Inhibition of connective tissue growth factor/CCN2 expression in human dermal fibroblasts by interleukin-1alpha and beta.

Connective tissue growth factor (CTGF/CCN2) is a matricellular protein induced by transforming growth factor (TGF)-beta and intimately involved with tissue repair and overexpressed in various fibrotic conditions. We previously showed that keratinocytes in vitro downregulate TGF-beta-induced expression of CTGF in fibroblasts by an interleukin (IL)-1 alpha-dependent mechanism. Here, we investigated further the mechanisms of this downregulation by both IL-1alpha and beta. Human dermal fibroblasts and NIH 3T3 cells were treated with IL-1alpha or beta in presence or absence of TGF-beta1. IL-1 suppressed basal and TGF-beta-induced CTGF mRNA and protein expression. IL-1alpha and beta inhibited TGF-beta-stimulated CTGF promoter activity, and the activity of a synthetic minimal promoter containing Smad 3-binding CAGA elements. Furthermore, IL-1alpha and beta inhibited TGF-beta-stimulated Smad 3 phosphorylation, possibly linked to an observed increase in Smad 7 mRNA expression. In addition, RNA interference suggested that TGF-beta activated kinase1 (TAK1) is necessary for IL-1 inhibition of TGF-beta-stimulated CTGF expression. These results add to the understanding of how the expression of CTGF in human dermal fibroblasts is regulated, which in turn may have implications for the pathogenesis of fibrotic conditions involving the skin.

Fibrinogen kinetics and protein turnover in obese non-diabetic males: effects of insulin.

BACKGROUND: Although hyperfibrinogenemia and insulin resistance are common in obesity and diabetes mellitus, the impact of obesity per se on fibrinogen turnover and the insulin effects on fibrinogen and protein kinetics is unknown. METHODS: We measured fibrinogen and albumin fractional (FSR) and absolute (ASR) synthesis rates, as well as protein turnover, in non-diabetic, obese and in control male subjects both before and following an euglycemic, euaminoacidemic, hyperinsulinemic clamp, using L-[(2)H(3)]-Leucine isotope infusion. RESULTS: In the obese, basal fibrinogen concentrations was approximately 25% greater (p < 0.035), and fibrinogen pool approximately 45% greater (p < 0.005), than in controls. Both FSR and ASR of fibrinogen were similar to control values. With hyperinsulinemia, although fibrinogen FSR and ASR were not significantly modified with respect to baseline in either group, fibrinogen ASR resulted to be approximately 50% greater in the obese than in controls (p < 0.015). Hyperinsulinemia equally stimulated albumin synthesis and suppressed leucine appearance from endogenous proteolysis in both groups. Amino acid clearance was also similar. In the obese, the insulin-mediated glucose disposal was approximately 50% lower (p < 0.03) than in controls, and it was inversely correlated with fibrinogen ASR during the clamp in both groups (r = - 0.58). CONCLUSIONS: In obese, non-diabetic males, post absorptive fibrinogen production is normal. Whole-body amino acid disposal, basal and insulin-responsive protein degradation, and albumin synthesis are also normal. However, the greater fibrinogen ASR in the obese with hyperinsulinemia, and the inverse relationship between insulin sensitivity and clamp fibrinogen production, suggest a role for hyperinsulinemia and/or insulin resistance on fibrinogen production in obesity.

Fibrinogen kinetics and protein turnover in hypertension: Effects of insulin.

