Alloxan-induced diabetes exacerbates coronary atherosclerosis and calcification in Ossabaw miniature swine with metabolic syndrome

Abstract

Background: There is a preponderance of evidence implicating diabetes with increased coronary artery disease (CAD) and calcification (CAC) in human patients with metabolic syndrome (MetS), but the effect of diabetes on CAD severity in animal models remains controversial. We investigated whether diabetes exacerbates CAD/CAC and intracellular free calcium ([Ca²⁺]_i) dysregulation in the clinically relevant Ossabaw miniature swine model of MetS.

Methods: Sixteen swine, eight with alloxan-induced diabetes, were fed a hypercaloric, atherogenic diet for 6 months. Alloxan-induced pancreatic beta cell damage was examined by immunohistochemical staining of insulin. The metabolic profile was confirmed by body weight, complete blood panel, intravenous glucose tolerance test (IVGTT), and meal tolerance test. CAD severity was assessed with intravascular ultrasound and histology. [Ca²⁺]_i handling in coronary smooth muscle (CSM) cells was assessed with fura-2 ratiometric imaging.

Results: Fasting and post-prandial blood glucose, total cholesterol, and serum triglycerides were elevated in MetS-diabetic swine. This group also exhibited hypoinsulinemia during IVGTT and less pancreatic beta cell mass when compared to lean and MetS-nondiabetic swine. IVUS analysis revealed that MetS-diabetic swine had greater percent wall coverage, percent plaque burden, and calcium index when compared to lean and MetS-nondiabetic swine. Fura-2 imaging of CSM [Ca²⁺]_i revealed that MetS-nondiabetic swine exhibited increased sarcoplasmic reticulum Ca²⁺ store release and Ca²⁺ influx through voltage-gated Ca²⁺ channels compared to lean swine. MetS-diabetic swine exhibited impaired Ca²⁺ efflux.

Conclusions: Diabetes exacerbates coronary atherosclerosis and calcification in Ossabaw miniature swine with MetS, accompanied by progression of [Ca²⁺]_i dysregulation in advanced CAD/CAC. These results recapitulate increased CAD in humans with diabetes and establish Ossabaw miniature swine as an animal model for future MetS/diabetes comorbidity studies.

Keywords: Atherosclerosis; Calcium dysregulation; Calcium index; Intravascular ultrasound; Metabolism; Obesity.

Fig. 1

Impaired glucose clearance after a meal tolerance test in MetS/D-A swine. a Plasma glucose was monitored for 7 h after a meal, which revealed the lean and MetS group had comparable glucose clearance and the MetS/D-A group had impaired glucose clearance. p values are compared to lean group. b Area under the curve analysis shows postprandial hyperglycemia in the MetS/D-A swine as compared to all other groups. *p < 0.05 compared with lean swine; ^†p < 0.05 compared with MetS swine; ^‡p < 0.05 compared with MetS-A swine. (Lean = 8; MetS = 3; MetS-A = 3; MetS/D-A = 3.)

Fig. 2

IVGTT supports the conclusion that MetS/D-A swine exhibit impaired glucose clearance. a Blood glucose levels were tested for 60 min after a bolus of glucose (0.5 mg/kg body weight). p values are compared to lean group. b Area under the curve analysis shows impaired glucose clearance in the MetS-A and MetS/D-A groups. c MetS/D-A swine exhibited a fourfold higher fasting blood glucose before the bolus of glucose was administered. *p < 0.05 compared with lean swine; ^†p < 0.05 compared with MetS swine; ^‡p < 0.05 compared with MetS-A swine. (Lean = 10; MetS = 5; MetS-A = 8; MetS/D-A = 8.)

Fig. 3

Serum insulin levels were lower in the MetS/D-A group than in the MetS-A group. a Insulin levels were measured at minutes 0, 10, 20, 40, and 60 during the IVGTT protocol. b Area under the curve analysis revealed that MetS/D-A swine exhibited hypoinsulinemia as compared to the swine in the lean and MetS groups. *p < 0.05 compared with lean swine; ^†p < 0.05 compared with MetS swine. (Lean = 10; MetS = 5; MetS-A = 8; MetS/D-A = 8.)

