Hypothalamic astroglial connexins are required for brain glucose sensing-induced insulin secretion

Abstract

Hypothalamic glucose detection participates in maintaining glycemic balance, food intake, and thermogenesis. Although hypothalamic neurons are the executive cells involved in these responses, there is increasing evidence that astrocytes participate in glucose sensing (GS); however, it is unknown whether astroglial networking is required for glucose sensitivity. Astroglial connexins 30 and 43 (Cx30 and Cx43) form hexameric channels, which are apposed in gap junctions, allowing for the intercellular transfer of small molecules such as glucose throughout the astroglial networks. Here, we hypothesized that hypothalamic glucose sensitivity requires these connexins. First, we showed that both Cxs are enriched in the rat hypothalamus, with highly concentrated Cx43 expression around blood vessels of the mediobasal hypothalamus (MBH). Both fasting and high glycemic levels rapidly altered the protein levels of MBH astroglial connexins, suggesting cross talk within the MBH between glycemic status and the connexins' ability to dispatch glucose. Finally, the inhibition of MBH Cx43 (by transient RNA interference) attenuated hypothalamic glucose sensitivity in rats, which was demonstrated by a pronounced decreased insulin secretion in response to a brain glucose challenge. These results illustrate that astroglial connexins contribute to hypothalamic GS.

Figure 1

Cx43 and Cx30 protein levels in various brain areas. (A,B). Relative protein quantification (upper panel) and representative western blots (lower panel) for Cx43 (n=4) and Cx30 (n=8) in rat mediobasal hypothalamus (MBH), cortex (Ctx), thalamus (Th), hippocampus (Hp), cerebellum (Cb), and hindbrain (Hb). The results are expressed as a percentage of relative MBH protein levels for Cx43 or Cx30 taken as references. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) signal (density ratio of Cx/GAPDH) was used as the control for protein loading. Comparison for each brain region relative to MBH was performed using a Student unpaired t-test for Cx30 and a Mann–Whitney test for Cx43, *P⩽0.05.

Figure 2

Hypothalamic Cx43 and Cx30 distribution. (A) Cx43 immunostaining in the hypothalamus and observations at higher magnification of the mediobasal area. Scale bar, 200 μm. (a, a′) Cx43 immunostaining in the mediobasal hypothalamus. Scale bar, 50 μm. (B) Double immunohistochemistry representing endothelial staining with von Willebrand Factor (in green, left panel), Cx43 staining (in red, central panel), and merged images (right panel). Scale bar, 20 μm. (C) Cx30 immunostaining in the hypothalamus and observations at higher magnification of the mediobasal area. Scale bar, 200 μm.

Figure 3

Changes in metabolic status and blood glucose concentration alter mediobasal hypothalamus (MBH) Cx43 and Cx30 protein levels. Relative protein level quantification (upper panel) and representative western blots (lower panel) of MBH Cx43 (left panel) and Cx30 (right panel) in panel A fed (black bar, n=8), 24-hour fasted (white bar, n=10) and 4-hour refed rats (gray bars, n=10), (B) in saline-injected (black bar, n=7) and 3-hour hyperglycemic rats (white bar, n=7; glycemia: 18.0±3.1 mmol/L), and (C) in saline-infused (black bars, n=11 for Cx43, n=5 for Cx30) versus 48-h hyperglycemic rats (white bars, n=15 for Cx43, n=8 for Cx30; glycemia: 15.1±0.7 mmol/L). The results are expressed as percentages of the respective control group after values have been normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) signal density (loading control). (A) One-way analysis of variance and (B,C) unpaired Student's t-test have been performed compared with respective control group, *P⩽0.05, **P⩽0.01 and ***P⩽0.001.

Figure 4

Transient mediobasal hypothalamus (MBH) Cx43 inhibition decreases protein levels of astroglial connexins Cx43 and Cx30, without altering main glucose and lactate transporters expression. (A) Relative protein quantification for MBH Cx43 (upper panel, siCtrl: n=16; siCx43: n=13) and representative western blots (lower panel). (B) Cx43 immunostaining in the MBH of siCx43 and siCtrl-injected rats after 72 hours. Scale bar, 200 μm. (C) Relative protein quantification for MBH Cx30 (upper panel, siCtrl: n=10; siCx43, n=11) and representative western blots (lower panel). (D) Relative Cx43 and Cx30 proteins quantification in the parietal cortex (Ctx) and hindbrain (Hb), compared with siCtrl (siCx43 and siCtrl: n=6). (E) Relative proteins quantification in MBH of 72 hours siCx43-treated rats, of astroglial transporters GLUT1, MCT1 (siCtrl, n=13; siCx43, n=11) and MCT4 (siCx43 and siCtrl: n=6); and neuronal transporters GLUT3 (siCx43 and siCtrl: n=6) and MCT2 (siCtrl: n=13; siCx43: n=12). The results are expressed as a percentage of siCtrl protein levels after the values were normalized to GAPDH signal density (loading control). Unpaired t-tests (or Mann–Whitney tests for the MCT1 western blot) have been performed compared with siCtrl group, **P⩽0.01, ***P⩽0.001.

Figure 5

Transient downregulation of mediobasal hypothalamus (MBH) Cx43 inhibits hypothalamic glucose sensing in vivo. (A) Peripheral blood glucose levels after 9 mg/kg intracarotid glucose injection in siCtrl (full black line; n=10) and siCx43 rats (dotted black line; n=7). (B) Delta plasma insulin levels (values compared with time 0 minutes before injection) after 9 mg/kg intracarotid glucose injection in siCtrl and siCx43 rats. Statistical analysis was performed using a repeated measures two-way analysis of variance followed by a Bonferroni post hoc test ** P⩽0.01.