The gene encoding the proprotein convertase subtilisin/kexin type 9 (PCSK9) is linked to familial hypercholesterolemia, as are those of the low-density lipoprotein receptor (LDLR) and apolipoprotein B. PCSK9 enhances LDLR degradation, resulting in low-density lipoprotein accumulation in plasma. To analyze the role of hepatic PCSK9, total and hepatocyte-specific knockout mice were generated. They exhibit 42% and 27% less circulating cholesterol, respectively, showing that liver PCSK9 was responsible for two thirds of the phenotype. We also demonstrated that, in liver, PCSK9 is exclusively expressed in hepatocytes, representing the main source of circulating PCSK9. The data suggest that local but not circulating PCSK9 regulates cholesterol levels. Although transgenic mice overexpressing high levels of liver and circulating PCSK9 led to the almost complete disappearance of the hepatic LDLR, they did not recapitulate the plasma cholesterol levels observed in LDLRdeficient mice. Single LDLR or double LDLR/PCSK9 knockout mice exhibited similar cholesterol profiles, indicating that PCSK9 regulates cholesterol homeostasis exclusively through the LDLR. Finally, the regenerating liver of PCSK9-deficient mice exhibited necrotic lesions, which were prevented by a high-cholesterol diet. However, lipid accumulation in hepatocytes of these mice was markedly reduced under both chow and high-cholesterol diets, revealing that PCSK9 deficiency confers resistance to liver steatosis. Conclusion: Although PCSK9 is a target for controlling hypercholesterolemia, our data indicate that upon hepatic damage, patients lacking PCSK9 could be at risk. (HEPATOLOGY 2008;48:646-654.) P roprotein convertase subtilisin/kexin type 9 (PCSK9) 1 is the ninth member of the proprotein convertase family. 2 The first seven members, including furin, cleave protein precursors of hormones, growth factors, receptors, or surface glycoproteins at basic sites (after Arg or Lys residues). The eighth member, SKI-1 3 or S1P, 4 is known to cleave membrane-bound transcription factors such as the SREBPs 5 in their luminal domains, resulting in the release of their DNA-binding domain. Proprotein convertases can also inactivate secreted substrates, such as endothelial lipase 6 and PCSK9. 7 PCSK9 is synthesized as a precursor that undergoes autocatalytic cleavage of its N-terminal prosegment in the ER, 1 a step required for its exit from this compartment and its efficient secretion. Secreted PCSK9 remains associated with its prosegment. 1 Different from the other proprotein convertases, this serine protease has no known substrate other than itself. In addition, the tight association of the prosegment with the active site 8 raises the question of the existence of an in trans PCSK9 protease

Dietary proteins and amino acids are important modulators of glucose metabolism and insulin sensitivity. Although high intake of dietary proteins has positive effects on energy homeostasis by inducing satiety and possibly increasing energy expenditure, it has detrimental effects on glucose homeostasis by promoting insulin resistance and increasing gluconeogenesis. Varying the quality rather than the quantity of proteins has been shown to modulate insulin resistance induced by Western diets and has revealed that proteins derived from fish might have the most desirable effects on insulin sensitivity. In vitro and in vivo data also support an important role of amino acids in glucose homeostasis through modulation of insulin action on muscle glucose transport and hepatic glucose production, secretion of insulin and glucagon, as well as gene and protein expression in various tissues. Moreover, amino acid signaling is integrated by mammalian target of rapamycin, a nutrient sensor that operates a negative feedback loop toward insulin receptor substrate 1 signaling, promoting insulin resistance for glucose metabolism. This integration suggests that modulating dietary proteins and the flux of circulating amino acids generated by their consumption and digestion might underlie powerful new approaches to treat various metabolic diseases such as obesity and diabetes.