BACKGROUND AND AIM: Hyperfibrinogenemia, a cardiovascular risk factor, is frequent in hypertension and largely unexplained. In this study, we measured fibrinogen production and whole-body protein turnover under both basal and hyperinsulinemic states, in hypertensive [H] and control [C] subjects, using a leucine stable isotope tracer and precursor-product relationships. METHODS AND RESULTS: Since hypertension is often a feature of the "metabolic", insulin resistance syndrome, which in turn affects both fibrinogen kinetics and whole-body protein turnover, we selected hypertensive subjects without the metabolic syndrome. Following basal measurements, an euglycemic, approximately euaminoacidemic, hyperinsulinemic clamp was performed, with plasma insulin raised to 700-900 pmol/L. In H, rates of the fractional and absolute synthesis (FSR and ASR, respectively) of fibrinogen were 30%-40% greater (p<0.05 or less) than in C in both states, whereas leucine turnover was normal. Hyperinsulinemia did not modify fibrinogen synthesis in either group with respect to baseline, whereas it suppressed leucine appearance from endogenous proteolysis by approximately 40% to same extent in both groups. Amino acid clearance was similar in both the H and C subjects. In H, the insulin-mediated glucose disposal (M) was approximately 25% lower, (although insignificantly) than in controls, showing no overall insulin resistance. There was an inverse correlation between M and fibrinogen FSR during the clamp. CONCLUSIONS: In essential hypertension fibrinogen production is increased, is not further stimulated by insulin, and is inversely related to insulin sensitivity at high-physiological insulin concentrations. Amino acid disposal and basal as well as insulin-responsive protein degradation rates are instead normal.

Diabetic nephropathy is associated with increased albumin and fibrinogen production in patients with type 2 diabetes.

AIMS/HYPOTHESIS: Hyperfibrinogenaemia and albuminuria are cardiovascular risk factors, often coexisting in diabetic and non-diabetic people. Albuminuria in turn is associated with a compensatory albumin overproduction in non-diabetic patients. It is not known whether the presence of albuminuria in patients with type 2 diabetes mellitus is associated with greater albumin and fibrinogen production rates than in normoalbuminuric patients. SUBJECTS, MATERIALS, AND METHODS: Using leucine isotope methods, we measured fractional and absolute synthesis rates (FSR, ASR) of albumin and fibrinogen in post-absorptive type 2 diabetic patients with either normal (n=11) or increased (n=10) urinary albumin excretion. RESULTS: In albuminuric patients, albumin FSR (16.2+/-1.5%/day) and ASR (20.5+/-1.9 g/day) were greater (p<0.02 and p<0.05, respectively) than in normoalbuminuric patients (FSR=11.5+/-1.1%/day; ASR=15.7+/-1.2 g/day). Fibrinogen FSR was similar between patients with normal and increased albumin excretion, but concentration, the circulating pool and ASR of fibrinogen were 40 to 50% greater (p<0.035) in patients with albuminuria. Albuminuria was positively correlated with albumin ASR, with fibrinogen concentration, the fibrinogen pool and ASR, whereas albumin synthesis was inversely correlated with calculated oncotic pressure. CONCLUSIONS/INTERPRETATION: Synthesis of albumin and fibrinogen is upregulated in type 2 diabetic patients with increased urinary albumin excretion. Albuminuria is associated with enhanced fibrinogen and albumin synthesis.

Visceral obesity is characterized by impaired nitric oxide-independent vasodilation.

BACKGROUND: Endothelial dysfunction has been described in obesity. This study examines the impact of visceral obesity on nitric oxide-independent relaxation in the human forearm. METHODS AND RESULTS: In ten viscerally obese and ten matched controls forearm blood flow (FBF) was measured by venous occlusion plethysmography during intrabrachial infusion of: (1) sodium nitroprusside; (2) bradykinin, before and after inhibition of vasoactive prostaglandins and nitric oxide; (3) potassium; (4) ouabain (Na(+)/K(+)ATPase inhibitor) alone or (5) in combination with BaCl(2)(K(IR)inhibitor). Baseline FBF and endothelium-independent vasodilatation were similar in the two groups. In obese patients, bradykinin-induced increase of FBF was significantly less than in controls (P<0.01). Irrespective of prostaglandins and nitric oxide inhibition, bradykinin response was lower in the viscerally obese. Intrabrachial potassium determined a significantly blunted response (P<0.05). Ouabain caused a similar, moderate decrease in basal FBF in the two groups; the coinfusion of BaCl(2)caused a more intense decline in FBF which was significantly relevant in obese (-24+/-5%, P<0.01). CONCLUSIONS: In obese patients there is a blunted nitric oxide-independent relaxation determined by a decreased response of inwardly rectifying potassium channels.

