Nesfatin-1 as a crucial mediator of glucose homeostasis in the reptile, Hemidactylus flaviviridis

Abstract

Nesfatin-1 is a crucial regulator of energy homeostasis in mammals and fishes, however, its metabolic role remains completely unexplored in amphibians, reptiles, and birds. Therefore, present study elucidates role of nesfatin-1 in glucose homeostasis in wall lizard wherein fasting stimulated hepatic nucb2/nesfatin-1, glycogen phosphorylase (glyp), phosphoenolpyruvate carboxykinase (pepck), and fructose 1,6-bisphosphatase (fbp), while feeding upregulated pancreatic nucb2/nesfatin-1 and insulin, suggesting towards tissue-specific dual role of nesfatin-1 in glucoregulation. The glycogenolytic/gluconeogenic role of nesfatin-1 was further confirmed by an increase in media glucose levels along with heightened hepatic pepck and fbp expression and concomitant decline in liver glycogen content in nesfatin-1-treated liver of wall lizard. Moreover, treatment with nesfatin-1 stimulated insulin expression in pancreas while insulin downregulated pancreatic nucb2/nesfatin-1. Further, prolonged fasting induced elevated nucb2/nesfatin-1, and lipolytic markers, adipose triglyceride lipase (atgl) and monoglyceride lipase (mgl) in adipose tissue implicate nesfatin-1 in lipolysis which is substantiated by nesfatin-1-mediated direct upregulation of atgl and mgl. Our study provides the first comprehensive overview of tissue-dependent role of nesfatin-1 in regulating energy homeostasis in a reptile.

Keywords: Carbohydrate metabolism; Glucoregulation; Lipolysis; Nesfatin-1; Wall lizard.

Fig. 1

Food deprivation-induced changes in mRNA expression of nucb2/nesfatin-1 and glucoregulatory genes in the liver of wall lizard. Effect of 3, 7, and 15 days of fasting was observed on hepatic mRNA expression of (a) nucb2/nesfatin-1, (b) glyp, (c) pepck, and (d) fbp. Each group consisted of five lizards (N = 5/group). The expression of nucb2/nesfatin-1 as well as glucoregulatory genes increased significantly during fasting in comparison to fed condition. Variation in gene expression between fed (shaded bars) and fasted (black bars) lizards of respective durations was analysed using Student’s unpaired t-test (P < 0.05). Significant difference between groups is indicated by asterisk (*). Data are represented as mean ± standard error of mean (SEM).

Fig. 2

Nesfatin-1-mediated regulation of glycogenolytic and gluconeogenic markers in the liver of Hemidactylus flaviviridis. Concentration-dependent effect of nesfatin-1 (0.1 nM, 1 nM, and 10 nM) on expression of (a) glyp, (b) pepck, and (c) fbp in wall lizard liver was investigated employing in vitro experiments (N = 5/group). Hepatic expression of glyp showed a concentration-dependent decline while pepck and fbp demonstrated a concentration-dependent incline upon treatment with nesfatin-1. Variation in gene expression were assessed using one-way ANOVA (P < 0.05) followed by Bonferroni test (P < 0.05). Bars with different superscripts (a, b, c, d) denote significant difference between groups. Data are represented as mean ± SEM.

Fig. 3

Effect of nesfatin-1 on hepatic glycogenolysis and glucose production (a) Hepatic glycogen content decreased significantly while (b) media glucose level showed marked increase upon treatment of wall lizard liver with varying concentrations of nesfatin-1 for 6 h. Liver incubated in medium without nesfatin-1 was used as control. Data were analysed using one-way ANOVA (P < 0.05) followed by Bonferroni test (P < 0.05) and represented as mean ± SEM (N = 5). Significant variation is represented by different superscript (a, b, c).

Fig. 4

Effect of feeding/fasting on the expression of nucb2/nesfatin-1 and lipolytic markers in the adipose tissue of wall lizard. Changes in mRNA expression of (a) nucb2/nesfatin-1, (b) atgl, and (c) mgl was evaluated in the adipose tissue of food-deprived (black bars) as well as well-fed (shaded bars) wall lizards (N = 5/group). Fasting-/feeding-induced variation in gene expression was analysed using Student’s unpaired t-test (P < 0.05) which showed significant stimulation of all three genes after 15 days of fasting. Data are presented as mean ± SEM. Significant difference between groups is denoted by asterisk (*).

