Characterization of Endogenous Nucleobindin-2/Nesfatin-1 in Rodents

Whole body energy homeostasis is regulated by the endocrine system. Nesfatin-1 is a newly identified multifunctional metabolic peptide with insulinotropic, endocrine, glucoregulatory, fat reducing and cardiovascular functions. While nesfatin-1 tissue expression and secretion are considered meal responsive, it is unknown whether macronutrients and/or development regulate its secretion. Is endogenous nesfatin-1 critical for energy balance? The central hypothesis of this thesis research is that the tissue specific expression of NUCB2/nesfatin-1 is regulated developmentally, and by nutrients, and that endogenous NUCB2/nesfatin-1 is critical for the maintenance of energy homeostasis. The specific objectives of this research were to determine the developmental, and nutrient regulated expression of NUCB2/nesfatin-1, and to characterize the metabolic phenotype of mice lacking NUCB2/nesfatin-1. Three key findings were made in this research. First, it was found that NUCB2/nesfatin-1 presents an ontogenic pattern of expression in the gastroenteropancreatic tissues and serum of rats. An age-dependent co-expression of related peptides, ghrelin and its processing enzyme, ghrelin-O-acyl transferase (GOAT), and nesfatin-1 processing prohormone convertases were also found in the endocrine pancreas. Second, it was determined that the NUCB2 mRNA expression and NUCB2/nesfatin-1 secretion in mice are influenced by nutrients in a tissue specific manner in vitro and in vivo, and it depends on the duration of exposure to specific diets tested. This research identified nutrients as major regulators of endogenous NUCB2/nesfatin-1. Third, a sexually dimorphic effect of NUCB2/nesfatin-1 disruption in mice was found, with alterations in body weight, food intake, insulin secretion, glucose homeostasis and energy homeostasis. These data support a metabolic role for endogenous nesfatin-1. Together, this research provides important new information on developmental and cell specific regulation of nesfatin-1 expression, nutrient modulation of its expression and secretion, and an essential role for endogenous nesfatin-1 in maintaining energy homeostasis. For example, endocrine pancreas is an abundant source of nesfatin-1. Absence of endogenous nesfatin-1 causes defects in insulin secretion from islet beta cells, and alters glucose homeostasis. Exogenous nesfatin-1 causes a reduction in body weight. NUCB2 gene disruption resulted in abnormal body weight in mice, further confirming that nesfatin-1 indeed influences body mass

A Pharmacokinetic Dose-Optimization Study of Cabotegravir and Bictegravir in a Mouse Pregnancy Model

Animal pregnancy models can be useful tools to study HIV antiretroviral safety and toxicity and to perform mechanistic studies that are not easily performed in humans. Utilization of clinically relevant dosing in these models improves the relevance of the findings. Cabotegravir and bictegravir are new integrase strand transfer inhibitors (INSTIs), recently approved for the treatment of people living with HIV. Studies of these drugs in pregnancy are very limited. The objective of this study was to perform a dose-optimization study of cabotegravir and bictegravir in a mouse pregnancy model with the goal of determining the dose that would yield plasma drug concentrations similar those observed in humans. Pregnant mice were administered increasing doses of cabotegravir or bictegravir in combination with emtricitabine and tenofovir by oral gavage from gestational day 11.5 to 15.5. Drug concentrations in the maternal plasma at 1 h and 24 h post drug administration and in the amniotic fluid at 1 h post drug administration were determined using high-performance liquid chromatography coupled with tandem mass spectrometry. A review of cabotegravir and bictegravir human pharmacokinetic studies are also reported. We hope these data will encourage studies of HIV antiretroviral safety/toxicity and mechanistic studies in animal pregnancy models

Metabolic implications and safety of dolutegravir use in pregnancy

Dolutegravir is recommended for all people living with HIV because of its efficacy, high barrier to resistance, favourable safety and tolerability profile, and affordability. Dolutegravir has the highest rates of viral suppression in pregnancy, therefore preventing perinatal HIV transmission. In view of these benefits, particularly for pregnant women, an important question is if dolutegravir is safe in pregnancy. Dolutegravir has been associated with metabolic complications, including weight gain and rare events of hyperglycaemia, that could affect maternal, fetal, and postnatal health. We review the current clinically and experimentally based literature on the implications of dolutegravir use for pregnant women and for developing embryos and fetuses. Possible effects on folate status, energy metabolism, adipogenesis, and oxidative stress are considered. In many instances, insufficient data are available, pointing to the need for additional research in this important area of HIV treatment

NUCB2 mRNA Expression in the Stomach (A), Small Intestine (Duodenum; B), Large Intestine (C) and Liver (D) of Mice gavaged with water, high protein, high carbohydrate, high fat and the No Gavage Group (n = 7 mice/group).

<p>No significant differences were found in NUCB2 mRNA in the stomach of mice gavaged with various liquid diets and no gavage group. Same letters (a) indicate no significant differences found between the various groups, using One Way ANOVA followed by Tukey’s Multiple Comparison Test.</p

NUCB2 mRNA Expression (A, C, E) and NUCB2/nesfatin-1 Secretion (B, D, F) from MGN3-1 Cells Incubated for 4 Hours with Different Concentrations of Linolenic Acid, Octanoic Acid and Oleic Acid (0 µM, 1 µM, 10 µM, 100 µM).

