Depression,Pain Intensity,and Interference in Acute Spinal Cord Injury

Background:

The high prevalence of pain and depression in persons with spinal cord injury (SCI) is well known. However the link between pain intensity, interference, and depression, particularly in the acute period of injury, has not received sufficient attention in the literature.

Objective:

To investigate the relationship of depression, pain intensity, and pain interference in individuals undergoing acute inpatient rehabilitation for traumatic SCI.

Methods:

Participants completed a survey that included measures of depression (PHQ-9), pain intensity (“right now”), and pain interference (Brief Pain Inventory: general activity, mood, mobility, relations with others, sleep, and enjoyment of life). Demographic and injury characteristics and information about current use of antidepressants and pre-injury binge drinking also were collected. Hierarchical multiple regression was used to test depression models in 3 steps: (1) age, gender, days since injury, injury level, antidepressant use, and pre-injury binge drinking (controlling variables); (2) pain intensity; and (3) pain interference (each tested separately).

Results:

With one exception, pain interference was the only statistically significant independent variable in each of the final models. Although pain intensity accounted for only 0.2% to 1.2% of the depression variance, pain interference accounted for 13% to 26% of the variance in depression.

Conclusion:

Our results suggest that pain intensity alone is insufficient for understanding the relationship of pain and depression in acute SCI. Instead, the ways in which pain interferes with daily life appear to have a much greater bearing on depression than pain intensity alone in the acute setting.Key words: depression, pain, spinal cord injuriesThe high incidence and prevalence of pain following spinal cord injury (SCI) is well established^1–6 and associated with numerous poor health outcomes and low quality of life (QOL).^1,7,8 Although much of the literature on pain in SCI focuses on pain intensity, there is emerging interest in the role of pain interference or the extent to which pain interferes with daily activities of life.^7,9 With prevalence as high as 77% in SCI, pain interference impacts life activities such as exercise, sleep, work, and household chores.^2,7,10–13 Pain interference also has been associated with disease management self-efficacy in SCI.¹⁴ There is a significant relationship between pain intensity and interference in persons with SCI.⁷ Like pain, the high prevalence of depression after SCI is well-established.^15–17 Depression and pain often co-occur,^18,19 and their overlap ranges from 30% to 60%.¹⁹ Pain is also associated with greater duration of depressed mood.²⁰ Pain and depression share common biological pathways and neurotransmitter mechanisms,¹⁹ and pain has been shown to attenuate the response to depression treatment.^21,22Despite the interest in pain and depression after SCI and implications for the treatment of depression, their co-occurrence has received far less attention in the literature.²³ Greater pain has been associated with higher levels of depression in persons with SCI,^16,24 although this is not a consistent finding.²⁵ Similarly, depression in persons with SCI who also have pain appears to be worse than for persons with non-SCI pain, suggesting that the link between pain and depression may be more intense in the context of SCI.²⁶ In one of the few studies of pain intensity and depression in an acute SCI rehabilitation setting, Cairns et al ²⁷ found a co-occurrence of pain and depression in 22% to 35% of patients. This work also suggested an evolution of the relationship between pain and depression over the course of the inpatient stay, such that they become associated by discharge. Craig et al²⁸ found that pain levels at discharge from acute rehabilitation predicted depression at 2-year follow-up. Pain interference also has been associated with emotional functioning and QOL in persons with SCI^1,7,29,30 and appears to mediate the relationship between ambulation and depression.³¹Studies of pain and depression in person with SCI are often limited methodologically to examine the independent contributions of pain intensity and interference to depression in an acute setting. For example, they include only pain intensity^{16,23,25,28,30}; classify subjects by either pain plus depression²³ or pain versus no pain^8,28,30; use pain intensity and interference as predictor and outcome, respectively¹; collapse pain interference domains into a single score¹; or use only univariate tests (eg, correlations).^7,8,25,30 In addition, the vast majority focus on the chronic period of injury. To fill a gap in knowledge, we examined the independent contributions of pain intensity and pain interference to depression, while accounting for injury and demographic characteristics, antidepressant treatment, and pre-injury binge drinking in a sample of persons with acute SCI. We hypothesized that when accounting for both pain intensity and interference in the model, interference would have an independent and significant relationship with depression, above and beyond pain intensity.     

Muscle Density and Bone Quality of the Distal Lower Extremity Among Individuals with Chronic Spinal Cord Injury

Background:

Understanding the related fates of muscle density and bone quality after chronic spinal cord injury (SCI) is an important initial step in determining endocrine-metabolic risk.

Objective:

To examine the associations between muscle density and indices of bone quality at the distal lower extremity of adults with chronic SCI.

Methods:

A secondary data analysis was conducted in 70 adults with chronic SCI (C2-T12; American Spinal Injury Association Impairment Scale [AIS] A-D; ≥2 years post injury). Muscle density and cross-sectional area (CSA) and bone quality indices (trabecular bone mineral density [TbBMD] at the distal tibia [4% site] and cortical thickness [CtTh], cortical area [CtAr], cortical BMD [CtBMD], and polar moment of inertia [PMI] at the tibial shaft [66% site]) were measured using peripheral quantitative computed tomography. Calf lower extremity motor score (cLEMS) was used as a clinical measure of muscle function. Multivariable linear regression analyses were performed to determine the strength of the muscle-bone associations after adjusting for confounding variables (sex, impairment severity [AIS A/B vs AIS C/D], duration of injury, and wheelchair use).

Results:

Muscle density was positively associated with TbBMD (b = 0.85 [0.04, 1.66]), CtTh (b = 0.02 [0.001, 0.034]), and CtBMD (b = 1.70 [0.71, 2.69]) (P < .05). Muscle CSA was most strongly associated with CtAr (b = 2.50 [0.12, 4.88]) and PMI (b = 731.8 [161.7, 1301.9]) (P < .05), whereas cLEMS was most strongly associated with TbBMD (b = 7.69 [4.63, 10.76]) (P < .001).

Conclusion:

Muscle density and function were most strongly associated with TbBMD at the distal tibia in adults with chronic SCI, whereas muscle size was most strongly associated with bone size and geometry at the tibial shaft.Key words: bone mineral density, bone quality, muscle density, muscle size, osteoporosis, peripheral quantitative computed tomography, spinal cord injurySpinal cord injury (SCI) is associated with sublesional muscle atrophy,^1–3 changes in muscle fiber type,^4,5 reductions in hip and knee region bone mineral density (BMD),^6–8 and increased central and regional adiposity after injury.^9,10 Adverse changes in muscle and bone health in individuals with SCI contribute to an increased risk of osteoporosis,^11–13 fragility fractures,¹⁴ and endocrine-metabolic disease (eg, diabetes, dyslipidemia, heart disease).^15–17 Crosssectional studies have shown a higher prevalence of lower extremity fragility fractures among individuals with SCI ranging from 1% to 34%.^18–20 Fragility fractures are associated with negative health and functional outcomes, including an increased risk of morbidity and hospitalization,^21,22 mobility limitations,²³ and a reduced quality of life.²⁴ Notably, individuals with SCI have a normal life expectancy, yet fracture rates increase annually from 1% per year in the first year to 4.6% per year in individuals greater than 20 years post injury.^25,26Muscle and bone are thought to function as a muscle-bone unit, wherein muscle contractions impose loading forces on bone that produce changes in bone geometry and structure.^27,28 A growing body of evidence has shown that individuals with SCI (predominantly those with motor complete injury) exhibit similar patterns of decline in muscle cross-sectional area (CSA) and BMD in the acute and subacute stages following injury.^4,11,29 Prospective studies have exhibited a decrease in BMD of 1.1% to 47% per year^6,7,30 and up to 73% in the 2 to 7 years following SCI.^8,14,31,32 Decreases in muscle CSA have been well-documented following SCI, with greater disuse atrophy observed after complete SCI versus incomplete SCI, presumably due to the absence of voluntary muscle contractions and associated mobility limitations.^1,2,16 Muscle quality is also compromised early after SCI, resulting in sublesional accumulation of adipose tissue in the chronic stage of injury^3,33,34; the exact time course of this event has been poorly elucidated to date. Adipose tissue deposition within and between skeletal muscle is linked to an increase in noncontractile muscle tissue and a reduction in muscle force-generating capacity on bone.^35,36 Skeletal muscle fat infiltration is up to 4 times more likely to occur in individuals with SCI,^1,16,37 contributing to metabolic complications (eg, glucose intolerance),¹⁶ reduced muscle strength and function,³⁸ and mobility limitations³ – all factors that may be associated with a deterioration in bone quality after SCI.The association between lean tissue mass and bone size (eg, BMD and bone mineral content) in individuals with SCI has been wellestablished using dual energy x-ray absorptiometry (DXA).^9,10,29,34 However, DXA is unable to measure true volumetric BMD (vBMD), bone geometry, and bone structure. Peripheral quantitative computed tomography (pQCT) is an imaging technique that improves our capacity to measure indices of bone quality and muscle density and CSA at fracture-prone sites (eg, tibia).^3,39 Recent evidence from cross-sectional pQCT studies has shown that muscle CSA and calf lower extremity motor score (cLEMS) were associated with indices of bone quality at the tibia in individuals with SCI.^13,40 However, neither study measured muscle density (a surrogate of fatty infiltration when evaluating the functional muscle-bone unit). Fatty infiltration of muscle is common after SCI^1,16,37 and may affect muscle function or the muscle-bone unit, but the association between muscle density and bone quality indices at the tibia in individuals with chronic SCI is unclear. Muscle density measured using pQCT may be an acceptable surrogate of muscle quality when it is difficult to assess muscle strength due to paralysis.^3,39 Additionally, investigating which muscle outcome (muscle density, CSA, cLEMS) is most strongly associated with vBMD and bone structure may inform modifiable targets for improving bone quality and reducing fracture risk after chronic SCI.The primary objective of this secondary analysis was to examine the associations between pQCTderived calf muscle density and trabecular vBMD at the tibia among adults with chronic SCI. The secondary objective was to examine the associations between calf muscle density, CSA, and function and tibial vBMD, cortical CSA and thickness, and polar moment of inertia (PMI). First, we hypothesize that calf muscle density will be a positive correlate of trabecular and cortical vBMD, cortical CSA and thickness, and PMI at the tibia in individuals with chronic SCI. Second, we hypothesize that of the key muscle variables (cLEMS, CSA and density), calf muscle density and cLEMS will be most strongly associated with trabecular vBMD, whereas calf muscle CSA will be most strongly associated with cortical CSA and PMI.     

