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. Author manuscript; available in PMC: 2014 Jul 18.
Published in final edited form as: Biochem Soc Trans. 2014 Apr;42(2):231–238. doi: 10.1042/BST20130283

Targeting mitochondria to restore failed adaptation to exercise in diabetes

Kate Geary *, Leslie A Knaub *,†, Irene E Schauer *,†, Amy C Keller *,†, Peter A Watson *,†, Matthew W Miller *, Chrystelle V Garat *, Kristen J Nadeau *,‡, Melanie Cree-Green *,‡, Subbiah Pugazhenthi *,†, Judith G Regensteiner *, Dwight J Klemm *, Jane EB Reusch *,†,1
PMCID: PMC4103429  NIHMSID: NIHMS602948  PMID: 24646223

Abstract

Our translational research group focuses on addressing the problem of exercise defects in diabetes with basic research efforts in cell and rodent models and clinical research efforts in subjects with diabetes mellitus. CREB (cAMP-response-element-binding protein) regulates cellular differentiation of neurons, β-cells, adipocytes and smooth muscle cells; it is also a potent survival factor and an upstream regulator of mitochondrial biogenesis. In diabetes and cardiovascular disease, CREB protein content is decreased in the vascular media, and its regulation in aberrant in β-cells, neurons and cardiomyocytes. Loss of CREB content and function leads to decreased vascular target tissue resilience when exposed to stressors such as metabolic, oxidative or sheer stress. This basic research programme set the stage for our central hypothesis that diabetes-mediated CREB dysfunction predisposes the diabetes disease progression and cardiovascular complications. Our clinical research programme revealed that diabetes mellitus leads to defects in functional exercise capacity. Our group has determined that the defects in exercise correlate with insulin resistance, endothelial dysfunction, decreased cardiac perfusion and diastolic dysfunction, slowed muscle perfusion kinetics, decreased muscle perfusion and slowed oxidative phosphorylation. Combined basic and clinical research has defined the relationship between exercise and vascular function with particular emphasis on how the signalling to CREB and eNOS [endothelial NOS (nitric oxide synthase)] regulates tissue perfusion, mitochondrial dynamics, vascular function and exercise capacity. The present review summarizes our current working hypothesis that restoration of eNOS/NOS dysfunction will restore cellular homoeostasis and permit an optimal tissue response to an exercise training intervention.

Keywords: cAMP-response-element-binding protein (CREB), cardiovascular disease, diabetes, exercise, mitochondrion, nitric oxide synthase (NOS)

Introduction

Diabetes is a leading cause of death and disability worldwide and a leading cause of blindness, end-stage renal disease, non-traumatic amputation and CVD (cardiovascular disease). Interventions to optimize glucose control, blood pressure and lipids dramatically decrease microvascular and macrovascular events, yet excess burden remains. For example, even with optimal risk factor reduction, cardiovascular events and death is 3–5-fold higher in people with diabetes [1]. The goal of the research ongoing in our laboratory is to define novel strategies for reduction of CVD in diabetes. In our larger clinical research groups, we made the concerning observation that people with diabetes have decreased functional exercise capacity [2]. Specifically, adults and adolescents with either Type 2 or Type 1 diabetes have decreased cardiovascular fitness as measured by O2 consumption (V̇o2 max) and slowed oxygen uptake kinetics [2–5]. The decreased V̇o2 peak is associated with excess CVD and all-cause mortality [6,7]. V̇o2 kinetics measures the acceleration of oxygen uptake and consumption at the onset of exercise and, when slowed, represents a defect in submaximal exercise function (relevant for activities of daily living). These defects in functional exercise capacity have implications for day-to-day life and longevity.

