Martin C. Carey

Hepatic Insulin Resistance Is Sufficient to Produce Dyslipidemia and Susceptibility to Atherosclerosis

Insulin resistance plays a central role in the development of the metabolic syndrome, but how it relates to cardiovascular disease remains controversial. Liver insulin receptor knockout (LIRKO) mice have pure hepatic insulin resistance. On a standard chow diet, LIRKO mice have a proatherogenic lipoprotein profile with reduced high-density lipoprotein (HDL) cholesterol and very low-density lipoprotein (VLDL) particles that are markedly enriched in cholesterol. This is due to increased secretion and decreased clearance of apolipoprotein B-containing lipoproteins, coupled with decreased triglyceride secretion secondary to increased expression of Pgc-1 beta (Ppargc-1b), which promotes VLDL secretion, but decreased expression of Srebp-1c (Srebf1), Srebp-2 (Srebf2), and their targets, the lipogenic enzymes and the LDL receptor. Within 12 weeks on an atherogenic diet, LIRKO mice show marked hypercholesterolemia, and 100% of LIRKO mice, but 0% of controls, develop severe atherosclerosis. Thus, insulin resistance at the level of the liver is sufficient to produce the dyslipidemia and increased risk of atherosclerosis associated with the metabolic syndrome.

Role of Gallbladder Mucus Hypersecretion in the Evolution of Cholesterol Gallstones

Because mucin glycoproteins may be important in the pathophysiology of gallstones, we studied the relationship among biliary lipids, gallbladder mucin secretion, and gallstone formation in cholesterol-fed prairie dogs. Organ culture studies of gallbladder explants revealed that the incorporation of [(3)H]glucosamine into tissue and secretory gallbladder glycoproteins was significantly increased at 3, 5, 8, and 14 d of feeding. Peak secretion of labeled mucin occurred at 5 d, when total tissue and secreted glycoprotein production was fivefold greater than control. Gel filtration of the secreted glycoprotein on Sepharose 4B indicated that the majority of radioactivity was present in a macromolecule of > 1 million molecular weight. The increased secretion of gallbladder mucin was organ specific, in that [(3)H]glucosamine incorporation into glycoproteins of stomach and colon was unaffected by cholesterol feeding. Similarly, the incorporation of [(3)H]mannose into gallbladder membrane glycoproteins was not altered by cholesterol feeding. The rate of glycoprotein synthesis and secretion returned to normal upon withdrawal of the cholesterol diet, and ligation of the cystic duct before cholesterol feeding prevented gallbladder mucin hypersecretion. Both results indicate that the stimulus to mucin secretion was a constituent of bile. Gallbladder bile after 5 d contained cholesterol in micelles, liquid crystals, and crystals, whereas hepatic bile remained a single micellar phase throughout cholesterol feeding. For this reason the cholesterol-saturation indices of gallbladder bile were compared in both homogenized and centrifuged samples. The micellar phase of gallbladder bile was appreciably less saturated than homogenized bile at 5 and 8 d, which reflects the continuous nucleation of cholesterol in the gallbladder. Purified human gallbladder mucin gels were shown to induce nucleation of lecithin-cholesterol liquid crystals from supersaturated hepatic bile. These in turn gave rise to cholesterol monohydrate crystals within 18 h. Control supersaturated hepatic bile could not be nucleated by the addition of other proteins, and was stable for days upon standing. These results suggest that the increase in cholesterol content of bile in cholesterolfed prairie dogs stimulates gallbladder mucus hypersecretion, and that gallbladder mucus gel is a nucleating agent for biliary cholesterol.

