Disrupted and Elevated Circadian Secretion of Glucagon-Like Peptide-1 in a Murine Model of Type 2 Diabetes

Abstract

Metabolism and circadian rhythms are intimately linked, with circadian glucagon-like peptide-1 (GLP-1) secretion by the intestinal L-cell entraining rhythmic insulin release. GLP-1 secretion has been explored in the context of obesogenic diets, but never in a rodent model of type 2 diabetes (T2D). There is also considerable disagreement regarding GLP-1 levels in human T2D. Furthermore, recent evidence has demonstrated decreased expression of the β-cell exocytotic protein secretagogin (SCGN) in T2D. To extend these findings to the L-cell, we administered oral glucose tolerance tests at 6 time points in 4-hour intervals to the high-fat diet/streptozotocin (HFD-STZ) mouse model of T2D. This revealed a 10-fold increase in peak GLP-1 secretion with a phase shift of the peak from the normal feeding period into the fasting-phase. This was accompanied by impairments in the rhythms of glucose, glucagon, mucosal clock genes (Arntl and Cry2), and Scgn. Immunostaining revealed that L-cell GLP-1 intensity was increased in the HFD-STZ model, as was the proportion of L-cells that expressed SCGN; however, this was not found in L-cells from humans with T2D, which exhibited decreased GLP-1 staining but maintained their SCGN expression. Gcg expression in isolated L-cells was increased along with pathways relating to GLP-1 secretion and electron transport chain activity in the HFD-STZ condition. Further investigation into the mechanisms responsible for this increase in GLP-1 secretion may give insights into therapies directed toward upregulating endogenous GLP-1 secretion.

Keywords: GIP; GLP-1; diurnal; enteroendocrine; glucose; insulin.

Figure 1.

HFD-STZ mice exhibit decreased pancreatic insulin and secretagogin. C57Bl/6J mice fed a high-fat diet (HFD) were treated with streptozotocin (STZ) as indicated or were fed regular chow (RC, control) followed by 2 oral glucose tolerance tests (OGTTs) separated by 1 week and then sacrificed (A, n = 15-20). Percent peripheral fat (B, n = 8) and overnight fasting blood glucose (C, n = 8) at 8 weeks. Representative images of INS and SCGN in pancreatic sections (D), β-cell area (E, n = 4-6), INS staining intensity (F, n = 4-6), SCGN area (G, n = 4-6) and SCGN staining intensity (H, n = 4-6). Light/dark calorie consumption at 8 weeks (I, n = 8). *P < 0.05, **P < 0.01, *** P < 0.001.

Figure 2.

HFD-STZ mice exhibit hyperglycemia and hyperglucagonemia as well as abnormal circadian rhythms in response to timed OGTTs. OGTTs were administered at 6 time points (ZT2, 6, 10, 14, 18, and 22; shaded area from ZT12 to ZT24 indicates lights-out) over the 24-hour clock to 4-hour fasted C57Bl/6J RC and HFD-STZ mice (n = 10), expressed as ∆AUC responses in plasma glucose (A), insulin (B) and glucagon (C). Peak time points of significant rhythms are shown in the boxed insets and are color-coded accordingly.*P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3.

HFD-STZ mice exhibit increased GLP-1 and GIP levels and altered rhythms in response to timed OGTTs. OGTTs were administered at 6 time points (ZT2, 6, 10, 14, 18, and 22; shaded area from ZT12 to ZT24 indicates lights-out) over the 24-hour clock to 4-hour fasted C57Bl/6J RC and HFD-STZ mice (n = 10), expressed as ∆AUC responses in GLP-1 (A) and GIP (B). Peak time points of significant rhythms are shown in the boxed insets and are color-coded accordingly. *P < 0.05, ***P < 0.001.

Figure 4.

HFD-STZ animals exhibit altered patterns in clock gene and Scgn expression in the ileal mucosa. mRNA was extracted from ileal mucosal scrapes at 6 time points (ZT2, 6, 10, 14, 18, and 22; shaded area from ZT12 to ZT24 indicates lights-out) over the 24-hour clock in 4-hour fasted C57Bl/6J RC (black) and HFD-STZ (red) mice (n = 4-5) and analyzed by qRT-PCR for Arntl (A), Per2 (B), Cry2 (C), Sirt1 (D), Scgn (E), and Gcg (F). Peak time points of significant rhythms are shown in the boxed insets and are color-coded accordingly. *P < 0.05.

Figure 5.

HFD-STZ mice exhibit an increase in L-cell GLP-1 and SCGN expression. Murine ileal sections were immunostained for GLP-1 and SCGN and the percentage of SCGN+/GLP-1- and SCGN+/GLP-1 + cells was determined in C57Bl/6J RC (A, n = 4-6) and HFD-STZ mice (B, n = 4-6); representative images are shown (DAPI: blue). (C-E) L-cell GLP-1 (C, n = 4-6) and SCGN (D, n = 4-6) intensity, and the number of L-cells per section (E, n = 6). *P < 0.05.

Figure 6.

Altered gene expression in L-cells from Gcg-Venus HFD-STZ mice. Volcano plot of the transcriptomes of L-cells isolated from Gcg-Venus RC and HFD-STZ mice at ZT14 (A). (B-C) Barcode plots of L-cell GLP-1 Secretion (B) and Electron Transport Chain (C) pathways. EnrichmentMap analysis comparing L-cells from Gcg-Venus RC and HFD-STZ mice (D). n = 8 (4 males + 4 females per condition).

Figure 7.

L-cells from HFD-STZ mice exhibit increased expression of unfolded protein response genes and CHOP intensity. Barcode plots (n = 8) of the Unfolded Protein Response pathway in L-cells isolated from Gcg-Venus RC and HFD-STZ mice at ZT14 (A). Representative ileal sections from C57Bl/6J mice co-stained for CHOP and GLP-1 SCGN (DAPI: blue) (B), Pearson’s Coefficient analysis of CHOP co-localization with GLP-1 (C, n = 4-6), and L-cell CHOP intensity (D, n = 4-6). *P < 0.05, **P < 0.01.

Figure 8.

Ileal sections from subjects with type 2 diabetes exhibit decreased GLP-1 expression. (A-D) Human ileal sections from healthy controls (CON) and subjects with T2D (DIA) were co-stained for GLP-1 and SCGN (n = 3-5). (A-B) Percentage of SCGN+/GLP-1- and SCGN+/GLP-1 + cells in control (A) and diabetic (B) subjects; representative images are shown (DAPI: blue). (C-D) L-cell GLP-1 (C) and SCGN (D) staining intensity. (E-H) Human ileal sections from healthy controls and subjects with T2D were analyzed by RNAscope for GCG and SCGN expression (n = 3-5). (E-F) Representative images are shown for control (E) and diabetic (F) subjects. (G-H) GCG (G) and SCGN (H) intensity. *P < 0.05.