Impairment of albumin and whole body postprandial protein synthesis in compensated liver cirrhosis.

To investigate the anabolic effects of feeding in cirrhosis, we measured albumin fractional synthesis rate (FSR) and whole body protein synthesis in six nondiabetic patients with stable liver cirrhosis (three in the Child-Pugh classification Class A, three in Class B) and in seven normal control subjects, before and after administration of a 4-h mixed meal. Leucine tracer precursor-product relationships and whole body kinetics were employed at steady state. Basal levels of postabsorptive albumin concentration and FSR, whole body leucine rate of appearance, oxidation, and nonoxidative leucine disposal (NOLD, approximately equal to protein synthesis) were similar in the two groups. However, after the meal, in the patients neither albumin FSR (from 8.5 +/- 1.5 to 8.8 +/- 1.8 %/day) nor NOLD (from 1.69 +/- 0.22 to 1.55 +/- 0.26 micromol x kg(-1) x min(-1)) changed (P = nonsignificant vs. basal), whereas they increased in control subjects (albumin FSR: from 10.9 +/- 1.5 to 15.9 +/- 1.9 %/day, P < 0.002; NOLD: from 1.80 +/- 0.14 to 2.10 +/- 0.19 micromol x kg(-1) x min(-1), P = 0.032). Thus mixed meal ingestion did not stimulate either albumin FSR or whole body protein synthesis in compensated liver cirrhosis. The mechanism(s) maintaining normoalbuminemia at this disease stage need to be further investigated.

Insulin infusion normalizes fasting and post-prandial albumin and fibrinogen synthesis in Type 1 diabetes mellitus.

AIMS: The effect of metabolic control on hepatic synthesis of plasma proteins in Type 1 diabetes mellitus (T1DM), in the post-absorptive and post-prandial state, is not known. METHODS: We measured fractional synthetic rates (FSR) of albumin and fibrinogen in six insulin-infused T1DM patients and in five to nine control subjects, before and for approx. 4 h after a mixed liquid meal. Phenylalanine tracer precursor/product relationships and steady-state conditions were used. In the post-absorptive state, patients were studied in near euglycaemic conditions after an overnight intravenous insulin infusion. During the meal (approx. 11 kcal/kg), the insulin infusion rate was increased to maintain plasma glucose concentrations below approx. 10 mmol/l. RESULTS: Post-absorptive FSR of albumin (5.7 +/- 0.6%/day) and fibrinogen (11.3 +/- 0.6%/day) in T1DM were similar to control values (6.4 +/- 0.9 and 13.1 +/- 1.1, respectively). After the meal, albumin FSR increased (P = 0.0032 by anova) in both groups (T1DM, to 14.4 +/- 2.7%/day; controls, to 18.2 +/- 3.7%/day). Fibrinogen FSR also increased (P = 0.0048 by anova) in both the T1DM (to 18.2 +/- 2.6) and the control subjects (to 27.3 +/- 6.2). There was no difference between T1DM and control subjects in the post-prandial FSR of both proteins. CONCLUSIONS: Albumin and fibrinogen FSR in T1DM can be maintained within near-normal ranges by insulin infusion under post-absorptive and post-prandial conditions.

Plasma protein synthesis in patients with low-grade nephrotic proteinuria.

