Gianfranco Guarnieri

ESPEN Guidelines on Parenteral Nutrition: adult renal failure.

Among patients with renal failure, those with ARF and critical illness represent by far the largest group undergoing artificial nutrition. ARF, especially in the ICU, seldom occurs as isolated organ failure but rather is a component of a much more complex metabolic environment, in the setting of the multiple organ failure. Nutritional programs for ARF patients must consider not only the metabolic derangements peculiar to renal failure and with the underlying disease process/associated complications, but also the relevant derangements in nutrient balance due to renal replacement therapies, especially when highly efficient renal replacement therapies (RRT) are used, such as continuous veno-venous hemofiltration (CVVH), or prolonged intermittent modalities such as sustained low-efficiency dialysis (SLED). Finally it is to be taken into account that nutrient requirements can change considerably during the course of illness itself (see also guidelines on PN in intensive care). From a metabolic point of view, patients with CKD or on chronic HD who develop a superimposed acute illness should be considered to be similar to patients with ARF. The same principles in respect of PN should therefore be applied.

Metabolic response to injury and sepsis: changes in protein metabolism

The metabolic response to trauma and sepsis involves an increased loss of body proteins. Specific sites of changes of protein and amino acid metabolism have been identified. In skeletal muscle, the rate of proteolysis is accelerated greatly. The rate of protein synthesis also may be increased but not enough to match the increase in degradation. Intramuscular glutamine concentration is decreased because of increased efflux and possibly decreased de novo synthesis. In the liver, the rate of synthesis of selected proteins (i.e., albumin, transferrin, prealbumin, retinol-binding protein, and fibronectin) is decreased, whereas acute phase protein synthesis is accelerated. Tissues characterized by rapidly replicating cells, such as enterocytes, immune cells, granulation tissue, and keratinocytes, exhibit early alterations in the case of decreased protein synthesis capacity. In these tissues, glutamine use is accelerated. Increased stress hormone (cortisol and glucagon) and cytokine secretion, as well as intracellular glutamine depletion, are potential mediators of altered protein metabolism in trauma and sepsis. However, the relative importance of these factors has not been clarified. Therapy of acute protein catabolism may include the use of biosynthetic human growth hormone, possibly in combination with insulin-like growth factor-1, and the administration of metabolites at pharmacologic doses. We recently studied the effects of carnitine and alanyl-glutamine administration in severely traumatized patients. We found that both carnitine and the glutamine dipeptide restrained whole-body nitrogen loss without affecting selected indices of protein metabolism in the skeletal muscle.

Atherosclerosis and inflammation. Patterns of cytokine regulation in patients with peripheral arterial disease

Inflammatory phenomena at sites of atherosclerotic plaques are increasingly thought to be major determinants of the progression and clinical outcome of atherosclerotic disease. Therefore, attention is being paid to systemic markers/mediators which may reflect the inflammatory activity in the plaques. This study evaluates the pattern of the main proinflammatory cytokines tumor necrosis factor-alpha (TNFalpha), interleukin-1beta (IL-1beta), and interleukin-6 (IL-6), their soluble receptors/antagonist, and a variety of inflammatory markers, in patients with peripheral arterial disease (PAD). Eight patients with PAD suffering from claudicatio intermittens (CI), eight with critical limb ischemia (CLI) and eight controls (C) were studied. Blood samples were collected at baseline in all groups and. for C and CI, immediately after and 4 h after a 30-min treadmill test. Baseline: no differences in cytokine plasma levels were detected among the three groups. In contrast, soluble receptors of TNF (type I and II) and of IL-6, and IL-1beta receptor antagonist (IL-1ra) were increased in CI and CLI patients, as compared to C. Of note, IL-Ira correlated with the occurrence and stage of the disease in a highly significant proportion of the patients, reaching a predictive value for the disease of P < 0.0001. The opposite trend was observed for the soluble receptor of IL-1beta. Notably, in the patients no alterations could be found in white blood cell counts, expression of CD11c adherence molecule by circulating monocytes or, in vitro. O2- release from zymosan-activated neutrophils. Moreover, plasma levels of platelet activating factor (PAF), of neutrophil elastase and of the acute phase reactants C-reactive protein (CRP) and alpha1-acid glycoprotein were not found to be significantly altered. In contrast, the acute-phase proteins alpha1-antitrypsin (alpha1AT) and haptoglobin (HG) were found to be increased. Effect of treadmill: IL-1beta and TNFalpha remained at baseline levels following exercise, and IL-6 dropped to undetectable levels. Among cytokine antagonists, again the most relevant changes concerned the IL-1ra, which was significantly increased immediately after the treadmill test, both in CI and C, and returned to baseline levels after 4 h. In contrast, soluble TNFalpha, IL-1beta and IL-6 receptors, PAF, and the other markers of leukocyte activation were not found to be altered. Soluble TNFalpha and IL-6 receptors were shown to inhibit the biological effects of their ligands. Similarly, IL-1ra and the acute phase proteins alpha1AT and HG have been reported to exert anti-inflammatory functions. The increased plasma levels of these agents, together with low levels of inflammatory cytokines and other pro-inflammatory mediators such as PAF and alpha1-acid glycoprotein, appear to draw an undescribed picture, so far, of upregulation of a composite systemic anti-inflammatory mechanism in atherosclerotic patients. IL-1ra appears to be a reliable marker of the state of activation of this mechanism. These results may provide a basis for developing new insights into the pathogenesis of the atherosclerotic disease.