Distinct Functions of Activated Protein C Differentially Attenuate Acute Kidney Injury

Administration of activated protein C (APC) protects from renal dysfunction, but the underlying mechanism is unknown. APC exerts both antithrombotic and cytoprotective properties, the latter via modulation of protease-activated receptor-1 (PAR-1) signaling. We generated APC variants to study the relative importance of the two functions of APC in a model of LPS-induced renal microvascular dysfunction. Compared with wild-type APC, the K193E variant exhibited impaired anticoagulant activity but retained the ability to mediate PAR-1-dependent signaling. In contrast, the L8W variant retained anticoagulant activity but lost its ability to modulate PAR-1. By administering wild-type APC or these mutants in a rat model of LPS-induced injury, we found that the PAR-1 agonism, but not the anticoagulant function of APC, reversed LPS-induced systemic hypotension. In contrast, both functions of APC played a role in reversing LPS-induced decreases in renal blood flow and volume, although the effects on PAR-1-dependent signaling were more potent. Regarding potential mechanisms for these findings, APC-mediated PAR-1 agonism suppressed LPS-induced increases in the vasoactive peptide adrenomedullin and infiltration of iNOS-positive leukocytes into renal tissue. However, the anticoagulant function of APC was responsible for suppressing LPS-induced stimulation of the proinflammatory mediators ACE-1, IL-6, and IL-18, perhaps accounting for its ability to modulate renal hemodynamics. Both variants reduced active caspase-3 and abrogated LPS-induced renal dysfunction and pathology. We conclude that although PAR-1 agonism is solely responsible for APC-mediated improvement in systemic hemodynamics, both functions of APC play distinct roles in attenuating the response to injury in the kidney.Acute kidney injury (AKI) leading to renal failure is a devastating disorder,¹ with a prevalence varying from 30 to 50% in the intensive care unit.² AKI during sepsis results in significant morbidity, and is an independent risk factor for mortality.^3,4 In patients with severe sepsis or shock, the reported incidence ranges from 23 to 51%^5–7 with mortality as high as 70% versus 45% among patients with AKI alone.^1,8The pathogenesis of AKI during sepsis involves hemodynamic alterations along with microvascular impairment.⁴ Although many factors change during sepsis, suppression of the plasma serine protease, protein C (PC), has been shown to be predictive of early death in sepsis models,⁹ and clinically has been associated with early death resulting from refractory shock and multiple organ failure in severe sepsis.¹⁰ Moreover, low levels of PC have been highly associated with renal dysfunction and pathology in models of AKI.¹¹ During vascular insult, PC becomes activated by the endothelial thrombin-thrombomodulin complex, and the activated protein C (APC) exhibits both antithrombotic and cytoprotective properties. We have previously demonstrated that APC administration protects from renal dysfunction during cecal ligation and puncture and after endotoxin challenge.^11,12 In addition, recombinant human APC [drotrecogin alfa (activated)] has been shown to reduce mortality in patients with severe sepsis at high risk of death.¹³ Although the ability of APC to protect from organ injury in vivo is well documented,^11,14,15 the precise mechanism mediating the response has not been ascertained.APC exerts anticoagulant properties via feedback inhibition of thrombin by cleavage of factors Va and VIIIa.¹⁶ However, APC bound to the endothelial protein C receptor (EPCR) can also exhibit direct potent cytoprotective properties by cleaving protease-activated receptor-1 (PAR-1).¹⁷ Various cell culture studies have demonstrated that the direct modulation of PAR-1 by APC results in cytoprotection by several mechanisms, including suppression of apoptosis,^18,19 leukocyte adhesion,^19,20 inflammatory activation,²¹ and suppression of endothelial barrier disruption.^22,23 In vivo, the importance of the antithrombotic activity of APC is well established in model systems^24,25 and in humans.²⁶ However, the importance of PAR-1-mediated effects of APC also has been clearly defined in protection from ischemic brain injury²⁷ and in sepsis models.²⁸ Hence, there has been significant debate whether the in vivo efficacy of APC is attributed primarily to its anticoagulant (inhibition of thrombin generation) or cytoprotective (PAR-1-mediated) properties.^17,29The same active site of APC is responsible for inhibition of thrombin generation by the cleavage of factor Va and for PAR-1 agonism. Therefore, we sought to generate point mutations that would not affect catalytic activity, but would alter substrate recognition to distinguish the two functions. Using these variants, we examined the relative role of the two known functions of APC in a model of LPS-induced renal microvascular dysfunction.     

Dietary Intake Relative to Cardiovascular Disease Risk Factors in Individuals With Chronic Spinal Cord Injury: A Pilot Study

Background:

The relationship between cardiovascular disease (CVD) risk factors and dietary intake is unknown among individuals with spinal cord injury (SCI).

Objective:

To investigate the relationship between consumption of selected food groups (dairy, whole grains, fruits, vegetables, and meat) and CVD risk factors in individuals with chronic SCI.

Methods:

A cross-sectional substudy of individuals with SCI to assess CVD risk factors and dietary intake in comparison with age-, gender-, and race-matched able-bodied individuals enrolled in the Coronary Artery Risk Development in Young Adults (CARDIA) study. Dietary history, blood pressure, waist circumference (WC), fasting blood glucose, high-sensitivity C-reactive protein (hs-CRP), lipids, glucose, and insulin data were collected from 100 SCI participants who were 38 to 55 years old with SCI >1 year and compared to 100 matched control participants from the CARDIA study.

Results:

Statistically significant differences between SCI and CARDIA participants were identified in WC (39.2 vs 36.2 in.; P < .001) and high-density lipoprotein cholesterol (HDL-C; 39.2 vs 47.5 mg/dL; P < .001). Blood pressure, total cholesterol, triglycerides, glucose, insulin, and hs-CRP were similar between SCI and CARDIA participants. No significant relation between CVD risk factors and selected food groups was seen in the SCI participants.

Conclusion:

SCI participants had adverse WC and HDL-C compared to controls. This study did not identify a relationship between consumption of selected food groups and CVD risk factors.Key words: cardiovascular disease risk factors, dietary intake, spinal cord injuryCardiovascular disease (CVD) is a leading cause of death in individuals with chronic spinal cord injuries (SCIs).^1–5 This is partly because SCI is associated with several metabolic CVD risk factors, including dyslipidemia,^6–10 glucose intolerance,^6,11–14 and diabetes.^15–17 In addition, persons with SCI exhibit elevated markers of inflammation^18,19 and endothelial activation²⁰ that are correlated with higher CVD prevalence.^21–23 Obesity, and specifically central obesity, another CVD risk factor,^24–26 is also common in this population.^12,27–29Dietary patterns with higher amounts of whole grains and fiber have been shown to improve lipid abnormalities,³⁰ glucose intolerance, diabetes mellitus,^31–34 hypertension,³⁵ and markers of inflammation³⁶ in the general population. These dietary patterns are also associated with lower levels of adiposity.³¹ Ludwig et al reported that the strong inverse associations between dietary fiber and multiple CVD risk factors – excessive weight gain, central adiposity, elevated blood pressure, hypertriglyceridemia, low high-density lipoprotein cholesterol (HDL-C), high low-density lipoprotein cholesterol (LDL-C), and high fibrinogen – were mediated, at least in part, by insulin levels.³⁷ Whole-grain food intake is also inversely associated with fasting insulin, insulin resistance, and the development of type 2 diabetes.^32,38,39Studies in the general population have also shown a positive association between the development of metabolic syndrome as well as heart disease and consumption of a Western diet, a diet characterized by high intake of processed and red meat and low intake of fruit, vegetables, whole grains, and dairy.^40,41 Red meat, which is high in saturated fat, has been shown to have an association with adverse levels of cholesterol and blood pressure and the development of obesity, metabolic syndrome, and diabetes.^40,42,43Numerous studies have shown that individuals with chronic SCI have poor diet quality.^44–49 A Canadian study found that only 26.7% of their sample was adherent to the recommendations about the consumption of fruit, vegetables, and grains from the “Eating Well with Canada’s Food Guide.”⁴⁴ Individuals with chronic SCI have also been found to have low fiber and high fat intakes when their diets were compared to dietary recommendations from the National Cholesterol Education Program,⁴⁶ the 2000 Dietary Guidelines for Americans,⁴⁹ and the recommended Dietary Reference Intakes and the Acceptable Macronutrient Distribution Range.^47,48However, unlike in the general population, the relationship between dietary intake and obesity and CVD risk factors is unknown in the chronic SCI population. If a dietary pattern consisting of higher intake of whole grains and dietary fiber is favorably associated with obesity and CVD risk factors in individuals with chronic SCI, then trials of increased intake of whole grains and fiber intake could be conducted to document health benefits and inform recommendations. The purpose of this pilot study is to investigate the association between selected food group intake and CVD risk factors in individuals with chronic SCI as compared to age-, gender-, and race-matched able-bodied individuals enrolled in the Coronary Artery Risk Development in Young Adults (CARDIA) study. Data will also be used to plan future studies in the relatively understudied field of CVD and nutrition in individuals with SCI.     

Restoring Voluntary Grasping Function in Individuals with Incomplete Chronic Spinal Cord Injury: Pilot Study

Background:

Functional electrical stimulation (FES) therapy has been shown to be one of the most promising approaches for improving voluntary grasping function in individuals with subacute cervical spinal cord injury (SCI).

Objective:

To determine the effectiveness of FES therapy, as compared to conventional occupational therapy (COT), in improving voluntary hand function in individuals with chronic (≥24 months post injury), incomplete (American Spinal Injury Association Impairment Scale [AIS] B-D), C4 to C7 SCI.

Methods:

Eight participants were randomized to the intervention group (FES therapy; n = 5) or the control group (COT; n = 3). Both groups received 39 hours of therapy over 13 to 16 weeks. The primary outcome measure was the Toronto Rehabilitation Institute-Hand Function Test (TRI-HFT), and the secondary outcome measures were Graded Redefined Assessment of Strength Sensibility and Prehension (GRASSP), Functional Independence Measure (FIM) self-care subscore, and Spinal Cord Independence Measure (SCIM) self-care subscore. Outcome assessments were performed at baseline, after 39 sessions of therapy, and at 6 months following the baseline assessment.

Results:

After 39 sessions of therapy, the intervention group improved by 5.8 points on the TRI-HFT’s Object Manipulation Task, whereas the control group changed by only 1.17 points. Similarly, after 39 sessions of therapy, the intervention group improved by 4.6 points on the FIM self-care subscore, whereas the control group did not change at all.

