M. Thomas Clandinin

Cancer Cachexia in the Age of Obesity: Skeletal Muscle Depletion Is a Powerful Prognostic Factor, Independent of Body Mass Index

PURPOSE Emerging evidence suggests muscle depletion predicts survival of patients with cancer. PATIENTS AND METHODS At a cancer center in Alberta, Canada, consecutive patients with cancer (lung or GI; N = 1,473) were assessed at presentation for weight loss history, lumbar skeletal muscle index, and mean muscle attenuation (Hounsfield units) by computed tomography (CT). Univariate and multivariate analyses were conducted. Concordance (c) statistics were used to test predictive accuracy of survival models. RESULTS Body mass index (BMI) distribution was 17% obese, 35% overweight, 36% normal weight, and 12% underweight. Patients in all BMI categories varied widely in weight loss, muscle index, and muscle attenuation. Thresholds defining associations between these three variables and survival were determined using optimal stratification. High weight loss, low muscle index, and low muscle attenuation were independently prognostic of survival. A survival model containing conventional covariates (cancer diagnosis, stage, age, performance status) gave a c statistic of 0.73 (95% CI, 0.67 to 0.79), whereas a model ignoring conventional variables and including only BMI, weight loss, muscle index, and muscle attenuation gave a c statistic of 0.92 (95% CI, 0.88 to 0.95; P < .001). Patients who possessed all three of these poor prognostic variables survived 8.4 months (95% CI, 6.5 to 10.3), regardless of whether they presented as obese, overweight, normal weight, or underweight, in contrast to patients who had none of these features, who survived 28.4 months (95% CI, 24.2 to 32.6; P < .001). CONCLUSION CT images reveal otherwise occult muscle depletion. Patients with cancer who are cachexic by the conventional criterion (involuntary weight loss) and by two additional criteria (muscle depletion and low muscle attenuation) share a poor prognosis, regardless of overall body weight.

Long chain polyunsaturated fatty acids are required for efficient neurotransmission in C. elegans.

The complex lipid constituents of the eukaryotic plasma membrane are precisely controlled in a cell-type-specific manner, suggesting an important, but as yet, unknown cellular function. Neuronal membranes are enriched in long-chain polyunsaturated fatty acids (LC-PUFAs) and alterations in LC-PUFA metabolism cause debilitating neuronal pathologies. However, the physiological role of LC-PUFAs in neurons is unknown. We have characterized the neuronal phenotype of C. elegans mutants depleted of LC-PUFAs. The C. elegans genome encodes a single Δ6-desaturase gene (fat-3), an essential enzyme for LC-PUFA biosynthesis. Animals lacking fat-3 function do not synthesize LC-PUFAs and show movement and egg-laying abnormalities associated with neuronal impairment. Expression of functional fat-3 in neurons, or application of exogenous LC-PUFAs to adult animals rescues these defects. Pharmacological, ultrastructural and electrophysiological analyses demonstrate that fat-3 mutant animals are depleted of synaptic vesicles and release abnormally low levels of neurotransmitter at cholinergic and serotonergic neuromuscular junctions. These data indicate that LC-PUFAs are essential for efficient neurotransmission in C. elegans and may account for the clinical conditions associated with mis-regulation of LC-PUFAs in humans.

Polyunsaturated fatty acids and T-cell function: implications for the neonate.

Infant survival depends on the ability to respond effectively and appropriately to environmental challenges. Infants are born with a degree of immunological immaturity that renders them susceptible to infection and abnormal dietary responses (allergies). T-lymphocyte function is poorly developed at birth. The reduced ability of infants to respond to mitogens may be the result, of the low number of CD45RO+ (memory/antigen-primed). T cells in the infant or the limited ability to produce cytokines [particularly interferon-γ, interleukin (IL)-4, and IL-10]. There have been many important changes in optimizing breast milk substitutes for infants; however, few have been directed at replacing factors in breast milk that convey immune benefits. Recent research has been directed at the neurological, retinal, and membrane benefits of adding 20∶4n−6 (arachidonic acid; AA) and 22∶6n−3 (docosahexaenoic acid; DHA) to infant formula. In addults and animals, feeding DHA affects T-cell function. However, the effect of these lipids on the development and function of the infants immune system is not known. We recently reported the effect of adding DHA+AA to a standard infant formula on several functional indices of immune development. Compared with standard formula, feeding a formula containing DHA+AA increased the proporition of antigen mature (CD45RO+) CD4+ cells, improved IL-10 production, and reduced IL-2 production to levels not different from those of human milk-fed infants. This review will briefly describe T-cell development and the potential immune effect of feeding long-chain polyunsaturated fatty acids to the neonate.