Hyperleptinemia prevents increased plasma ghrelin concentration during short-term moderate caloric restriction in rats

BACKGROUND & AIMS Ghrelin is an orexigenic hormone secreted by the stomach. Increased plasma ghrelin concentration was reported during diet-induced weight loss in obese humans, suggesting that ghrelin contributes to adaptive increment in appetite associated with caloric restriction. Leptin reduces spontaneous food intake and body weight in rodents. The current study tested the hypothesis that increased plasma leptin prevents the potential increase in plasma ghrelin concentration during moderate caloric restriction in lean rats. METHODS Six-month-old male rats (body weight, 367 +/- 9 grams) were randomly assigned to one of the following treatments (8 rats each) for 1 week: (1) leptin subcutaneous infusion to induce moderate hyperleptinemia and moderate caloric restriction (-26% of ad libitum), (2) vehicle infusion and pair feeding, and (3) vehicle infusion and ad libitum feeding. RESULTS Leptin-treated (-19 +/- 5 grams) and pair-fed (-19 +/- 2) rats lost weight compared with ad libitum-fed rats (-3 +/- 1, P < 0.05). Compared with control (6.8 +/- 0.7 ng/mL), plasma leptin was higher in leptin-treated (18.6 +/- 0.9 ng/mL, P < 0.01) rats and lower in pair-fed rats (4.3 +/- 0.4 ng/mL, P < 0.05). Plasma ghrelin was substantially higher in calorie-restricted than control rats (2505 +/- 132 pg/mL vs. 1790 +/- 134 pg/mL, P < 0.01), and leptin treatment (1625 +/- 117 pg/mL) completely prevented this change. Plasma ghrelin concentration was negatively correlated with body weight changes in calorie-restricted and control (r = -0.75, P < 0.01) but not in leptin-treated rats (P > 0.8). CONCLUSIONS Moderate hyperleptinemia prevents an increase of plasma ghrelin during moderate short-term caloric restriction. Satiety-inducing effects of leptin include suppression of gastric orexigenic signals and disruption of a potential feedback mechanism between body weight changes and plasma ghrelin in lean adult rats.

Short‐term bed rest impairs amino acid‐induced protein anabolism in humans

Diminished muscular activity is associated with alterations of protein metabolism. The aim of this study was to evaluate the effect of short‐term muscle inactivity on regulation of whole‐body protein deposition during amino acid infusion to simulate an experimental postprandial state. We studied nine healthy young volunteers at the end of 14 day periods of strict bed rest and of controlled ambulation using a cross‐over design. Subjects received a weight‐maintaining diet containing 1 g protein kg−1 day−1. l[1‐13C]leucine was used as a marker of whole‐body protein kinetics in the postabsorptive state and during a 3 h infusion of an amino acid mixture (0.13 g amino acid (kg lean body mass)−1 h−1). In the postabsorptive state, bed rest decreased (P < 0.05) the rate of leucine disposal (Rd) to protein synthesis and tended to decrease leucine rate of appearance (Ra) from proteolysis, whereas the rate of leucine oxidation did not change significantly. Amino acid infusion increased leucine Rd to protein synthesis and oxidation and decreased leucine Ra from proteolysis in both the bed rest and ambulatory conditions. Changes from basal in leucine Rd to protein synthesis were lower (P < 0.05) during bed rest than those in the ambulatory period, whereas changes in leucine Ra from proteolysis and oxidation were not significantly different. During amino acid infusion, net leucine deposition into body protein was 8 ± 3% lower during bed rest than during the ambulatory phase. In conclusion, short‐term bed rest leads to reduced stimulation of whole‐body protein synthesis by amino acid administration. Results of this study were, in part, presented at the meeting, Experimental Biology, 2004, Washington DC.

