M. Harold Laughlin

Mitochondrial dysfunction precedes insulin resistance and hepatic steatosis and contributes to the natural history of non-alcoholic fatty liver disease in an obese rodent model

BACKGROUND & AIMS In this study, we sought to determine the temporal relationship between hepatic mitochondrial dysfunction, hepatic steatosis and insulin resistance, and to examine their potential role in the natural progression of non-alcoholic fatty liver disease (NAFLD) utilising a sedentary, hyperphagic, obese, Otsuka Long-Evans Tokushima Fatty (OLETF) rat model. METHODS OLETF rats and their non-hyperphagic control Long-Evans Tokushima Otsuka (LETO) rats were sacrificed at 5, 8, 13, 20, and 40 weeks of age (n=6-8 per group). RESULTS At 5 weeks of age, serum insulin and glucose and hepatic triglyceride (TG) concentrations did not differ between animal groups; however, OLETF animals displayed significant (p<0.01) hepatic mitochondrial dysfunction as measured by reduced hepatic carnitine palmitoyl-CoA transferase-1 activity, fatty acid oxidation, and cytochrome c protein content compared with LETO rats. Hepatic TG levels were significantly elevated by 8 weeks of age, and insulin resistance developed by 13 weeks in the OLETF rats. NAFLD progressively worsened to include hepatocyte ballooning, perivenular fibrosis, 2.5-fold increase in serum ALT, hepatic mitochondrial ultrastructural abnormalities, and increased hepatic oxidative stress in the OLETF animals at later ages. Measures of hepatic mitochondrial content and function including beta-hydroxyacyl-CoA dehydrogenase activity, citrate synthase activity, and immunofluorescence staining for mitochondrial carbamoyl phosphate synthetase-1, progressively worsened and were significantly reduced at 40 weeks in OLETF rats compared to LETO animals. CONCLUSIONS Our study documents that hepatic mitochondrial dysfunction precedes the development of NAFLD and insulin resistance in the OLETF rats. This evidence suggests that progressive mitochondrial dysfunction contributes to the natural history of obesity-associated NAFLD.

Induction of nitric oxide synthase mRNA in coronary resistance arteries isolated from exercise-trained pigs

The purpose of this study was to develop a method by which endothelial cell nitric oxide synthase (ecNOS) mRNA expression could be measured in single coronary resistance arteries and to test the hypothesis that ecNOS gene expression is upregulated by exercise training. Yucatan miniature swine were randomly assigned to exercise-trained (ET; n = 5) or sedentary (Sed; n = 4) groups for 16 wk. Individual coronary resistance arteries (50-100 microns) were dissected, frozen in liquid nitrogen, and homogenized in a LiCl buffer, mRNA was isolated from each vessel, and ecNOS gene expression was assessed using reverse transcriptase (RT)-polymerase chain reaction (PCR) standardized by coamplifying ecNOS with glyceraldehyde 3-phosphate dehydrogenase (GAPHD). The ecNOS-to-GAPDH amplicon ratio was significantly greater in coronary resistance arteries isolated from ET pigs than in Sed controls. On the basis of these data, it is concluded that RT-PCR can be used on single coronary resistance arteries to assess cell-specific mRNA expression and that ecNOS gene expression is upregulated by exercise training in porcine coronary resistance arteries.The purpose of this study was to develop a method by which endothelial cell nitric oxide synthase (ecNOS) mRNA expression could be measured in single coronary resistance arteries and to test the hypothesis that ecNOS gene expression is upregulated by exercise training. Yucatan miniature swine were randomly assigned to exercise-trained (ET; n = 5) or sedentary (Sed; n = 4) groups for 16 wk. Individual coronary resistance arteries (50-100 μm) were dissected, frozen in liquid nitrogen, and homogenized in a LiCl buffer. mRNA was isolated from each vessel, and ecNOS gene expression was assessed using reverse transcriptase (RT)-polymerase chain reaction (PCR) standardized by coamplifying ecNOS with glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The ecNOS-to-GAPDH amplicon ratio was significantly greater in coronary resistance arteries isolated from ET pigs than in Sed controls. On the basis of these data, it is concluded that RT-PCR can be used on single coronary resistance arteries to assess cell-specific mRNA expression and that ecNOS gene expression is upregulated by exercise training in porcine coronary resistance arteries.