Insulin Administered Intranasally as an Insulin-Bile Salt Aerosol: Effectiveness and Reproducibility in Normal and Diabetic Subjects

Efficacy and reproducibility of insulin administered intranasally as an insulin-deoxycholate 1% (w/v) aerosol to normal and diabetic subjects were assessed by measurements of blood glucose and serum insulin levels. Following administration of 0.5 U insulin/kg with the unconjugated bile salt to fasting volunteers (N = 29), peak serum insulin levels of 103 ± 49 μM/ml above baseline were observed at 10 min. Blood glucose concentration began to fall by 10 min, reaching 54 ± 14% of control levels by 30 min, and returning to baseline by 60–80 min. Blood glucose response and peak serum insulin levels were reproducible when the same aerosol dose was repeatedly administered to the same subjects; however, intersubject variations were noted. By comparing serum insulin levels after i.v. and nasal routes of administration, nasal insulin absorption was approximately 10% as efficient as intravenous insulin. Dose response studies revealed that peak serum insulin concentrations were a linear function of the administered dose. In subjects with type I and type II diabetes mellitus, serum insulin levels increased in a manner similar to controls, and resulted in a prompt reduction of blood glucose concentration. However, in contrast to normal subjects, the duration of the glucose response was more prolonged, lasting as long as 5 h. Nasal administration of insulin as an aerosol with bile salts or bile salt analogs should be further evaluated as a possible nonparenteral approach to insulin therapy.

Micellar properties of dihydroxy and trihydroxy bile salts: Effects of counterion and temperature☆

Abstract The shift of the wavelength of maximum absorption (λ-max) of a dye, Rhodamine 6G, was used to determine the critical micelle concentration (CMC) of two bile salts, sodium taurocholate (NaTC), a trihydroxy bile salt, and sodium taurodeoxycholate (NaTDC), a dihydroxy bile salt. The micellar properties of these salts and equimolar mixtures of both were studied in water and with added counterion (NaCl) over a temperature range of 10–80°C. The CMC range of the bile salts was: NaTC 0.6–6.0 mM, NaTDC 0.8–3.3 mM and NaTC NaTDC mixture 0.9–5.6 mM. As predicted on molecular grounds both salts have both nonionic and anionic surfactant properties. The hydroxyl groups clustered on one side of the bile salt nucleus prevent precipitation of dye bile salt complexes at low bile salt concentrations. Thus, bile salts behave as nonionic detergents with dyes of opposite charge and allow the first break point in a plot of the λ-max shift vs. log concentration of bile salt to be taken as an estimate of the CMC. The CMC of both salts and mixtures first fell slightly (about 10% decrease) and then increased with increases of temperature. Added counterion (NaCl) had little effect on the CMC of NaTC until very high concentrations (1–3 M). The CMC of NaTDC was decreased slightly by added NaCl. An assessment of micellar charge, determined from the slope of the log CMC vs. log NaCl plot showed that the micelles are highly charged and bind little or no counterions in water. The CMC values obtained in this study, when compared with values in the literature, were found to be somewhat lower. Finally, the standard thermodynamic functions of micellization were calculated on the assumption that micelles and monomers can be treated mathematically as a mass action equilibrium. The free energy changes (ΔF) were negative and varied little with temperature. The difference in ΔF between NaTC and NaTDC on micellization was −310 cal/mole and is approximately equivalent to half the ΔF value of micelle formation when a classical alkyl detergent has its hydrocarbon chain lengthened by one methylene group. At low temperatures micelle formation in bile salt solutions is an entropy-directed process while above 60°C a negative enthalpy change appears to be the predominant driving force.

Hepatic insulin resistance directly promotes formation of cholesterol gallstones

Despite the well-documented association between gallstones and the metabolic syndrome, the mechanistic links between these two disorders remain unknown. Here we show that mice solely with hepatic insulin resistance, created by liver-specific disruption of the insulin receptor (LIRKO mice) are markedly predisposed toward cholesterol gallstone formation due to at least two distinct mechanisms. Disinhibition of the forkhead transcription factor FoxO1, increases expression of the biliary cholesterol transporters Abcg5 and Abcg8, resulting in an increase in biliary cholesterol secretion. Hepatic insulin resistance also decreases expression of the bile acid synthetic enzymes, particularly Cyp7b1, and produces partial resistance to the farnesoid X receptor, leading to a lithogenic bile salt profile. As a result, after twelve weeks on a lithogenic diet, all of the LIRKO mice develop gallstones. Thus, hepatic insulin resistance provides a crucial link between the metabolic syndrome and increased cholesterol gallstone susceptibility.