Calorie Restriction Modulates Inactivity-Induced Changes in the Inflammatory Markers C-Reactive Protein and Pentraxin-3

CONTEXT Energy balance and physical activity potentially influence systemic inflammation. OBJECTIVE Our objective was to test the hypothesis that moderate energy restriction may prevent activation of inactivity-induced inflammatory response. DESIGN Participants were studied four times at the end of 14-d periods of experimental bed rest or controlled ambulation, after receiving eucaloric or hypocaloric diets. SETTING The study was conducted at the clinical research center of the German Space Agency. SUBJECTS Nine healthy young volunteers participated. INTERVENTIONS Energy intake was calibrated to physical activity and decreased by about 20% in hypocaloric conditions. MAIN OUTCOME MEASURES Changes in body fat by dual-energy x-ray absorptiometry as well as plasma inflammatory markers and cytokine mRNA levels in blood cells were measured. RESULTS Fat mass did not change significantly in eucaloric conditions and decreased in hypocaloric periods (-1.0 +/- 0.3 and -1.0 +/- 0.3 kg in ambulatory and bed rest, respectively). Bed rest in eucaloric conditions increased plasma C-reactive protein (CRP) (+143 +/- 53%) and both the ratios between plasma IL-6 and IL-10 (4+/-1 times) and white blood cell IL-6 and IL-10 mRNAs (5 +/- 1 times). Energy restriction prevented bed-rest-mediated increases in CRP and the IL-6 to IL-10 ratio. Bed rest increased (P = 0.03) long pentraxin-3 (PTX3) plasma concentration, without significant activity-by-diet interaction. In all conditions (n = 36), CRP and PTX3 were inversely correlated (r = -0.61; P < 0.001). Changes in fat mass, leptin, and IL-6 directly correlated with CRP and inversely correlated with PTX3. IL-10 inversely correlated with CRP and directly correlated with PTX3 (r = 0.52; P < 0.01). CONCLUSIONS Calorie restriction prevents the inflammatory response induced by 14 d of bed rest. We suggest an inverse regulation of CRP and PTX3 in response to changes in energy balance.

Caloric restriction improves endothelial dysfunction during vascular aging: effects on nitric oxide synthase isoforms and oxidative stress in rat aorta.

Aging is characterized by activation of inducible over endothelial nitric oxide synthase (iNOS and eNOS), impaired antioxidant activity and increased oxidative stress, which reduces nitric oxide bioavailability and causes endothelial dysfunction. Caloric restriction (CR) blunts oxidative stress. We investigated whether CR impacts endothelial dysfunction in aging and the underlying mechanisms. Aortas from young (YC, 6 months of age) and old (OC, 24 months of age) rats ad-libitum fed and from old rats caloric-restricted for 3-weeks (OR, 26%) were investigated. Endothelium-dependent vasorelaxation was impaired in OC, associated with reduced eNOS and increased iNOS expression (P<0.05). Aortic nitrite was similar in OC and YC, but the contribution of calcium-independent NOS to total NOS activity was increased whereas that of calcium-dependent NOS was reduced (p≤0.0003). Plasma thiobarbituric acid-reactive substances (TBARS) were elevated in OC as well as aortic nitrotyrosine (P<0.05). Expression of manganese superoxide dismutase (MnSOD) and total SOD activity were impaired in OC (P<0.05 vs. YC), whereas copper-zinc (CuZn) SOD expression was similar in OC and YC. CR restored endothelial dysfunction in old rats, reduced iNOS expression, total nitrite and calcium-independent NOS activity in aorta (P<0.05) without changes in eNOS expression and calcium-dependent NOS activity. Sirtuin-1 expression did not differ among groups. Plasma TBARS and aortic nitrotyrosine were reduced (P<0.05) in OR compared with OC. In OR CuZnSOD protein and SOD activity increased (P<0.05) without changes in MnSOD expression. Short-term CR improves age-related endothelial dysfunction. Reversal of altered iNOS/eNOS ratio, reduced oxidative stress and increased SOD enzyme activity rather than enhanced NO production appear to be involved in this effect.

