Transthyretin contributes to insulin resistance and diminishes exercise-induced insulin sensitivity in obese mice by inhibiting AMPK activity in skeletal muscle.

Exercise improves obesity-induced insulin resistance and metabolic disorders via mechanisms that remain unclear. Here, we show that the levels of the hepatokine transthyretin (TTR) in circulation are elevated in insulin-resistant individuals including high-fat diet (HFD)-induced obese mice, db/db mice, and patients with metabolic syndrome. Liver Ttr mRNA and circulating TTR levels were reduced in mice by treadmill training, as was the TTR levels in quadriceps femoris muscle; however, AMP-activated protein kinase (AMPK) signaling activity was enhanced. Transgenic overexpression of TTR or injection of purified TTR triggered insulin resistance in mice fed on regular chow (RC). Furthermore, TTR overexpression reduced the beneficial effects of exercise on insulin sensitivity in HFD-fed mice. TTR was internalized by muscle cells via the membrane receptor Grp78 and the internalization into the quadriceps femoris was reduced by treadmill training. The TTR/Grp78 combination in C2C12 cells was increased, whereas the AMPK activity of C2C12 cells was decreased as the TTR concentration rose. In addition, Grp78 silencing prevented the TTR internalization and reversed its inhibitory effect on AMPK activity in C2C12 cells. Our study suggests that elevated circulating TTR may contribute to insulin resistance and counteract the exercise-induced insulin sensitivity improvement; the TTR suppression might be an adaptive response to exercise through enhancing AMPK activity in skeletal muscles.NEW & NOTEWORTHY Exercise improves obesity-induced insulin resistance via mechanisms that remain unclear. The novel findings of the study are that circulating TTR (a hepatokine) level is decreased by exercise, and the elevated circulating TTR, as was the elevated transthyretin internalization mediated by Grp78, counteracts the exercise-induced insulin sensitivity by downregulating AMPK activity in skeletal muscle of obese mice. These data suggest that TTR suppression might be an adaptive response to exercise through the crosstalk between liver and muscle.

Exploring the Physiological Role of Transthyretin in Glucose Metabolism in the Liver.

Transthyretin (TTR), a 55 kDa evolutionarily conserved protein, presents altered levels in several conditions, including malnutrition, inflammation, diabetes, and Alzheimer's Disease. It has been shown that TTR is involved in several functions, such as insulin release from pancreatic ß-cells, recovery of blood glucose and glucagon levels of the islets of Langerhans, food intake, and body weight. Here, the role of TTR in hepatic glucose metabolism was explored by studying the levels of glucose in mice with different TTR genetic backgrounds, namely with two copies of the TTR gene, TTR+/+; with only one copy, TTR+/-; and without TTR, TTR-/-. Results showed that TTR haploinsufficiency (TTR+/-) leads to higher glucose in both plasma and in primary hepatocyte culture media and lower expression of the influx glucose transporters, GLUT1, GLUT3, and GLUT4. Further, we showed that TTR haploinsufficiency decreases pyruvate kinase M type (PKM) levels in mice livers, by qRT-PCR, but it does not affect the hepatic production of the studied metabolites, as determined by 1H NMR. Finally, we demonstrated that TTR increases mitochondrial density in HepG2 cells and that TTR insufficiency triggers a higher degree of oxidative phosphorylation in the liver. Altogether, these results indicate that TTR contributes to the homeostasis of glucose by regulating the levels of glucose transporters and PKM enzyme and by protecting against mitochondrial oxidative stress.

In Vitro and In Vivo Effects of SerpinA1 on the Modulation of Transthyretin Proteolysis.

