Hepatic denervation impairs the assembly and secretion of VLDL-TAG

VLDL secretion is a regulated process that depends on the availability of lipids, apoB and MTP. Our aim was to investigate the effect of liver denervation upon the secretion of VLDL and the expression of proteins involved in this process. Denervation was achieved by applying a 85% phenol solution onto the portal tract, while control animals were treated with 9% NaCl. VLDL secretion was evaluated by the Tyloxapol method. The hepatic concentration of TAG and cholesterol, and the plasma concentration of TAG, cholesterol, VLDL-TAG, VLDL-cholesterol and HDL-cholesterol were measured, as well as mRNA expression of proteins involved in the process of VLDL assembly. Hepatic acinar distribution of MTP and apoB was evaluated by immunohistochemistry. Denervation increased plasma concentration of cholesterol (125.3 +/- 10.1 vs. 67.1 +/- 4.9 mg dL(-1)) and VLDL-cholesterol (61.6 +/- 5.6 vs. 29.4 +/- 3.3 mg dL(-1)), but HDL-cholesterol was unchanged (45.5 +/- 6.1 vs. 36.9 +/- 3.9 mg dL(-1)). Secretion of VLDL-TAG (47.5 +/- 23.8 vs. 148.5 +/- 27.4 mg dL h(-1)) and mRNA expression of CPT I and apoB were reduced (p < 0.01) in the denervated animals. MTP and apoB acinar distribution was not altered in the denervated animals, but the intensity of the reaction was reduced in relation to controls. Copyright (C) 2008 John Wiley & Sons, Ltd.

Modulation of Bone Morphogenetic Protein-9 Expression and Processing by Insulin, Glucose, and Glucocorticoids: Possible Candidate for Hepatic Insulin-Sensitizing Substance

Bone morphogenetic protein 9 (BMP-9), a member of the TGF-beta superfamily predominantly expressed in nonparenchymal liver cells, has been demonstrated to improve glucose homeostasis in diabetic mice. Along with this therapeutic effect, BMP-9 was proposed as a candidate for the hepatic insulin-sensitizing substance ( HISS). Whether BMP-9 plays a physiological role in glucose homeostasis is still unknown. In the present study, we show that BMP-9 expression and processing is severely reduced in the liver of insulin-resistant rats. BMP-9 expression and processing was directly stimulated by in situ exposition of the liver to the combination of glucose and insulin and oral glucose in overnight fasted rats. Additionally, prolonged fasting ( 72 h) abrogated refeeding-induced BMP-9 expression and processing. Previous exposition to dexamethasone, a known inductor of insulin resistance, reduced BMP-9 processing stimulated by the combination of insulin and glucose. Finally, we show that neutralization of BMP-9 with an anti-BMP-9 antibody induces glucose intolerance and insulin resistance in 12-h fasted rats. Collectively, the present results demonstrate that BMP-9 plays an important role in the control of glucose homeostasis of the normal rat. Additionally, BMP-9 is expressed and processed in an HISS-like fashion, which is impaired in the presence of insulin resistance. BMP-9 regulation according to the feeding status and the presence of diabetogenic factors reinforces the hypothesis that BMP-9 might exert the role of HISS in glucose homeostasis physiology. ( Endocrinology 149: 6326-6335, 2008)

Fluorescence spectroscopy to diagnose hepatic steatosis in a rat model of fatty liver

Steatosis is diagnosed on the basis of the macroscopic aspect of the liver evaluated by the surgeon at the time of organ extraction or by means of a frozen biopsy. In the present study, the applicability of laser-induced fluorescence (LIF) spectroscopy was investigated as a method for the diagnosis of different degrees of steatosis experimentally induced in rats. Rats received a high-lipid diet for different periods of time. The animals were divided into groups according to the degree of induced steatosis diagnosis by histology. The concentration of fat in the liver was correlated with LIF by means of the steatosis fluorescence factor (SFF). The histology classification, according to liver fat concentration was, Severe Steatosis, Moderate Steatosis, Mild Steatosis and Control (no liver steatosis). Fluorescence intensity could be directly correlated with fat content. It was possible to estimate an average of fluorescence intensity variable by means of different confidence intervals (P=95%) for each steatosis group. SFF was significantly higher in the Severe Steatosis group (P < 0.001) compared with the Moderate Steatosis, Mild Steatosis and Control groups. The various degrees of steatosis could be directly correlated with SFF. LIF spectroscopy proved to be a method capable of identifying the degree of hepatic steatosis in this animal model, and has the potential of clinical application for non-invasive evaluation of the degree of steatosis.

