Dipeptidylpeptidase inhibition is associated with improvement in blood pressure and diastolic function in insulin-resistant male Zucker obese rats

Abstract

Diastolic dysfunction is a prognosticator for future cardiovascular events that demonstrates a strong correlation with obesity. Pharmacological inhibition of dipeptidylpeptidase-4 (DPP-4) to increase the bioavailability of glucagon-like peptide-1 is an emerging therapy for control of glycemia in type 2 diabetes patients. Accumulating evidence suggests that glucagon-like peptide-1 has insulin-independent actions in cardiovascular tissue. However, it is not known whether DPP-4 inhibition improves obesity-related diastolic dysfunction. Eight-week-old Zucker obese (ZO) and Zucker lean rats were fed normal chow diet or diet containing the DPP-4 inhibitor, linagliptin (LGT), for 8 weeks. Plasma DPP-4 activity was 3.3-fold higher in ZO compared with Zucker lean rats and was reduced by 95% with LGT treatment. LGT improved echocardiographic and pressure volume-derived indices of diastolic function that were impaired in ZO control rats, without altering food intake or body weight gain during the study period. LGT also blunted elevated blood pressure progression in ZO rats involving improved skeletal muscle arteriolar function, without reducing left ventricular hypertrophy, fibrosis, or oxidative stress in ZO hearts. Expression of phosphorylated- endothelial nitric oxide synthase (eNOS)(Ser1177), total eNOS, and sarcoplasmic reticulum calcium ATPase 2a protein was elevated in the LGT-treated ZO heart, suggesting improved Ca(2+) handling. The ZO myocardium had an abnormal mitochondrial sarcomeric arrangement and cristae structure that were normalized by LGT. These studies suggest that LGT reduces blood pressure and improves intracellular Cai(2+) mishandling and cardiomyocyte ultrastructure, which collectively result in improvements in diastolic function in the absence of reductions in left ventricular hypertrophy, fibrosis, or oxidative stress in insulin-resistant ZO rats.

Figure 1.

Linagliptin (LGT) reduces DPP-4 activity in the plasma (A) and myocardium (B) of Zucker Lean (ZL) and Obese rats. C, DPP-4 protein expression is elevated in control- and LGT-treated ZO rat hearts compared to lean counterparts. D, Twenty four hour ambulatory mean arterial pressure (MAP) of Zucker rats. Symbols indicate differences in MAP between treatment groups within a single time period. P < .05, *, ZLC vs ZOC; ‡, ZLC vs ZLL; †, ZOC vs ZOL; and Δ, ZOC vs ZLL.

Figure 2.

Zucker Obese (ZO) rats exhibit diastolic dysfunction (A–E) and impaired vasomotor reactivity of skeletal muscle arterioles (F and G) which are ameliorated by the DPP-4 inhibitor, linagliptin (LGT). A, Representative tissue doppler images are shown of the early and late (E′ and A′) motion of the septal annulus during diastole and during early systole (S′). Mean group values for E′, A′ and E′/A′ are listed in Table 2. B, Representative pressure-volume loops are shown of ZOC and ZOL during pre-load reduction. C–E, Regression analysis indicates significant relationships between tau (t), the time constant of isovolumic relaxation, generated by PV analysis and echocardiography-derived parameters of early diastolic relaxation. Vp = propagation velocity of mitral inflow; IVRT = isovolumic relaxation time; E′ = velocity of early septal annular wall motion determined by pulse wave echocardiography for the same rats. Data points were plotted from ZOC and ZOL rats that had both cardiac procedures. Lintagliptin treatment improved vasomotor reactivity of skeletal muscle arterioles. Concentration-response curves of gastrocnemius 1A arterioles to acetylcholine (F) and sodium nitroprusside (G). Values are mean ± SE, sample size in parentheses. *, P < .05 for ZOC versus all other groups (F) and ZLC & ZLL versus all other groups (G).

Figure 3.

Linagliptin (LGT) effects on myocardial Ca+2 handling proteins. Bar graphs show β-actin normalized protein levels of phosphorylated-PLBser16 (A), total PLB (B), SERCA2a (C), total PLB/SERCA2a ratio (D), phosphorylated-SERCA2aTyr294/295 (E), and glutathiolated SERCA2a (F) at the 674 residue and from LV homogenates control and LGT treated ZL and ZO rats. Representative protein bands with corresponding β-actin bands are shown above bar graphs. P < .05; †, ZOC vs ZOL.

Figure 4.

Linagliptin (LGT) abrogates excessive numbers of intermyofibrillar mitochondria in Zucker Obese (ZO) Rats. Panel A illustrates the normal mitochondrial morphology (arrows) in the intermyofibrillar regions between sarcomeres (S) in the Zucker Lean control (ZLC) rats. Panel B reveals a similar mitochondrial morphology and sarcomere arrangement in the Zucker Lean linagliptin (ZLL) compared to ZLC. Panel C depicts mitochondrial biogenesis in the intermyofibrillar regions between sarcomeres. Note that the marked expansion of intermyofibrillar mitochondria (Mt) distorts the normal sarcomere morphology compared to A, B, and D. Insert in panel C depicts the abnormal mitochondrial morphology consisting of loss of matrix electron density and abnormal cristae compared to the cristae shown in the insert in panel D of linagliptin treated models (ZOL). Panel D demonstrates the normalization – abrogation of mitochondrial biogenesis in the ZO rats treated with linagliptin (ZOL) models. Insert depicts a more electron dense matrix and improved cristae. Magnification ×2000; bar = 0.5μm panels A–D. Magnification ×4000; bar = 0.2μm in insert panels C and D.

Figure 5.

Cardiac insulin signaling is improved in the Zucker Obese (ZO) heart. Bar graphs show levels of total IRS-1/actin in myocardial protein extracts from control or LGT-treated Zucker lean (ZL) and obese rats (A). Bar graphs show levels of total Akt (B), phosphorylated-Aktthr308 (C), phosphorylated-Aktser473 (E) and D and F show the ratios of each phosphorylated-Akt to total Akt. Representative bands are shown above bar graphs. Values are mean ± SE. P < .05; *, ZLC vs ZOC; †, ZOC vs ZOL.

Figure 6.

Linagliptin (LGT) treatment increases myocardial eNOS expression in the Zucker Obese (ZO) heart. Bar graphs show levels of phosphorylated-eNOSser1177 (A) and total eNOS protein (B) normalized to actin in myocardial protein extracts. Representative bands are shown above bar graphs. Values are mean ± SE. P < .05; *, ZLC vs ZOC; †, ZOC vs ZOL.