Conclusion:

The results of the pilot data justify a clinical trial to compare FES therapy and COT alone to improve voluntary hand function in individuals with chronic incomplete tetraplegia.Key words: chronic patients, functional electrical stimulation, grasping, therapy, upper limbIn the United States and Canada, there is a steady rate of incidence and an increasing rate of prevalence of individuals living with spinal cord injury (SCI). For individuals with tetraplegia, hand function is essential for achieving a high level of independence in activities of daily living.^1–5 For the majority of individuals with tetraplegia, the recovery of hand function has been rated as their highest priority.⁵Traditionally, functional electrical stimulation (FES) has been used as a permanent neuroprosthesis to achieve this goal.^6–14 More recently, researchers have worked toward development of surface FES technologies that are meant to be used as shortterm therapies rather than permanent prosthesis. This therapy is frequently called FES therapy or FET. Most of the studies published to date, where FES therapy was used to help improve upper limb function, have been done in both the subacute and chronic stroke populations^15–23 and 2 have been done in the subacute SCI population.^1–3 With respect to the chronic SCI population, there are no studies to date that have looked at use of FES therapy for retraining upper limb function. In a review by Kloosterman et al,²⁴ the authors have discussed studies that have used various combinations of therapies for improving upper extremity function in chronic SCI individuals; however, the authors found that the only study that showed significant improvements before and after was the study published by Needham-Shropshire et al.²⁵ This study examined the effectiveness of neuromuscular stimulation (NMS)–assisted arm ergometry for strengthening triceps brachii. In this study, electrical stimulation was used to facilitate arm ergometry, and it was not used in the context of retraining reaching, grasping, and/or object manipulation.Since 2002, our team has been investigating whether FES therapy has the capacity to improve voluntary hand function in complete and incomplete subacute cervical SCI patients who are less than 180 days post injury at the time of recruitment in the study.^1–3 In randomized controlled trials (RCTs) conducted by our team, we found that FES therapy is able to restore voluntary reaching and grasping functions in individuals with subacute C4 to C7 incomplete SCI.^1–3 The changes observed were transformational; individuals who were unable to grasp at all were able to do so after only 40 one-hour sessions of the FES therapy, whereas the control group showed significantly less improvement. Inspired by these results, we decided to conduct a pilot RCT with chronic (≥24 months following injury) C4 to C7 SCI patients (American Spinal Injury Association Impairment Scale [AIS] B-D), which is presented in this article. The purpose of this pilot study was to determine whether the FES therapy is able to restore voluntary hand function in chronic tetraplegic individuals. Based on the results of our prior phase I¹ and phase II^2,3 RCTs in the subacute SCI population, we hypothesized that individuals with chronic tetraplegia who underwent the FES therapy (intervention group) may have greater improvements in voluntary hand function, especially in their ability to grasp and manipulate objects, and perform activities of daily living when compared to individuals who receive similar volume and duration of conventional occupational therapy (COT: control group).     

Predictors of Subclinical Atherosclerosis in Women With Spinal Cord Injury

Background:

Chronic spinal cord injury (SCI) is associated with an increase in risk factors for cardiovascular disease (CVD). In the general population, atherosclerosis in women occurs later than in men and usually presents differently. Associations between risk factors and incidence of CVD have not been studied in women with SCI.

Objective:

To determine which risk factors for CVD are associated with increased carotid intima-media thickness (CIMT), a common indicator of atherosclerosis, in women with SCI.

Methods:

One hundred and twenty-two females older than 18 years with traumatic SCI at least 2 years prior to entering the study were evaluated. Participants were asymptomatic and without evidence of CVD. Exclusion criteria were acute illness, overt heart disease, diabetes, and treatment with cardiac drugs, lipid-lowering medication, or antidiabetic agents. Measures for all participants were age, race, smoking status, level and completeness of injury, duration of injury, body mass index, serum lipids, fasting glucose, hemoglobin A1c, and ultrasonographic measurements of CIMT. Hierarchical multiple linear regression was conducted to predict CIMT from demographic and physiologic variables.

Results:

Several variables were significantly correlated with CIMT during univariate analyses, including glucose, hemoglobin A1c, age, and race/ethnicity; but only age was significant in the hierarchical regression analysis.

Conclusions:

Our data indicate the importance of CVD in women with SCI.Key words: age, cardiovascular disease, carotid intima-media thickness, hemoglobin A1c, risk factors, smokingThe secondary conditions of metabolic syndrome and cardiovascular disease (CVD) resulting from spinal cord injury (SCI ) are not well understood. In particular, persons with SCI have an increase in metabolic risk factors for cardiovascular disease (CVD),^1–5 but researchers have not determined whether this increase is associated with an increased incidence of CVD. The association has not been shown in reports on mortality or prevalence rates for CVD in people with SCI^6–12 or in the few studies that have appraised CVD in people with SCI using physiologic assessments.^13–18 Either the question was not addressed, or the evidence is insufficient due to low sample sizes and a lack of objective, prospective epidemiological studies assessing this question. Nevertheless, studies consistently show that metabolic syndrome is prevalent among individuals with SCI.^1–5,12 Metabolic syndrome consists of multiple interrelated risk factors that increase the risk for atherosclerotic heart disease by 1.5- to 3-fold.^19,20Compounding the uncertainty about the association of metabolic risk factors with CVD in SCI are possible gender differences.^21–24 Findings from studies of men with SCI might not apply to women with SCI. For example, the correlation between physical activity and high-density lipoprotein (HDL) levels in men with SCI is not found for women with SCI.^25,26 Furthermore, able-bodied women develop atherosclerosis later than do able-bodied men, and they usually present differently.²⁷ Some studies indicate that abnormal glucose metabolism may play a particularly important role in CVD in women²⁷; data from our group suggest that this is the case in women with SCI as well.¹⁵ Although women constitute 18% to 20% of the SCI population, no studies have evaluated cardiovascular health in women with chronic SCI.Carotid intima-media thickness (CIMT) is the most robust, highly tested, and often used noninvasive endpoint for assessing the progression of subclinical atherosclerosis in men and women of all ages.^28–46 For people with SCI, CIMT is a reliable surrogate measure of asymptomatic CVD.^15,47 The incidence of asymptomatic CVD appears to increase with the duration of SCI,¹⁵ where duration of injury is a cardiac risk factor independent of age.¹⁷ Moreover, CIMT is greater in men with SCI than in matched able-bodied controls,⁴⁸ indicating a subclinical and atypical presentation of CVD. A variety of studies have confirmed the usefulness of high-resolution B-mode ultrasound measurement of CIMT for quantitation of subclinical atherosclerosis.⁴⁹To better discern the association of risk factors with measures of subclinical atherosclerotic disease in women with SCI, we performed blood tests and ultrasonographic measurements of CIMT on 122 females with chronic SCI who were free of overt CVD. We tested for the 3 metabolic risk factors that are consistently identified in the varied definitions of metabolic syndrome: abnormal carbohydrate metabolism, abnormally high triglycerides, and abnormally low HDL cholesterol. We also tested for 4 other CVD risk factors: high levels of low-density lipoprotein (LDL), high total cholesterol, high body mass index (BMI), and a history of smoking.     

Slit Diaphragms Contain Tight Junction Proteins

Slit diaphragms are essential components of the glomerular filtration apparatus, as changes in these junctions are the hallmark of proteinuric diseases. Slit diaphragms, considered specialized adherens junctions, contain both unique membrane proteins (e.g., nephrin, podocin, and Neph1) and typical adherens junction proteins (e.g., P-cadherin, FAT, and catenins). Whether slit diaphragms also contain tight junction proteins is unknown. Here, immunofluorescence, immunogold labeling, and cell fractionation demonstrated that rat slit diaphragms contain the tight junction proteins JAM-A (junctional adhesion molecule A), occludin, and cingulin. We found these proteins in the same protein complexes as nephrin, podocin, CD2AP, ZO-1, and Neph1 by cosedimentation, coimmunoprecipitation, and pull-down assays. PAN nephrosis increased the protein levels of JAM-A, occludin, cingulin, and ZO-1 several-fold in glomeruli and loosened their attachment to the actin cytoskeleton. These data extend current information about the molecular composition of slit diaphragms by demonstrating the presence of tight junction proteins, although slit diaphragms lack the characteristic morphologic features of tight junctions. The contribution of these proteins to the assembly of slit diaphragms and potential signaling cascades requires further investigation.Slit diaphragms are specialized cell-cell junctions located between mature podocytes that have fascinated cell biologists and nephrologists for more than 40 yr.¹ In contrast to podocytes, most other epithelial cells have junctional complexes composed of tight junctions and adherens junctions. Slit diaphragms originate from typical apical junctional complexes between primordial epithelia of the early S-shaped body. These junctional complexes migrate in a zipper-like fashion to the base of the cell where tight junctions persist as interdigitation of the foot processes begins.^2,3 Slit diaphragms appear during the capillary loop stage and gradually replace tight junctions. In many diseases associated with proteinuria and foot process loss or effacement, there is a rerun in reverse of this developmental sequence, and tight junctions reappear between adjoining foot processes.^4–6Major progress has been made recently in establishing the molecular make-up of the slit diaphragms. Several integral membrane proteins, including nephrin,⁷ podocin,⁸ and Neph1,⁹ not found in other junctions, have been identified as slit diaphragm components. Slit diaphragms are currently looked upon as signaling platforms in which nephrin and Neph1 transduce major signals that serve to maintain the filtration slits and to regulate podocyte shape through interaction of slit diaphragm proteins with the actin cytoskeleton.¹⁰ Mutations in nephrin,⁷ Neph1,⁹ and podocin⁸ have been linked to diseases associated with foot process effacement and proteinuria. In addition to these specialized slit diaphragm proteins, a number of other proteins that are associated with junctions in other locations are also concentrated at the slit diaphragms, including the adherens junction proteins P-cadherin,¹¹ FAT,¹² β-catenin,¹¹ and p120 catenin;¹³ scaffold proteins such as ZO-1,^14,15 CD2AP,¹⁶ MAGI-2,¹⁷ and CASK;¹³ and actin binding proteins, including IQGAP¹⁷ and α-actinin 4.^17,18 Because slit diaphragms share some morphologic features with adherens junctions and contain P-cadherin and catenins, slit diaphragms are assumed to represent modified adherens junctions.¹¹ However, several scaffold proteins that are often associated with tight junctions (i.e., ZO-1,^14,15 MAGI-1,¹⁹ MAGI-2,¹⁷ and CASK¹³) are present at slit diaphragms and have been shown to associate with nephrin. Based on their derivation from typical tight junctions² and the fact that they are replaced by tight junctions in nephrosis,^4,6 we reasoned that slit diaphragms might also contain membrane proteins normally associated with tight junctions.In this paper, we used morphological, biochemical, and bioinformatics techniques to investigate the expression of representative tight junction proteins in glomeruli in situ and in slit diaphragm-enriched fractions. Here, we document the presence of several tight junction proteins in slit diaphragms and demonstrate their interactions with slit diaphragm proteins in both normal and PAN nephrotic rats. The presence of tight junction proteins in slit diaphragms adds a new dimension to understanding the organization and functions of these junctions.     