Diet-induced changes in membrane gangliosides in rat intestinal mucosa, plasma and brain.

Objectives: The objective of this study was to determine if dietary gangliosides induce changes in the ganglioside content of intestinal mucosa, plasma and brain and to identify where GM3 and GD3 are localized in the enterocyte membrane. Methods: Male 18-day-old Sprague-Dawley rats were fed a semipurified diet containing 20% (w/w) fat. The control diet contained triglyceride, reflecting the fat formulation of an existing infant formula. Two experimental diets were formulated by adding sphingomyelin (1% w/w of total fat) or a ganglioside-enriched lipid (0.1% w/w of total fat) to the control diet fat. The ganglioside fraction of ganglioside-enriched lipid diet contained more than 80% GD3. After 2 weeks of feeding, the total and individual ganglioside and cholesterol content was measured in small intestinal mucosa, plasma and brain. Results: The ganglioside-enriched lipid diet significantly increased total gangliosides in the intestinal mucosa, plasma and brain compared with the control diet. The ganglioside-enriched lipid diet significantly increased the level of GD3 (7.5% w/w) in the intestine compared with control (3.2% w/w) while decreasing the level of GM3, the major ganglioside in the intestine. The ratio of cholesterol to ganglioside in the intestinal mucosa, plasma and brain decreased significantly in rats fed the ganglioside-enriched lipid diet compared with controls. Confocal microscopy showed that GM3 is exclusively localized in the apical membrane of the enterocyte whereas GD3 is primarily localized in the basolateral membrane. Conclusions: The authors conclude that dietary ganglioside is absorbed in the small intestine and transported to different membrane sites, altering ganglioside levels in the intestinal mucosa, plasma and brain and thus possibly having the potential to change developing enterocyte function (and possibly that of other cell lines).

Phosphatidylethanolamine methyltransferase: evidence for influence of diet fat on selectivity of substrate for methylation in rat brain synaptic plasma membranes

Male weanling rats were fed diets containing 20% (w/w) fat differing in fatty acid composition for 24 days. Synaptic plasma membranes were isolated from the brain and the fatty acid composition of phosphatidylethanolamine and phosphatidylcholine was determined. In vitro assays of phosphatidylethanolamine methyl-transferase activity were performed on fresh membrane samples to assess effect of dietary fat on the rate of phosphatidylethanolamine methylation for phosphatidylcholine synthesis via the phosphatidylethanolamine methyltransferase pathway. Dietary level of n-6 and ratio of n-6 to n-3 fatty acids influenced membrane phospholipid fatty acid composition and activity of the lipid-dependent phosphatidylethanolamine methyltransferase pathway. Rats fed a diet rich in n-6 fatty acids produced a high ratio of n-6/n-3 fatty acids in synaptosomal membrane phosphatidylethanolamine, and elevated rates of methylation of phosphatidylethanolamine to phosphatidylcholine by phosphatidylethanolamine methyltransferases, suggesting that the pathway exhibits substrate selectivity for individual species of phosphatidylethanolamine containing long-chain homologues of dietary n-6 and n-3 fatty acids (20:4(n-6), 22:4(n-6), 22:5(n-6) and 22:6(n-3). It may be concluded that diet alters the membrane content of n-6, n-3 and monounsaturated fatty acids, and that change in phosphatidylethanolamine species available for methylation to phosphatidylcholine alters the rate of product synthesis in vivo by the phosphatidylethanolamine methyltransferase pathway.

