Low Intrinsic Aerobic Exercise Capacity and Systemic Insulin Resistance are not Associated with Changes in Myocardial Substrate Oxidation or Insulin Sensitivity

Abstract

BACKGROUND

In patients, inactivity, obesity and insulin resistance are associated with increased incidence of heart failure. Rats selectively bred for low (LCR) intrinsic aerobic exercise capacity show signs of the metabolic syndrome inducing insulin resistance, compared to their counterparts bred for high intrinsic aerobic capacity (HCR).

HYPOTHESIS

We reasoned that systemic insulin resistance in LCR should translate to impaired substrate oxidation and reduced insulin sensitivity in the heart.

METHODS

Isolated hearts were perfused in the working mode to analyze cardiac function, substrate oxidation patterns, insulin response, and oxygen consumption.

RESULTS

After 22 generations of selective breeding, LCR displayed reduction of exercise capacity (LCR vs. HCR: distance 280±12vs.1968±63m, time 19.5±0.6vs.71.7±1.4min, speed 19.2±0.3vs.45.3±0.7m/min; all p<0.05). At 21 weeks, body weight (+34%), tibia length (+6%), heart weight (+31%), and heart weight to tibia length ratio (+24%; all p<0.05) were increased. LCR display higher random glucose, higher fasting glucose, and higher insulin levels in serum than HCR indicating the presence of insulin resistance in LCR. Here, in contrast, isolated hearts showed no differences in glucose (0.22±0.02µmol/min/gdry) or fatty acid oxidation (0.79±0.10µmol/min/gdry), oxygen consumption (28.3±4.1nmolO₂/min/gdry) or cardiac power (18.6±1.6mW/gdry). Furthermore, sensitivity to insulin (Δglucose oxidation: +0.57±0.095µmol/min/gdry) was not different between the two populations.

CONCLUSION

Low intrinsic exercise capacity and systemic insulin resistance in rats is not associated with changes in cardiac substrate oxidation, insulin sensitivity, oxygen consumption, or cardiac function. The lack of cardiac insulin resistance in the face of systemic insulin resistance supports a concept of different pathomechanisms for these two conditions.

Introduction

Type 2 diabetes (T2DM) is one of the most prevalent metabolic disorders and a major health burden. One cardinal feature of diabetes mellitus is insulin resistance which is also an independent risk factor for heart failure [5, 15]. A causal relationship between systemic insulin resistance and heart failure has not been proven, but we recently demonstrated that pressure overload induces cardiac insulin resistance [25]. Cardiac insulin resistance precedes the onset of contractile dysfunction, and others have shown that systemic insulin resistance may cause cardiac insulin resistance [4].

Insulin resistance and T2DM depend on environmental risk factors and a genetic predisposition, and are considered complex (polygenic) diseases [31, 34]. Therefore, most animal studies relate to pharmacologial interventions or genetically modified animal models. As a suitable wild type model for complex diseases, we selectively bred normal N:NIH rats for high (HCR) and low (LCR) intrinsic oxidative running capacity for several generations. LCR display a reduced life span compared to HCR [17] and dysregulation of lipid metabolism in skeletal muscle [27]. These animals exhibit a cardiovascular phenotype with high blood pressure and endothelial dysfunction [33]. LCR display elevated fasting glucose and insulin levels [27, 33] compared to HCR and impaired response to oral and intraperitoneal glucose challenge [20, 22].

We reasoned that this systemic insulin resistance in LCR should translate to impaired substrate oxidation and reduced insulin sensitivity of the heart.

We therefore assessed functional and metabolic characteristics in hearts of rats bred for high and low intrinsic exercise capacity.

Materials and Methods

MATERIALS

Chemicals were obtained from Sigma Aldrich (Deisenhofen, Germany), Merck (Darmstadt, Germany), Serva (Heidelberg, Germany), Essex (München, Germany), Bayer (Leverkusen, Germany), Narkodorm-n (Neumünster, Germany) and BIO-RAD (München, Germany).