Splanchnic blood flow and hepatic glucose production in exercising humans : role of renin-angiotensin system

The study examined the implication of the renin-angiotensin system (RAS) in regulation of splanchnic blood flow and glucose production in exercising humans. Subjects cycled for 40 min at 50% maximal O2 consumption (VO2 max) followed by 30 min at 70% VO2 max either with [angiotensin-converting enzyme (ACE) blockade] or without (control) administration of the ACE inhibitor enalapril (10 mg iv). Splanchnic blood flow was estimated by indocyanine green, and splanchnic substrate exchange was determined by the arteriohepatic venous difference. Exercise led to an ~20-fold increase (P < 0.001) in ANG II levels in the control group (5.4 ± 1.0 to 102.0 ± 25.1 pg/ml), whereas this response was blunted during ACE blockade (8.1 ± 1.2 to 13.2 ± 2.4 pg/ml) and in response to an orthostatic challenge performed postexercise. Apart from lactate and cortisol, which were higher in the ACE-blockade group vs. the control group, hormones, metabolites, VO2, and RER followed the same pattern of changes in ACE-blockade and control groups during exercise. Splanchnic blood flow (at rest: 1.67 ± 0.12, ACE blockade; 1.59 ± 0.18 l/min, control) decreased during moderate exercise (0.78 ± 0.07, ACE blockade; 0.74 ± 0.14 l/min, control), whereas splanchnic glucose production (at rest: 0.50 ± 0.06, ACE blockade; 0.68 ± 0.10 mmol/min, control) increased during moderate exercise (1.97 ± 0.29, ACE blockade; 1.91 ± 0.41 mmol/min, control). Refuting a major role of the RAS for these responses, no differences in the pattern of change of splanchnic blood flow and splanchnic glucose production were observed during ACE blockade compared with controls. This study demonstrates that the normal increase in ANG II levels observed during prolonged exercise in humans does not play a major role in the regulation of splanchnic blood flow and glucose production.

Intravenous AICAR administration reduces hepatic glucose output and inhibits whole body lipolysis in type 2 diabetic patients

Aims/hypothesis: The 5′-AMP-activated protein kinase (AMPK) pathway is intact in type 2 diabetic patients and is seen as a target for diabetes treatment. In this study, we aimed to assess the impact of the AMPK activator 5-aminoimidazole-4-carboxamide riboside (AICAR) on both glucose and fatty acid metabolism in vivo in type 2 diabetic patients.

Methods: Stable isotope methodology and blood and muscle biopsy sampling were applied to assess blood glucose and fatty acid kinetics following continuous i.v. infusion of AICAR (0.75 mg kg−1 min−1) and/or NaCl (0.9%) in ten male type 2 diabetic patients (age 64 ± 2 years; BMI 28 ± 1 kg/m2).
Results Plasma glucose rate of appearance (R a) was reduced following AICAR administration, while plasma glucose rate of disappearance (R d) was similar in the AICAR and control test. Consequently, blood glucose disposal (R d expressed as a percentage of R a) was increased following AICAR infusion (p < 0.001). Accordingly, a greater decline in plasma glucose concentration was observed following AICAR infusion (p < 0.001). Plasma NEFA R a and R d were both significantly reduced in response to AICAR infusion, and were accompanied by a significant decline in plasma NEFA concentration. Although AMPK phosphorylation in skeletal muscle was not increased, we observed a significant increase in acetyl-CoA carboxylase phosphorylation (p < 0.001).

Conclusions/interpretation: The i.v. administration of AICAR reduces hepatic glucose output, thereby lowering blood glucose concentrations in vivo in type 2 diabetic patients. Furthermore, AICAR administration stimulates hepatic fatty acid oxidation and/or inhibits whole body lipolysis, thereby reducing plasma NEFA concentration.

Computer-assisted image analysis of liver collagen: relationship to Ishak scoring and hepatic venous pressure gradient