Overt nephrotic syndrome is characterized by albumin and fibrinogen hyperproduction and reduced very low density lipoprotein apolipoprotein B-100 (VLDL apoB-100) clearance. Whether similar changes also occur in low-grade proteinuria is not known. Thus we measured albumin, fibrinogen, and VLDL apoB-100 kinetics in six patients with modest proteinuria and normal creatinine clearance (P) and in ten control subjects (C) by leucine tracer infusion and precursor-product relationships. In P, plasma albumin concentration was decreased (P < 0.003), whereas concentrations of fibrinogen and VLDL apoB-100 were increased (P < 0.001). In P, albumin fractional secretion rate (FSR) was increased (P < 0.01), fibrinogen FSR was normal, and VLDL apoB-100 FSR was decreased (P < 0.03). As a result, in P, absolute secretion rates (ASR) of albumin and fibrinogen were increased (P < 0.03), whereas VLDL apoB-100 ASR was normal. Albumin FSR was inversely correlated to oncotic pressure in P but not in C. These findings suggest that low-grade nephrotic proteinuria is characterized by simultaneous multiple alterations in turnover rates of albumin, fibrinogen, and VLDL apoB-100. Their pathogenesis, however, appears to be multifactorial.

Glucose kinetics and splanchnic uptake following mixed meal ingestion in cirrhotic-diabetic subjects.

Although glucose intolerance and/or overt diabetes are common in cirrhotic subjects, the mechanism(s) that lead to post-prandial hyperglycemia in cirrhosis are not entirely known. To this aim, we measured whole-body rates of glucose appearance (Ra) and of disappearance (Rd) in cirrhotic-diabetic subjects and in controls, before and following a 4-hr administration of a mixed meal. In the post-prandial phase, endogenous and dietary glucose Ra, as well as first-pass splanchnic uptake of dietary glucose, were measured using a double (ie oral and intravenous) glucose tracer technique. In the fasting state, the cirrhotic patients were hyperglycemic (12.0 +/- 1.4 vs 4.4 +/- 0.2 mmol/l in controls, p < 0.001), had a higher glucose Ra (17.0 +/- 2.7 vs 10.2 +/- 0.5 micromol x kg(-1) x min(-1), p < 0.05) and a lower clearance rate (1.51 +/- 0.19 vs 2.32 +/- 0.06 ml x kg x min, p < 0.02). Following the meal, plasma glucose increased to greater values (p < 0.002) in the patients (to 16.8 +/- 2 mmol/l, mean values of the last 40 min) than in the controls (to 7.2 +/- 0.4 mmol/l). Insulin increased in both groups but it was 35% lower (p > 0.05) in the patients. Post-prandial total glucose Ra (cirrhotics: 21.3 +/- 2.6; controls: 19.2 +/- 1.4 pmol x kg(-1) x min(-1)), endogenous Ra (cirrhotics: 7.3 +/- 1.5; controls: 7.0 +/- 1.3 micromol x kg(-1) x min(-1)) and first-pass splanchnic uptake of dietary glucose (cirrhotics: 9.8 +/- 2.6; controls: 11.5 +/- 1.6 micromol x kg x min(-1)), were not different between the 2 groups, whereas glucose clearance remained lower (p<0.001) in the patients (1.31 +/- 0.25 ml x kg(-1) x min)-1)) than in the controls (2.72 +/- 0.26). These data demonstrate that, in cirrhotic-diabetic patients, post-pran-dial hyperglycemia is not due to a reduced extraction of dietary glucose nor to an increased endogenous production, but rather to a defect in peripheral glucose clearance, secondary to either insulin-resistance and/or relative insulin deficiency.

Increased fibrinogen production in type 2 diabetic patients without detectable vascular complications: correlation with plasma glucagon concentrations.

