Analysis of lumbar spine stenosis specimens for identification of amyloid.

BACKGROUND: Lumbar spinal stenosis (LSS) is a common reason for spine surgery in which ligamentum flavum is resected. Transthyretin (TTR) amyloid is an often unrecognized and potentially modifiable mechanism for LSS that can also cause TTR cardiac amyloidosis. Accordingly, older adult patients undergoing lumbar spine (LS) surgery were evaluated for amyloid and if present, the precursor protein, as well as comprehensive characterization of the clinical phenotype. METHODS: A prospective, cohort study in 2 academic medical centers enrolled 47 subjects (age 69 ± 7 years, 53% male) undergoing clinically indicated LS decompression. The presence of amyloid was evaluated by Congo Red staining and in those with amyloid, precursor protein was determined by laser capture microdissection coupled to mass spectrometry (LCM-MS). The phenotype was assessed by disease-specific questionnaires (Swiss Spinal Stenosis Questionnaire and Kansas City Cardiomyopathy Questionnaire) and the 36-question short-form health survey, as well as biochemical measures (TTR, retinol-binding protein, and TTR stability). Cardiac testing included technetium-99m-pyrophosphate scintigraphy, electrocardiograms, echocardiograms, and cardiac biomarkers as well as measures of functional capacity. RESULTS: Amyloid was detected in 16 samples (34% of participants) and was more common in those aged ≥ 75 years of age (66.7%) compared with those <75 years (22.3%, p < 0.05). LCM-MS demonstrated TTR as the precursor protein in 62.5% of participants with amyloid while 37.5% had an indeterminant type of amyloid. Demographic, clinical, quality-of-life measures, electrocardiographic, echocardiographic, and biochemical measures did not differ between those with and without amyloid. Among those with TTR amyloid (n = 10), one subject had cardiac involvement by scintigraphy. CONCLUSIONS: Amyloid is detected in more than a third of older adults undergoing LSS. Amyloid is more common with advancing age and is particularly common in those >75 years old. No demographic, clinical, biochemical, or cardiac parameter distinguished those with and without amyloid. In more than half of subjects with LS amyloid, the precursor protein was TTR indicating the importance of pathological assessment.

Retinol-binding protein 2 (RBP2): biology and pathobiology.

Retinol-binding protein 2 (RBP2; originally cellular retinol-binding protein, type II (CRBPII)) is a 16 kDa cytosolic protein that in the adult is localized predominantly to absorptive cells of the proximal small intestine. It is well established that RBP2 plays a central role in facilitating uptake of dietary retinoid, retinoid metabolism in enterocytes, and retinoid actions locally within the intestine. Studies of mice lacking Rbp2 establish that Rbp2 is not required in times of dietary retinoid-sufficiency. However, in times of dietary retinoid-insufficiency, the complete lack of Rbp2 gives rise to perinatal lethality owing to RBP2 absence in both placental (maternal) and neonatal tissues. Moreover, when maintained on a high-fat diet, Rbp2-knockout mice develop obesity, glucose intolerance and a fatty liver. Unexpectedly, recent investigations have demonstrated that RBP2 binds long-chain 2-monoacylglycerols (2-MAGs), including the canonical endocannabinoid 2-arachidonoylglycerol, with very high affinity, equivalent to that of retinol binding. Crystallographic studies establish that 2-MAGs bind to a site within RBP2 that fully overlaps with the retinol binding site. When challenged orally with fat, mucosal levels of 2-MAGs in Rbp2 null mice are significantly greater than those of matched controls establishing that RBP2 is a physiologically relevant MAG-binding protein. The rise in MAG levels is accompanied by elevations in circulating levels of the hormone glucose-dependent insulinotropic polypeptide (GIP). It is not understood how retinoid and/or MAG binding to RBP2 affects the functions of this protein, nor is it presently understood how these contribute to the metabolic and hormonal phenotypes observed for Rbp2-deficient mice.

Retinol-binding protein 2 (RBP2) binds monoacylglycerols and modulates gut endocrine signaling and body weight.

Expressed in the small intestine, retinol-binding protein 2 (RBP2) facilitates dietary retinoid absorption. Rbp2-deficient (Rbp2-/- ) mice fed a chow diet exhibit by 6-7 months-of-age higher body weights, impaired glucose metabolism, and greater hepatic triglyceride levels compared to controls. These phenotypes are also observed when young Rbp2-/- mice are fed a high fat diet. Retinoids do not account for the phenotypes. Rather, RBP2 is a previously unidentified monoacylglycerol (MAG)-binding protein, interacting with the endocannabinoid 2-arachidonoylglycerol (2-AG) and other MAGs with affinities comparable to retinol. X-ray crystallographic studies show that MAGs bind in the retinol binding pocket. When challenged with an oil gavage, Rbp2-/- mice show elevated mucosal levels of 2-MAGs. This is accompanied by significantly elevated blood levels of the gut hormone GIP (glucose-dependent insulinotropic polypeptide). Thus, RBP2, in addition to facilitating dietary retinoid absorption, modulates MAG metabolism and likely signaling, playing a heretofore unknown role in systemic energy balance.