Histopathological scoring of disease stage uses descriptive categories without measuring the amount of fibrosis. Collagen, the major component of fibrous tissue, can be quantified by computer-assisted digital image analysis (DIA) using histological sections. We determined relationships between DIA, Ishak stage, and hepatic venous pressure gradient (HVPG) reflecting severity of fibrosis. One hundred fifteen patients with hepatitis C virus (HCV) who had undergone transplantation had 250 consecutive transjugular liver biopsies combined with HVPG (median length, 22 mm; median total portal tracts, 12), evaluated using the Ishak system and stained with Sirus red for DIA. Liver collagen was expressed as collagen proportionate area (CPA). Median CPA was 6% (0.2-45), correlating with Ishak stage (stage 6 range, 13%-45%), and with HVPG (r = 0.62; P < 0.001). Median CPA was 4.1% when HVPG was less than 6 mm Hg and 13.8% when HVPG was 6 mm Hg or more (P < 0.0001) and 6% when HVPG was less than 10 mm Hg and 17.3% when HVPG was 10 mm Hg or higher (P < 0.0001). Only CPA, not Ishak stage/grade, was independently associated by logistic regression, with HVPG of 6 mm Hg or more [odds ratio, 1.206; 95% confidence interval (CI), 1.094-1.331; P < 0.001], or HVPG of 10 mm Hg or more (odds ratio, 1.105; 95% CI, 1.026-1.191; P = 0.009). CPA increased by 50% (3.6%) compared with 20% in HVPG (1 mm Hg) in 38 patients with repeated biopsies. Conclusion: CPA assessed by DIA correlated with Ishak stage scores and HVPG measured contemporaneously. CPA was a better histological correlate with HVPG than Ishak stage, had a greater numerical change when HVPG was low, and resulted in further quantitation of fibrosis in cirrhosis.

Modulating the interaction of CXCR4 and CXCL12 by low-molecular-weight heparin inhibits hepatic metastasis of colon cancer

Liver metastasis is the major obstacle for prolonging the survival of colon cancer patients. Low-molecular-weight heparin (LMWH), a common drug for venous thromboembolism, has displayed beneficial effects in improving the survival of cancer patients, though the mechanism remains unclear. This study aimed to investigate the effects of LMWH on hepatic metastasis of colon cancer and its underlying molecular mechanism by targeting the interaction of the chemokine receptor CXCR4 and its ligand CXCL12 (formerly known as stromal cell-derived factor 1α, SDF-1α), as the CXCR4-CXCL12 axis has been shown to regulate the interaction of cancer cells and stroma. Experimental results revealed that LMWH (Enoxaparin, 3500-5500 Da) inhibited the CXCL12-stimulated proliferation, adhesion and colony formation of human colon cancer HCT-116 cells that highly expressed CXCR4. Interestingly, LMWH or an anti-CXCR4 blocking antibody diminished the migrating and invading abilities of HCT116 cells stimulated by the recombinant CXCL12 protein or liver homogenates which contained endogenous CXCL12 protein. Although LMWH did not significantly inhibit the growth of subcutaneous colon tumors, it significantly suppressed the formation of hepatic metastasis established by intrasplenic injection of colon cancer cells in nude Balb/c mice and also downregulated the expression of CXCL12 in hepatic sinusoidal endothelial cells. The results suggest that LMWH inhibits the formation of hepatic metastasis of colon cancer by disrupting the interaction of CXCR4 and CXCL12, supporting that perioperative administration of LMWH may help to prevent the seeding and subsequent growth of hepatic metastases of colon cancer cells.

Inflammation, hepatic enzymes and resistance training in individuals with metabolic risk factors

Aims Increases in inflammatory markers, hepatic enzymes and physical inactivity are associated with the development of the metabolic syndrome (MetS). We examined whether inflammatory markers and hepatic enzymes are correlated with traditional risk factors for MetS and studied the effects of resistance training (RT) on these emerging risk factors in individuals with a high number of metabolic risk factors (HiMF, 2.9 ± 0.8) and those with a low number of metabolic risk factors (LoMF, 0.5 ± 0.5).

Methods Twenty-eight men and 27 women aged 50.8 ± 6.5 years (mean ± sd) participated in the study. Participants were randomized to four groups, HiMF training (HiMFT), HiMF control (HiMFC), LoMF training (LoMFT) and LoMF control (LoMFC). Before and after 10 weeks of RT [3 days/week, seven exercises, three sets with intensity gradually increased from 40–50% of one repetition maximum (1RM) to 75–85% of 1RM], blood samples were obtained for the measurement of pro-inflammatory cytokines, C-reactive protein (CRP), -glutamyltransferase (GGT) and alanine aminotransferase (ALT).

Results At baseline, HiMF had higher interleukin-6 (33.9%), CRP (57.1%), GGT (45.2%) and ALT (40.6%) levels, compared with LoMF (all P < 0.05). CRP, GGT and ALT correlated with the number of risk factors (r = 0.48, 0.51 and 0.57, respectively, all P < 0.01) and with other anthropometric and clinical measures (r range from 0.26 to 0.60, P < 0.05). RT did not significantly alter inflammatory markers or hepatic enzymes (all P > 0.05).