Fig. 5

Nesfatin-1-mediated regulation of lipolytic markers in the adipose tissue of wall lizard. Expression of (a) atgl and (b) mgl in control (without nesfatin-1) and experimental (nesfatin-1-treated) adipose tissue showed a significant increase in expression of both the genes upon direct treatment with the peptide. The qRT-PCR data were analysed using one-way ANOVA followed by Bonferroni test (P < 0.05). Data is represented as mean ± SEM (N = 5/group) and significant variation is denoted by different alphabets (a, b).

Fig. 6

Fasting-induced variation in pancreatic nucb2/nesfatin-1 and insulin expression in wall lizard. Significant decline in mRNA expression of (a) nucb2/nesfatin-1 and (b) insulin in wall lizard pancreas (N = 5/group) upon 3, 7, and 15 days of fasting (black bars) in comparison to fed (shaded bars) condition. Data were assessed using Student’s unpaired t-test (P < 0.05). Data are represented as mean ± SEM and significant difference is denoted by asterisk (*).

Fig. 7

Insulinotropic action of nesfatin-1 in pancreas of wall lizard: in vitro study. Wall lizard pancreas incubated (1 h) with varying concentrations of nesfatin-1 (0.1 nM, 1 nM and 10 nM) in the presence of low (2 mM) as well as high glucose (16.7 mM) were assessed for insulin mRNA expression. Nesfatin-1 demonstrated an insulinotropic effect on pancreas which was not affected by low or high glucose levels. Data were analysed by two-way ANOVA (P < 0.05) followed by Bonferroni test (P < 0.05). Different alphabets (a, b) indicate significant difference in pancreatic insulin after treatment with varying concentrations of nesfatin-1 in low glucose condition while different numerals (1, 2) denote significant change in gene expression upon treatment with nesfatin-1 in the presence of high media glucose.

Fig. 8

Regulation of pancreatic nucb2/nesfatin-1 by insulin. A significant decline in wall lizard pancreatic nucb2/nesfatin-1 was observed after 6 h incubation with varying concentrations of insulin (N = 4/group). Variation in the expression of nucb2/nesfatin-1 upon treatment with insulin was analysed using one-way ANOVA followed by Bonferroni test (P < 0.05). Bars with different superscript (a, b) denote significant difference between groups. Data are presented as mean ± SEM.

Fig. 9

Diagrammatic representation of nesfatin-1- mediated regulation of glucose/energy homeostasis in wall lizard, Hemidactylus flaviviridis. Under fasting condition when glucose availability is low, nesfatin-1 initiates hepatic glucose production by activating both gluconeogenesis and glycogenolysis. This is evidenced by the upregulation of glyp, pepck, and fbp gene expression along with decline in liver glycogen stores. Nesfatin-1 also stimulates lipolysis by upregulating the expression of atgl and mgl which may be instrumental in providing substrate for gluconeogenesis as well as energy production. However, under well fed condition nesfatin-1 might induce insulin-dependent glucose uptake in the liver. Thus, nesfatin-1 appears to exert a tissue-specific and fed/fasted state-dependent dual role in maintaining glucose homeostasis depending upon the energy status of wall lizard. Moreover, a regulatory feedback loop between nesfatin-1 and insulin is evident in pancreas, as insulin downregulates pancreatic nucb2/nesfatin-1 expression. In the visual representation, yellow arrows indicate upregulation of gene expression, solid black and red arrows denote experimentally confirmed pathway while dotted arrows illustrate nesfatin-1-mediated possible mechanisms in wall lizard that warrant further investigation. Abbreviations: glyp- glycogen phosphorylase, pepck- phosphoenolpyruvate carboxykinase, fbp- fructose-1,6-bisphosphatase, atgl- adipose triglyceride lipase, and mgl- monoacylglycerol lipase.