Transthyretin (TTR) proteolysis has been recognized as a complementary mechanism contributing to transthyretin-related amyloidosis (ATTR amyloidosis). Accordingly, amyloid deposits can be composed mainly of full-length TTR or contain a mixture of both cleaved and full-length TTR, particularly in the heart. The fragmentation pattern at Lys48 suggests the involvement of a serine protease, such as plasmin. The most common TTR variant, TTR V30M, is susceptible to plasmin-mediated proteolysis, and the presence of TTR fragments facilitates TTR amyloidogenesis. Recent studies revealed that the serine protease inhibitor, SerpinA1, was differentially expressed in hepatocyte-like cells (HLCs) from ATTR patients. In this work, we evaluated the effects of SerpinA1 on in vitro and in vivo modulation of TTR V30M proteolysis, aggregation, and deposition. We found that plasmin-mediated TTR proteolysis and aggregation are partially inhibited by SerpinA1. Furthermore, in vivo downregulation of SerpinA1 increased TTR levels in mice plasma and deposition in the cardiac tissue of older animals. The presence of TTR fragments was observed in the heart of young and old mice but not in other tissues following SerpinA1 knockdown. Increased proteolytic activity, particularly plasmin activity, was detected in mice plasmas. Overall, our results indicate that SerpinA1 modulates TTR proteolysis and aggregation in vitro and in vivo.

Vitamin A Transport and Cell Signaling by the Retinol-Binding Protein Receptor STRA6.

Vitamin A, retinol, circulates in blood bound to retinol binding protein (RBP). In some tissues, the retinol-RBP complex (holo-RBP) is recognized by a membrane receptor, termed STRA6, which mediates uptake of retinol into cells. Recent studies have revealed that, in addition to serving as a retinol transporter, STRA6 is a ligand-activated cell surface signaling receptor that, upon binding of holo-RBP activates JAK/STAT signaling, culminating in the induction of STAT target genes. It has further been shown that retinol transport and cell signaling by STRA6 are critically interdependent and that both are coupled to intracellular vitamin A metabolism. The molecular mechanism of action of STRA6 and its associated machinery is beginning to be revealed, but further work is needed to identify and characterize the complete range of genes and associated signaling cascades that are regulated by STRA6 in different tissues. An understanding of STRA6 is clinically relevant, as for example, it has been shown to be hyper- activated in obese animals, leading to insulin resistance. A potential role for STRA6 in other pathologies, including cancer, awaits further investigation.

Transthyretin and Normal Human Pregnancy: Mini Review.

Since transthyretin (TTR) was discovered, it has been regarded as a serum protein carrier of thyroid hormones and retinol. However, many other important functions of TTR have been found recently, and current evidence suggests that it plays a role in human receptivity and normal pregnancy. TTR is abundant in the uterine cavity, uterine secretion, placenta, and serum of pregnant females in the peri-implantation uterus and the first trimester of pregnancy. It may be involved in the delivery of maternal thyroid hormones to the fetus. In addition, it appears to play a key role in the preeclampsia mechanism and may be involved in spiral artery remodeling. This review will summarize what is currently known about TTR and normal pregnancy; it will focus on our findings regarding the role of TTR in the spiral artery remodeling process and the additional research required in the future.

Current and future treatment of amyloid diseases.

There are more than 30 human proteins whose aggregation appears to cause degenerative maladies referred to as amyloid diseases or amyloidoses. These disorders are named after the characteristic cross-ß-sheet amyloid fibrils that accumulate systemically or are localized to specific organs. In most cases, current treatment is limited to symptomatic approaches and thus disease-modifying therapies are needed. Alzheimer's disease is a neurodegenerative disorder with extracellular amyloid ß-peptide (Aß) fibrils and intracellular tau neurofibrillary tangles as pathological hallmarks. Numerous clinical trials have been conducted with passive and active immunotherapy, and small molecules to inhibit Aß formation and aggregation or to enhance Aß clearance; so far such clinical trials have been unsuccessful. Novel strategies are therefore required and here we will discuss the possibility of utilizing the chaperone BRICHOS to prevent Aß aggregation and toxicity. Type 2 diabetes mellitus is symptomatically treated with insulin. However, the underlying pathology is linked to the aggregation and progressive accumulation of islet amyloid polypeptide as fibrils and oligomers, which are cytotoxic. Several compounds have been shown to inhibit islet amyloid aggregation and cytotoxicity in vitro. Future animal studies and clinical trials have to be conducted to determine their efficacy in vivo. The transthyretin (TTR) amyloidoses are a group of systemic degenerative diseases compromising multiple organ systems, caused by TTR aggregation. Liver transplantation decreases the generation of misfolded TTR and improves the quality of life for a subgroup of this patient population. Compounds that stabilize the natively folded, nonamyloidogenic, tetrameric conformation of TTR have been developed and the drug tafamidis is available as a promising treatment.