ANIMAL PROTOCOL

All animal procedures were approved by the the Animal Welfare Committee of the University of Leipzig, Germany. Animals were handled and housed in accordance with National Institutes of Health (NIH) guidelines. The creation of the high-/low-capacity rat (HCR/LCR) model of high and low intrinsic aerobic capacity has been described previously [16]. Briefly, bidirectionally selected lines were generated from a founder population of 80 male and 88 female N: NIH stock rats based on intrinsic aerobic treadmill running capacity. Thirteen families for each line were set up for a within-family rotational breeding paradigm that keeps the inbreeding at < 1% per generation. At each generation, young adult rats (11 weeks of age) were tested for their inherent ability to perform forced speed-ramped treadmill running until exhausted. This test was performed daily over 5 consecutive days. The greatest distance in meters achieved out of the 5 trials was considered the best estimate of an individual’s aerobic exercise capacity [16]. The highest scored female and male from each of the 13 families were selected as breeders for the next generation of high-capacity runners (HCR). The same process was used with lowest scored females and males to generate low-capacity runners (LCR). Male HCR and LCR rats (generation 22, 21 weeks of age) used for this investigation were housed in pairs in a temperature-controlled environment with a 12-hour light/dark cycle.

ISOLATED WORKING HEART PERFUSION

The preparation has been described in detail before by us [10, 24] and others [11]. Rats were anesthetized with sodium pentobarbital (5 mg/100 g body wt i.p.). After injection of heparin (200 IU) into the inferior vena cava, the heart was rapidly removed and placed in ice cold Krebs-Henseleit bicarbonate buffer. The aorta was freed of excess tissue and cannulated. A brief period of retrograde perfusion (less than 5 min) with oxygenated buffer containing glucose (10 mM) was necessary to wash out any blood from the heart and to perform left atrial cannulation. Hearts were then perfused as working hearts at 37° C with recirculating Krebs-Henseleit buffer (200 ml) containing 1 % bovine serum albumin, Cohn fraction V, fatty acid free (Celliance, Toronto, Canada). Perfusate calcium concentration was 2.5 mM. Hearts were perfused with glucose (5,0 mmol/l) and oleate (0,4 mmol/l) as substrates and insulin was present (1mU/ml). The perfusate was gassed with 95% O₂ - 5% CO₂, and recirculated. All experiments were carried out with a preload of 15 cm H₂O and an afterload of 100 cm H₂O. The hearts were beating spontaneously at a rate of approximately 250 beats/min. After stabilization hearts were perfused for a 30 minutes period, in which all samples were withdrawn and measurements performed. Aortic flow and coronary flow were measured every five minutes by timing the rise of the fluid meniscus in a calibrated glass tube [28]. Cardiac output was calculated as the sum of aortic and coronary flow. Heart rate as well as systolic and diastolic aortic pressure was measured continuously with a Hewlett-Packard transducer and recording system (Hewlett Packard, Waltham, Mass.). Mean aortic pressure (cm H₂O) was calculated as (systolic + diastolic pressure × 2) / 3. Heart rate was measured as beats per minute and cardiac output as ml/min. Cardiac power was determined as described before [6]. All hearts were perfused without and with insulin (1mU/ml) added. At the end of perfusion, hearts were freeze clamped and weighed. The wet to dry ratio was determined and the dry weight calculated.

SUBSTRATE OXIDATION RATES AND OXYGEN CONSUMPTION

Samples of the coronary effluent (2 ml) were withdrawn every 5 min for the assessment of glucose and fatty acid oxidation rates determined as the production of ¹⁴CO₂ from [U-¹⁴C]-glucose and ³H₂O from [9,10-³H]-oleate [7]. Oxygen consumption (MVO₂) was calculated by the following equation: MVO2 = [PO2 (oxygenated perfusate) − PO₂ (coronary effluent)] × Bunsen solubility coefficient of O2 × coronary flow. PO2 was measured by the use of a fiber-optic oxygen sensor (FOXY-AL300; Ocean Optics), which was connected to a spectrophotometer (USB2000-FL-450; Ocean Optics) [35]. Oxygen saturation of the coronary effluent was measured with the sensor in the samples drawn every 5 minutes.