Conclusions HiMF was associated with increased inflammatory markers and hepatic enzyme concentrations. RT did not reduce inflammatory markers and hepatic enzymes in individuals with HiMF.

The effect of cocoa supplementation on hepatic steatosis, reactive oxygen species and LFABP in a rat model of NASH

Background: Non alcoholic steatohepatitis is hypothesised to develop via a mechanism involving fat accumulation and oxidative stress. The current study aimed to investigate if an increase in oxidative stress was associated with changes in the expression of liver fatty acid binding protein in a rat model of non alcoholic steatohepatitis and whether cocoa supplementation attenuated those changes.

Methods: Female Sprague Dawley rats were fed a high fat control diet, a high fat methionine choline deficient diet, or one of four 12.5% cocoa supplementation regimes in combination with the high fat methionine choline deficient diet.

Results: Liver fatty acid binding protein mRNA and protein levels were reduced in the liver of animals with fatty liver disease when compared to controls. Increased hepatic fat content was accompanied by higher levels of oxidative stress in animals with fatty liver disease when compared to controls. An inverse association was found between the levels of hepatic liver fatty acid binding protein and the level of hepatic oxidative stress in fatty liver disease. Elevated NADPH oxidase protein levels were detected in the liver of animals with increased severity in inflammation and fibrosis. Cocoa supplementation was associated with partial attenuation of these pathological changes, although the severity of liver disease induced by the methionine choline deficient diet prevented complete reversal of any disease associated changes. Red blood cell glutathione was increased by cocoa supplementation, whereas liver glutathione was reduced by cocoa compared to methionine choline deficient diet fed animals.

Conclusion: These findings suggest a potential role for liver fatty acid binding protein and NADPH oxidase in the development of non alcoholic steatohepatitis. Furthermore, cocoa supplementation may have be of therapeutic benefit in less sever forms of NASH.

Management of advanced neuroendocrine tumors with hepatic metastasis

Neuroendocrine tumors (NETs) in general and specifically these gastroenteropancreatic-neuroendocrine tumors often present a considerable diagnostic and therapeutic challenge, especially those that behave in an aggressive fashion. The majority of tumors are diagnosed at a stage that the only curative treatment, radical surgical intervention, is no longer an option and thus long-term therapy with somatostatin analogs is focused on symptom amelioration and in the improvement of quality of life. Although biotherapy is currently the most efficient treatment to achieve palliation, conventional chemotherapy may have some utility in undifferentiated or highly proliferating neuroendocrine carcinomas and pancreatic NETs. Hepatic metastases, depending on size, location, and number may be amenable to surgical resection or radiofrequency ablation. If surgery is not feasible, embolization either alone (bland), in combination with chemotherapeutic agents, or using radioactive microspheres can be used. Peptide receptor targeted radiotherapy using radiolabeled octapeptide analogs (⁹⁰Yttrium or ¹⁷⁷Lutetium-octreotide) may lead to reduction in tumor size, but in most circumstances has a tumor stabilizing effect. A variety of antiangiogenesis and growth factor-targeted agents have been evaluated, but to date, the results have failed to meet our expectations.

Methazolamide is a new hepatic insulin sensitizer that lowers blood glucose in vivo

We previously used Gene Expression Signature technology to identify methazolamide (MTZ) and related compounds with insulin sensitizing activity in vitro. The effects of these compounds were investigated in diabetic db/db mice, insulin-resistant diet-induced obese (DIO) mice, and rats with streptozotocin (STZ)-induced diabetes. MTZ reduced fasting blood glucose and HbA1c levels in db/db mice, improved glucose tolerance in DIO mice, and enhanced the glucose-lowering effects of exogenous insulin administration in rats with STZ-induced diabetes. Hyperinsulinemic-euglycemic clamps in DIO mice revealed that MTZ increased glucose infusion rate and suppressed endogenous glucose production. Whole-body or cellular oxygen consumption rate was not altered, suggesting MTZ may inhibit glucose production by different mechanism(s) to metformin. In support of this, MTZ enhanced the glucose-lowering effects of metformin in db/db mice. MTZ is known to be a carbonic anhydrase inhibitor (CAI); however, CAIs acetazolamide, ethoxyzolamide, dichlorphenamide, chlorthalidone, and furosemide were not effective in vivo. Our results demonstrate that MTZ acts as an insulin sensitizer that suppresses hepatic glucose production in vivo. The antidiabetic effect of MTZ does not appear to be a function of its known activity as a CAI. The additive glucose-lowering effect of MTZ together with metformin highlights the potential utility for the management of type 2 diabetes.