Adipocyte-specific expression of a retinoic acid receptor α dominant negative form causes glucose intolerance and hepatic steatosis in mice.

All-trans-retinoic acid (ATRA) has been well described as a positive regulator for early stage of adipocyte differentiation and lipid metabolism and also linked to an in vivo fat-lowering effect in mice. However, not all studies support this association. Our objective was to characterize the action of ATRA in mature adipocytes of mice by ablating RAR signaling through overexpression of a well-characterized dominant negative RARα mutant (RARdn) form specifically in adipocytes. Altered RAR signaling in adipocytes resulted in a significant decrease in ATRA levels in visceral and brown adipose tissues as well as liver tissue. This was linked to significant impairments in glucose clearance and elevated hepatic lipid accumulation for chow diet fed mice, indicating the development of metabolic disease, including hepatic steatosis. In addition, we found that adipose RARdn expression in mice fed a chow diet decreased thermogenesis. We conclude that altered RAR signaling and ATRA levels in adipocytes impacts glucose and lipid metabolism in mice.

Vitamin A Absorption, Storage and Mobilization.

It is well established that chylomicron remnant (dietary) vitamin A is taken up from the circulation by hepatocytes, but more than 80 % of the vitamin A in the liver is stored in hepatic stellate cells (HSC). It presently is not known how vitamin A is transferred from hepatocytes to HSCs for storage. Since retinol-binding protein 4 (RBP4), a protein that is required for mobilizing stored vitamin A, is synthesized solely by hepatocytes and not HSCs, it similarly is not known how vitamin A is transferred from HSCs to hepatocytes. Although it has long been thought that RBP4 is absolutely essential for delivering vitamin A to tissues, recent research has proven that this notion is incorrect since total RBP4-deficiency is not lethal. In addition to RBP4, vitamin A is also found in the circulation bound to lipoproteins and as retinoic acid bound to albumin. It is not known how these different circulating pools of vitamin A contribute to the vitamin A needs of different tissues. In our view, better insight into these three issues is required to better understand vitamin A absorption, storage and mobilization. Here, we provide an up to date synthesis of current knowledge regarding the intestinal uptake of dietary vitamin A, the storage of vitamin A within the liver, and the mobilization of hepatic vitamin A stores, and summarize areas where our understanding of these processes is incomplete.

Retinoids in the pancreas.

Retinoids (vitamin A and its natural and synthetic analogs) are required by most tissues for maintaining the normal health of the tissue. This is certainly true for the pancreas. The recent literature is convincing that retinoids are needed by the adult to assure normal pancreatic endocrine functions, especially those of the α- and ß-cells. It is also well established that retinoids are required to insure normal pancreas development in utero, including the development of the endocrine pancreas. The actions of retinoids for maintaining normal pancreatic islet functions has drawn considerable research interest from investigators interested in understanding and treating metabolic disease. Pancreatic retinoids are also of interest to investigators studying the origins of pancreatic disease, including the development of pancreatic fibrosis and its sequelae. This research interest is focused on pancreatic stellate cells (PSCs) which store retinoids and possess the metabolic machinery needed to metabolize retinoids. The literature on pancreatic disease and retinoids suggests that there is an association between impairments in pancreatic retinoid storage and metabolism and the development of pancreatic disease. These topics will be considered in this review.

DGAT1-deficiency affects the cellular distribution of hepatic retinoid and attenuates the progression of CCl4-induced liver fibrosis.