The proprotein convertase subtilisin kexin-9 (PCSK9) circulates in plasma as mature and furin-cleaved forms. A polyclonal antibody against human PCSK9 was used to develop an ELISA that measures total plasma PCSK9 rather than only the mature form. A cross-sectional study evaluated plasma levels in normal (n = 254) and hypercholesterolemic (n = 200) subjects treated or untreated with statins or statin plus ezetimibe. In controls, mean plasma PCSK9 (89.5 ± 31.9 ng/ml) correlated positively with age, total cholesterol, LDL-cholesterol (LDL-C), triglycerides, and fasting glucose. Sequencing PCSK9 from individuals at the extremes of the normal PCSK9 distribution identifi ed a new loss-of-function R434W variant associated with lower levels of circulating PCSK9 and LDL-C. In hypercholesterolemic subjects, PCSK9 levels were higher than in controls (99.3 ± 31.7 ng/ml, P < 0.04) and increased in proportion to the statin dose, combined or not with ezetimibe. In treated patients (n = 139), those with familial hypercholesterolemia (FH; due to LDL receptor gene mutations) had higher PCSK9 values than non-FH (147.01 ± 42.5 vs . 127.2 ± 40.8 ng/ml, P < 0.005), but LDL-C reduction correlated positively with achieved plasma PCSK9 levels to a similar extent in both subsets ( r = 0.316, P < 0.02 in FH and r = 0.275, P < 0.009 in non-FH). The detection of circulating PCSK9 in both FH and non-FH subjects means that this assay could be used to monitor response to therapy in a wide range of patients.-Dubuc, G., M. Tremblay, G. Paré, H. Jacques, J. Hamelin, S. Benjannet, L. Boulet, J. Genest, L. Bernier, N. G. Seidah, and J. Davignon. A new method for measurement of total plasma PSCK9: clinical applications.

The aim of the present study was to determine the effects of feeding various dietary proteins on insulin sensitivity and glucose tolerance in rats. Male Wistar rats were fed for 28 days with isoenergetic diets containing either casein, soy protein, or cod protein. Cod protein-fed and soy protein-fed rats had lower fasting plasma glucose and insulin concentrations compared with casein-fed animals. After intravenous glucose bolus, cod protein- and soy protein-fed rats induced lower incremental areas under glucose curves compared with casein-fed animals. Improved peripheral insulin sensitivity was confirmed by higher glucose disposal rates in cod protein- and soy protein-fed rats (15.2 +/- 0.3 and 13.9 +/- 0.6 mg. kg(-1). min(-1), respectively) compared with casein-fed animals (6.5 +/- 0.7 mg. kg(-1). min(-1), P < 0.05). Moreover, test meal experiments revealed that, in the postprandial state, the lower plasma insulin concentrations in cod protein- and soy protein-fed animals could be also due to decreased pancreatic insulin release and increased hepatic insulin removal. In conclusion, the metabolic responses to three common dietary proteins indicate that cod and soy proteins, when compared with casein, improve fasting glucose tolerance and peripheral insulin sensitivity in rats.

The cellular mechanism by which high-fat feeding induces skeletal muscle insulin resistance was investigated in the present study. Insulin-stimulated glucose transport was impaired (ϳ40 -60%) in muscles of high fat-fed rats. Muscle GLUT4 expression was significantly lower in these animals (ϳ40%, P < 0.05) but only in type IIa-enriched muscle. Insulin stimulated the translocation of GLUT4 to both the plasma membrane and the transverse (T)-tubules in chow-fed rats. In marked contrast, GLUT4 translocation was completely abrogated in the muscle of insulin-stimulated high fat-fed rats. High-fat feeding markedly decreased insulin receptor substrate (IRS)-1-associated phosphatidylinositol (PI) 3-kinase activity but not insulin-induced tyrosine phosphorylation of the insulin receptor and IRS proteins in muscle. Impairment of PI 3-kinase function was associated with defective Akt/protein kinase B kinase activity (؊40%, P < 0.01) in insulin-stimulated muscle of high fat-fed rats, despite unaltered phosphorylation (Ser473/Thr308) of the enzyme. Interestingly, basal activity of atypical protein kinase C (aPKC) was elevated in muscle of high fat-fed rats compared with chow-fed controls. Whereas insulin induced a twofold increase in aPKC kinase activity in the muscle of chowfed rats, the hormone failed to further increase the kinase activity in high fat-fed rat muscle. In conclusion, it was found that GLUT4 translocation to both the plasma membrane and the T-tubules is impaired in the muscle of high fat-fed rats. We identified PI 3-kinase as the first step of the insulin signaling pathway to be impaired by high-fat feeding, and this was associated with alterations in both Akt and aPKC kinase activities.

Phytoestrogens are increasingly incorporated into the diet of menopausal women. However, there are limited data on the efficacy of flaxseed on the consequences of estrogen deficiency in menopausal women. The purpose of the study was to assess the effects of flaxseed incorporation into the diet of healthy menopausal women. One hundred and ninety-nine menopausal women were randomly assigned to consume 40 g flaxseed/d (n = 101) or wheat germ placebo (n = 98) for 12 months. At baseline and at month 12, serum levels of lipids, bone mineral density (BMD), and menopausal symptoms were evaluated. Statistical analysis was performed under the intention to treat principle. Flaxseed reduced serum total (-0.20 +/- 0.51 mmol/liter; P = 0.012) and high-density lipoprotein (-0.08 +/- 0.24 mmol/liter; P = 0.031) cholesterol concentrations compared with wheat germ placebo. BMD did not differ significantly between the two arms. Both flaxseed and wheat germ reduced (P < 0.0001) the severity scores of menopausal symptoms, but no statistical difference was found between the two arms. Our findings suggest that 1-yr incorporation of flaxseed into the diet produced a favorable, but not clinically significant, effect on blood cholesterol and caused no significant change in BMD or symptoms in healthy menopausal women.