Hepatic stearoyl-CoA desaturase (SCD)-1 activity and diacylglycerol but not ceramide concentrations are increased in the nonalcoholic human fatty liver

OBJECTIVE—To determine whether 1) hepatic ceramide and diacylglycerol concentrations, 2) SCD1 activity, and 3) hepatic lipogenic index are increased in the human nonalcoholic fatty liver.RESEARCH DESIGN AND METHODS—We studied 16 subjects with (n = 8) and without (n = 8) histologically determined nonalcoholic fatty liver (NAFL⁺ and NAFL⁻) matched for age, sex, and BMI. Hepatic concentrations of lipids and fatty acids were quantitated using ultra-performance liquid chromatography coupled to mass spectrometry and gas chromatography.RESULTS—The absolute (nmol/mg) hepatic concentrations of diacylglycerols but not ceramides were increased in the NAFL⁺ group compared with the NAFL⁻ group. The livers of the NAFL⁺ group contained proportionally less long-chain polyunsaturated fatty acids as compared with the NAFL⁻ group. Liver fat percent was positively related to hepatic stearoyl-CoA desaturase 1 (SCD1) activity index (r = 0.70, P = 0.003) and the hepatic lipogenic index (r = 0.54, P = 0.030). Hepatic SCD1 activity index was positively related to the concentrations of diacylglycerols (r = 0.71, P = 0.002) but not ceramides (r = 0.07, NS).CONCLUSIONS—We conclude that diacylglycerols but not ceramides are increased in NAFL. The human fatty liver is also characterized by depletion of long polyunsaturated fatty acids in the liver and increases in hepatic SCD1 and lipogenic activities.Nonalcoholic fatty liver disease (NAFLD) is characterized by lipid accumulation in the liver (≥10% of liver weight), which cannot be attributed to alcohol consumption or any other liver disease (1). NAFLD covers a range from simple nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH) and fibrosis (1). The fatty liver is resistant to the action of insulin to inhibit hepatic glucose (2,3) and VLDL (4) production, resulting in hyperglycemia and hypertriglyceridemia. The mechanisms underlying insulin resistance in human NAFLD are unclear. While triacylglycerols themselves are inert, lipid intermediates may act as important regulators of both oxidative stress (5) and insulin signaling (6). In vitro studies as well as studies in animals suggest that diacylglycerols, which are immediate precursors of triacylglycerols (7), can induce insulin resistance by activating specific isoforms of protein kinase C (PKC) (8,9). The concentrations of diacylglycerols have recently been shown to be increased in human NAFLD compared with subjects with normal liver histology (10). Ceramides are another class of reactive lipids that mediate saturated fat–induced insulin resistance (6). There are no data comparing ceramide and diacylglycerol concentrations in the human liver or relating them to hepatic fat content.Sources of hepatic lipids include dietary chylomicron remnants, free fatty acids released from either adipose tissue triacylglycerols or chylomicrons hydrolyzed at a rate in excess of what can be taken up by tissues (spillover), and de novo lipogenesis (11). Increased lipolysis is a major contributor to hepatic fat accumulation (12–14). In addition, when estimated using tracer techniques, de novo lipogenesis has been found to be significantly increased in subjects with NAFLD compared with normal subjects (12,15,16). De novo lipogenesis produces saturated fatty acids (17,18). Stearoyl-CoA desaturase 1 (SCD1) converts saturated fatty acids to monounsaturated fatty acids, which are major substrates for synthesis of triacylglycerols and other lipids (19). SCD1 knockout mice are resistant to the development of obesity and hepatic steatosis (20,21), whereas the activity of SCD1 is significantly increased in the fatty livers of ob/ob mice (20,22). These data thus suggest that hepatic SCD1 activity may contribute to lipid accumulation in NAFLD. There are, however, no data on hepatic SCD1 activity in human NAFLD.To address the above questions, we quantified the full range of lipids and fatty acids using ultra-performance liquid chromatography (UPLC) coupled to mass spectrometry (MS) and gas chromatography in the human liver. These analyses were performed in two groups of subjects matched for age, sex, and BMI but with either a normal liver fat content (≤10% macrovesicular steatosis) or a nonalcoholic fatty liver (NAFL) (≥20% macrovesicular steatosis [1]).     

Population-specific risk of type 2 diabetes conferred by HNF4A P2 promoter variants: a lesson for replication studies

OBJECTIVE—Single nucleotide polymorphisms (SNPs) in the P2 promoter region of HNF4A were originally shown to be associated with predisposition for type 2 diabetes in Finnish, Ashkenazi, and, more recently, Scandinavian populations, but they generated conflicting results in additional populations. We aimed to investigate whether data from a large-scale mapping approach would replicate this association in novel Ashkenazi samples and in U.K. populations and whether these data would allow us to refine the association signal.RESEARCH DESIGN AND METHODS—Using a dense linkage disequilibrium map of 20q, we selected SNPs from a 10-Mb interval centered on HNF4A. In a staged approach, we first typed 4,608 SNPs in case-control populations from four U.K. populations and an Ashkenazi population (n = 2,516). In phase 2, a subset of 763 SNPs was genotyped in 2,513 additional samples from the same populations.RESULTS—Combined analysis of both phases demonstrated association between HNF4A P2 SNPs (rs1884613 and rs2144908) and type 2 diabetes in the Ashkenazim (n = 991; P < 1.6 × 10⁻⁶). Importantly, these associations are significant in a subset of Ashkenazi samples (n = 531) not previously tested for association with P2 SNPs (odds ratio [OR] ∼1.7; P < 0.002), thus providing replication within the Ashkenazim. In the U.K. populations, this association was not significant (n = 4,022; P > 0.5), and the estimate for the OR was much smaller (OR 1.04; [95%CI 0.91–1.19]).CONCLUSIONS—These data indicate that the risk conferred by HNF4A P2 is significantly different between U.K. and Ashkenazi populations (P < 0.00007), suggesting that the underlying causal variant remains unidentified. Interactions with other genetic or environmental factors may also contribute to this difference in risk between populations.The presence of type 2 diabetes susceptibility genes on chromosome 20 has been suggested by linkage scans in several populations. The 20q12–q13 region (Online Mendelian Inheritance in Man [OMIM] 603694) is the best replicated and harbors the gene HNF4A, mutations that lead to type 1 maturity-onset diabetes of the young (OMIM 125850). Evidence for association between SNPs in the β-cell P2 promoter region of HNF4A has been recognized in Finnish (1) and Ashkenazi (2) populations, with data suggesting that the HNF4A P2 SNPs (or variants in strong linkage disequilibrium with them) contribute to the linkage signal on chromosome 20q (1,2). Association with HNF4A promoter SNPs has been replicated in some (3–7) but not all (8–12) populations tested. In other populations, there was evidence for association with SNPs or haplotypes in the HNF4A region other than the P2 SNPs (10,13–15). More recently, the association between HNF4A promoter SNPs and type 2 diabetes has been confirmed in Scandinavians but not in a broader meta-analysis with additional populations (16), suggesting that P2 SNPs confer varying risk effects in different populations, possibly due to the underlying causal variant not having been identified. We investigated a 10-Mb interval (38.1–48.2 Mb National Center for Biotechnology Information build 35) centered around HNF4A, including genotypes from 4,608 nonredundant (r² < 1) SNPs (one SNP per 2 Kb, on average) in five type 2 diabetic case-control populations, to evaluate whether we could confirm and refine the association signal in Ashkenazim and whether this association was also present in U.K. populations. We were also interested in assessing whether there was evidence for additional association signals within this broader interval. We tested an Ashkenazi type 2 diabetes case-control study (n = 998), including novel samples (n = 531) not previously tested for linkage or association with HNF4A P2 SNPs (2); two U.K. population-based case-control studies where linkage and association studies with HNF4A P2 had not be carried out (n = 2,189); and two additional U.K. case-control collections (n = 1,842), with one enriched for earlier-onset type 2 diabetes where linkage studies had not been done but that showed suggestive association with HNF4A P2 SNPs (4) and one that included samples where, despite no evidence of linkage to chromosome 20q, association of HNF4A P2 SNPs with type 2 diabetes risk had previously been suggested (4,17).     