Our translational research group focuses on addressing the problem of exercise defects in diabetes with basic research efforts in cell and rodent models and clinical research efforts in subjects with diabetes mellitus. The present review outlines the basic observations that have led to our current working model starting with our observation that the transcription factor CREB (cAMP-response-element-binding protein), is a crucial modulator of cellular homoeostasis. CREB regulates cellular differentiation of neurons, β-cells, adipocytes and SMCs (smooth muscle cells); it is also a potent survival factor and an upstream regulator of mitochondrial biogenesis. In diabetes and CVD, CREB protein content is decreased in the vascular media and its regulation in aberrant in β-cells, neurons and cardiomyocytes [8–12]. Loss of CREB content and function leads to decreased vascular target tissue resilience when exposed to stressors such as metabolic, oxidative or sheer stress. This basic research programme set the stage for our central hypothesis that diabetes-mediated CREB dysfunction predisposes the diabetes disease progression and cardiovascular complications. The second line of research revealed that diabetes mellitus (Type 1 and Type 2 in youth and adults) leads to defects in functional exercise capacity [13]. This defect manifests in a 20–30% decrease in maximal O2 consumption and slowed oxygenation and heart rate kinetics at submaximal exercise. Our group has determined that the defects in exercise correlate with insulin resistance, endothelial dysfunction, decreased cardiac perfusion and diastolic dysfunction, slowed muscle perfusion kinetics, decreased muscle perfusion [4,13–15] and slowed oxidative phosphorylation (M. Cree-Green and K.J. Nadeau, unpublished work). In the last few years, we have begun to weave these two threads together to define the relationship between exercise and vascular function with particular emphasis on how the signalling to CREB and eNOS [endothelial NOS (nitric oxide synthase)] regulates tissue perfusion, mitochondrial dynamics, vascular function and exercise capacity. This review summarizes our published work on CREB, mitochondrial function, cell-specific eNOS/NOS and diabetes mediated exercise impairments to set the stage for our current working hypothesis that restoration of eNOS/NOS dysfunction will restore cellular homoeostasis and permit optimal response to an exercise training intervention.

CREB regulates vascular SMC phenotype and is degraded in vascular disease

CREB is a transcription factor that plays an integral role in cellular proliferation, differentiation and adaptive responses [16–18]. CREB is regulated by G-protein-coupled receptors, tyrosine kinase receptors, oxidant and osmotic stress and Ca2+. Pivotal targets of CREB include genes important for survival, differentiation and metabolic adaptation (Figure 1). CREB is an integrator or hub protein that plays a role in life/death decisions of the cell and it is dysfunctional in the vasculature, heart, brain and β-cell in diabetes [8,11,12,20]. Our laboratory first identified CREB as a determinant of the quiescent SMC phenotype in a series of companion papers [10,11]. We demonstrated that overexpression of active CREB in SMCs decreased proliferative potential, whereas dominant-negative CREB increased SMC activation [10]. Diabetes mellitus induces phenotypic modulation of SMCs in the vasculature, leading to aberrant proliferation and migration of SMCs and vascular dysfunction [11]. Vascular CREB content is decreased in a spectrum of rodent models of CVD risk (hypertension, obesity, diabetes, the metabolic syndrome, pulmonary hypertension and dyslipidaemia) [21]. Using an atherosclerosis model of LDL (low-density lipoprotein) receptor-null mice and in vitro studies of SMCs exposed to LDL and oxLDL (oxidized LDL), we showed that both forms of LDL induce an acute activation of CREB. However, only oxLDL leads to CREB down-regulation [21]. We showed further that SMCs exposed to a panel of non-esterified (‘free’) fatty acids exhibited an acute activation of CREB via PKC (protein kinase C) activation. Only saturated fatty acids triggered the down-regulation of CREB [22]. CREB protein content is also reduced in the SMCs of hypertensive pulmonary arteries (PA SMCs) in animals exposed to chronic hypoxia. Hypoxia-induced PA SMCs produce a growth factor called PDGF (platelet-derived growth factor)-BB. We defined that CREB down-regulation by chronic PDGF-BB is mediated through chronic activation of PI3K (phosphoinositide 3-kinase)/Akt and induction of a novel downstream target: protein kinase CK2 [23]. CK2 augments CREB phosphorylation at Ser103 and Ser107, enhancing the nuclear export and proteasomal degration of CREB [23]. In the systemic vasculature, TZDs (thiazolidinediones) prevent arterial remodelling and vasoconstriction. TZDs block induction of CK2 and interfere with PDGF-mediated CREB degradation [24]. The physiological relevance of the TZD/Akt/CK2/CREB SMC protection pathway is supported by our recent publications demonstrating the ability of rosiglitazone, PI3K inhibitors and antioxidants to block the proliferation of PA SMCs and stimulate regression of arterial remodelling [24–26]. Collectively, these data support a model wherein CREB serves as a regulator of the quiescent SMC phenotype. Models of vascular disease including diabetes mellitus, hyperlipidaemia, aging and pulmonary hypertension consistently show that loss of SMC CREB via degradation or nuclear export is permissive for the proliferative SMC phenotype, ultimately promoting disease progression.