Simvastatin impairs exercise training adaptations.

OBJECTIVES This study sought to determine if simvastatin impairs exercise training adaptations. BACKGROUND Statins are commonly prescribed in combination with therapeutic lifestyle changes, including exercise, to reduce cardiovascular disease risk in patients with metabolic syndrome. Statin use has been linked to skeletal muscle myopathy and impaired mitochondrial function, but it is unclear whether statin use alters adaptations to exercise training. METHODS This study examined the effects of simvastatin on changes in cardiorespiratory fitness and skeletal muscle mitochondrial content in response to aerobic exercise training. Sedentary overweight or obese adults with at least 2 metabolic syndrome risk factors (defined according to National Cholesterol Education Panel Adult Treatment Panel III criteria) were randomized to 12 weeks of aerobic exercise training or to exercise in combination with simvastatin (40 mg/day). The primary outcomes were cardiorespiratory fitness and skeletal muscle (vastus lateralis) mitochondrial content (citrate synthase enzyme activity). RESULTS Thirty-seven participants (exercise plus statins: n = 18; exercise only: n = 19) completed the study. Cardiorespiratory fitness increased by 10% (p < 0.05) in response to exercise training alone, but was blunted by the addition of simvastatin resulting in only a 1.5% increase (p < 0.005 for group by time interaction). Similarly, skeletal muscle citrate synthase activity increased by 13% in the exercise-only group (p < 0.05), but decreased by 4.5% in the simvastatin-plus-exercise group (p < 0.05 for group-by-time interaction). CONCLUSIONS Simvastatin attenuates increases in cardiorespiratory fitness and skeletal muscle mitochondrial content when combined with exercise training in overweight or obese patients at risk of the metabolic syndrome. (Exercise, Statins, and the Metabolic Syndrome; NCT01700530).

Exercise increases blood flow to locomotor, vestibular, cardiorespiratory and visual regions of the brain in miniature swine

1 The purpose of these experiments was to use radiolabelled microspheres to measure blood flow distribution within the brain, and in particular to areas associated with motor function, maintenance of equilibrium, cardiorespiratory control, vision, hearing and smell, at rest and during exercise in miniature swine. Exercise consisted of steady‐state treadmill running at intensities eliciting 70 and 100 % maximal oxygen consumption (V̇O2,max). 2 Mean arterial pressure was elevated by 17 and 26 % above that at rest during exercise at 70 and 100 %V̇O2,max, respectively. 3 Mean brain blood flow increased 24 and 25 % at 70 and 100 %V̇O2,max, respectively. Blood flow was not locally elevated to cortical regions associated with motor and somatosensory functions during exercise, but was increased to several subcortical areas that are involved in the control of locomotion. 4 Exercise elevated perfusion and diminished vascular resistance in several regions of the brain related to the maintenance of equilibrium (vestibular nuclear area, cerebellar ventral vermis and floccular lobe), cardiorespiratory control (medulla and pons), and vision (dorsal occipital cortex, superior colliculi and lateral geniculate body). Conversely, blood flow to regions related to hearing (cochlear nuclei, inferior colliculi and temporal cortex) and smell (olfactory bulbs and rhinencephalon) were unaltered by exercise and associated with increases in vascular resistance. 5 The data indicate that blood flow increases as a function of exercise intensity to several areas of the brain associated with integrating sensory input and motor output (anterior and dorsal cerebellar vermis) and the maintenance of equilibrium (vestibular nuclei). Additionally, there was an intensity‐dependent decrease of vascular resistance in the dorsal cerebellar vermis.

Vascular Effects of Exercise: Endothelial Adaptations Beyond Active Muscle Beds

Endothelial adaptations to exercise training are not exclusively conferred within the active muscle beds. Herein, we summarize key studies that have evaluated the impact of chronic exercise on the endothelium of vasculatures perfusing nonworking skeletal muscle, brain, viscera, and skin, concluding with discussion of potential mechanisms driving these endothelial adaptations.