Fig. 4

IHC shows diminished pancreatic beta cell mass in MetS-A and MetS/D-A swine. Immunohistochemical analysis using antibodies against insulin revealed that beta cell mass was decreased in MetS-A swine as compared to lean swine, and even less in MetS/D-A swine. Taken together, these data show that swine in the MetS/D-A group were indeed diabetic, due to alloxan-induced beta-cell damage. *p < 0.05 compared with lean swine; ^‡p < 0.05 compared with MetS-A swine. (Lean = 7; MetS-A = 7; MetS/D-A = 8.)

Fig. 5

MetS/D-A swine had more advanced disease than lean and MetS-A swine. a Angiogram showing the LAD and CFX coronary arteries. All IVUS still frames collected for analysis were from the proximal 45 mm of the LAD. b Representative IVUS image of the LAD of a MetS/D-A swine showing 100% wall coverage, the percent of the circumference of the arterial wall covered by intimal thickening (original lumen highlighted in red, intimal thickening highlighted in yellow). c Representative IVUS image of the LAD of a MetS/D-A swine illustrating plaque burden, the percent of original lumen that is now occupied by a lesion (original lumen highlighted in red, new lumen with lesion highlighted in yellow). d The MetS/D-A swine had significantly greater wall coverage compared to MetS-A and lean swine. e The MetS/D-A swine had significantly greater plaque burden, compared to MetS-A and lean swine. *p < 0.05 compared with lean swine; ^‡p < 0.05 compared with MetS-A swine. (Lean = 6; MetS-A = 7; MetS/D-A = 7.)

Fig. 6

MetS/D-A swine showed greater spotty calcification compared to lean swine and MetS-A swine. a Representative IVUS image from the proximal LAD of a MetS/D-A swine. IVUS images were analyzed for calcification severity by using the calcium index measurement. The lumen is outlined in red, with the arc of spotty calcification and acoustic shadowing outlined in yellow. b Representative Von Kossa-stained histological section from the proximal LAD of a MetS/D-A swine, with noticeable spotty calcification in the neointimal layer (blue arrows). c Calcium index is higher in MetS/D-A swine as compared to lean and MetS-A swine. d Findings from analysis of IVUS data correlate to the Von Kossa histological staining analysis. *p < 0.05 compared with lean swine; ^‡p < 0.05 compared with MetS-A swine. (Lean = 6; MetS-A = 7; MetS/D-A = 7.)

Fig. 7

MetS-A swine showed greater SR store and VGCC function compared to lean and MetS/D-A swine. a After depolarizing the cell with an 80 mM K⁺ solution to induce calcium influx and maximal loading of the sarcoplasmic reticulum (SR) store, the SR store was released by activating ryanodine receptors with 5 mM caffeine. The time to half signal recovery was measured during caffeine wash-out and corresponds to the activity of calcium extrusion mechanisms. b A similar protocol was used with an additional exposure to a 2 mM barium solution, which enters the cell via voltage-gated calcium channels and binds to fura-2, but is not transported by Ca²⁺ ATPases. Barium as a [Ca²⁺]_i surrogate thereby provides a more selective measure of Ca entry. c The measured SR store release from CSM in MetS-A swine is greater than that seen in MetS/D-A and lean swine. This is consistent with our previous findings which show biphasic calcium handling alterations in CSM as CAD progresses in severity. d Extrusion mechanism activity as measured by time to half recovery is impaired in MetS/D-A swine. e The activity of voltage-gated calcium channels as measured by barium influx is increased in CSM from MetS-A swine and decreases back to baseline in CSM from MetS/D-A swine. This, again, is consistent with the biphasic pattern seen in CAD progression. *p < 0.05 compared with lean swine; ^‡p < 0.05 compared with MetS-A swine. (Lean = 6; MetS-A = 8; MetS/D-A = 8.)