The Aß-clearance protein transthyretin, like neprilysin, is epigenetically regulated by the amyloid precursor protein intracellular domain.

Proteolytic cleavage of the amyloid precursor protein (APP) by the successive actions of ß- and γ-secretases generates several biologically active metabolites including the amyloid ß-peptide (Aß) and the APP intracellular domain (AICD). By analogy with the Notch signalling pathway, AICD has been proposed to play a role in transcriptional regulation. Among the cohort of genes regulated by AICD is the Aß-degrading enzyme neprilysin (NEP). AICD binds to the NEP promoter causing transcriptional activation by competitive replacement with histone deacetylases (HDACs) leading to increased levels of NEP activity and hence increased Aß clearance. We now show that the Aß-clearance protein transthyretin (TTR) is also epigenetically up-regulated by AICD. Like NEP regulation, AICD derived specifically from the neuronal APP isoform, APP695 , binds directly to the TTR promoter displacing HDAC1 and HDAC3. Cell treatment with the tyrosine kinase inhibitor Gleevec (imatinib) or with the alkalizing agent NH4 Cl causes an accumulation of 'functional' AICD capable of up-regulating both TTR and NEP, leading to a reduction in total cellular Aß levels. Pharmacological regulation of both NEP and TTR might represent a viable therapeutic target in Alzheimer's disease.

Novel drugs targeting transthyretin amyloidosis.

Transthyretin amyloidosis (ATTR) is either a hereditary disease related to a mutation in the transthyretin gene that leads to neuropathy and/or cardiomyopathy or an acquired disease of the elderly that leads to restrictive cardiomyopathy. The prevalence of this disease is higher than once thought and awareness is likely to increase amongst physicians and in particular cardiologists. Until recently there have been no treatment options for this disease except to treat the heart failure with diuretics and the neuropathy symptomatically. However, there are several emerging pharmacologic therapies designed to slow or stop the progression of ATTR. This article reviews novel therapeutic drugs that work at different points in the pathogenesis of this disease attempting to change its natural history and improve outcomes.

Presence of N-glycosylated transthyretin in plasma of V30M carriers in familial amyloidotic polyneuropathy: an escape from ERAD.

Familial amyloid polyneuropathy (FAP) is an autosomal dominant disease characterized by deposition of amyloid related to the presence of mutations in the transthyretin (TTR) gene. TTR is mainly synthesized in liver, choroid plexuses of brain and pancreas and secreted to plasma and cerebrospinal fluid (CSF). Although it possesses a sequon for N-glycosylation N-D-S at position 98, it is not secreted as a glycoprotein. The most common FAP-associated mutation is TTR V30M. In a screening for monoclonal antibodies developed against an amyloidogenic TTR form, we detected a distinct TTR with slower electrophoretic mobility in Western of plasma from carriers of the V30M mutation, not present in normal plasma. Mass spectrometry analyses of this slower migrating TTR (SMT) identified both wild-type and mutant V30M; SMT was undetectable upon N-glycosidase F treatment. Furthermore, SMT readily disappeared in the plasma of V30M - FAP patients after liver transplantation and appeared in plasma of transplanted domino individuals that received a V30M liver. SMT was also detected in plasma, but not in CSF of transgenic mice for the human V30M mutation. A hepatoma cell line transduced to express human V30M did not present the SMT modification in secretion media. Glycosylated TTR was absent in fibrils extracted from human kidney V30M autopsy tissue or in TTR aggregates extracted from the intestine of human TTR transgenic mice. Studies on the metabolism of this novel, glycosylated TTR secreted from FAP liver are warranted to provide new mechanisms in protein quality control and etiopathogenesis of the disease.

Amyloid fibrils trigger the release of neutrophil extracellular traps (NETs), causing fibril fragmentation by NET-associated elastase.