Whither biliary sludge

A mong the growing list of pathogenic events implicated in the formation of human gallstones, gelation of gallbladder mucin with entrapment of “particulate matter” nucleated from bile, also known as biliary sludge, is unquestionably assuming a pivotal role (1,2). The peculiar phrase “biliary sludge” is, of course, jargon. In classic American fashion, the notion is borrowed from sanitary engineering (“sludge thickening tanks”) and, presumably because of its graphic pungency, seems to have become firmly entrenched in both radiologic and surgical nomenclature. Our intent is not to proscribe this appellation, but rather to anoint it. What follows is an attempt to place this common form of biliary tract dysfunction in pathophysiologic context. We will probe the metabolic and physicalchemical origins of biliary sludge, explore how Lee and colleagues, in a recent issue of this journal (3), have furthered our knowledge of its origins and fate, and attempt to provide a unifying hypothesis for further investigation of biliary sludge in the pathogenesis and possible prevention of human gallstones. In our understanding of the evolution of macroscopic cholesterol gallstones, many pieces of the jigsaw puzzle have been fitted into place over the past 20 yr (4). Investigators from diverse disciplines now continue to focus upon the earliest identifiable evolutionary events, such as the metabolic origin (57) and physical-chemical nature (8) of cholesterolsupersaturated bile, gallbladder mucin accumulation and gelation (g-12), biliary nucleation (13,14) and antinucleation factors (l5-l8), nucleation stages, physical chemistry and kinetics (19-21), and gallbladder dysmotility (11,22-29). But the picture is still incomplete; we have broad brush strokes of the secondary colors without a palette that displays the primary colors. Both in gallstone-prone American Indians in the United States and in western populations, gallstones are distinctly rare before the third decade of life (4) yet, during early adolescence, the lipid composition of bile changes from being unsaturated with cholesterol to being supersaturated with cholesterol (30,31). This alteration is most dramatic in women and correlates with enhanced urinary output of endogenous estrogens (3 1). Contrary to previously held beliefs (32) that this resulted generally from hyposecretion of bile salts beginning in adolescence, it is now becoming clear that hypersecretion of biliary cholesterol is the major cause of supersaturated bile in both obese and nonobese humans in the western hemisphere (5,33-35). The pathogenic wisdom of the 1970s was that deficient secretion of bile salts from a contracted bile salt pool was the causative cornerstone in the formation of lithogenic bile (reviewed in Reference 36). This belief now requires reexamination. Hyposecretion of bile salts into the upper small intestine in earlier secretion studies apparently resulted from suboptimal cholecystokinin release during formula perfusion of the duodenum, with diversion of hepatic bile into a partially relaxed gallbladder (37). Furthermore, the presence of gallstones per se in the gallbladder results in an apparent contraction of the bile salt pool when measured by isotope-labeled bile salt dilution kinetics (38). Nevertheless, adult American Indians in the United States may be a genetic and biologically distinct group in this regard. Pima Indians, in addition to being hypersecretors of cholesterol, exhibit true enterohepatic bile salt deficiency before the formation of cholesterol gallstones (39). Hypersecretion of biliary cholesterol is caused by many factors (5). These putatively include the following.