Ghrelin Enhances in Vivo Skeletal Muscle But Not Liver AKT Signaling in Rats

Objective: Ghrelin administration can induce fat weight gain and hyperglycemia (potentially through ghrelin‐induced hepatic glucose production), but plasma ghrelin is positively associated with whole‐body insulin sensitivity (mainly reflecting muscle insulin action) being increased in lean individuals or after diet‐induced weight loss and reduced in obesity or after diet‐induced weight gain. To investigate potential mechanisms, we measured in vivo effects of sustained ghrelin administration at a non‐orexigenic dose on skeletal muscle and liver insulin signaling at the AKT level and adipokine expression changes.

Bortezomib arrests the proliferation of hepatocellular carcinoma cells HepG2 and JHH6 by differentially affecting E2F1, p21 and p27 levels.

Despite the broad anti-tumour potential of the proteasome inhibitor bortezomib, partial information is available with regard to its effects on hepatocellular carcinoma (HCC) cells. Here we studied the effects of bortezomib on two human HCC cell lines displaying a different phenotype, hepatocyte-like for HepG2 and undifferentiated for JHH6. Bortezomib induced a dose- and time-dependent increase in cell toxicity and decrease of cell viability, with JHH6 being less sensitive than HepG2. Moreover, a differential influence on major cell cycle regulatory genes was responsible for the observed decrease of S and increase of G(2)-M phase cells. In HepG2, bortezomib induced a post-transcriptional increase of cyclin E1 together with a transcriptional-mediated decrease of the transcription factor E2F1. This in turn resulted in the reduction of the hyper-phosphorylated form of pRB and in the transcriptional down-regulation of the E2F1 targets cyclin D1, cyclin A2 and CdK2 but not cyclin E1. Up-regulation of LRH1, a liver specific cyclin E1 transcription factor, accounted for the unvaried cyclin E1 mRNA levels. Additionally, bortezomib induced both transcriptional and post-translational increase of p21(waf1/cip1) and p27(kip1). In JHH6, an overall more contained variation in cell cycle mediators was observed with the reduction of E2F1, cyclin A2, LRH1 and the increase of p21(waf1/cip1) being the most evident. In conclusion, the presented data show the mechanisms regulating cell proliferation inhibition by bortezomib in two different HCC cell lines. Despite a certain phenotype-dependent effect, the potent action exerted by bortezomib makes this drug attractive for future experimentation in animal models, possibly leading to novel treatments for HCC.

Treating hyperglycemia improves skeletal muscle protein metabolism in cancer patients after major surgery

Objective:Cancer and surgical stress interact to aggravate insulin resistance, protein catabolism, and glutamine depletion in skeletal muscle. We compared the effects of insulin-mediated euglycemia and moderate hyperglycemia on kinetics of protein and selected amino acids in skeletal muscle of female cancer patients after major surgery. Design:In each patient, a 24-hr period of insulin-mediated tight euglycemia (mean blood glucose, 5.8 ± 0.4 mmol/L) preceded or followed a 24-hr control period of moderate hyperglycemia (mean blood glucose, 9.6 ± 0.6 mmol/L) on the first and second day after surgery, in randomized order, according to a crossover experimental design. Setting:Intensive care unit, cancer hospital. Patients:Cancer patients after abdominal radical surgery combined with intraoperative radiation therapy. Interventions:Intensive (57 ± 11 units/24 hrs) and conventional (25 ± 5 units/24 hrs) insulin treatment during total parenteral nutrition. Measurements and Main Results:Muscle metabolism was assessed at the end of each 24-hr period of euglycemia and of hyperglycemia by leg arteriovenous catheterization with stable isotopic tracers. We found that euglycemia as compared with hyperglycemia was associated with higher (p < .05) fractional glucose uptake (16% ± 4% vs. 9% ± 3%); higher (p < .05) muscle protein synthesis and neutral net protein balance (−3 ± 3 vs. −11 ± 3 nmol phenylalanine·100 mL−1·min−1, respectively); lower (−52% ± 12%, p < .01) muscle nonprotein leucine disposal (an index of leucine oxidation) and higher (p < .05) plasma leucine concentrations; and higher (3.6 ± 1.7 times, p < .01) net de novo muscle glutamine synthesis and plasma glutamine concentrations (p < .05). Euglycemia was associated with higher (23% ± 7%, p < .05) plasma concentrations of arginine but did not affect either arginine release from muscle or plasma concentration and muscle flux of asymmetrical dimethylarginine. Rate of muscle proteolysis correlated (p < .05) with muscle release of asymmetrical dimethylarginine. Conclusions:Treating hyperglycemia improves skeletal muscle protein and amino acid metabolism in cancer patients after major surgery.