A Recessive Gene for Primary Vesicoureteral Reflux Maps to Chromosome 12p11-q13

Primary vesicoureteral reflux (pVUR) is one of the most common causes of pediatric kidney failure. Linkage scans suggest that pVUR is genetically heterogeneous with two loci on chromosomes 1p13 and 2q37 under autosomal dominant inheritance. Absence of pVUR in parents of affected individuals raises the possibility of a recessive contribution to pVUR. We performed a genome-wide linkage scan in 12 large families segregating pVUR, comprising 72 affected individuals. To avoid potential misspecification of the trait locus, we performed a parametric linkage analysis using both dominant and recessive models. Analysis under the dominant model yielded no signals across the entire genome. In contrast, we identified a unique linkage peak under the recessive model on chromosome 12p11-q13 (D12S1048), which we confirmed by fine mapping. This interval achieved a peak heterogeneity LOD score of 3.6 with 60% of families linked. This heterogeneity LOD score improved to 4.5 with exclusion of two high-density pedigrees that failed to link across the entire genome. The linkage signal on chromosome 12p11-q13 originated from pedigrees of varying ethnicity, suggesting that recessive inheritance of a high frequency risk allele occurs in pVUR kindreds from many different populations. In conclusion, this study identifies a major new locus for pVUR and suggests that in addition to genetic heterogeneity, recessive contributions should be considered in all pVUR genome scans.Vesicoureteral reflux (VUR; OMIM no. 193000) is the retrograde flow of urine from the bladder to the ureters and the kidneys during micturation. Uncorrected, VUR can lead to repeated urinary tract infections, renal scarring and reflux nephropathy, accounting for up to 25% of pediatric end stage renal disease.^1,2 VUR is commonly seen as an isolated disorder (primary VUR; pVUR), but it can also present in association with complex congenital abnormalities of the kidney and urinary tract or with specific syndromic disorders, such as renal-coloboma and branchio-oto-renal syndromes.^3–8pVUR has a strong hereditary component, with monozygotic twin concordance rates of 80%.^9–12 Sibling recurrence rates of 30% to 65% have suggested segregation of a single gene or oligogenes with large effects.^9,12–14 Interestingly however, the three published genome-wide linkage scans of pVUR have strongly suggested multifactorial determination.^15–17 Two pVUR loci have been identified with genome-wide significance on chromosomes 1p13 and 2q37 under an autosomal dominant transmission with locus heterogeneity.^15,16 Multiple suggestive signals have also been reported, but remarkably, these studies show little overlap.^15–17 These data suggest that pVUR may be extremely heterogeneous, with mutations in different genes each accounting for a fraction of cases. The genes underlying pVUR loci have not yet been identified, but two recent studies have reported segregating mutations in the ROBO2 gene in up to 5% of pVUR families.^18,19Despite evidence for genetic heterogeneity and different subtypes of disease, genetic studies have all modeled pVUR as an autosomal dominant trait.^15–17,20 Recessive inheritance has generally not been considered because the absence of affected parents can be explained by spontaneous resolution of pVUR with older age. However, many pVUR cohorts are composed of affected sibships or pedigrees compatible with autosomal recessive transmission, suggesting the potential for alternative modes of inheritance.^{9–12,16,17,20–22} Systematic family screening to clarify the mode of inheritance is not feasible for pVUR because the standard diagnostic tool, the voiding cystourethrogram (VCUG), is invasive and would expose participants to radiation. Formal assessment of a recessive contribution in sporadic pVUR has also been difficult because studies have been conducted in populations with low consanguinity rates.^{9–12,16,17,20–22} However, recent studies have identified an unexpected recessive contribution to several complex traits such as ductus arteriosus or autism.^23,24 Thus, in addition to genetic heterogeneity, genes with alternative modes of transmission may segregate among pVUR families, and misspecification of the inheritance model may complicate mapping studies of this trait.Several approaches can be considered to address the difficulties imposed by complex inheritance, variable penetrance, and genetic heterogeneity. Studying large, well characterized cohorts with newer single-nucleotide polymorphism (SNP)-based technologies can maximize inheritance information across the genome and increase the power of linkage studies.²⁵ In addition, in the setting of locus heterogeneity and uncertainty about the mode of transmission, analysis under a dominant and a recessive model has greater power compared with nonparametric methods and more often results in detection of the correct mode of transmission without incurring a significant penalty for multiple testing.^26–29 We combined these approaches in this study and successfully localized a major gene for VUR, which unexpectedly demonstrates autosomal recessive transmission.     

Plasmin in Nephrotic Urine Activates the Epithelial Sodium Channel

Proteinuria and increased renal reabsorption of NaCl characterize the nephrotic syndrome. Here, we show that protein-rich urine from nephrotic rats and from patients with nephrotic syndrome activate the epithelial sodium channel (ENaC) in cultured M-1 mouse collecting duct cells and in Xenopus laevis oocytes heterologously expressing ENaC. The activation depended on urinary serine protease activity. We identified plasmin as a urinary serine protease by matrix-assisted laser desorption/ionization time of-flight mass spectrometry. Purified plasmin activated ENaC currents, and inhibitors of plasmin abolished urinary protease activity and the ability to activate ENaC. In nephrotic syndrome, tubular urokinase-type plasminogen activator likely converts filtered plasminogen to plasmin. Consistent with this, the combined application of urokinase-type plasminogen activator and plasminogen stimulated amiloride-sensitive transepithelial sodium transport in M-1 cells and increased amiloride-sensitive whole-cell currents in Xenopus laevis oocytes heterologously expressing ENaC. Activation of ENaC by plasmin involved cleavage and release of an inhibitory peptide from the ENaC γ subunit ectodomain. These data suggest that a defective glomerular filtration barrier allows passage of proteolytic enzymes that have the ability to activate ENaC.Nephrotic syndrome is characterized by proteinuria, sodium retention, and edema. Increased renal sodium reabsorption occurs in the cortical collecting duct (CCD),^1,2 where a rate-limiting step in transepithelial sodium transport is the epithelial sodium channel (ENaC), which is composed of the three homologous subunits: α, β, γ.³ENaC activity is regulated by hormones, such as aldosterone and vasopressin (AVP)^4,5; however, adrenalectomized rats and AVP-deficient Brattleboro rats are capable of developing nephrotic syndrome,^1,6 and nephrotic patients do not consistently display elevated levels of sodium-retaining hormones,^7,8 suggesting that renal sodium hyper-reabsorption is independent of systemic factors. Consistent with this, sodium retention is confined to the proteinuric kidney in the unilateral puromycin aminonucleoside (PAN) nephrotic model.^2,9,10There is evidence that proteases contribute to ENaC activation by cleaving the extracellular loops of the α- and γ-subunits.^11–13 Proteolytic activation of ENaC by extracellular proteases critically involves the cleavage of the γ subunit,^14–16 which probably leads to the release of a 43-residue inhibitory peptide from the ectodomain.¹⁷ Both cleaved and noncleaved channels are present in the plasma membrane,^18,19 allowing proteases such as channel activating protease 1 (CAP1/prostasin),²⁰ trypsin,²⁰ chymotrypsin,²¹ and neutrophil elastase²² to activate noncleaved channels from the extracellular side.^23,24 We hypothesized that the defective glomerular filtration barrier in nephrotic syndrome allows the filtration of ENaC-activating proteins into the tubular fluid, leading to stimulation of ENaC. The hypothesis was tested in the PAN nephrotic model in rats and with urine from patients with nephrotic syndrome.     

Prevalence of melanocortin-4 receptor deficiency in Europeans and their age-dependent penetrance in multigenerational pedigrees

OBJECTIVE— Melanocortin-4 receptor (MC4R) deficiency is the most frequent genetic cause of obesity. However, there is uncertainty regarding the degree of penetrance of this condition, and the putative impact of the environment on the development of obesity in MC4R mutation carriers is unknown.RESEARCH DESIGN AND METHODS— We determined the MC4R sequence in 2,257 obese individuals and 2,677 nonobese control subjects of European origin and established the likely functional impact of all variants detected. We then included relatives of probands carriers and studied 25 pedigrees, including 97 carriers and 94 noncarriers from three generations.RESULTS— Of the MC4R nonsynonymous mutations found in obese subjects, 68% resulted in a loss of function in vitro. They were found in 1.72% of obese versus 0.15% of nonobesed subjects (P = 6.9 × 10⁻¹⁰). Among the families, abnormal eating behavior was more frequent in both MC4R-deficient children and adults than in noncarriers. Although BMI was inversely associated with educational status in noncarrier adults, no such relationship was seen in MC4R mutation carriers. We observed a generational effect, with a penetrance of 40% in MC4R-deficient adults aged >52 years, 60% in 18- to 52-year-old adults, and 79% in children. The longitudinal study of adult carriers showed an increasing age-dependent penetrance (37% at 20 years versus 60% at >40 years).CONCLUSIONS— We have established a robust estimate of age-related penetrance for MC4R deficiency and demonstrated a generational effect on penetrance, which may relate to the development of an “obesogenic” environment. It remains to be seen whether appropriate manipulation of environmental factors may contribute to preventing the development of obesity even in those strongly genetically predisposed to it.The leptin-melanocortin axis controls human energy homeostasis, and the melanocortin-4 receptor (MC4R) is a key player in its central regulation (1). Loss-of-function mutations in MC4R cause severe familial forms of obesity (2,3), and infrequent gain-of-function polymorphisms have been associated with protection against obesity (4,5). At least 72 nonsynonymous mutations have been discovered so far, but some have no obvious functional consequences (6,7), highlighting the importance of functional characterization of MC4R mutations in the determination of potential pathogenicity. MC4R is a membrane-bound G-protein–coupled receptor that activates adenylate cyclase. Loss-of-function mutations result in a partial or complete loss of function as measured by cAMP production. The majority of missense mutations in MC4R result in intracellular retention of the mutated protein, whereas some primarily affect ligand binding or ligand/receptor activation (8,9). In some cases, the alteration of the basal activity of the receptor (8,10) has been reported.The prevalence of loss-of-function MC4R mutations ranges from 0.5 to 5.8% in childhood-onset obesity (11–14). The contribution of MC4R mutations to late-onset obesity is still debated (13,15–18). Obesity due to MC4R mutations has been extensively studied, and although heterozygous loss-of-function mutations can clearly cause familial obesity, they can be found in individuals who are not obese (19). There is a need for reliable estimates of penetrance. Furthermore, no study has thoroughly assessed the effect of loss-of-function MC4R mutations in elderly subjects. Previous studies using part of our French cohort evidenced the first mutation in MC4R and demonstrated that most of them lead to an intracellular retention of the receptor (2,13,18).Although hyperphagia is a key feature of MC4R deficiency, with increased food intake at an ad libitum test meal reported in severely obese MC4R-deficient children (10), an apparent amelioration of obesity and food intake disturbances has been suggested in adulthood in some studies (6,11). Obesity is a complex trait, and MC4R mutations offer a unique opportunity to analyze the effects of both aging and shared environment on the evolution of body mass in this paradigm. In this extensive study of 2,257 unrelated obese subjects, 2,677 control subjects of European descent, and 25 multigenerational pedigrees with several MC4R mutations carriers, we provide a comprehensive picture of the prevalence of this condition and of factors that determine the expression of the obesity phenotype and support previous observations reported in a German familial study (20).     

EMG Biofeedback and Exercise for Treatment of Cervical and Shoulder Pain in Individuals with a Spinal Cord Injury: A Pilot Study

Background:

Chronic or recurrent musculoskeletal pain in the cervical and shoulder region is a common secondary problem after spinal cord injury (SCI), reported by 30% to 70% of individuals.

Objective:

The purpose of this study was to investigate the effect of electromyographic (EMG) biofeedback training, in addition to a standard exercise program, on reducing shoulder pain in manual wheelchair users with SCI.

Methods:

Fifteen individuals with SCI, C6 or lower, who were manual wheelchair users with shoulder pain were randomly assigned to 1 of 2 interventions. The Exercise group (n = 7) received instruction on a standard home-based exercise program. The EMG Biofeedback plus Exercise group (n = 8) received identical exercise instruction plus EMG biofeedback training to improve muscle balance and muscle relaxation during wheelchair propulsion. Shoulder pain was assessed by the Wheelchair Users Shoulder Pain Index (WUSPI) at baseline, at posttest 10 weeks after the start of intervention, and at follow-up 16 weeks after posttest.

Results:

The number of participants per group allowed only within-group comparisons; however, the findings indicated a beneficial effect from EMG biofeedback training. Shoulder pain, as measured by WUSPI, decreased 64% from baseline to posttest for the EMG Biofeedback plus Exercise group (P = .02). Shoulder pain for the Exercise group decreased a nonsignificant 27%. At follow-up, both groups showed continued improvement, yet the benefit of EMG biofeedback training was still discernible. The EMG Biofeedback plus Exercise group had an 82% reduction in shoulder pain from baseline to follow-up (P = .004), while the Exercise group showed a 63% reduction (P = .03) over the same time period.

