The heparan sulfate-fibroblast growth factor signaling system in liver growth and function

The heparan sulfate (HS)-fibroblast growth factor (FGF) signaling system is a ubiquitous regulator that senses local environmental changes and mediates cell-to-cell communication. This system consists of three mutually interactive components. These are regulatory polypeptides (FGF), FGF receptor (FGFR) and heparan sulfate proteoglycans (FGFRHS). All four FGFR genes are expressed in the adult liver. Expression of the FGFR1–3 genes is generally associated with non-parenchymal cells while expression of the FGFR4 gene is associated with parenchymal hepatocytes. We showed that livers of mice lacking FGFR4 exhibited normal morphology and regenerated normally in response to partial hepatectomy. However, the FGFR4 (−/−) mice exhibited depleted gallbladders, an elevated bile acid pool and elevated excretion of bile acids. Cholesterol- and bile acid-controlled liver cholesterol 7α-hydroxylase (Cyp7a), the limiting enzyme for bile acid synthesis, was elevated, unresponsive to dietary cholesterol, but repressed normally by dietary cholate. These results indicated that FGFR4 was not directly involved in liver growth but exerted negative control on liver bile acid synthesis. This was confirmed in transgenic mice overexpressing the constitutively active human FGFR4 in livers. The transgenic mice exhibited decreased fecal bile acid excretion, bile acid pool size, and expression of Cyp7a. Introduction of this constitutively active human FGFR4 into FGFR4 (−/−) mice restored the inhibition of bile acid synthesis. Activation of the c-Jun N-terminal Kinase (JNK) pathway by FGFR4 correlated with the repressive effect on bile acid synthesis. ^ To determine whether FGFR4 played a broader role in liver-specific metabolic function, we examined the impact of both acute and chronic exposure to CCl 4 in FGFR4 (−/−) mice. Following acute CCl4 exposure, the FGFR4 (−/−) mice exhibited accelerated liver injury, a significant increase in liver mass and delayed hepatolobular repair, with no apparent effect on liver cell proliferation and restoration of cellularity. Chronic CCl4 exposure resulted in severe fibrosis in livers of FGFR4 (−/−) mice compared to normal mice. Analysis at both mRNA and protein levels indicated an 8 hr delay in FGFR4-deficient mice in the down-regulation of cytochrome P450 2E1 (CYP2E1) protein, the major enzyme whose products underlie CCl 4-induced injury. These results show that hepatocyte FGFR4 protects against acute and chronic insult to the liver and prevents accompanying fibrosis. ^ Of the 23 FGF polypeptides, FGF1 and FGF2 are present at significant levels in the liver. To determine whether FGF1 and FGF2 played a role in CCl 4-induced liver injury and fibrosis, we examined the impact of both acute and chronic exposure to CCl4 in both wild-type and FGF1-FGF2 double-knockout mice. Following acute CCl4 exposure, FGF1(−/−)FGF2(−/−) mice exhibited accelerated liver injury, overall normal liver growth and repair, and decreased liver collagen α1(I) induction. Liver fibrosis resulting from chronic CCl4 exposure was markedly decreased in livers of FGF1(−/−)FGF2(−/−) mice compared to wild-type mice. This study suggests a role for FGF1 and FGF2 in hepatic fibrogenesis. ^ In summary, our three part study shows that specific components of the ubiquitous HS-FGF signaling family in the liver context interfaces with metabolite- and xenobiotic-controlled networks to regulate liver function, but has no apparent direct effect on liver cell growth. ^

Physiologic and psychosocial stage-based differences for dietary fat consumption in women with type 2 diabetes