FXR and ABCG5/ABCG8 as determinants of cholesterol gallstone formation from quantitative trait locus mapping in mice

BACKGROUND & AIMS Cholesterol gallstone formation is a complex genetic trait. To identify additional cholesterol gallstone susceptibility loci, we performed a quantitative trait locus analysis using an intercross of PERA/Ei and I/LnJ inbred strains of mice. METHODS Mice of both sexes were examined for gallstone weight and evaluated according to a scoring system for the physical chemistry of cholelithiasis during feeding of a lithogenic diet. Intercross offspring were genotyped, and linkage analysis was performed by interval mapping. Differences in messenger RNA expression of positional candidate genes were determined using reverse-transcription and real-time polymerase chain reaction. RESULTS We identified significant loci associated with gallstone weight on chromosomes 10 and 4, named Lith7 and Lith8, respectively (both susceptibility alleles conferred by strain I/LnJ). Positional candidate genes with higher expression in I/LnJ mice are Fxr (official symbol, Nr1h4), encoding the nuclear bile salt receptor, on chromosome 10 and Shp1 (official symbol, Nr0b2), encoding the small heterodimer partner 1, on chromosome 4. A significant locus associated with gallstone score on chromosome 17, named Lith9 (susceptibility allele conferred by strain PERA/Ei), colocalizes with the genes Abcg5 and Abcg8 that encode the canalicular cholesterol transporter. Higher hepatic messenger RNA expression of Abcg5 and Abcg8 in strain PERA/Ei correlates positively with higher biliary cholesterol levels. CONCLUSIONS Our findings suggest a primary role of the nuclear bile salt receptor FXR and the canalicular cholesterol transporter ABCG5/ABCG8 in the genetic susceptibility and pathogenesis of cholesterol cholelithiasis in these strains of inbred mice.

Pathogenesis of cholesterol gallstones: a parsimonious hypothesis.

Three decades of fundamental research on cholesterol gallstones have borne witness to a continuous growth of basic and clinical information on all aspects of the disease [1–5]. Unfortunately, with respect to pathogenesis our understanding is imperfect and often unintentionally confusing and conflicting [1,2]. Furthermore, according to the current paradigm [6] the simultaneous occurrence of several independent defects to explain all pathophysiological elements in the disease appears excessive [7]. In this article we propose a unified hypothesis for cholesterol gallstone pathogenesis that is realistic on the basis of recent biophysical [8] and genetic [9] insights. Our hope is that eventually Occam’s razor or the law of parsimony (1836), can be put to use in understanding this common disease. The current working hypothesis on pathogenesis [6– 8,10] involves a tetralogy of simultaneous defects. They are (1) cholesterol hypersecretion with unphysiological supersaturation of bile, (2) gallbladder hypomotility (‘biliary stasis’), (3) accelerated cholesterol crystallization in bile and (4) mucus hypersecretion with gel formation in the gallbladder [11]. The severity of each of these defects may vary markedly [12] and they have not been considered to be interrelated to date. As first demonstrated 20 years ago [13], bile becomes supersaturated with cholesterol because of hypersecretion of hepatic cholesterol into bile [5]. Cholesterol monohydrate crystal formation occurs rapidly in the lithogenic gallbladder because of accelerated nucleation and phase transitions [14]. A poorly contractile and compliant gallbladder that neither fills nor empties adequately in the lithogenic state [15] allows cholesterol crystals to become entrapped in a mucin gel (‘biliary sludge’) [11]. After months to years, crystals agglomerate to form macroscopic gallstones [8]. It is inconceivable that such a large number of hepatobiliary abnormalities [8] could have independent causes simultaneously [7]. Furthermore, in the modern era of cholesterol gallstone research, the earliest metabolic defect discovered was a small but rapidly circulating bile salt pool accompanied by increased primary bile salt turnover rates [16]. As primary bile salts are synthesized in the liver from cholesterol [5], the dilemma for most investigators was to understand how a liver as well as an intestinal defect could be related to explain the enterohepatic circulatory abnormalities in gallstone disease. In this review, we attempt to marshall the available evidence to demonstrate that hepatic hypersecretion of cholesterol may be the major trigger for the entire panoply of pathophysiologic abnormalities responsible for cholesterol gallstone disease [7,8]. Accumulating evidence with respect to gallbladder muscle injury [17–19], qualitative and quantitative alterations in specific biliary proteins [2,11,20–22] and possibly pronucleating critical nuclei of colloidal dimensions [23,24] suggest that molecular cholesterol may in fact touch off the other abnormalities in this disease [7,8]. Furthermore, the combined influences of these secondary defects will be shown to explain the perturbations of the enterohepatic circulation in lithogenesis and which may amplify many of the primary and secondary defects depending on their severity (Fig. 1). The high prevalence rates of cholesterol gallstones in Pima Indians suggested that an inherited defect in cholesterol homeostasis was responsible for the disease [25]. Only recently has genetic analysis of the cholesterol gallstone-susceptible inbred laboratory mouse [9] revealed that at least two Lith genes affect cholesterol gallstone formation. The protein encoded by Lith 1, the European Journal of Clinical Investigation (1996)26, 343–352