The accumulation of amyloid fibrils is a feature of amyloid diseases, where cell toxicity is due to soluble oligomeric species that precede fibril formation or are formed by fibril fragmentation, but the mechanism(s) of fragmentation is still unclear. Neutrophil-derived elastase and histones were found in amyloid deposits from patients with different systemic amyloidoses. Neutrophil extracellular traps (NETs) are key players in a death mechanism in which neutrophils release DNA traps decorated with proteins such as elastase and histones to entangle pathogens. Here, we asked whether NETs are triggered by amyloid fibrils, reasoning that because proteases are present in NETs, protease digestion of amyloid may generate soluble, cytotoxic species. We show that amyloid fibrils from three different sources (α-synuclein, Sup35, and transthyretin) induced NADPH oxidase-dependent NETs in vitro from human neutrophils. Surprisingly, NET-associated elastase digested amyloid fibrils into short species that were cytotoxic for BHK-21 and HepG2 cells. In tissue sections from patients with primary amyloidosis, we also observed the co-localization of NETs with amyloid deposits as well as with oligomers, which are probably derived from elastase-induced fibril degradation (amyloidolysis). These data reveal that release of NETs, so far described to be elicited by pathogens, can also be triggered by amyloid fibrils. Moreover, the involvement of NETs in amyloidoses might be crucial for the production of toxic species derived from fibril fragmentation.

Delayed development of specific thyroid hormone-regulated events in transthyretin null mice.

Thyroid hormones (THs) are vital for normal postnatal development. Extracellular TH distributor proteins create an intravascular reservoir of THs. Transthyretin (TTR) is a TH distributor protein in the circulatory system and is the only TH distributor protein synthesized in the central nervous system. We investigated the phenotype of TTR null mice during development. Total and free 3',5',3,5-tetraiodo-L-thyronine (T(4)) and free 3',3,5-triiodo-L-thyronine (T(3)) in plasma were significantly reduced in 14-day-old (P14) TTR null mice. TTR null mice also displayed a delayed suckling-to-weaning transition, decreased muscle mass, delayed growth, and retarded longitudinal bone growth. In addition, ileums from postnatal day 0 (P0) TTR null mice displayed disordered architecture and contained fewer goblet cells than wild type. Protein concentrations in cerebrospinal fluid from P0 and P14 TTR null mice were higher than in age-matched wild-type mice. In contrast to the current literature based on analyses of adult TTR null mice, our results demonstrate that TTR has an important and nonredundant role in influencing the development of several organs.

Amyloid neuropathies.

PURPOSE OF REVIEW: As amyloid neuropathies have benefited from recent major progress, this review is timely and relevant. RECENT FINDINGS: The main recent articles on amyloid neuropathy cover its description, methods for diagnosis and therapies. Varied clinical presentations are described in transthyretin (TTR)-familial amyloidosis with polyneuropathy (FAP) and light chain amyloid neuropathy. Mass spectrometry is able to identify the biochemical nature of amyloidogenic protein in nerve biopsy and skin biopsy samples for diagnosis of small fiber polyneuropathy. Both nerve biopsy and TTR gene sequencing are important to identify sporadic cases of amyloid neuropathy. Nerve biopsy is useful in demonstrating the amyloid origin of neuropathies developing after domino liver transplant recipients. Liver transplantation improves long-term survival in Met30 TTR-FAP. Factors recognized as leading to cardiomyopathy progression or heart involvement after liver transplantation are late disease onset and fibril composition. Combined heart and liver transplantation is recommended in severe restrictive cardiomyopathy. Antiamyloid drugs are emerging: tafamidis, a TTR stabilizer, showed in a phase III controlled study its ability to slow stage 1 FAP progression. Other strategies are emerging for TTR-FAP (combination doxycycline-tauroursodeoxycholic acid, small interfering RNA, antisense oligonucleotide, monoclonal antibody antiserum amyloid P component). For light chain neuropathy, intensive chemotherapy may be helpful. SUMMARY: There is better recognition of amyloid neuropathies, and hope for enrolling patients with FAP in future clinical trials testing new antiamyloid drugs.

Transthyretin-related familial amyloidotic polyneuropathy: description of a cohort of patients with Leu64 mutation and late onset.

