Proteinase activity and receptor cleavage: mechanism for insulin resistance in the spontaneously hypertensive rat

Abstract

Arterial hypertension is associated with organ dysfunctions, but the mechanisms are uncertain. We hypothesized that enhanced proteolytic activity in the microcirculation of spontaneously hypertensive rats (SHRs) may be a pathophysiological mechanism causing cell membrane receptor cleavage and examine this for 2 different receptors. Immunohistochemistry of matrix-degrading metalloproteinases (matrix metalloproteinase [MMP]-9) protein shows enhanced levels in SHR microvessels, mast cells, and leukocytes compared with normotensive Wistar-Kyoto rats. In vivo microzymography shows cleavage by MMP-1 and -9 in SHRs that colocalizes with MMP-9 and is blocked by metal chelation. SHR plasma also has enhanced protease activity. We demonstrate with an antibody against the extracellular domain that the insulin receptor-alpha density is reduced in SHRs, in line with elevated blood glucose levels and glycohemoglobin. There is also cleavage of the binding domain of the leukocyte integrin receptor CD18 in line with previously reported reduced leukocyte adhesion. Blockade of MMPs with a broad-acting inhibitor (doxycycline, 5.4 mg/kg per day) reduces protease activity in plasma and microvessels; blocks the proteolytic cleavage of the insulin receptor, the reduced glucose transport; normalizes blood glucose levels and glycohemoglobin levels; and reduces blood pressure and enhanced microvascular oxidative stress of SHRs. The results suggest that elevated MMP activity leads to proteolytic cleavage of membrane receptors in the SHR, eg, cleavage of the insulin receptor-binding domain associated with insulin resistance.

Figure 1

(A) MMP-9 levels as detected by labeling with primary antibody and Vector NovaRED substrate in selected circulating leukocytes of SHR and WKY before (left panels) and after treatment (right panels) with doxycycline. (B) Light absorption (see Methods) due to MMP 9 labeling over individual leukocytes in control (C) and doxycycline treated rats (D). The number of measurements in each group is 90 cells derived from 3 animals in each animal group. *p<0.05 versus WKY, **p<0.05 versus WHY without doxycycline treatment (control), †† p<0.05 versus SHR without doxycycline.

Figure 2

(A) Selected micrographs of microvessels and interstitium of WKY and SHR mesentery after MMP-9 immunolabeling with Vector NovaRED substrate. Note labeling in SHR endothelial cells of arterioles (A) and venules (V) as well as in interstitial mast cells (arrows). (B) MMP-9 protein levels as detected by light absorption measurement of Vector NovaRED substrate without (control, C) and with doxycycline (D) treatment. The number of measurements in each microvessel type and avascular area is 90 derived from 3 rats in each animal group. *p<0.05 versus WKY. p<0.05 WKY control vs WKY after doxycycline; SHR control vs SHR after doxycycline. ‡ p<0.05 WKY vs SHR after doxycycline.

Figure 3

(A) Digital fluorescent micrographs of WKY and SHR mesenteric microvessels labeled with fluorogenic peptide substrate showing matrix metalloproteinase (MMP-1, 9) enzymatic activity. Arterioles (A) and venules (V) are visible. Note the enhanced fluorescent emission over the endothelial cells and mast cells in the SHR, an affect that is less detectable after the doxycycline treatment. (B) MMP-9 activity levels as detected by fluorescent substrate intensity in mesentery of control WKY and SHR without (C) and with doxycycline (D) treatment. The number of measurements in each vessel type and in the avascular area is 90 derived from 3 mesenteries in each animal group. *p<0.05 versus WKY, ††p<0.05 versus same strain without doxycycline treatment.

Figure 4

Plasma protease activity values as determined by fluorescent units without (control, C) and with doxycycline (D) treatment. The number of measurements is n = 3 in control WKY and SHR and n = 4 in the doxycycline treated WKY and SHRs. *p< 0.05 versus WKY, **p<0.05 versus WHY without doxycycline treatment (C), and ††p<0.05 versus SHR without doxycycline.