Figure 1. Targets of CREB regulation.

Figure 1

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CREB is a transcriptional hub integrating signals from G-protein-coupled receptors, tyrosine kinase receptors, Ca2+ and oxidant injury to regulate genes essential for survival, differentiation and metabolic adaptation. BDNF, brain-derived neurotrophic factor; CBP, CREB-binding protein; CRE, cAMP-response element; IAP-2, inhibitor of apoptosis 2; IRS-2, insulin receptor substrate 2; NRF-2, nuclear factor-erythroid 2-related factor 2; POLII, RNA polymerase II; TFAM, transcription factor A, mitochondrial; TFIID, transcription factor IID.

CREB regulation of mitochondrial function

Mitochondria are critical sensors of cellular environment involved in cellular homoeostatic decision making. In the context of cellular stress (either toxic or physiological), mitochondrial adaptation is at the centre of cell fate. The decision to increase or decrease metabolism, adjust fuel partitioning and efficiency, and support survival are each, in part, regulated by the mitochondria. Early work from our group and others demonstrated that CREB is a critical regulator of cell survival and mitochondrial integrity via stimulation of Bcl-2 expression [27]. We reported redundant signalling downstream of the insulin receptor via p38 MAPK (mitogen-activated protein kinase), Akt and ERK (extracellular-signal-regulated kinase) to CREB and Bcl-2 [27–29]. In addition, we demonstrated a potent role for CREB in the context of diabetes-mediated oxidant or cytokine stress [8,20,30]. More recently, we and others reported that CREB is an upstream regulator of mitochondrial biogenesis via regulation of PGC1α (peroxisome-proliferator-activated receptor γ co-activator 1α) and ERRα (oestrogen-related receptor α) [31]. We demonstrated that exercise stimulated CREB activity and Bcl-2 decreased caspase 3 activation and decreased heart failure in the SHHF (spontaneous hypertensive heart failure) rat [12]. Consistent with this observation, CREB activity (not protein content) is decreased across a spectrum of cardiac heart failure models [32]. Fentzke et al. [33] generated a cardiac-specific transgenic mouse with dominant-negative CREB under the MHC promoter (MHC DNCREB). This mouse develops dilated cardiomyopathy [33]. Watson et al. [31] showed abnormal mitochondrial structure and function plus abnormal fuel partitioning in the MHC DNCREB mouse. In addition, this mouse fails to respond to exercise training with physiological remodelling [34]. Taken together, this study indicates that CREB is essential for heart mitochondrial function and contributes to physiological remodelling. These observations led us to postulate that CREB dysregulation in the diabetic heart and vasculature may contribute to, or exacerbate, the well-established mitochondrial dysfunction in diabetes. We are currently pursuing aspects of this question in our basic and clinical studies. In the next section, we provide an overview of our work in the diabetic vasculature evaluating the mitochondrial adaptation to exercise intervention and close with a summary of our ongoing and published clinical trials targeting insulin action, CREB and the mitochondria as strategies to improve exercise capacity in diabetes and enhance the physiological impact of exercise in the diabetes mellitus host.

Mitochondrial adaptation

CREB content and nuclear localization are decreased in vascular diseases including diabetes, dyslipidaemia, hypertension, aging and pulmonary hypertension [9,21,35]. In the light of the impact of CREB on mitochondrial function, we evaluated the mitochondrial content and function in diabetic rodent models and observed decreased mitochondrial protein profiles and enzyme activities [30]. We next tested whether we could restore vascular CREB content or CREB mitochondrial targets with exercise intervention [36]. In control rats, we observed a significant increase in mitochondrial protein profiles with a short-term exercise intervention which was, unexpectedly, absent from hypertensive or hypertensive diabetes mellitus rats [36,37] (Figure 2). These unanticipated results led to a series of studies to define the molecular determinants of vascular mitochondrial response to exercise and to evaluate these targets in diabetes mellitus.

Figure 2. Aortic mitochondrial protein content with exercise intervention.