Gene targeting with zinc finger nucleases to produce cloned eGFP knockout pigs

We report using zinc finger nucleases (ZFNs) and somatic cell nuclear transfer (SCNT) to generate pigs with a knockout (KO) mutation of an enhanced green fluorescent protein (eGFP) transgene. ZFNs are synthetic modular proteins composed of a FokI endonuclease domain linked to a sequence-specific zinc finger DNA-binding domain. Pairs of ZFNs bind to the target region, allowing FokI dimerization and subsequent DNA cleavage. Mutation of an eGFP transgene by using ZFNs has been demonstrated in rats (Geurts et al., 2009); however, there are no reports of ZFN-mutated livestock offspring. Using ZFNs to increase the efficiency of gene modification may advance the production of agriculturally and clinically relevant animal models, particularly in species where modification is difficult. All animal procedures followed an approved IACUC protocol. Adult porcine ear fibroblasts hemizygous for the eGFP transgene (Whitworth et al., 2009), from a grandson of the founder animal, were cultured to 75% confluency, trypsinized, and cotransfected (Ross et al., 2010) with a pair of ZFN plasmids that bind to opposing strands at the eGFP target site (Sigma–Aldrich CompoZr®) and a red fluorescent CAG-tomato plasmid as a transient selectable fluorophore reporting transfection efficiency. Transfected fibroblasts were cultured for 96 h and selected for red fluorescence by automated cell sorting (FACS; Fig. 1A,B) and seeded into 96-well plates (100 cells/well) based on CAG-tomato expression (2% of total cells sorted). A second round of FACS using negative selection of eGFP fluorescence enriched for eGFP KO cells (~5% of sorted cells). PCR of genomic DNA from fibroblasts in wells containing predominantly non-green cells was used to amplify a fragment bracketing the ZFN targeting site. The PCR product was cloned into E. coli, and 16 colonies showed mutations by Sanger sequencing. These mutations at the ZFN cleavage site included a 6-bp deletion, a 333-bp deletion, and a 222-bp deletion replaced with a 113-bp inversion of the deleted sequence (Supplemental Information http://animalsciences.missouri.edu/faculty/prather/). Fibroblast colonies determined to carry ZFN-induced mutations were used as donor cells for SCNT, and embryo transfer (n = 3; Whitworth et al., 2009). One Day-12 embryo collection recovered 7 of 9 embryos that did not fluoresce. Quality sequence was obtained from four embryos, and confirmed that the two embryos that fluoresced had an intact eGFP and two embryos that did not fluoresce were mutated. One of seven piglets recovered via Cesarean section at term for the other two pregnancies fluoresced (Fig. 1C). DNA sequencing confirmed that this piglet had unaltered sequence at the predicted ZFN cut site, while the non-fluorescing piglets had various deletions and insertions. While a direct comparison to a 12% mutation rate in the rat made by microinjection (Geurts et al., 2009) cannot be made because of our FACS preselection and SCNT methods, six out of seven founders being mutated is consistent with their results. This study establishes ZFN-based gene modification in a large animal model. The methods used are anticipated to be useful in selective knockout of endogenous swine genes of agricultural and biomedical importance without introducing any transgenic sequence into the genome. Figure 1 A: Control porcine ear fibroblasts; eGFP and Hoechst DNA stain fluorescence. B: Porcine ear fibroblasts treated with eGFP-specific ZFNs; eGFP and Hoechst DNA stain fluorescence. GFP fluorescence is absent in a high proportion of the treated fibroblasts ...

Increased muscle sympathetic nerve activity acutely alters conduit artery shear rate patterns

Escalating evidence indicates that disturbed flow patterns, characterized by the presence of retrograde and oscillatory shear stress, induce a proatherogenic endothelial cell phenotype; however, the mechanisms underlying oscillatory shear profiles in peripheral conduit arteries are not fully understood. We tested the hypothesis that acute elevations in muscle sympathetic nerve activity (MSNA) are accompanied by increases in conduit artery retrograde and oscillatory shear. Fourteen healthy men (25 +/- 1 yr) performed three sympathoexcitatory maneuvers: graded lower body negative pressure (LBNP) from 0 to -40 Torr, cold pressor test (CPT), and 35% maximal voluntary contraction handgrip followed by postexercise ischemia (PEI). MSNA (microneurography; peroneal nerve), arterial blood pressure (finger photoplethysmography), and brachial artery velocity and diameter (duplex Doppler ultrasound) in the contralateral arm were recorded continuously. All maneuvers elicited significant increases in MSNA total activity from baseline (P < 0.05). Retrograde shear (-3.96 +/- 1.2 baseline vs. -8.15 +/- 1.8 s(-1), -40 LBNP, P < 0.05) and oscillatory shear index (0.09 +/- 0.02 baseline vs. 0.20 +/- 0.02 arbitrary units, -40 LBNP, P < 0.05) were progressively augmented during graded LBNP. In contrast, during CPT and PEI, in which MSNA and blood pressure were concomitantly increased (P < 0.05), minimal or no changes in retrograde and oscillatory shear were noted. These data suggest that acute elevations in MSNA are associated with an increase in conduit artery retrograde and oscillatory shear, an effect that may be influenced by concurrent increases in arterial blood pressure. Future studies should examine the complex interaction between MSNA, arterial blood pressure, and other potential modulatory factors of shear rate patterns.