Effect of providing a formula supplemented with long-chain polyunsaturated fatty acids on immunity in full-term neonates

To determine the effect of feeding formula containing long-chain PUFA (LCP) on immune function, healthy term infants were randomised at age 2 weeks to either a standard term formula (Formula; n 14) or the same formula supplemented with the LCP 20 : 4n-6 and 22 : 6n-3 (Formula+LCP; n 16). Peripheral blood was collected at 2 and 6 weeks to measure immune cell response (the rate of [3H]thymidine uptake and cytokine production after stimulation with phytohaemagglutinin (PHA)). Compared with cells from infants receiving only human milk (HM), the rate of [3H]thymidine uptake in response to PHA, but not IL-2 production, was lower for Formula+LCP infants (P < 0.05). Compared with HM-fed infants, Formula-fed infants (but not Formula+LCP infants) produced more TNF-alpha (unstimulated) and had a fewer CD3+CD44+ cells before stimulation and fewer CD11c+ cells post-stimulation (P < 0.05). However, compared with Formula-fed infants, the Formula+LCP infants had an immune cell distribution (higher percentage CD3+CD44+ and CD4+CD28+ cells) and cytokine profile (lower production of TNF-alpha post-stimulation) that did not differ from HM infants. Additionally, it was found that feeding infants formula during the first 10 d of life influenced immune function. These infants had a higher percentage of CD3+, CD4+CD28+, and lower percentage of CD14+ cells and produced more TNF-alpha and interferon-gamma after PHA stimulation than HM-fed infants (P < 0.05). These results demonstrate that early diet influences both the presence of specific cell types and function of infant blood immune cells. Since many diseases have a strong immunological component, these immune changes may be of physiological importance to the developing infant.

Dietary cholesterol and/or n − 3 fatty acid modulate Δ9-desaturase activity in rat liver microsomes

delta 9-Desaturase activity and fatty acid composition of liver microsomal phospholipids in rats fed diets enriched with either saturated (hydrogenated beef tallow) or alpha-linolenic (linseed oil) or eicosapentaenoic and docosahexaenoic (fish oil) acids with or without 2% cholesterol supplementation were investigated. Both the linseed oil and the fish oil diets inhibited delta 9-desaturase activity in the rat liver microsomes. The inhibition was greater when feeding fish oil (90%) compared with the linseed oil (60%) diet. Dietary cholesterol feeding accelerated conversion of palmitic (16:0) to palmitoleic (16:1) acid, irrespective of the fatty acid supplement. Feeding the linseed oil diet decreased, while feeding the fish oil diet increased synthesis of the monounsaturated fatty acids of n-7 series (palmitoleic and vaccenic acid) and decreased 18:1(n-9) in microsomal membrane lipids when compared with animals fed beef tallow. Addition of 2% cholesterol to the otherwise low cholesterol diets led to accumulation of 16:1(n-7), and 18:1(n-9) in microsomal membranes. These results suggest that delta 9-desaturase activity is dependent on the cholesterol contents as well as the n-3 fatty acid content of microsomal membranes on which it is localized.

Dietary 20:4n-6 and 22 :6n-3 modulates the profile of long- and very-long-chain fatty acids, rhodopsin content, and kinetics in developing photoreceptor cells