CALCULATION OF CARDIAC EFFICIENCY

Cardiac efficiency is the ratio between cardiac hydraulic work and MVO2 [2]. Cardiac efficiency was calculated essentially as described previously [21] where the respiratory chain energy was taken to be 0,421 J/µmol⁻¹ [23]:

$$\text{Cardiac efficiency}=\frac{\text{Cardiac Power}[\frac{\mathrm{W}}{\text{gdry}}]*60}{\text{Oxygen Consumption}[\frac{\mathrm{\mu }\text{mol}}{\text{min}*\text{gdry}}]*\text{Respiratory Chain Energy}[\frac{\mathrm{J}}{\mathrm{\mu }\text{mol}}]}$$

DENATURING GEL ELECTROPHORESIS AND WESTERN BLOT

Proteins were isolated from frozen tissue samples. Western blots were performed on a Semidry-Western blot-Apparatus. After SDS-PAGE electrophoresis, proteins were blotted to a PVDF membran and incubated with primary and secondary antibodies. Bands were visualized and semi-quantified by chemiluminescence using AIDA software. Antibodies against Akt, phospho Ser473-Akt, IRS-1 and phospho Ser636/639-IRS-1 were from Cell Signaling, and secondary anitbodies from Amersham Biosciences or Sigma.

STATISTICAL ANALYSIS

Data are presented as mean ± SEM. Data were analyzed using a one-way analysis of variance or a student t-test where appropriate. Post-hoc comparisons among the groups were performed using Tukey`s test. Differences among groups were considered statistically significant if p < 0.05.

Results

Table 1 shows baseline characteristics and differences between LCR and HCR selectively bred for 22 generations. At the age of 21 weeks LCR showed higher body weight, heart weight, and tibia length. Heart weight to tibia length ratio was elevated as was the body mass to tibia index.

Table 1.

Baseline characteristics of HCR and LCR in generation 22

	HCR	LCR
Age [weeks]	20,8 ± 0,2	20,9 ± 0,2
Body weight [g]	321 ± 8	430 ± 13***
Heart weight dry [mg]	245 ± 8	321 ± 20***
Heart weight dry/body weight [g/Kg]	0,76 ± 0,03	0,74 ± 0,03
Tibia length [mm]	39,7 ± 0,7	41,9 ± 0,4***
Heart weight dry/tibia length [g/m]	6,18 ± 0,19	7,65 ± 0,45***
Body mass to tibia index [g/cm2]	20,4 ± 0,4	24,4 ± 0,5***

Data are mean ± SEM;

^***p<0,001, compared to HCR; HCR – high capacity runners, LCR – low capacity runners, n= 7–8 per group.

Figure 1 displays the differences in intrinsic exercise capacity as a result of selective breeding. HCR displayed a more athletic phenotype with a sevenfold greater distance run to exhaustion than LCR rats. HCR were also able to run faster and for an extended time period.

Figure 1.

Maximal distance run to exhaustion (A), maximal speed (B), and distance run to exhaustion (C) of HCR and LCR after 22 generations of divergent selection. Data are mean ± SEM, n = 10.

Figure 2 shows data from a separate set of experiments [33] for fasting glucose and insulin levels of both HCR and LCR. LCR had significantly elevated glucose and fasting insulin levels, indicating the presence of systemic insulin resistance. These findings were confirmed by five other studies assessing glucose and insulin levels in LCR and HCR. [1, 13, 20, 22, 29]

Figure 2.

Fasting glucose levels (A) and insulin levels (B) in HCR and LCR. Data are mean ± SEM, n = 8. Data from Wisloff et al., (32) and used with permission from Science.