OBJECTIVE -The purpose of this article was to compare the effects of cod protein to those of other animal proteins on insulin sensitivity in insulin-resistant human subjects.RESEARCH DESIGN AND METHODS -Insulin sensitivity (M/I) was assessed using a hyperinsulinemic-euglycemic clamp in 19 insulin-resistant subjects fed a cod protein diet and a similar diet containing lean beef, pork, veal, eggs, milk, and milk products (BPVEM) for 4 weeks in a crossover design study. Both diets were formulated to differ only in protein source, thus providing equivalent amounts of dietary fibers and monounsaturated, polyunsaturated (including n-3), and saturated fatty acids (1.1:1.8:1.0). ␤-Cell function, estimated by oral glucose tolerance test-derived parameters, was also assessed.RESULTS -There was a significant improvement in insulin sensitivity (P ϭ 0.027) and a strong tendency for a better disposition index (␤-cell function ϫ M/I) (P ϭ 0.055) in subjects consuming the cod protein diet compared with those consuming the BPVEM diet. When median baseline M/I (4.8 ϫ 10 Ϫ3 mg ⅐ kg Ϫ1 ⅐ min Ϫ1 ⅐ pmol Ϫ1 ) was taken into account, an interaction on the 30-min C-peptide-to-30-min glucose ratio, used as an index of ␤-cell function, was observed between diet and M/I status (P ϭ 0.022). Indeed, this ratio strongly tended to increase in subjects with low M/I consuming the cod protein diet compared with those consuming the BPVEM diet (P ϭ 0.065).CONCLUSIONS -Dietary cod protein improves insulin sensitivity in insulin-resistant individuals and thus could contribute to prevention of type 2 diabetes by reducing the metabolic complications related to insulin resistance. Diabetes Care 30:2816-2821, 2007I nsulin resistance contributes to the pathophysiology of type 2 diabetes (1). Studies to reduce insulin resistance using insulin-sensitizing agents, such as thiazolidinediones, suggest that such therapies may delay or prevent progression from insulin resistance to type 2 diabetes (2,3) and preserve ␤-cell function (4). Among other therapeutic approaches, dietary interventions are also being studied.An increasing number of studies have been performed to investigate the metabolic effects of dietary proteins on insulin and glucose homeostasis. According to von Post-Skagegard et al. (5), a cod protein meal, compared with a milk protein meal, lowered insulin levels and reduced insulin-to-C-peptide and insulin-toglucose ratios in healthy women. In a previous study, both cod and soy proteins reduced fasting and postprandial glucose and insulin responses and increased peripheral insulin sensitivity compared with casein in rats fed a high-sucrose diet (6). Furthermore, dietary cod protein, compared with soy protein and casein, prevented the development of skeletal muscle insulin resistance (7) by normalizing insulin activation of the phosphatidylinositol 3-kinase/Akt-protein kinase B pathway and by improving GLUT4 translocation in skeletal muscle of high-fat-fed obese rats (8). Studies in humans also showed that including lean fish, whose major componen...

In the present study, we tested the hypothesis that fish protein may represent a key constituent of fish with glucoregulatory activity. Three groups of rats were fed a high-fat diet in which the protein source was casein, fish (cod) protein, or soy protein; these groups were compared with a group of chow-fed controls. High-fat feeding led to severe whole body and skeletal muscle insulin resistance in casein- or soy protein-fed rats, as assessed by the euglycemic clamp technique coupled with measurements of 2-deoxy-D-[(3)H]glucose uptake rates by individual tissues. However, feeding cod protein fully prevented the development of insulin resistance in high fat-fed rats. These animals exhibited higher rates of insulin-mediated muscle glucose disposal that were comparable to those of chow-fed rats. The beneficial effects of cod protein occurred without any reductions in body weight gain, adipose tissue accretion, or expression of tumor necrosis factor-alpha in fat and muscle. Moreover, L6 myocytes exposed to cod protein-derived amino acids showed greater rates of insulin-stimulated glucose uptake compared with cells incubated with casein- or soy protein-derived amino acids. These data demonstrate that feeding cod protein prevents obesity-induced muscle insulin resistance in high fat-fed obese rats at least in part through a direct action of amino acids on insulin-stimulated glucose uptake in skeletal muscle cells.