Fibrinogen is a strong cardiovascular risk factor in the general population, and increased fibrinogen plasma concentrations have been reported in type 2 diabetic patients. However, the mechanisms leading to hyperfibrinogenemia in type 2 diabetes are not known. It is also not known whether possible alterations of fibrinogen turnover may precede clinical diabetic micro- and macrovascular complications and therefore potentially contribute to their onset. To address these questions, fibrinogen production was determined in six male type 2 diabetic patients without detectable micro- and macrovascular complications (age, 45 +/- 4 yr; body mass index, 27 +/- 0.9 kg/m2) and in seven nondiabetic matched controls using leucine isotope precursor-product relationships. Plasma glucose (P < 0.001), insulin (P < 0.05), and glucagon concentrations (P < 0.01) were increased in the patients. Diabetic patients also had increased plasma fibrinogen concentration (+ approximately 50%; P < 0.01) and pool (+ approximately 40%; P < 0.01) as well as fractional (+ approximately 35%; P = 0.08) and absolute (+ approximately 100%; P < 0.01) synthetic rates. The plasma glucagon concentration was positively related (P < 0.005 or less) to the fibrinogen concentration as well as to fractional and absolute synthetic rates. Thus, fibrinogen production is markedly enhanced, and this alteration is likely to determine the observed hyperfibrinogenemia in type 2 diabetic patients. Hyperglucagonemia may contribute to the increased fibrinogen production. These findings in normoalbuminuric patients without clinical complications support the hypothesis that increased fibrinogen production and plasma concentrations may precede and possibly contribute to the onset of clinical cardiovascular complications in type 2 diabetes.

Effects of branched-chain-enriched amino acids and insulin on forearm leucine kinetics.

Although amino acid mixtures enriched in branched-chain amino acids (BCAA) and deficient in aromatic amino acids (AAA) are often used together with insulin and glucose in clinical nutrition, their physiological effects on muscle protein anabolism are not known. To this aim, we studied forearm leucine kinetics in post-absorptive volunteers, before and after the systemic infusion of BCAA-enriched, AAA-deficient amino acids along with insulin and the euglycaemic clamp. The results were compared with the effects of insulin infusion alone. A compartmental leucine forearm model was employed at steady state. Hyperaminoacidaemia with hyperinsulinaemia (to approximately 80-100 micro-units/ml) increased the leucine plasma concentration (+70%; P<0.001), inflow into the forearm cell (+150%; P<0.01), disposal into protein synthesis (+100%; P<0.01), net intracellular retention (P<0.01), net forearm balance (by approximately 6-fold; P<0.01) and net deamination to alpha-ketoisocaproate (4-methyl-2-oxopentanoate) (+9%; P<0.05). Leucine release from forearm proteolysis and outflow from the forearm cell were unchanged. In contrast, hyperinsulinaemia alone decreased plasma leucine concentrations (-35%; P<0.001) and leucine inflow (-20%; P<0.05) and outflow (-30%; P<0.01) into and out of forearm cell(s), it increased net intracellular leucine retention (P<0.03), and it did not change leucine release from forearm proteolysis (-20%; P=0.138), net leucine deamination to alpha-ketoisocaproate, leucine disposal into protein synthesis or net forearm protein balance. By considering all data together, leucine disposal into protein synthesis was directly correlated with leucine inflow into the cell (r=0.71; P<0.0001). These data indicate that the infusion of BCAA-enriched, AAA-deficient amino acids along with insulin is capable of stimulating forearm (i.e. muscle) protein anabolism in normal volunteers by enhancing intracellular leucine transport and protein synthesis. These effects are probably due to hyperaminoacidaemia and/or its interaction with hyperinsulinaemia, since they were not observed under conditions of hyperinsulinaemia alone.

Evidence for acute stimulation of fibrinogen production by glucagon in humans.