BACKGROUND: Diacylglycerol O-acyltransferase 1 (DGAT1) catalyzes the final step of triglyceride synthesis, transferring an acyl group from acyl-CoA to diacylglycerol. DGAT1 also catalyzes the acyl-CoA-dependent formation of retinyl esters in vitro and in mouse intestine and skin. Although DGAT1 is expressed in both hepatocytes and hepatic stellate cells (HSCs), we reported genetic and nutritional studies that established that DGAT1 does not contribute to retinyl ester formation in the liver. METHODS: We now have explored in more depth the role(s) of DGAT1 in hepatic retinoid metabolism and storage. RESULTS: Our data show that DGAT1 affects the cellular distribution between hepatocytes and HSCs of stored and newly absorbed dietary retinol. For livers of Dgat1-deficient mice, a greater percentage of stored retinyl ester is present in HSCs at the expense of hepatocytes. This is also true for newly absorbed oral [(3)H]retinol. These differences are associated with significantly increased expression, by 2.8-fold, of cellular retinol-binding protein, type I (RBP1) in freshly isolated HSCs from Dgat1-deficient mice, raising the possibility that RBP1, which contributes to retinol uptake into cells and retinyl ester synthesis, accounts for the differences. We further show that the retinyl ester-containing lipid droplets in HSCs are affected in Dgat1-null mice, being fewer in number but, on average, larger than in wild type (WT) HSCs. Finally, we demonstrate that DGAT1 affects experimentally induced HSC activation in vivo but that this effect is independent of altered retinoic acid availability or effects on gene expression. CONCLUSIONS: Our studies establish that DGAT1 has a role in hepatic retinoid storage and metabolism, but this does not involve direct actions of DGAT1 in retinyl ester synthesis.

Retinoic acid receptor signaling is required to maintain glucose-stimulated insulin secretion and ß-cell mass.

Retinoic acid signaling is required for maintaining a range of cellular processes, including cell differentiation, proliferation, and apoptosis. We investigated the actions of all-trans-retinoic acid (atRA) signaling in pancreatic ß-cells of adult mice. atRA signaling was ablated in ß-cells by overexpressing a dominant-negative retinoic acid receptor (RAR)-α mutant (RARdn) using an inducible Cre-Lox system under the control of the pancreas duodenal homeobox gene promoter. Our studies establish that hypomorphism for RAR in ß-cells leads to an age-dependent decrease in plasma insulin in the fed state and in response to a glucose challenge. Glucose-stimulated insulin secretion was also impaired in islets isolated from mice expressing RARdn. Among genes that are atRA responsive, Glut2 and Gck mRNA levels were decreased in isolated islets from RARdn-expressing mice. Histologic analyses of RARdn-expressing pancreata revealed a decrease in ß-cell mass and insulin per ß-cell 1 mo after induction of the RARdn. Our results indicate that atRA signaling mediated by RARs is required in the adult pancreas for maintaining both ß-cell function and mass, and provide insights into molecular mechanisms underlying these actions.

Retinoids: Potent regulators of metabolism.

Retinoids (vitamin A and its analogs) are highly potent regulators of cell differentiation, cell proliferation, and apoptosis. Because of these activities, retinoids have been most extensively studied in the contexts of embryonic development and of proliferative diseases, especially cancer and skin disease. Recently, there has been considerable new research interest focused on gaining understanding of the roles that retinoids and/or retinoid-related proteins may have in the development of metabolic diseases, primarily obesity, diabetes, and dyslipidemia. This review will summarize recent advances that have been made in these areas, focusing on the role of retinoids in modulating adipogenesis, the roles of retinoids and retinoid-related proteins as signaling molecules linking obesity with the development of type II diabetes, the roles of retinoids in pancreatic ß-cell biology/insulin secretion, and the actions of retinoids in hepatic steatosis.

Altered lipid catabolism in the vitamin A deficient liver.

The transcriptome pattern of metabolic genes in vitamin A deficient (VAD) liver has been compared to the vitamin A-sufficient (VAS) state using the Mouse 32k oligonucleotide (70mer) array. In VAD liver there was a decrease in expression of genes encoding enzymes of mitochondrial fatty acid (FA) oxidation; these genes included fatty acyl CoA ligase, carnitine o-palmitoyl transferase 1, medium chain acyl-CoA dehydrogenase, 3-ketoacyl CoA thiolase, and citrate synthase. Particularly affected was peroxisome metabolism, as genes encoding enzymes of peroxisomal FA oxidation and transport proteins were differentially expressed. These genes included those encoding acyl-CoA oxidase 1, the peroxisomal bifunctional enzyme, peroxisomal thiolase, and carnitine o-octanoyl transferase, the enzyme involved in shuttling FAs from the peroxisome to the mitochondrion. Most genes that were differentially expressed with chronic vitamin A depletion were responsive to treatment with all-trans retinoic acid (RA). Consistent with the decreased expression of genes involved in FA oxidation, we found an increase in hepatic macrocytic lipid accumulation and triglyceride levels. The relevant nuclear receptor gene that was differentially expressed in the VAD liver was that encoding the peroxisome proliferator-activated receptor (PPAR) alpha, the mRNA levels for which were decreased in VAD liver and increased with all-trans RA treatment. Down regulation of the PPAR alpha gene is the likely cause of the altered expression pattern of the above metabolic genes in VAD liver.