Conclusions:

This study provides preliminary evidence that EMG biofeedback has value when added to an exercise intervention to reduce shoulder pain in manual wheelchair users with SCI. These findings indicate that EMG biofeedback may be valuable in remediating musculoskeletal pain as a secondary condition in SCI. This preliminary conclusion will need to be studied and verified through future work.Key words: biofeedback, exercise, pain, spinal cord injuryChronic or recurrent musculoskeletal pain in the cervical and shoulder region is a common secondary problem after spinal cord injury (SCI) that interferes with daily activities and reduces quality of life (QOL).¹ Reported prevalence ranges from 30% to 70%.^2–9 Both incidence and intensity increase with duration of injury and become an added concern for SCI individuals as they age.^8,10,11 For manual wheelchair users, who rely on their upper extremities for mobility (wheelchair propulsion), 35% report shoulder pain. Shoulder pain increases to 60% for wheelchair athletes.^12,13Muscle imbalance plays a role in the development of musculoskeletal pain in manual wheelchair users, as muscles used for the forceful push phase strengthen relative to muscles used for the recovery phase.^13,14 Recent studies have reported effective use of exercise programs designed to address this problem.^15–18 Two of these studies were randomized trials that included an untreated control group to rule out spontaneous recovery.^17,18 As a result of this research, exercise has become a standard intervention for reducing shoulder pain in manual wheelchair users with SCI.In addition to muscle imbalance, muscle fatigue likely plays a significant role. Research on work-related musculoskeletal pain (WRMP) has demonstrated that disruption of normal work/rest cycles can promote localized muscle fatigue and pain.^19–21 Electromyographic studies have found that repetitive tasks consist of alternating periods of muscle contraction and muscle relaxation; a muscle that is slow to relax – bypassing the rest portion of work/rest cycles – is at risk of developing WRMP.^22–24 Clinical studies have reported slow and incomplete relaxation of the upper trapezius muscle during repetitive tasks, compared to pain-free controls, in musicians with occupational upper extremity pain²⁵ and individuals with cervical pain and headache.^{19, 24} Manual wheelchair propulsion, with its highly repetitive muscle activity, puts the wheelchair user at risk of muscle fatigue and eventually pain.This research suggests that EMG biofeedback interventions designed to improve work/ rest cycling may be an effective addition to rehabilitation programs for cervical and shoulder pain in manual wheelchair users with SCI. At present, EMG biofeedback procedures are widely used for muscle training in rehabilitation, athletics, and the workplace and have been effective in training overactive muscles to relax quickly during functional activities.^20,26–30 In the present study, EMG biofeedback training is designed to improve both muscle balance and work/rest cycles during functional wheelchair propulsion. The purpose of this study was to determine the extent to which EMG biofeedback training is associated with a reduction in shoulder pain, above and beyond the effects of a standard exercise program.     

Reciprocal regulation of plasma apelin and vasopressin by osmotic stimuli

Apelin is a neuropeptide that co-localizes with vasopressin (AVP) in magnocellular neurons and is involved in body fluid homeostasis. Osmotic stimuli have opposite effects on the regulation of apelin and AVP secretion in animal models, but whether this is true in humans is unknown. This study investigated the relationship among osmolality, apelin, and AVP in 10 healthy men after infusion of hypertonic saline or loading with water to increase and decrease plasma osmolality, respectively. Increasing plasma osmolality was accompanied by a parallel, linear increase in plasma AVP concentration and by a decrease in plasma apelin concentration. In contrast, decreasing plasma osmolality by water loading reduced plasma AVP concentration and rapidly increased plasma apelin concentration. These findings suggest that regulation of apelin secretion contributes to the maintenance of body fluid homeostasis.The osmotic pressure of body fluids is maintained within a remarkably narrow range in healthy adults. Body fluid homeostasis depends on neuronal pathways bearing very sensitive osmoreceptors,¹ located along the lamina terminalis, including the circumventricular organs, such as the subfornical organ and the organum vasculosum of the lamina terminalis as well as the median preoptic nucleus.² The subfornical organ and organum vasculosum of the lamina terminalis are neuronally interconnected with each other as well as with the median preoptic nucleus and the hypothalamic paraventricular and supraoptic nuclei.³ These neuronal pathways convert small changes in osmolality into a neuronal signal to neurons that influence sensations of thirst and systemic arginine vasopressin (AVP) release,² thereby adjusting the intake or output of water to counteract changes in solute concentration.^4,5A recently discovered peptide, apelin, may also play a major role in the maintenance of body fluid homeostasis. Apelin, initially isolated from bovine stomach extracts,⁶ is the endogenous ligand of the human orphan G protein–coupled receptor APJ (putative receptor protein related to the angiotensin receptor AT1).^6,7 It is a 36–amino acid peptide (apelin 36) derived from a single 77–amino acid precursor, proapelin.^6,8,9 Proapelin has a fully conserved C-terminal 17–amino acid sequence, apelin 17 (K17F), including the pyroglutamyl form of apelin 13 (pE13F). K17F and pE13F both are present in rat brain and plasma,¹⁰ and apelin 36 is present in testis and uterus.¹¹ All peptides exhibit a high affinity for the human^8,12,13 and the rat apelin receptors.¹⁴ Apelin possesses various cardiovascular functions (for reviews,^15–17). Apelin and its receptor have been detected in the endothelial cells of large conduit arteries, coronary vessels, and the endocardium of the right atrium.^18–20 Apelin injection decreases BP in animals,^9,21–23 via nitric oxide production.²¹ Apelin has a positive inotropic effect both on isolated perfused rat hearts ex vivo²⁴ and on normal and postmyocardial infarction rat hearts in vivo.²⁵ Apelin administration in mice reduces left ventricular preload and afterload and increases contractile reserve and cardiac output.²⁶ Finally, apelin-deficient mice develop heart failure with aging.²⁷Apelin and its receptor both are widely distributed in the brain^9,14,28,29 but are particularly abundant in the supraoptic nucleus and paraventricular nucleus, where they co-localize with AVP in magnocellular neurons.^10,29–31 Intracerebroventricular injection of K17F inhibits the typical phasic firing pattern of AVP neurons in lactating rats, resulting in decreased systemic AVP release and increased aqueous diuresis.¹⁰ Moreover, water deprivation in rats while increasing systemic AVP release and causing depletion of hypothalamic AVP stores decreases plasma apelin concentrations and results in hypothalamic accumulation of the peptide. Thus, the two peptides are conversely regulated to optimize AVP release into the blood circulation and prevent additional water loss through the kidney.¹⁰ Whether such opposite regulation of apelin and AVP secretion in response to osmotic stimuli is found in humans remains unknown.The aim of this clinical investigation was to examine the relationship between plasma osmolality, plasma apelin, and plasma AVP in healthy adults in various states of hydration. Hydration was modified by administration of a hypertonic saline infusion and by water loading to increase and decrease plasma osmolality, respectively.     

Connexin 40 Mediates the Tubuloglomerular Feedback Contribution to Renal Blood Flow Autoregulation

Connexins are important in vascular development and function. Connexin 40 (Cx40), which plays a predominant role in the formation of gap junctions in the vasculature, participates in the autoregulation of renal blood flow (RBF), but the underlying mechanisms are unknown. Here, Cx40-deficient mice (Cx40-ko) had impaired steady-state autoregulation to a sudden step increase in renal perfusion pressure. Analysis of the mechanisms underlying this derangement suggested that a marked reduction in tubuloglomerular feedback (TGF) in Cx40-ko mice was responsible. In transgenic mice with Cx40 replaced by Cx45, steady-state autoregulation and TGF were weaker than those in wild-type mice but stronger than those in Cx40-ko mice. Nω-Nitro-L-arginine-methyl-ester (L-NAME) augmented the myogenic response similarly in all genotypes, leaving autoregulation impaired in transgenic animals. The responses of renovascular resistance and arterial pressure to norepinephrine and acetylcholine were similar in all groups before or after L-NAME inhibition. Systemic and renal vasoconstrictor responses to L-NAME were also similar in all genotypes. We conclude that Cx40 contributes to RBF autoregulation by transducing TGF-mediated signals to the afferent arteriole, a function that is independent of nitric oxide (NO). However, Cx40 is not required for the modulation of the renal myogenic response by NO, norepinephrine-induced renal vasoconstriction, and acetylcholine- or NO-induced vasodilation.Connexins (Cx''s) are important in vascular development, cardiovascular function, and arterial pressure (AP) control.^1,2 Four Cx isoforms (Cx37, Cx40, Cx43, and Cx45) form gap junctions to facilitate intercellular communication in the vasculature.^1,2 Among these, Cx40 plays a prominent role. Cx40 is abundantly expressed in endothelial cells in most vascular beds.^1,2 Genetic ablation of Cx40 causes severe impairment of conducted vasodilator responses in arterioles,^3,4 uncoordinated vasomotion,⁵ and hypertension.^5–7 Furthermore, vascular expression of Cx40 is reduced in genetically hypertensive rats.⁸Within the kidney, gap junctions are prevalent in the juxtaglomerular apparatus (JGA).⁹ The JGA is a unique structure coordinating tubular function to the regulation of preglomerular vasomotor tone and renin release. Cx40 is the predominant connexin in the JGA, with expression in endothelial and renin-producing cells of afferent arterioles and glomerular mesangial cells.^6,10–12 Cx40 is thus strategically localized for impacting GFR, tubuloglomerular feedback (TGF), and renin secretion. Indeed, deletion of Cx40 leads to increased production of renin, ectopic renin expression, and loss of pressure- and angiotensin II (Ang II)-dependent control of renin release.^6,7,13 A rise in plasma renin concentration is also seen after administration of a putative Cx40-inhibiting peptide.¹² However, Cx40 expression is increased in response to a chronic reduction of renal perfusion pressure, a common stimulus for renin synthesis.¹⁰Our knowledge of the role of Cx40 in the regulation of organ blood flow and vascular resistance in vivo is limited. In the kidney, intrarenal infusion of peptides designed to inhibit Cx37, Cx40, or both Cx40 and Cx43 reduces basal renal blood flow (RBF) and increases AP.^12,14 Steady-state autoregulation of RBF and GFR is reported to be partially inhibited by peptides directed against Cx37 or Cx40.¹² Not known, however, is which of the three mechanisms responsible for renal autoregulation (TGF, myogenic response (MR), and an undefined third mechanism^15,16) is affected. In isolated JGAs, TGF responses¹⁷ and associated calcium waves¹⁸ are inhibited by nonspecific pharmacologic gap junction disrupters (e.g., heptanol). Such interventions also attenuate the MR to changes in vascular pressure in isolated cerebral¹⁹ and mesenteric arteries,²⁰ as is the case for inhibitory peptides against Cx37 and Cx43.²⁰ However, the functional significance of genetic deletion of Cx40 for blood flow regulation and autoregulation in vivo in any vascular bed, including the kidney, is not known. We postulated that Cx40 is required for complete autoregulation and TGF activity.Also poorly understood is the importance of gap junctions in vivo in vasoconstrictor and vasodilator responses of resistance arterioles. α-Adrenergically induced vasoconstriction is blunted by pharmacologic gap junction inhibitors in isolated arteries.^20,21 Gap junctions are also implicated in vasodilation,^14,22 although it is unclear whether or not Cx40 is involved.^3,14,23 Few studies have tested the participation of connexins in vasodilation in vivo.^5,14 To our knowledge, no data exist regarding connexins in vasoconstrictor responses in an animal.Nitric oxide (NO) may interact with connexin function. NO is thought to modulate connexin conductance acutely²⁴ and expression chronically.²⁵ NO attenuates the magnitude of acute vasoconstriction elicited by receptor agonists such as Ang II and NE.²⁶ In addition, NO blunts the strength of the pressure-induced MR in RBF autoregulation, an effect specific to the kidney and dependent on JGA function and TGF.^27,28 Considering the possible involvement of Cx40 in JGA function, we postulated that Cx40 is critical for NO modulation of agonist-induced renal vascular responses and pressure-induced RBF autoregulation.The present study tested the hypotheses that (a) Cx40 is essential to RBF autoregulation and is involved in MR, TGF, or both responses to an acute change in renal perfusion pressure, (b) Cx40 contributes to agonist-induced vasodilator and constrictor responses in the renal and systemic circulation, and (c) Cx40 is involved in the signaling pathway of NO that blunts the renal MR. To this end, we conducted RBF studies on mice with genetic ablation of Cx40. Parallel studies were performed on mice with the coding region for Cx40 replaced by Cx45 regulated by the Cx40 promotor. In the latter, we postulated that Cx45 can assume Cx40 function, at least in part.     