Figure 5

(A) Typical micrographs of immunolabel (Vector NovaRed ) for the extracellular domain of the insulin receptor-α on fresh leukocytes (neutrophils and monocytes) from WKY and SHR. The left panels show leukocytes from control rats and the right panels from rats after doxycycline treatment. (B) Insulin receptor-α density measured by light absorption after labeling with a primary antibody against the extracellular domain of the receptor and Vector NovaRed substrate. Notation is the same as in Figure 1B. Mean ± standard deviation in each group is derived from 30 cells per rat with 3 rats in each animal group. *p<0.05 versus WKY, †† p<0.05 versus same strain without doxycycline treatment (control, C).

Figure 6

(A) Micrographs of a typical fresh neutrophil from a donor Wistar rat (left image) on a blood smear after labeling with antibody against the extracellular domain of insulin receptor-α. The Wistar cells were exposed for 30 min to plasma from WKY (second from left) and SHR without (middle image) and with plasma for a WKY (WKY-Doxy image) and SHR (SHR-Doxy image) after chronic doxycycline treatment. (B) Insulin receptor-α density measured by light absorption after labeling with a primary antibody against the extracellular domain of the receptor and Vector NovaRed substrate. Groups are the same as in part A, without (C) and with doxycycline (D) treatment. Mean ± standard deviation in each group is derived from 30 cells per rat with 3 rats in each animal group. *p<0.05 versus Wistar (not shown at values = 1), †† p<0.05 versus control SHR without doxycycline treatment p<0.05 versus WKY with doxycycline treatment.

Figure 7

(A) Micrographs of a typical fresh neutrophil from a donor Wistar rat (left image) on a blood smear after labeling with an antibody against extracellular domain of CD18 integrin. The Wistar cells were exposed for 30 min to plasma from WKY (second from left) and SHR without (middle image) and with plasma for a WKY (WKY-Doxy image) and SHR (SHR-Doxy image) after chronic doxycycline treatment. Note that the antibody labels predominantly CD18 in the cell membrane. (B) Normalized light intensity of CD18 label on leukocytes versus values on a naïve Wistar donor (normalized to 1, not shown). The intensity measurements were made in a ring region over individual leukocytes with CD18 label. Groups are the same as in part A, without (C) and with doxycycline (D) treatment. Mean ± standard deviation in each group is derived from 30 cells per rat with 3 rats in each group. *p<0.05 versus values in Wistar rat leukocytes, †† p<0.05 versus SHR without doxycycline treatment.

Figure 8

(A) Bright field micrographs of mesentery after TNBT labeling in WKY and SHR before and after doxycyline treatment. Note the reduction of dark (blue/red) tetrazolium deposits in all microvessel types, arterioles (A), capillaries (C), and venules (V), after doxycycline treatment. (B) Light absorption measurements of zymogen deposits in the rat mesentery of SHR and WKY rats without (C) and with treatment by doxycycline (D). Mean ± standard deviation in each blood vessel type and in the avascular area is 30 per rat with 3 rats in each animal group. *p<0.05 versus WKY, †† p<0.05 versus same rat strain without doxycycline treatment, **p<0.05 versus WKY with doxycycline treatment.

Figure 9

(A) Bright field micrographs of typical SHR and WKY leukocytes before and after doxycyline treatment after immunolabeling for NF-κB. (B) Mean ± standard deviation of substrate density due to NF-κB immunolabeling in leukocytes of SHR and WKY rats without (C) and with treatment by doxycycline (D). Absorption values are derived from 90 randomly selected cells of three rats in each group and 3 separate measurements per cell. †p<0.05 versus control (C), p<0.05 versus control SHR without doxycycline treatment, *p<0.05 versus WKY with doxycycline treatment (D).