Fibrinogen, an acute-phase protein, and glucagon, a stress hormone, are often elevated in many conditions of physical and metabolic stress, including uncontrolled diabetes. However, the possible mechanisms for this association are poorly known. We have studied the acute effects of selective hyperglucagonemia (raised from -200 to -350 pg/ml for 3 h) on fibrinogen fractional secretion rate (FSR) in eight normal subjects during infusion of somatostatin and replacement doses of insulin, glucagon, and growth hormone. Fibrinogen FSR was evaluated by precursor-product relationships using either Phe (n = 8) or Leu (n = 2) tracers. Hyperglucagonemia did not change either plasma Phe or Tyr specific activity. After hyperglucagonemia, fibrinogen FSR increased by approximately 65% (from 12.9 +/- 3.6 to 21.5 +/- 6.1% per day, P < 0.025) using plasma Phe specific activity as the precursor pool. FSR increased by approximately 80% (from 16.6 +/- 4.8 to 29.4 +/- 8.8% per day, P < 0.025) if plasma Phe specific activity was corrected for the ketoisocaproate/Leu enrichment (or specific activity) ratio to obtain an approximate estimate of intrahepatic Phe specific activity. FSR increased by approximately 60% when using plasma Tyr specific activity as precursor pool (n = 8) (P < 0.05), as well as when using the Leu tracer precursor-product relationship (n = 2). In conclusion, selective hyperglucagonemia for approximately 3 h acutely stimulated fibrinogen FSR using a Phe tracer method. Thus, glucagon may be involved in the increase of fibrinogen concentration and FSR observed under stressed or pathologic conditions.

The role of substrates in the regulation of protein metabolism.

Substrates are powerful modulators of amino acid and protein turnover in vivo (Table 4). Intravenous infusions of amino acids exert a protein-anabolic effect, because they directly inhibit endogenous protein degradation and stimulate protein synthesis at the whole-body level. A stimulation of protein synthesis has been observed also at the forearm level. These changes resulted in an improvement of body and tissue protein balance, which is the ultimate goal of any nutritional intervention aimed at preserving body protein stores. In humans acute intravenous infusions of carbohydrates do not appear to affect either protein degradation or leucine oxidation. However, animal studies support the view that glucose availability spares essential amino acids at least in the fetus. The effects of hypercaloric refeeding with high-carbohydrate diets may, however, result in increased protein turnover. Lipids, in the form of long-chain fatty acids, inhibit endogenous protein breakdown and may suppress leucine oxidation in the whole body. They do not affect protein synthesis. In contrast, medium-chain fatty acids apparently increased leucine oxidation, and therefore increased net protein catabolism. Ketone bodies may be anabolic provided that fatty acid concentrations are not concurrently decreased.

Hyperglucagonemia stimulates phenylalanine oxidation in humans.

Glucagon stimulates in vitro liver phenylalanine (Phe) degradation, thus inducing net protein catabolism. Whether these effects occur also in vivo in humans is not known. Therefore, we studied the effects of physiological hyperglucagonemia on Phe rate of appearance (Ra), hydroxylation, and oxidation in seven normal volunteers during infusions of somatostatin with replacement doses of insulin and growth hormone. Steady-state Phe kinetics were evaluated using the L-[1-14C]Phe tracer both at the end of a 3-h basal glucagon replacement period (glucagon concentration: 212 +/- 115 ng/l) and after a 3-h hormone infusion at the rate of approximately 3 ng x kg-1 x min-1 (--> 654 +/- 280 ng/l). Hyperglucagonemia did not change plasma Phe concentration and Ra but increased Phe oxidation by approximately 30% (P < 0.01). Oxidation was also increased by approximately 24% (P < 0.01) using plasma [14C]tyrosine (Tyr) specific activity as a precursor pool. Phe hydroxylation to Tyr estimated by assuming a fixed ratio of Tyr to Phe Ra (0.73) did not change. Nonhydroxylated Phe disposal decreased by approximately 6% (P = 0.08). These data show that in humans in the postabsorptive state, hyperglucagonemia, with near maintenance of basal insulin and growth hormone concentrations, stimulates Phe oxidation but not Phe hydroxylation, suggesting a different regulation of these two Phe catabolic steps. Glucagon may also reduce Phe availability for protein synthesis.