Figure 2

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Mitochondrial protein expression profile in SD (Sprague–Dawley) and SHHF lean and obese rats with and without exercise training. Aortic lysates were generated from SD (n = 8 and 9 for sedentary and exercise respectively), SHHF lean (n = 8 and 10 for sedentary and exercise respectively) and SHHF obese (n = 9 for both sedentary and exercise) rats as labelled: 30 µg of protein was run on SDS/PAGE (10% gels), transferred on to nitrocellulose membranes and Western blot analysis was carried out. Results are means±S.E.M. *P < 0.05; †P=0.05–0.10 (Student’s t test). Reproduced with permission from [36]: Knaub, L.A., McCune, S., Chicco, A.J., Miller, M., Moore, R.L., Birdsey, N., Lloyd, M.I., Villarreal, J., Keller, A.C., Watson, P.A. and Reusch, J.E. (2013) Impaired response to exercise intervention in the vasculature in metabolic syndrome. Diab. Vasc. Dis. Res. 10, 222–238. © 2013 SAGE Publications.

Mitochondrial dynamics

Mitochondrial content, structure and function are coordinated by an agile intracellular machinery regulating mitochondrial biogenesis, mitochondrial dynamics (fission and fusion), mitochondrial localization and autophagy (reviewed in [38]). This machinery enables the homoeostatic plasticity needed for rapid adaptation to physiological challenges. In the vasculature, mitochondria serve a number of functions in addition to ATP production, including the regulation of Ca2+ sparks and currents, regulation of ROS (reactive oxygen species), and regulation of cell proliferation and survival [39,40]. Early studies by Taggart and Wray [41] and Sward et al. [42] demonstrated a seminal role for mitochondria in control of vascular contractile function (constriction and relaxation); more recent work defined their importance for calcium regulation and control of vascular tone (stiffness) [43]. We were interested in defining the determinants of basal and adaptive mitochondrial function in the vasculature.

Role of NOS in vascular mitochondrial content and adaptation

To better understand the underpinnings of decreased vascular mitochondrial content and adaptation in diabetes, we explored the connections between known mitochondrial regulatory pathways and established vascular consequences of diabetes. Endothelial dysfunction, defined by decreased flow-mediated vasodilatation and vascular insulin resistance (specifically to eNOS regulation) are established vascular abnormalities in diabetes [44]. The work of Nisoli’s group firmly established the role of NO [generated by eNOS and nNOS (neuronal NOS)] as an upstream regulator of mitochondrial biogenesis [45]. We therefore examined the impact of eNOS deletion or pharmacological inhibition of NOS on baseline and exercise-stimulated vascular mitochondrial biogenesis and dynamics. Miller et al. [37] reported decreased aortic mitochondrial protein profiles in aortic tissues of eNOS-null and l-NAME (NG-nitro-l-arginine methyl ester)-treated rodents (similar to our observations in diabetes mellitus) [37]. In addition to decreased CREB and PGC1α in these models, we also observed increased expression of fission proteins (Fis1 and Drp1) and decreased expression of fusion proteins (Opa1, Mfn1 and Mfn2) [37] (Figure 3). These data are consistent with NOS as a biologically important modulator of basal vascular mitochondrial function and structure. In addition, we demonstrated that exercise increased Mfn2 and decreased Fis1 in control rats [37]. This pattern would predict a more fused and networked mitochondrial structure. In contrast, l-NAME prohibited the increase in mitochondrial protein expression and the changes in Mfn2 and Fis1 with exercise [37]. These findings have informed our current working hypothesis that diabetes mellitus-mediated vascular NOS dysfunction (particularly with exercise) contributes to failed mitochondrial plasticity and vascular dysfunction.

Figure 3. Impact of NOS/eNOS on aortic mitochondrial protein content and dynamics baseline and with exercise intervention.

Figure 3

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eNOS deletion and NOS disruption mimic diabetes and disrupt adaptive mitochondrial dynamics. (A) eNOS deletion or pharmacological NOS inhibition decrease aortic mitochondrial content. (B) NOS inhibition decreases mitochondrial regulator PGC1α without changing SIRT1 (sirtuin 1) protein content. (C) NOS inhibition decreases mitochondrial fusion proteins (Mfn1, Mfn2 and OPA1) and increases mitochondrial fission proteins (Fis1 and Drp1). (D) Exercise intervention increases mitochondrial protein and Mfn2 and decreases Fis1 in control rat aorta, whereas no change is observed in rats exercising with the NOS inhibitor treatment. Adapted from [37]: Miller, M.W., Knaub, L.A., Olivera-Fragoso, L.F., Keller, A.C., Balasubramaniam, V., Watson, P.A. and Reusch, J.E. (2013) Nitric oxide regulates vascular adaptive mitochondrial dynamics. Am. J. Physiol. Heart Circ. Physiol. 304, H1624–H1633. © 2013 The American Physiological Society.