Transthyretin-related familial amyloidotic polyneuropathy (TTR-FAP) usually presents itself as a progressive sensorimotor polyneuropathy with severe autonomic dysfunction and cardiomyopathy. Eighteen patients carrying the Leu64 mutation underwent a series of regular follow-ups, including: neurological examination, electroneurography, electromyography, electrocardiography and echocardiography, blood analysis, a questionnaire on autonomic symptoms, cardiovascular autonomic tests and a 99mTc-DPD examination study. A late onset of a slowly progressive disease which reached its terminal stage after about 10 years was observed. The onset was mainly a length-dependent sensory neuropathy, although a focal onset with carpal tunnel syndrome was detected in three patients. At the onset of the disease, autonomic dysfunction was present in a small number of patients, but, within a few years, this had manifested in all members of the sample group. The only extra-neurological manifestations were cardiac related. It is reasonable to consider Southern Italy as an endemic focus of TTR-FAP. An underestimation of disease prevalence could be caused by a late onset of FAP, which can manifest in patients up to their late 70s. Follow-up of asymptomatic individuals may permit the early detection of symptoms and signs, allowing a detailed record of the natural history of the disease from the beginning and facilitating prompt treatment.

Neurodegeneration in familial amyloidotic polyneuropathy.

Familial amyloid polyneuropathies (FAP) constitute a group of inherited amyloidoses that affect peripheral nerves. One common form of FAP is caused by transthyretin (TTR) misfolding and deposition in the peripheral nervous system, leading to neuronal toxicity and death. The molecular mechanisms responsible for this toxicity are unclear; however, there is good biochemical and histopathological evidence that the toxicity of TTR mutations is correlated to their aggregation state. In addition, neuronal calcium dysregulation is a mechanism that has been suggested to drive the pathogenesis of FAP. Amyloidogenic TTR mutations cause significant calcium influx via L-type calcium channels in neuronal cell lines, while in primary sensory neurons, TTR mediates a calcium influx via a novel mechanism of transient receptor potential melanostatin (TRPM8) and voltage-gated sodium and calcium channel activation. Significantly, calcium dysregulation is a pathological hallmark of other neurodegenerative diseases involving amyloidosis, for example Alzheimer's disease, and this mechanism could explain the molecular events that drive amyloid toxicity in other neurodegenerative diseases.

[Amyloid polyneuropathies--biochemical and genetic aspects]. / Les polyneuropathies amyloïdes: aspects biochimiques et génétiques.

Familial amyloid polyneuropathies (FAP) are among the most frequent hereditary amyloidoses. These are serious, most often fatal diseases with autosomal dominant inheritance. FAP can be caused by mutations in four genes, namely those encoding transthyretin, Al-apoliprotein, gelsolin, and beta-2 microglobulin. Transthyretin is a tetramer composed of four identical subunits linked by non covalent bonds and bearing binding sites for thyroxine (T4) and retinol-binding protein (RBP). More than 120 transthyretin gene sequence variations have been characterized, of which only 80% seem to be pathogenic. Gene mutations can induce tetramer destabilization, thereby generating misfolded monomers that aggregate into insoluble amyloidfibrils. The mutation spectrum is highly variable across countries. For example, while the Val30Met mutation is found in 95% of the Portuguese and Swedish patient populations, high mutational heterogeneity is observed in France. Age of onset and clinical signs are influenced by numerous factors, especially the mutation type and the country, but the mechanisms underlying this variability are not fully clear. The three-dimensional structure of the normal transthyretin protein and a dozen mutants is now known, providing insights into the deleterious effects of mutations. A better understanding of the mechanisms involved in amyloid fibril formation has led to the development of drugs that inhibit transthyretin tetramer destabilization. It is hoped that, within afew years, such drugs will replace liver transplantation, which is currently the only curative treatment.

[Familial amyloidotic polyneuropathies]. / Neuropathies amyloïdes héréditaires: aspects cliniques et neuropathologiques.

