decarboxylase and insulinoma-associated protein-2 antibodies were negative. She was diagnosed with fulminant type 1 diabetes mellitus (F1DM). Remarkably, her anti-insulin antibody was positively converted, and her Sialylated Carbohydrate Antigen, Krebs von den Lungen-6 levels increased after ipilimumab therapy. She possessed F1DM-susceptible Human Leukocyte Antigen-DR4. A fluorescence activated cell sorting analysis showed an altered T-cell population. This case of F1DM highlights specific mechanisms underlying pancreatic beta cell immunity.