Figure 3 shows the results of isolated heart perfusions both in the presence and absence of insulin. Cardiac power (3A) was not different between HCR and LCR and was not affected by insulin stimulation. Basal glucose oxidation was not different between HCR and LCR (3B). Insulin induced a shift in substrate oxidation towards glucose oxidation. This significant increase in glucose oxidation (3B) was also comparable in both strains. Fatty acid oxidation (3C) was again similar in HCR and LCR and a significant decrease in fatty acid oxidation was observed in both groups with insulin addition. The insulin-induced shift in substrate oxidation was also comparable in HCR and LCR and the ratio of glucose to fatty acid oxidation changed correspondingly in both groups (3D). The switch in substrate utilization from fatty acid to glucose led to a significant reduction in oxygen consumption (3E) which was again comparable in both HCR and LCR. As a consequence, cardiac efficiency (3F) increased in response to insulin stimulation. In summary, none of the parameters determined in the isolated heart differed between the two strains.

Figure 3.

Basal (open bars) and insulin stimulated (hatched bars) cardiac power (A), glucose oxidation (B), fatty acid oxidation (C), ratio of glucose to fatty acid oxidation (D), oxygen consumption (E), and cardiac efficiency (F) in isolated working rat hearts of rats with high (HCR) and low (LCR) intrinsic aerobic capacity. Data are mean ± SEM, n = 7–8; * p<0,05; ** p<0,01; *** p<0,001, compared to resp. basal conditions.

We also assessed baseline expression and phosphorylation status of two major members of the insulin signaling cascade. Figure 4 shows protein expression (4A+D) and phospho-protein expression (4B+E) of Akt (4A-C) and IRS-1 (4D-F) in HCR and LCR after stimulation with insulin (1mU/ml). Total protein was not different between the groups. The phosphorylation status of Akt (4C) and serine-phosphorylated IRS-1 (4F) was also not different, supporting normal insulin signaling in these hearts.

Figure 4.

Relative protein expression and phosphorylation of Akt and IRS-1 after insulin stimulation in rat hearts of rats with high (HCR) and low (LCR) intrinsic aerobic capacity. Expression of Akt protein (A), phophorylated Akt protein (B), and ratio of phosphorylated Akt to total Akt (C). Expression of IRS-1 protein (D), serine-phosphorylated IRS-1 protein (E), and ratio of serine-phosphorylated IRS-1 to total IRS-1 (F). Arrows in (D, E) indicate IRS-1 protein. Data are mean ± SEM, n = 7–8.

Discussion

We show here, that low aerobic exercise capacity associated with systemic insulin resistance is not directly linked to cardiac insulin resistance or changes in substrate oxidation rates. The lack of cardiac insulin resistance in the face of systemic insulin resistance supports a concept of different pathomechanisms for these two conditions.

Diabetes mellitus is associated with increased cardiovascular risks [15]. It is a common notion that systemic insulin resistance affects the heart. Cardiac insulin resistance has been shown in genetically modified obese animals with systemic insulin resistance and it was suggested that the two conditions interact or may be the same (db/db [4] and ob/ob [19]). In patients, normal insulin sensitivity has been suggested in both type I and type II diabetes mellitus based on FDG-PET studies [30, 32]. However, the validity of these assessements is questionable because FDG-PET is a semiquantitative method for the assessment of glucose metabolism, and the kinetic properties of glucose and deoxyglucose change in the presence of insulin [8, 9]. Thus, this issue remains controversial. This controversy continues in mice, where Hafstad et al [12] demonstrated near normal cardiac insulin response in db/db mice, while Boudina et al established a convincing connection between systemic and cardiac insulin resistance [3]. These animals are characterised by a genetic modification. We used a strain of “wild type” rats which have only been selectively bred from “normal” stock. One may argue that examples for such selection procedure may be found in the human world as well. In this study, we demonstrated a clear distinction between systemic and cardiac insulin resistance. These findings suggest that systemic and cardiac insulin resistance may be based on different pathomechanisms. We demonstrated that cardiac insulin resistance develops in response to pressure overload in rat hearts developing hypertrophy and failure at near normal systemic glucose levels. Boudina et al established a link between systemic insulin resistance, fatty acid overload, cardiac mitochondrial dysfunction and diabetic cardiomyopathy in db/db mice [3]. We therefore reasoned that both pressure overload and fatty acid overload may cause cardiac insulin resistance and contribute to the onset of heart failure. In light of the present findings, our interpretation would require that cardiac insulin resistance still develops in the LCR rats. Interestingly, LCR have a lower life expectancy but the survival curves separate at 18 month of age (i.e., much later than our 21 week time point of investigation (unpublished observation 2009)