Transthyretin familial amyloid polyneuropathy (TTRFAP) is an autosomal dominant neuropathy that is fatal within about 10 years after symptom onset. TTRFAP is observed worldwide, albeit with a higher frequency of the most common variant, Val30met, in Portugal, Sweden and Japan. Various phenotypic differences are observed. TTRFAP should be considered in patients with a progressive axonal polyneuropathy of unknown origin, especially when associated with autonomic nervous system dysfunction. A positive family history is found in most cases when onset begins around 30 years of age, while late-onset FAP is often sporadic and may be confused with chronic inflammatory demyelinating polyneuropathy. Nerve biopsy is often used to confirm the presence of extracellular amyloid deposits in interstitial tissue of the endoneurial space, although amyloid can also befound in muscle, salivary gland and abdominal fat. It is important to stress that biopsy negativity does not rule out amyloidosis. Genetic testing for TTR gene mutations should be performed in case of progressive length-dependent axonal polyneuropathy predominantly involving small nerve fibers.

Transthyretin internalization by sensory neurons is megalin mediated and necessary for its neuritogenic activity.

Mutated transthyretin (TTR) causes familial amyloid polyneuropathy, a neurodegenerative disorder characterized by TTR deposition in the peripheral nervous system (PNS). The origin/reason for TTR deposition in the nerve is unknown. Here we demonstrate that both endogenous mouse TTR and TTR injected intravenously have access to the mouse sciatic nerve. We previously determined that in the absence of TTR, both neurite outgrowth in vitro and nerve regeneration in vivo were impaired. Reinforcing this finding, we now show that local TTR delivery to the crushed sciatic nerve rescues the regeneration phenotype of TTR knock-out (KO) mice. As the absence of TTR was unrelated to neuronal survival, we further evaluated the Schwann cell and inflammatory response to injury, as well as axonal retrograde transport, in the presence/absence of TTR. Only retrograde transport was impaired in TTR KO mice which, in addition to the neurite outgrowth impairment, might account for the decreased regeneration in this strain. Moreover, we show that in vitro, in dorsal root ganglia neurons, clathrin-dependent megalin-mediated TTR internalization is needed for TTR neuritogenic activity. Supporting this observation, we demonstrate that in vivo, decreased levels of megalin lead to decreased nerve regeneration and that megalin's action as a regeneration enhancer is dependent on TTR. In conclusion, our work unravels the mechanism of TTR action during nerve regeneration. Additionally, TTR presence in the nerve, as is here shown, may underlie its preferential deposition in the PNS of familial amyloid polyneuropathy patients.

Transthyretin: the servant of many masters.

Transthyretin (TTR) (formerly, thyroxine binding prealbumin) is an evolutionarily conserved serum and cerebrospinal fluid protein that transports holo-retinol-binding protein and thyroxine. Its serum concentration has been widely used to assess clinical nutritional status. It is also well known that wild-type transthyretin and approximately 100 different mutants give rise to a variety of forms of systemic amyloid deposition. It has been suspected and recently established that TTR can suppress the Alzheimer's disease phenotype in transgenic animal models of cerebral Abeta deposition. Thus, while TTR is a systemic amyloid precursor, in the brain it seems to have an anti-amyloidogenic effect. TTR is found in other organs as a result of local synthesis or transport, suggesting that it may have other, as yet undiscovered, functions. It is possible that its capacity to bind many classes of compounds allows it to serve as an endogenous detoxifier of molecules with potential pathologic effects.

[Transthyretin: it's miracle function and pathogenesis].

Transthyretin (TTR) was previously called prealbumin because the band it formed on agarose gel electrophoresis at pH 8.6 was at the prealbumin position. However, it has been well documented that TTR of rodents does not show a prealbumin position on electrophoresis. Now, its name describes its function, binding to retinol binding protein (RBP) and T4. The serum concentration of the protein is 20-40 mg/dl, and TTR forms a tetramer. The plasma half life of the protein is 1.9 days. TTR is synthesized by the liver, retina, pancreas, and choroid plexus. In cerebro-spinal fluid (CSF), it is the second most abundant protein, and is considered as an important protein in the pathogenesis of Alzheimer's disease, depression, and lead intoxication. In addition, TTR is a tryptophan-rich protein, it is used as one of the nutrition assessment proteins, it acts as an anti acute phase protein, and its plasma concentration decreases during inflammation and bacterial infection. Since TTR is a highly amyloidogenic protein because it contains a beta-sheet structure, it becomes a precursor protein in familial amyloidotic polyneuropathy(FAP). Moreover, TTR plays important roles in various CNS disorders, diabetes melitus, and lipid metabolism.