Bridging clinical therapeutics to new molecular targets

Type 2 diabetes affects more than 300 million people worldwide and its pathogenesis is a combination of insulin resistance and relative β-cell deficiency. Many agents exist to manage Type 2 diabetes, including agents that augment or replace defective insulin secretion and agents that improve or mimic insulin action on target tissues (decrease hepatic glucose output or improve glucose utilization). We are intrigued that the incretin class of insulin secretogogues, GLP1 (glucagon-like peptide 1) receptor analogues or DPP4 (dipeptidyl peptidase 4) inhibitors act via G-protein-coupled receptors that are highly expressed in the vasculature [46,47]. In addition, we closely follow the work of Lui’s group who has demonstrated that incretin agents stimulate eNOS and PKA (protein kinase A) and enhance endothelial function and tissue perfusion leading to improved muscle glucose utilization [48,49]. We therefore examined the impact of the DPP4 inhibitor saxagliptin on vascular mitochondrial adaptation in diabetes. In these studies, which have been published only in abstract form to date [50], we observed restoration of the exercise-mediated increase in mitochondrial protein profiles in the diabetes mellitus aorta, increased expression of eNOS, nNOS and PGC1α. Studies are underway to examine further the impact of the incretins on target tissue response to exercise in health and diabetes.

Clinical implications

People with diabetes have decreased functional exercise capacity that may be important in the premature mortality in diabetes. We have defined a set of physiological parameters that predict or correlate with exercise defects. As outlined in the Introduction, our groups have determined that the defects in exercise correlate with insulin resistance, endothelial dysfunction, diastolic dysfunction, decreased cardiac perfusion, slowed muscle perfusion kinetics [3–5,14,15] and slowed oxidative phosphorylation (J. Reusch, K. Nadeau and M. Cree-Green, unpublished work). Over the last decade, we have sought to target physical inactivity, insulin-sensitivity and excess oxidant burden as ways to improve exercise function in people with diabetes. As expected, exercise training can improve fitness in people with diabetes, but it is not normalized to the level of their matched non-diabetic counterparts [3]. In a series of proof-of-concept studies, we and others demonstrated that improving insulin-sensitivity and endothelial function with TZDs enhances exercise capacity in Type 2 diabetes [50]. Similarly, vitamin E plus C also improved endothelial function and exercise capacity (published so far only in abstract form [52]). Ongoing investigations (see http://www.clintrials.gov) are examining the impact of the incretin class of drugs on these end points.

Summary

Diabetes, via many pathways including glucose elevation, insulin resistance, oxidant burden and low-grade inflammation, generates persistent target organ stress that overwhelms the innate homoeostatic machinery and leads to target organ dysfunction. Our work has defined a unique consequence of this homoeostatic failure: loss of adaptive mitochondrial plasticity in the vasculature. Exploration of this defect has revealed the interesting roles of eNOS and nNOS that may be amenable to pharmacological targeting.

Acknowledgments

Funding

Our work is funded by a Veterans’ Administration Merit Award (Basic and Clinical), Denver Research Institute, National Institutes of Health [grant numbers RO1-DK064741, T32HL007171, P01HL014985 and UL1RR025780], Bristol-Myers Squibb, the University of Colorado Center for Women’s Health Research, American Diabetes Association, American Heart Association and Juvenile Diabetes Research Foundation International.

Abbreviations

CREB

cAMP-response-element-binding protein

CVD

cardiovascular disease

DPP4

dipeptidyl peptidase 4

eNOS

endothelial nitric oxide synthase

LDL

low-density lipoprotein

l-name

NG-nitro-l-arginine methyl ester

MHC DNCREB

dominant-negative CREB under the MHC promoter

nNOS

neuronal NOS

NOS

nitric oxide synthase

oxLDL

oxidized LDL

PA SMC

SMC of hypertensive pulmonary arteries

PDGF

platelet-derived growth factor

PGC1α

peroxisome-proliferator-activated receptor γ co-activator 1α

PI3K

phosphoinositide 3-kinase

SHHF

spontaneous hypertensive heart failure

SMC

smooth muscle cell

TZD

thiazolidinedione.

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