Decreased cardiac glutathione peroxidase levels and enhanced mandibular apoptosis in malformed embryos of diabetic rats

OBJECTIVE— To characterize normal and malformed embryos within the same litters from control and diabetic rats for expression of genes related to metabolism of reactive oxygen species (ROS) or glucose as well as developmental genes.RESEARCH DESIGN AND METHODS— Embryos from nondiabetic and streptozotocin-induced diabetic rats were collected on gestational day 11 and evaluated for gene expression (PCR) and distribution of activated caspase-3 and glutathione peroxidase (Gpx)-1 by immunohistochemistry.RESULTS— Maternal diabetes (MD group) caused growth retardation and an increased malformation rate in the embryos of MD group rats compared with those of controls (N group). We found decreased gene expression of Gpx-1 and increased expression of vascular endothelial growth factor-A (Vegf-A) in malformed embryos of diabetic rats (MDm group) compared with nonmalformed littermates (MDn group). Alterations of messenger RNA levels of other genes were similar in MDm and MDn embryos. Thus, expression of copper zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD), and sonic hedgehog homolog (Shh) were decreased, and bone morphogenetic protein-4 (Bmp-4) was increased, in the MD embryos compared with the N embryos. In MDm embryos, we detected increased activated caspase-3 immunostaining in the first visceral arch and cardiac area and decreased Gpx-1 immunostaining in the cardiac tissue; both findings differed from the caspase/Gpx-1 immunostaining of the MDn and N embryos.CONCLUSIONS— Maternal diabetes causes growth retardation, congenital malformations, and decreased general antioxidative gene expression in the embryo. In particular, enhanced apoptosis of the first visceral arch and heart, together with decreased cardiac Gpx-1 levels, may compromise the mandible and heart and thus cause an increased risk of developing congenital malformation.The cellular and molecular mechanisms of diabetic embryopathy are not completely clear. Previous experimental studies have suggested that the teratological impact of a diabetic environment partly depends on excess of reactive oxygen species (ROS) in the embryo (1) as a consequence of either increased free oxygen radical formation (2–4), decreased capacity of ROS-scavenging enzymes (5–9), or both. Furthermore, previous work has demonstrated that supplementation of antioxidative agents such as copper zinc superoxide dismutase (CuZnSOD) (1,2), N-acetylcysteine (10), vitamins E and C (8), and folic acid (11) in vitro, as well as butylated hydroxytoluene (12), vitamin E (13–19), vitamin C (18,20), N-acetylcysteine (21), and folic acid (11) in vivo, attenuate malformation rate and diminish markers of oxidative stress (e.g., by normalizing tissue levels of thiobarbituric acid reactive substances [15], isoprostane 8-iso-PGF_2α [22,23], and carbonylated proteins [24]).The driving cellular force behind diabetes-induced oxidative stress is likely associated with enhanced glucose metabolism (25–27) in the embryonic/fetal cells exposed to increased ambient levels of glucose. One putative primary source of reactive radical compounds is mitochondria receiving a high influx of pyruvate and oxygen and, subsequently, producing a large amount of ROS (mainly superoxide) (3) in the oxidative processes of the electron transport chain. The ensuing leakage of superoxide into other compartments of the mitochondria and the cytosol, and the further formation of hydrogen peroxide and hydroxyl radicals, should yield mitochondrial alterations (28) as well as lipid peroxidation (22) and DNA damage (29) in the embryo. There are several observations in support of this notion. The structural alterations, mainly high-amplitude swelling of the embryonic mitochondria (28), are diminished by maternal antioxidative treatment (16), thereby supporting the notion of a ROS-related etiology of the structural changes. Enhanced lipid peroxidation, as evidenced by increased levels of the isoprostane 8-iso-PGF_2α (22,23,30), may induce several teratogenic pathways in addition to the developmental disturbance caused by peroxidation of structural lipids in mitochondrial and cellular membranes. For instance, it has been demonstrated that 8-iso-PGF_2α, which is produced nonenzymatically by ROS-mediated oxidation of arachidonic acid in the offspring (30), has its own teratogenic potency (23). In addition, an excessive peroxidation of arachidonic acid may hamper prostaglandin biosynthesis by depleting precursor pools and, in particular, yield decreased concentration of prostaglandin E2 (31), which could obstruct neural tube closure (22).ROS-induced DNA damage (29) may directly disrupt development via altered expression of key genes. In addition, cellular DNA repair processes may activate poly(ADP-ribose) polymerase (PARP), which may cause glyceraldehyde-3-phosphate dehydrogenase (GAPDH) inhibition by poly(ADP-ribosylation) (32). The net result would be diminished embryonic GAPDH activity, which has been demonstrated in rat embryos subjected to diabetes in vivo and high glucose in vitro (33). Furthermore, decreased glycolytic flux proximal to GAPDH (32) and the presence of increased ambient glucose levels will yield enhanced flux in the polyol (34,35) and hexosamine (36) pathways. An increased availability of proximal glycolytic intermediaries would increase diacylglycerol production and cause activation of several protein kinase C isoforms (37,38), as well as enhance the flux in the advanced glycosylation end product pathway (39). All of the consequences of inhibited GAPDH activity may thus contribute to the teratogenic outcome of diabetic pregnancy. Evidently, there are multiple ways for a diabetes-induced state of oxidative stress in the embryo to disturb embryonic/fetal development, several of which enjoy considerable experimental support.Another consequence of a state of oxidative stress would be enhanced apoptosis in embryonic/fetal tissue (40), which has been described (26,41–43) and has been suggested to be mediated by enhanced Jun-amino-terminal kinase-1 and -2 activity (19,44). It has also been suggested that maternal diabetes induces an inflammatory state in the embryo, where proinflammatory cytokines (i.e., tumor necrosis factor-α [TNF-α] [45,46]) act to downregulate the principal ROS-scavenging enzymes via increased activity of mitogen-activated protein kinases (47). The exact relation between enhanced apoptosis and induction of malformations is still unclear, mainly since we do not fully comprehend the specific transmission of a general increase in programmed cell death into precisely restricted developmental damage to embryonic organs or organ systems (42).Based on earlier studies (7,48,49) and on a question that has been raised several times—which genes are involved in diabetes-induced embryonic dysmorphogenesis? (5,8,26,42,50)—we wanted to add to the teratological knowledge by identifying differences in gene expression between the malformed and nonmalformed offspring within the same litters of diabetic animals.Our working hypothesis was that genes of the malformed offspring with an expression pattern different from that of genes in the nonmalformed offspring within the same litter may be associated, either directly or indirectly, with the teratogenic process. We thus decided to compare gene expression of embryos that were morphologically normal and those that were malformed in litters of normal and diabetic rats. By comparing gene expression and protein distribution in embryos of the same age and with exposure to an identical intrauterine milieu, we aimed to control for possible confounding factors of the teratogenic process. From earlier work, we knew that the two tissues of the rat fetuses that are particularly vulnerable to the diabetic state are the mandible and the heart (17) (Fig. 1). We also knew that a susceptible period in diabetic rat pregnancy occurs during gestational days 6–10 (51), which corresponds to weeks 2–4 in human pregnancy. Bearing in mind that the size and relative immaturity of day-10 embryos makes them difficult to evaluate for developmental defects, we chose to interrupt the rat pregnancies on gestational day 11 in order to be able to clearly distinguish between malformed and nonmalformed embryos. We decided to relate embryonic (mal)development to expression of the major oxidative defense genes (SODs, glutathione peroxidase [Gpx]-1, Gpx-2, and catalase), to key genes of glucose metabolism (aldose reductase [AR] and GAPDH), to developmental/teratological genes [poly(ADP-ribose) polymerase (PARP)], to tumor protein 53 (p53), to bone morphogenetic protein-4 (Bmp-4), to Ret proto-oncogene (Ret), to sonic hedgehog homolog (Shh), to vascular endothelial growth factor-A (VEGF-A), to TNF-α, to interleukin-6 (IL-6), and to the tissue distribution of activated caspase-3 (denoting apoptotic rate) and Gpx-1.Open in a separate windowFIG. 1.A: Fetuses displaying micrognathia (left fetus) and normal morphology (right fetus). B: Outcome of pregnancy in the control (N) and manifestly diabetic (MD) groups, distributed as normal (□), malformed (▪), and resorbed (▒) embryos on gestational day 11. *P < 0.05 vs. N (χ² statistics). (Please see http://dx.doi.org/10.2337/db08-0830 for a high-quality digital representation of this figure.)     