<p>No change in NUCB2 mRNA expression (A, C) and nesfatin-1 secretion (B, D) when MGN3-1 cells were incubated with different concentrations of linolenic acid and octanoic acid. MGN3-1 cells incubated at 1, 10, 100 µM oleic acid had a significant decrease in NUCB2 mRNA expression (E; p<0.05), but no significant difference was found in nesfatin-1 secretion at 4 hours post-incubation (F; p<0.05). n = 9 wells/concentration pooled from 3 different studies. Different letters (a and b) shows significant differences found between control and various treatment groups, using One Way ANOVA followed by Tukey’s Multiple Comparison Test. There are no significant differences between groups marked by same letters.</p

Blood Glucose and Serum NUCB2/Nesfatin-1 in mice during acute administration of various liquid diets and in no-gavage group.

<p>(<b>A</b>) Fold change in blood glucose measured during an oral gavage study in mice gavaged with a specific liquid diet, divided into 5 groups: high protein (n = 7), High fat (n = 7), High carbohydrate (n = 7), water (n = 7) and a no gavage group (n = 7). Mice gavaged with the high carbohydrate liquid diet had high concentrations of blood glucose compared to the no-gavage group and the groups gavaged with water, high protein and high fat liquid diets (p<0.05, ANOVA followed by Tukey’s Multiple Comparison Test). Blood glucose concentration - Area under the Curve from 0–30 measured during an oral gavage study in mice gavaged with a specific liquid diet, divided into 4 groups: high protein, high fat, high carbohydrate, and water (n = 7 mice/group). Letter <i>a</i> denotes no difference between the groups gavaged with water, high protein and high fat liquid diet. Letter b denotes that the high carbohydrate group had significantly elevated glucose levels compared to high protein, high carbohydrate, high fat and water gavaged mice during 0–30 minutes post-administration (p<0.05, One-Way ANOVA followed by Tukey’s Multiple Comparison Test). (<b>B</b>) Secretion profile representing circulating levels of NUCB2/Nesfatin-1 in mice gavaged with a specific liquid diet, divided into 4 groups: high protein, high fat, high carbohydrate, and water (n = 7 mice/group). Letter a denotes no difference in serum NUCB2/Nesfatin-1 levels between mice gavaged with water, high protein, high carbohydrate, high fat liquid diets and the no-gavage group within the four time points tested. Letter b denotes that the serum nesfatin-1/NUCB2 levels in the high fat diet fed group was significantly higher to the levels in the control mice or mice fed other diets at all time points (p<0.05, One-Way ANOVA followed by Tukey’s Multiple Comparison Test).</p

NUCB2 mRNA Expression (A, C, E) and NUCB2/nesfatin-1 Secretion (B, D, F) from MGN3-1 Cells Incubated with Different Concentrations of Glucose (5.6 mM, 25 mM, 50 mM and 100 mM) at Various Incubation Periods (1 Hour and 2 Hours), and with Different Concentrations of L-Tryptophan (0.07 mM, 1 mM, 10 mM; 4 Hours).

<p>MGN3-1 cells incubated at 100 mM glucose had a significant increase in NUCB2 mRNA expression at 1 hour post-incubation (A; p<0.05), but no significant differences was found in nesfatin-1 secreted into the media from the same cells (B). Similarly, glucose caused a dose dependent increase in NUCB2 mRNA expression at 2 hours post-incubation (C; p<0.5) without causing any changes in nesfatin-1 secretion (D). n = 9 wells/concentration pooled from 3 different studies. MGN3-1 cells incubated at 10 mM L-Tryptophan had a significant increase in NUCB2 mRNA expression than cells incubated at 0.07 mM and 1 mM L-Tryptophan (E; p<0.05). Nesfatin-1 secretion significantly increased from cells incubated at 1 mM and 10 mM L-Tryptophan than 0.07 mM L-Tryptophan (F; p<0.05). n = 12 wells/concentration pooled from 3 different studies. Letters b and c denote significant differences found between control (a) and various treatment groups, using One Way ANOVA followed by Tukey’s Multiple Comparison Test. There are no significant differences between groups marked by same letters.</p

MGN3-1 Cells are Immunopositive for NUCB2/Nesfatin-1, Ghrelin, PC 1/3 and PC 2.

<p>Immunocytochemical staining of MGN3-1 cells for ghrelin immunoreactivity (A; FITC-Green), nesfatin-1 immunoreactivity (B; Texas-Red) and the nuclear stain DAPI. A merged image showing co-localization of nesfatin-1 and ghrelin immunoreactivity is shown in (C; Yellow). Immunocytochemical staining of MGN3-1 cells for PC 1/3 immunoreactivity (D; Texas-red), PC 2 immunoreactivity (E; Texas-red) and the nuclear stain DAPI. No primary antibody controls are shown in (F) for nesfatin-1 and ghrelin, respectively. Images taken at 40X magnification. Scale bar = 20 µm.</p

NUCB2/Nesfatin-1 co-localizes with ghrelin in MGN-3 cells.

<p>Confocal micrographs of MGN-3 cells stained for NUCB2/nesfatin-1 (A; Texas-Red) and ghrelin (B; FITC-Green). Merged image of A and B showing co-localization of NUCB2/nesfatin-1 and ghrelin immunoreactive cells (C; Yellow). No primary antibody negative control labeled only with secondary antibodies (D). Nuclei are stained with DAPI (Blue; A, B and C). Images were merged using Image J PC-based software. Scale bar = 25 µm.</p

Nutrients Differentially Regulate Nucleobindin-2/Nesfatin-1 <i>In Vitro</i> in Cultured Stomach Ghrelinoma (MGN3-1) Cells and <i>In Vivo</i> in Male Mice

<div><p>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.</p></div