Purpose. This cross-sectional, observational study explored differences among groups staged for intent to decrease dietary fat intake in women with type 2 diabetes in relation to demographic, weight concern, physiological, and psychosocial variables. ^ Methods. A sample of 100 community-dwelling, English-speaking women, who were over age 30 and had type 2 diabetes for at least a year, was accessed through a culturally diverse endocrinology clinic. Subjects completed 7 self-report instruments: demographic sheet, with 11-point weight satisfaction scale; staging algorithm; fat intake (MEDFICTS); depression (CES-D); diabetes-specific dietary knowledge (ADKnowl), social support and self-efficacy scales (SE-Type 2). Physiological variables were abstracted from the medical record (HbA 1c, blood pressure, serum cholesterol and triglycerides). ^ Results. The women's average age was 57.69 years ( SD = 3.07); 50% were married. Subjects were well-educated ( M = 14 years; SD = 3.33), with average diabetes duration of 10.57 years (SD = 9.11), high body mass index (M = 35.72; SD = 8.36), low diabetes-specific dietary knowledge, low weight satisfaction, but in good diabetes control. Racial/ethnic composition was 44% non-Hispanic-White-American, 18% Hispanic-White-American, 15% non-Hispanic-African-American, 16% Hispanic-African-American and 5% other. Fat intake was low and differed by racial/ethnic demographics. The highest fat intake scores were for non-Hispanic-African-Americans (M = 53), followed by Hispanic-White-Americans (M = 51), non-Hispanic-White-Americans (M = 45), and Hispanic-African-Americans (M = 32), who had the lowest fat intake scores. ^ MANOVA analyses revealed no significant differences between stages of behavior change in relation to psychosocial or weight concern variables, age, education, HbA1c, or cholesterol levels. Single women were more likely to be in the three preaction stages (precontemplation, contemplation, and preparation); married women were equally distributed across stages (the preaction stages plus action and maintenance). African-American women (Hispanic and non-Hispanic) were more likely in contemplation and preparation. Triglycerides were higher in women in the action stage than contemplation or preparation. Systolic blood pressure was higher in action than preparation; diastolic blood pressure was higher in action than preaction. ^ Conclusions. Healthcare professionals should consider race, ethnicity, and marital status in client interactions. Dietary intake can vary according to both race and ethnicity; collapsing racial/ethnic groups can alter means and distributions, generating faulty conclusions. Further research is warranted to explore relationships between dietary self-care and marital status, race, ethnicity, and physiological variables. ^

Blood pressure and dietary factors in Mexican-Americans of Starr County, Texas

This study examined the cross-sectional associations between blood pressure, hypertension and dietary factors among 580 Mexican-American adults residing in Starr County, Texas. The data were collected as part of Gallbladder Disease Study between April, 1985 and December, 1986.^ Dietary intake was assessed for the month previous to the interview by means of a 38 food item quantified frequency questionnaire representing foods and mixed dishes commonly consumed in the community. From the dietary information intake of calcium, cholesterol, total kilocalories, and percent of kilocalories contributed by total fat, saturated fat, monounsaturated fatty acids, polyunsaturated fatty acids, protein, carbohydrates were calculated. The effect of other factors associated with blood pressure, such as age, body mass index, body fat distribution, smoking, and drinking were controlled.^ Age was the most important predictor of both systolic and diastolic blood pressure and hypertension. For both males and females, systolic and diastolic blood pressure levels were consistently positively associated with body mass index but were not associated with waist hip ratio. However, a strong positive relationship between hypertension and waist hip ratio but not body mass index was observed.^ After controlling for age and body mass index it was noted that for males there were no significant associations between the dietary variables and systolic blood pressure. For diastolic blood pressure there were significant associations with percent fat, percent monounsaturated fatty acids, percent protein and percent carbohydrates.^ For females, there were significant associations between systolic blood pressure and percent protein, percent carbohydrates and cholesterol. There were no significant associations between dietary variables and diastolic blood pressure.^ After controlling for age and waist hip ratio significant associations between hypertension and percent fat, percent saturated fat, percent monounsaturated fatty acids, percent carbohydrate and percent protein were observed in males. Significant associations between hypertension and percent polyunsaturated fatty acids and percent protein were noted in females. ^

Altered cholesterol metabolism in a hypercholesterolemia -resistant rabbit colony