Fatty acid synthase inhibitors modulate energy balance via mammalian target of rapamycin complex 1 signaling in the central nervous system

OBJECTIVE—Evidence links the hypothalamic fatty acid synthase (FAS) pathway to the regulation of food intake and body weight. This includes pharmacological inhibitors that potently reduce feeding and body weight. The mammalian target of rapamycin (mTOR) is an intracellular fuel sensor whose activity in the hypothalamus is also linked to the regulation of energy balance. The purpose of these experiments was to determine whether hypothalamic mTOR complex 1 (mTORC1) signaling is involved in mediating the effects of FAS inhibitors.RESEARCH DESIGN AND METHODS—We measured the hypothalamic phosphorylation of two downstream targets of mTORC1, S6 kinase 1 (S6K1) and S6 ribosomal protein (S6), after administration of the FAS inhibitors C75 and cerulenin in rats. We evaluated food intake in response to FAS inhibitors in rats pretreated with the mTOR inhibitor rapamycin and in mice lacking functional S6K1 (S6K1^−/−). Food intake and phosphorylation of S6K1 and S6 were also determined after C75 injection in rats maintained on a ketogenic diet.RESULTS—C75 and cerulenin increased phosphorylation of S6K1 and S6, and their anorexic action was reduced in rapamycin-treated rats and in S6K1^−/− mice. Consistent with our previous findings, C75 was ineffective at reducing caloric intake in ketotic rats. Under ketosis, C75 was also less efficient at stimulating mTORC1 signaling.CONCLUSIONS—These findings collectively indicate an important interaction between the FAS and mTORC1 pathways in the central nervous system for regulating energy balance, possibly via modulation of neuronal glucose utilization.Energy balance is achieved when caloric intake is matched to expenditure. A complex neuroendocrine system underlies this process and regulates energy homeostasis in mammals. In addition to sensing hormonal signals of stored fuels, such as leptin (1), specific populations of neurons in the central nervous system (CNS), and particularly within the hypothalamus, have the ability to sense locally available nutrients, including glucose (2), fatty acids (3), and amino acids (4,5).Recent evidence has highlighted the role of intracellular fuel sensing in the regulation of energy balance (6). In particular, the biochemical pathway underlying fatty acid metabolism has been involved in the regulation of both feeding and glucose homeostasis (6–10). Fatty acid synthase (FAS) catalyzes the condensation of malonyl-CoA and acetyl-CoA to generate long-chain fatty acids (LCFAs). Acetyl-CoA carboxylase (ACC) and FAS are expressed in the hypothalamus (7), where malonyl-CoA (11) and LCFA-CoA levels (8) decrease during fasting and increase after refeeding. Studies using FAS inhibitors and other pharmacological or genetic tools that modify the activity of different enzymes regulating fatty acid metabolism support a role for this pathway in the regulation of feeding (9,12–15).Peripheral administration of the natural FAS inhibitor cerulenin (2,3-epoxy-4-oxo-6-dodecadienoylamide) or the synthetic inhibitor C75 (trans-4-carboxy-5-octyl-3-methylenebutyrolactone) causes profound dose-dependent anorexia and weight loss in several rodent models (12,13,15,16). Reduced food intake is also observed with central administration of much lower doses of C75, suggesting that the brain is the key site of action (12). Increased hypothalamic malonyl-CoA is necessary for the anorexic and weight-reducing effects of FAS inhibitors (9,15,17). Interestingly, the ability of leptin to reduce food intake depends on increased hypothalamic malonyl-CoA (8) and possibly palmitoyl-CoA (18), which are achieved through the concomitant inhibition of AMP-activated protein kinase (AMPK) and activation of ACC (18). Therefore, the hypothalamic fatty acid synthesis pathway appears to process different fuel signals and convert them into efferent outputs that prevent further consumption of nutrients.The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that controls critical aspects of cell growth (19). mTOR is a component of at least two multiprotein complexes: mTOR complex 1 (mTORC1), which includes raptor, and mTOR complex 2 (mTORC2), which includes rictor. Whereas mTORC2 regulates phosphorylation of Akt, mTORC1 modulates the activity of S6 kinase 1 (S6K1) and 4E binding protein 1 (20). Notably, the phosphorylation of S6K1 at Thr 389 is one of the markers commonly used to evaluate mTORC1 activity in vivo (21). Insulin, IGF, amino acids, and glucose all activate intracellular cascades that lead to activation of mTORC1 (22). We reported that the anorexic action of central leptin and leucine are both dependent on the activation of mTORC1 signaling in the hypothalamus (4). Given the ability of mTORC1 to sense and integrate fuel signals, and the role it plays in controlling food intake (4), we hypothesized that mTORC1 signaling is involved in monitoring the biochemical changes induced by the modulation of hypothalamic fatty acid metabolism.     

Which Pain Coping Strategies and Cognitions Are Associated with Outcomes of a Cognitive Behavioral Intervention for Neuropathic Pain after Spinal Cord Injury?

Background:

Chronic neuropathic pain is one of the most difficult problems to manage after spinal cord injury (SCI). Pain coping and pain cognitions are known to be associated with the patient’s experience of neuropathic pain, but they have not been studied in the context of a cognitive behavioral treatment program for coping with neuropathic pain after SCI.

Objective:

To explore associations of pain coping strategies and cognitions with pain intensity and pain-related disability and changes in pain coping strategies and cognitions with changes in pain intensity and pain-related disability.

Methods:

Forty-seven persons who participated in the CONECSI (COping with NEuropathiC Spinal cord Injury pain) trial completed questionnaires before the intervention (baseline) and 3 months after of the intervention (follow-up).

Results:

Compared to baseline, participants showed more favorable scores on 2 pain coping scales (Pain Transformation and Worrying), the subtotal score Active Coping, and 3 pain cognitions scales (Catastrophizing, Optimism, and Reliance on Health Care) at follow-up. Baseline Reliance on Health Care was associated with change in pain intensity and pain-related disability. Change in Catastrophizing and change in Restriction cognitions were associated with change in pain-related disability.

Conclusions:

Our findings suggest that modifying pain coping strategies and cognitions by a cognitive behavioral intervention for chronic neuropathic pain after SCI may have some beneficial effects on pain intensity and pain-related disability. Further research should show how dysfunctional pain coping strategies and cognitions can be most effectively modified.Key words: chronic pain, cognitive behavioral therapy, pain intensity, pain-related disability, psychologicalPain is a significant problem for many persons with spinal cord injury (SCI). About 65% to 85% of them report the presence of pain,^1,2 and around a third of them experience severe pain.¹ Chronic neuropathic SCI pain (CNSCIP) at the level of the injury is present in 41% of all persons at 5 years after SCI, and 34% of them have CNSCIP below the level of injury.¹ Pain contributes to poorer rehabilitation outcomes and reduced quality of life,³ and it is one of the most difficult problems to manage, even in the presence of other problems that interfere with daily life.⁴ The longterm prognosis for pain resolution following SCI is poor.^1,3 Many persons affected report that SCI pain persists or even worsens over time.⁵Although biomedical changes associated with the disability itself may play a primary role in the presence and severity of pain, psychosocial factors, such as attitudes and cognitions, coping behavior, and the social environment play a major role in the perceived intensity and impact of pain in many persons with physical disabilities and chronic pain.^6–10 Psychosocial factors may perpetuate chronic pain¹¹ and predict pain and functioning in persons with physical disabilities.⁹ Several studies identified pain cognitions such as catastrophizing^7,12–15 and pain coping strategies such as passive coping^7,14 as predictors of increased pain intensity and pain-related disability. Interventions to modify dysfunctional pain cognitions and pain coping strategies therefore hold promise for persons with CNSCIP.^7,16 In particular, cognitive behavior therapy (CBT) has been used for a variety of chronic pain problems and is expected to change patient cognitions and coping strategies.¹⁷Studies to investigate the effect of CBT-based treatment programs for persons with chronic SCI pain have been rare,^18,19 especially among persons with CNSCIP.²⁰ Although these studies showed some promising results, in terms of changes in pain intensity and life satisfaction,²⁰ anxiety,^18,19,20 and depression,^18,20 they failed to prove the effectiveness of CBT as compared to placebo or usual care.Changes in pain coping strategies and cognitions were not examined,²⁰ or associations with changes in the outcome measures were not studied.^18,19 Kennedy and colleagues¹⁸ found no changes in coping strategies compared to controls, whereas Nicholson Perry and colleagues¹⁹ found a significant improvement in pain catastrophizing in the treatment group but no differences with the usual care group.In any case, persons vary in their response to CBT, and little is known about patient characteristics that predict or moderate the effects of CBT on patient outcomes following SCI.^17,21 A better understanding of how baseline pain coping strategies and cognitions, and changes in such strategies and cognitions, are associated with treatment outcomes could help direct limited resources to those persons most likely to benefit, match patients with the most appropriate treatments, and tailor interventions to patient characteristics.¹⁷Recently, Heutink and colleagues^22,23 reported the design and results of the CONECSI (COping with NEuropathiC Spinal cord Injury pain) trial, the first randomized controlled trial (RCT) on the effectiveness of a multidisciplinary CBT program for coping with CNSCIP compared with a waiting list control group. No significant intervention effects on the primary outcomes pain intensity and pain-related disability were found. There was a trend (P = .059) toward an effect of the intervention on pain-related disability immediately after the intervention, and the intervention group showed a significant decrease in pain intensity and pain-related disability that was not found in the control group. Significant treatment effects were found on the secondary outcomes anxiety and participation in activities.²³The current study utilized data from the CONECSI trial to explore associations between baseline pain coping strategies and pain cognitions on the one hand and pain intensity and pain-related disability on the other, and to study the hypothesis that changes in pain coping strategies and pain cognitions are associated with outcome variables after CBT intervention. The specific aims were to explore changes in pain coping strategies and cognitions during the CBT intervention and to explore (a) associations between baseline pain coping strategies and cognitions and baseline pain and pain-related disability, (b) associations between baseline pain coping strategies and cognitions and the changes in pain and pain-related disability during the RCT, and (c) associations between changes in pain coping strategies and cognitions and changes in pain intensity and pain-related disability during the RCT. We hypothesized that more catastrophizing,^7,12–15 less active coping, and more passive coping^7,14 at baseline would be associated with higher pain intensity and pain-related disability at baseline and greater changes in pain intensity and pain-related disability. Further it was hypothesized that favorable changes in pain coping strategies and pain cognitions would be associated with favorable changes in pain intensity and pain-related disability.