Daily exercise vs. caloric restriction for prevention of nonalcoholic fatty liver disease in the OLETF rat model

The maintenance of normal body weight either through dietary modification or being habitually more physically active is associated with reduced incidence of nonalcoholic fatty liver disease (NAFLD). However, the means by which weight gain is prevented and potential mechanisms activated remain largely unstudied. Here, we sought to determine the effects of obesity prevention by daily exercise vs. caloric restriction on NAFLD in the hyperphagic, Otsuka Long-Evans Tokushima Fatty (OLETF) rat. At 4 wk of age, male OLETF rats (n = 7-8/group) were randomized to groups of ad libitum fed, sedentary (OLETF-SED), voluntary wheel running exercise (OLETF-EX), or caloric restriction (OLETF-CR; 70% of SED) until 40 wk of age. Nonhyperphagic, control strain Long-Evans Tokushima Otsuka (LETO) rats were kept in sedentary cage conditions for the duration of the study (LETO-SED). Both daily exercise and caloric restriction prevented obesity and the development of type 2 diabetes observed in the OLETF-SED rats, with glucose tolerance during a glucose tolerance test improved to a greater extent in the OLETF-EX animals (30-50% lower glucose and insulin areas under the curve, P < 0.05). Both daily exercise and caloric restriction also prevented excess hepatic triglyceride and diacylglycerol accumulation (P < 0.001), hepatocyte ballooning and nuclear displacement, and the increased perivenular fibrosis and collagen deposition that occurred in the obese OLETF-SED animals. However, despite similar hepatic phenotypes, OLETF-EX rats also exhibited increased hepatic mitochondrial fatty acid oxidation, enhanced oxidative enzyme function and protein content, and further suppression of hepatic de novo lipogenesis proteins compared with OLETF-CR. Prevention of obesity by either daily exercise or caloric restriction attenuates NAFLD development in OLETF rats. However, daily exercise may offer additional health benefits on glucose homeostasis and hepatic mitochondrial function compared with restricted diet alone.

Flow regulation of ecNOS and Cu/Zn SOD mRNA expression in porcine coronary arterioles