The objective of this study was to determine whether addition of dietary 20:4n-6 and 22:6n-3 to a conventional infant formula fat blend influences membrane long-chain and very-long-chain fatty acid composition, rhodopsin content, and rhodopsin kinetics in developing rat photoreceptor cells. The dietary fats were formulated based on the fat composition of a conventional infant formula providing an 18:2n-6/18:3n-3 ratio of 7:1 (SMA, Wyeth Nutritionals), which served as the control fat blend. This dietary fat blend was modified to contain 20:4n-6 [arachidonic acid (AA)], 22:6n-3 [docosahexaenoic acid (DHA)], AA + DHA, or an 18:2n-6/18:3n-3 ratio of 4:1 (α-linolenic acid). Dams were fed diets from birth, and rat pups were fed the same diet after weaning. Retinas and rod outer segments were prepared in the dark from pups at 2, 3, and 6 wk of age for fatty acid analysis of individual phospholipids, rhodopsin content, and rhodopsin disappearance kinetics after light exposure. Feeding AA + DHA in the diet increased 22:6n-3 levels in phosphatidylcholine and phosphatidylethanolamine. In phosphatidylcholine, total n-6 tetraenoic very-long-chain fatty acids and total n-3 pentaenoic and n-3 hexaenoic very-long-chain fatty acids increased after feeding AA and DHA, respectively. Developmental changes were characterized by a decrease in 20:4n-6 in the major phospholipids, whereas 22:6n-3 increased with age in rod outer segments. The highest rhodopsin content occurred in the retina of rats fed diets containing AA and/or DHA. The kinetics of rhodopsin disappearance after light exposure was highest in rats fed DHA at 6 wk of age. This study demonstrates that small manipulations of the dietary level of 20:4n-6 and 22:6n-3 are important determinants of fatty acid composition of membrane lipid and visual pigment content and kinetics in the developing photoreceptor cell.

Dietary fat alters membrane composition in rod outer segments in normal and diabetic rats: Impact on content of very-long-chain (C ⩾ 24) polyenoic fatty acids

The effect of high n - 3 (5.8%, w/w) vs. a low n - 3 (1.2%, w/w) fatty acids in a diet with a low ratio of polyunsaturated/saturated fatty acids (P/S = 0.27) content was investigated to determine the effect of diet on the level of long- and very-long-chain fatty acids (VLCFA C > or = 24) in phospholipids of rod outer segments (ROS) of normal and diabetic rats. After 6 weeks of feeding, diets high in n - 3 fatty acids increased the levels of 22:5(n - 3) and 22:6(n - 3), while decreasing the 22:5(n - 6) level in all major phospholipid classes. n - 6 and n - 3 VLCFA of C24 to C34 with 4, 5 and 6 double bonds were found only in phosphatidylcholine (PC) while other phospholipid classes contained only C24 fatty acids as minor components. The content of VLCFA in PC was approx. 6.7% (w/w) of total fatty acids in the ROS. Feeding a high n - 3 fatty acid diet significantly reduced n - 6 tetraenoic VLCFA in all phospholipids. In the diabetic state, the levels of n - 6 tetraenes and pentaenes in individual phospholipids were different from control animals. This study demonstrates that the VLCFA content of photoreceptor cells reflects the dietary level of n - 3 fatty acids fed. The unique polyenoic n - 6 and n - 3 VLCFA appear to be synthesized from shorter chain precursors which respond to altering the ratio of n - 6/n - 3 fatty acids fed.

20:5n-3 but not 22:6n-3 is a Preferred Substrate for Synthesis of n-3 Very-Long- Chain Fatty Acids (C24–C36) in Retina

The objective of this study was to determine if 20:5n-3 or 22:6n-3 is the primary precursor of very-long-chain fatty acids (VLCFAs; C24–C36) synthesized in retina. Rats were fed semisynthetic, nutritionally complete diet containing 20% (w/w) fat with 3% (w/w) of 22:6n-3. After 6 weeks feeding, the vitreal fluid of each eye was injected with [3H]20:5n-3 or [3H]22:6n-3. Rats were then maintained under constant light (330 lux) or dark conditions for 48 hr. After 48 hr in vivo metabolism, the amount of label present in individual fatty acids was determined in major phospholipids in retina. For [3H]22:6n-3, 90% of total incorporation remained in 22:6n-3, whereas for [3H]20:5n-3 the label was actively incorporated into pentaenoic and hexaenoic VLCFAs up to 34 carbon chain length. 22:5n-3 derived from [3H]20:5n-3 was among the most highly labeled fatty acids. These observations suggest that 22:6n-3 is incorporated directly into retinal phospholipids without further metabolism, whereas 20:5n-3 and 22:5n-3 are metabolically active precursors for synthesis of VLCFAs.