A colony of rabbits has been developed at the University of Texas Medical School at Houston that is resistant to dietary-induced hypercholesterolemia. The liver of resistant rabbits had higher levels of ($\sp{125}$I) $\beta$-VLDL binding and 3-hydroxy-3-methylglutaryl (HMGCoA) reductase activity, but lower acyl coenzyme A:cholesterol acyltransferase (ACAT) activity than normal rabbits. Direct quantitation of intracellular cholesterol content of the liver revealed that the resistant rabbits had $<$10% of the intracellular free cholesterol present in normal rabbits. Fibroblasts isolated from normal and resistant rabbits exhibited differences in ($\sp{125}$I) LDL binding, HMGCoA reductase activity and ACAT activity that were similar to those found in the liver. No structural differences were found in the LDL receptor of normal and resistant fibroblasts that would account for the increased binding capacity of the resistant cells. The regulation of LDL receptor levels by exogenous oxygenated sterols was similar in normal and resistant fibroblasts. The regulation of LDL receptor binding capacity by LDL was attenuated in the resistant compared to normal fibroblasts, suggesting that the resistant fibroblasts have an alternate pathway for processing lipoprotein-derived cholesterol. Sterol-balance studies revealed that the cholesterol-fed resistant rabbits increased lithocholic acid excretion compared to the basal state, and had higher levels of deoxycholic acid excretion than cholesterol-fed normal rabbits. In addition, the specific activity and mRNA levels of cholesterol 7$\alpha$-hydroxylase (C7$\alpha$H) were higher in resistant rabbits than normal rabbits, suggesting that the increased bile acid excretion was due to an increase in bile acid synthesis. Increased clearance of cholesterol relieves the negative feedback inhibition cholesterol exerts on expression of the LDL receptor. The number of cell surface LDL receptors is then increased in resistant rabbits and allows rapid clearance of lipoproteins from the plasma compartment, thereby reducing plasma cholesterol levels. The low intracellular cholesterol level also relieves the negative feedback inhibition cholesterol exerts on HMGCoA reductase. Increased synthesis of cholesterol from acetate provides cells with cholesterol for bile acid synthesis and/or homeostasis. The activity of ACAT is then decreased due to the flux of cholesterol through the bile acid synthetic pathways. ^

Characterization of the cholesterol 7$\alpha$-hydroxylase promoter in hypercholesterolemia -resistant rabbits

Our laboratory has developed and partially characterized a strain of New Zealand white rabbits that are resistant to the hypercholesterolemia which typically occurs in normal rabbits when fed a cholesterol-enriched diet. This phenotype is most likely attributed to an increase in bile acid excretion by hypercholesterolemia-resistant (CRT) rabbits as a result of elevated enzyme activity of cholesterol 7$\alpha$-hydroxylase (C7$\alpha$H), the rate-limiting enzyme in bile acid synthesis. Northern analysis revealed that CRT rabbits, in comparison to normal rabbits, have a 7-fold greater steady-state C7$\alpha$H mRNA levels irrespective of dietary regimen. The C7$\alpha$H gene in both phenotypes was determined to be a single copy gene. The hypothesis was that the elevated C7$\alpha$H mRNA levels in CRT rabbits, in comparison to normal animals, was due to an increase in the transcription rate of the C7$\alpha$H gene as a result of a mutation in a cis-acting element and/or a trans-acting factor within the hepatocyte. To isolate the C7$\alpha$H gene from both normal and CRT rabbits, genomic libraries were prepared from both phenotypes into $\lambda$GEM12 vectors using conventional techniques. Three CRT and one normal phage clones that contained the C7$\alpha$H gene were identified by screening the library with a series of probes located within different exons of the C7$\alpha$H cDNA. Sequencing analysis confirmed that approximately 1100 bp of the C7$\alpha$H 5'-flanking region from both normal and CRT phenotypes was identical. The increase in C7$\alpha$H mRNA levels was not attributed to a cis-acting mutation within this region. Liver nuclear extracts were prepared from normal and CRT rabbits maintained either on a basal or 0.25% cholesterol-enriched diet and incubated with several radiolabeled DNA fragments from the C7$\alpha$H gene. A 37 basepair region, located between nucleotides $-$452 to $-$416 was identified that had altered binding patterns between normal and CRT rabbits as a function of diet. Two additional regions, $-$747 to $-$575 and $-$580 to $-$442, produced banding patterns which were identical, irrespective of phenotype or diet. In conclusion, these studies suggested that the increase in C7$\alpha$H mRNA in CRT rabbits was due to differences in binding of a cholesterol-responsive transcription factor to the C7$\alpha$H promoter. ^