The purpose of this study was to test the hypothesis that increased flow through coronary arterioles increases endothelial cell nitric oxide synthase (ecNOS) and Cu/Zn superoxide dismutase (SOD) mRNA expression. Single porcine coronary arterioles (ID 100-160 μm; pressurized) were cannulated, perfused, and exposed to intraluminal flow sufficient to produce maximal flow-induced dilation of coronary arterioles (high flow; 7.52 ± 0.22 μl/min), low flow (0.84 ± 0.05 μl/min), or no flow for 2 or 4 h. Mean shear stress was calculated to be 5.7 ± 1.0 dyn/cm2 for high-flow arterioles and 1.6 ± 1.0 dyn/cm2 for low-flow arterioles. At the end of the treatment period, mRNA was isolated from each vessel, and ecNOS and SOD mRNA expression was assessed using a semiquantitative RT-PCR. All data were standardized by coamplifying ecNOS or SOD with glyceraldehyde-3-phosphate dehydrogenase. The results indicate that ecNOS mRNA expression is increased in arterioles exposed to 2 or 4 h of high flow. In contrast, SOD mRNA expression was increased only after 4 h of high flow. Neither gene is induced by exposure to low flow. On the basis of these data, we concluded that ecNOS and SOD mRNA expression is regulated by flow in porcine coronary arterioles. In addition, we concluded that a threshold level of flow and shear stress must be sustained to elicit the upregulation of ecNOS and SOD mRNA expression.The purpose of this study was to test the hypothesis that increased flow through coronary arterioles increases endothelial cell nitric oxide synthase (ecNOS) and Cu/Zn superoxide dismutase (SOD) mRNA expression. Single porcine coronary arterioles (ID 100-160 micrometers; pressurized) were cannulated, perfused, and exposed to intraluminal flow sufficient to produce maximal flow-induced dilation of coronary arterioles (high flow; 7.52 +/- 0.22 microliter/min), low flow (0.84 +/- 0.05 microliter/min), or no flow for 2 or 4 h. Mean shear stress was calculated to be 5.7 +/- 1.0 dyn/cm2 for high-flow arterioles and 1. 6 +/- 1.0 dyn/cm2 for low-flow arterioles. At the end of the treatment period, mRNA was isolated from each vessel, and ecNOS and SOD mRNA expression was assessed using a semiquantitative RT-PCR. All data were standardized by coamplifying ecNOS or SOD with glyceraldehyde-3-phosphate dehydrogenase. The results indicate that ecNOS mRNA expression is increased in arterioles exposed to 2 or 4 h of high flow. In contrast, SOD mRNA expression was increased only after 4 h of high flow. Neither gene is induced by exposure to low flow. On the basis of these data, we concluded that ecNOS and SOD mRNA expression is regulated by flow in porcine coronary arterioles. In addition, we concluded that a threshold level of flow and shear stress must be sustained to elicit the upregulation of ecNOS and SOD mRNA expression.

The coronary circulation in exercise training

Exercise training (EX) induces increases in coronary transport capacity through adaptations in the coronary microcirculation including increased arteriolar diameters and/or densities and changes in the vasomotor reactivity of coronary resistance arteries. In large animals, EX increases capillary exchange capacity through angiogenesis of new capillaries at a rate matched to EX-induced cardiac hypertrophy so that capillary density remains normal. However, after EX coronary capillary exchange area is greater (i.e., capillary permeability surface area product is greater) at any given blood flow because of altered coronary vascular resistance and matching of exchange surface area and blood flow distribution. The improved coronary capillary blood flow distribution appears to be the result of structural changes in the coronary tree and alterations in vasoreactivity of coronary resistance arteries. EX also alters vasomotor reactivity of conduit coronary arteries in that after EX, α-adrenergic receptor responsiveness is blunted. Of interest, α- and β-adrenergic tone appears to be maintained in the coronary microcirculation in the presence of lower circulating catecholamine levels because of increased receptor responsiveness to adrenergic stimulation. EX also alters other vasomotor control processes of coronary resistance vessels. For example, coronary arterioles exhibit increased myogenic tone after EX, likely because of a calcium-dependent PKC signaling-mediated alteration in voltage-gated calcium channel activity in response to stretch. Conversely, EX augments endothelium-dependent vasodilation throughout the coronary arteriolar network and in the conduit arteries in coronary artery disease (CAD). The enhanced endothelium-dependent dilation appears to result from increased nitric oxide bioavailability because of changes in nitric oxide synthase expression/activity and decreased oxidant stress. EX also decreases extravascular compressive forces in the myocardium at rest and at comparable levels of exercise, mainly because of decreases in heart rate and duration of systole. EX does not stimulate growth of coronary collateral vessels in the normal heart. However, if exercise produces ischemia, which would be absent or minimal under resting conditions, there is evidence that collateral growth can be enhanced. While there is evidence that EX can decrease the progression of atherosclerotic lesions or even induce the regression of atherosclerotic lesions in humans, the evidence of this is not strong due to the fact that most prospective trials conducted to date have included other lifestyle changes and treatment strategies by necessity. The literature from large animal models of CAD also presents a cloudy picture concerning whether EX can induce the regression of or slow the progression of atherosclerotic lesions. Thus, while evidence from research using humans with CAD and animal models of CAD indicates that EX increases endothelium-dependent dilation throughout the coronary vascular tree, evidence that EX reverses or slows the progression of lesion development in CAD is not conclusive at this time. This suggests that the beneficial effects of EX in CAD may not be the result of direct effects on the coronary artery wall. If this suggestion is true, it is important to determine the mechanisms involved in these beneficial effects.