In contrast, Stanley et al [26] argued that normal cardiac insulin response in the presence of systemic insulin resistance may result in chronic cardiac overstimulation with insulin. Thus, it is still possible that the development of cardiac insulin resistance contributes to contractile dysfunction, and that this mechanism plays a role in the LCR rats. Insulin may thereby cause hypertrophy and may ultimately contribute to the development of heart failure. We are not able to provide the answers to this controversy. Due to the unexpected normal findings of our heart perfusions in both strains including normal Akt phosphorylation in response to insulin, we consider it unlikely that the above described insulin overstimulation hypothesis applies. In contrast, an alternative interpretation of the results would allow making the argument that insulin sensitivity may even be reuced in the low capacity hearts. This alternative suggestion is based on the normal finding ex vivo (substrate oxidation rates, Akt phosphorylation) and the threefold higher insulin levels in vivo. However, if increased insulin levels would be able to overstimulate the heart, we would expect either increased glucose oxidation rates or increased Akt phosphorylation status. Our findings do not support any of these prerequisites and we therefore believe that overstimulation through insulin does not apply. However, full evaluation of cardiac insulin sensitivity in vivo would require the use of a different method (e.g. hyperinsulinemic-euglycemic clamp) and comparison of the activity status of the insulin signalling cascade in heart and peripheral tissues (primarily skeletal muscle and adipose tissue). Unfortunately, we have not performed these experiments in this investigation.

Our results offer the additional interesting observation that insulin reduces oxygen consumption by 30–40% at normal cardiac power and therefore improves cardiac efficiency (i.e., oxygen consumption related to cardiac power). Such an increase in efficiency has previously been reported by others [18] and has been attributed to the shift from primarily fatty acid to the less oxygen expensive glucose oxidation. Theoretically, such a shift from 100% use of fatty acid to 100% use of glucose as sole oxidation substrate would result in a calculatory increase in efficiency of 25–30% (based on 36 mol ATP produced per mol glucose, 118,5 mol ATP per mol oleate, 6 and 25,5 mol oxygen per mol glucose and fatty acid respective). Our finding of 30–40% reduction in oxygen consumption would be consistent with the calculated number. However, our oxidative substrate composition before and after insulin represents a clear mix of both glucose and fatty acid oxidation. Thus, our reduction in oxygen consumption and increase in efficiency cannot be explained by a substrate switch alone. Since our measured rates of oxygen consumption correlate tightly with oxygen consumption from exogenous substrate use it is unlikely that methodological limitations may serve as an explanation. It is therefore reasonable to speculate, that other insulin activated mechanisms possibly improved respiratory coupling may serve as an explanation. However, such an investigation was beyond the scope of this study.

Another interesting observation was the fact that cardiac power of isolated hearts did not differ in HCR and LCR, because a previous investigation assessing cardiac power of isolated hearts from the third generation of selective HCR-LCR breeding demonstrated decreased cardiac power in LCR [14]. While the exact reason for this difference is not clear, it is interesting to note that next to methodological differences (Hussain et al measured only with glucose as substrate) the time point of investigation was at 30 weeks of age. Consistent with our above described hypothesis, it is possible that insulin resistance was already present in these hearts. However insulin sensitivity was not assessed.

Our conclusions are limited by the fact that assessment of substrate oxidation rates and insulin resistance was performed ex vivo. However most of the currently available noninvasive methods are unable to truly quantitate these parameters, prerequisites to assess cardiac substrate oxidation and insulin resistance in vivo.

In summary, we conclude that low aerobic exercise capacity associated with systemic insulin resistance is not directly linked to cardiac insulin resistance or changes in substrate oxidation rates ex vivo. We suggest that the lack of cardiac insulin resistance in the face of systemic insulin resistance supports a concept of different pathomechanisms for these two conditions.