Spontaneous cholecysto‐ and hepatolithiasis in Mdr2−/− mice: A model for low phospholipid‐associated cholelithiasis

Previously, we identified needle‐like and filamentous, putatively “anhydrous” cholesterol crystallization in vitro at very low phospholipid concentrations in model and native biles. Our aim now was to address whether spontaneous gallstone formation occurs in Mdr2 (Abcb4) knockout mice that are characterized by phospholipid‐deficient bile. Biliary phenotypes and cholesterol crystallization sequences in fresh gallbladder biles and non‐fixed liver sections were determined by direct and polarizing light microscopy. The physical chemical nature and composition of crystals and stones were determined by sucrose density centrifugation and before mass and infrared spectroscopy. Gallbladder biles of Mdr2−/− mice precipitate needle‐like cholesterol crystals at 12 weeks of age on chow. After 15 weeks, more than 50% of Mdr2−/− mice develop gallbladder stones, with female mice displaying a markedly higher gallstone‐susceptibility. Although gallbladder biles of Mdr2−/− mice contain only traces (≤ 1.1 mM) of phospholipid and cholesterol, they become supersaturated with cholesterol and plot in the left 2‐phase zone of the ternary phase diagram, consistent with “anhydrous” cholesterol crystallization. Furthermore, more than 40% of adult female Mdr2−/− mice show intra‐ and extrahepatic bile duct stones. In conclusion, spontaneous gallstone formation is a new consistent feature of the Mdr2−/− phenotype. The Mdr2−/− mouse is therefore a model for low phospholipid‐associated cholelithiasis recently described in humans with a dysfunctional mutation in the orthologous ABCB4 gene. The mouse model supports the concept that this gene is a monogenic risk factor for cholesterol gallstones and a target for novel therapeutic strategies. (HEPATOLOGY 2004;39:117–128.)

Enterohepatic cycling of bilirubin: A putative mechanism for pigment gallstone formation in ileal Crohn's disease

BACKGROUND & AIMS Patients with ileal disease, bypass, or resection are at increased risk for developing gallstones. In ileectomized rats, bilirubin secretion rates into bile are elevated, most likely caused by increased colonic bile salt levels, which solubilize unconjugated bilirubin, prevent calcium complexing, and promote its absorption and enterohepatic cycling. The hypothesis that ileal disease or resection engenders the same pathophysiology in humans was tested. METHODS Sterile gallbladder bile samples were obtained intraoperatively from 29 patients with Crohns disease and 19 patients with ulcerative colitis. Bilirubin, total calcium, biliary lipids, beta-glucuronidase activities, and cholesterol saturation indices in bile were measured, and markers of hemolysis and ineffective erythropoiesis in blood were assessed. RESULTS Bilirubin conjugates, unconjugated bilirubin, and total calcium levels were increased 3-10-fold in bile of patients with ileal disease and/or resection compared with patients with Crohns colitis or ulcerative colitis. Biliary bilirubin concentrations correlated positively with the anatomic length and duration of ileal disease. Endogenous biliary beta-glucuronidase activities were comparable in all groups, and both the hemogram and serum vitamin B12 levels were normal. CONCLUSIONS This study establishes that increased bilirubin levels in bile of patients with Crohns disease are caused by lack of functional ileum, supporting the hypothesis that enterohepatic cycling of bilirubin occurs.