The beneficial effects of thyroid hormone (TH) on lipid levels are primarily due to its action at the thyroid hormone receptor β (THR-β) in the liver, while adverse effects, including cardiac effects, are mediated by thyroid hormone receptor α (THR-α). A pyridazinone series has been identified that is significantly more THR-β selective than earlier analogues. Optimization of this series by the addition of a cyanoazauracil substituent improved both the potency and selectivity and led to MGL-3196 (53), which is 28-fold selective for THR-β over THR-α in a functional assay. Compound 53 showed outstanding safety in a rat heart model and was efficacious in a preclinical model at doses that showed no impact on the central thyroid axis. In reported studies in healthy volunteers, 53 exhibited an excellent safety profile and decreased LDL cholesterol (LDL-C) and triglycerides (TG) at once daily oral doses of 50 mg or higher given for 2 weeks.
Liver regeneration stimulated by a loss of liver mass leads to hepatocyte and nonparenchymal cell proliferation and rapid restoration of liver parenchyma. Mice with targeted disruption of the interleukin-6 (IL-6) gene had impaired liver regeneration characterized by liver necrosis and failure. There was a blunted DNA synthetic response in hepatocytes of these mice but not in nonparenchymal liver cells. Furthermore, there were discrete G 1 phase (prereplicative stage in the cell cycle) abnormalities including absence of STAT3 (signal transducer and activator of transcription protein 3) activation and depressed AP-1, Myc, and cyclin D1 expression. Treatment of IL-6-deficient mice with a single preoperative dose of IL-6 returned STAT3 binding, gene expression, and hepatocyte proliferation to near normal and prevented liver damage, establishing that IL-6 is a critical component of the regenerative response.
Following hepatic injury or stress, gluconeogenic and acute-phase response genes are rapidly upregulated to restore metabolic homeostasis and limit tissue damage. Regulation of the liver-restricted insulin-like growth factor binding protein 1 (IGFBP-1) gene is dramatically altered by changes in the metabolic state and hepatectomy, and thus it provided an appropriate reporter to assess the transcriptional milieu in the liver during repair and regeneration. The cytokine interleukin-6 (IL-6) is required for liver regeneration and repair, and it transcriptionally upregulates a vast array of genes during liver growth by unknown mechanisms. Evidence for a biologic role of IL-6 in IGFBP-1 upregulation was demonstrated by increased expression of hepatic IGFBP-1 in IL-6 transgenic and following injection of IL-6 into nonfasting animals and its reduced expression in IL-6−/− livers posthepatectomy. In both hepatic and nonhepatic cells, IL-6 -mediated IGFBP-1 promoter activation was via an intact hepatocyte nuclear factor 1 (HNF-1) site and was dependent on the presence of endogenous liver factor HNF-1 and induced factors STAT3 and AP-1 (c-Fos/c-Jun). IL-6 acted through the STAT3 pathway, as dominant negative STAT3 completely blocked IL-6-mediated stimulation of the IGFBP-1 promoter via the HNF-1 site. HNF-1/c-Fos and HNF-1/STAT3 protein complexes were detected in mouse livers and in hepatic and nonhepatic cell lines overexpressing STAT3/c-Fos/HNF-1. Similar regulation was demonstrated using glucose-6-phosphatase and α-fibrinogen promoters, indicating that HNF-1/IL-6/STAT3/AP-1-mediated transactivation of hepatic gene expression is a general phenomenon after liver injury. These results demonstrate that the two classes of transcription factors, growth induced (STAT3 and AP-1) and tissue specific (HNF-1), can interact as an adaptive response to liver injury to amplify expression of hepatic genes important for the homeostatic response during organ repair.
Liver regeneration provides one of the few systems for analysis of mitogenesis in the fully developed, intact animal. Several proteins have been identified as part of the primary growth response in regenerating liver and in mitogen-stimulated cells. Some of these proteins, such as the Jun and Fos families of transcription factors, are thought to have a role in activating transcription of genes expressed subsequently in the growth response. Through differential screening of a regenerating-liver cDNA library, we have identified a rapidly and highly induced gene encoding a 21-kDa leucine-zipper-containing protein that we have designated liver regeneration factor 1 (LRF-1). LRF-1 has no homology with other leucine-zipper proteins outside the basic and leucine-zipper domains. LRF-1 alone can bind DNA, but it preferentially forms heteromeric complexes with c-Jun and Jun-B and does not interact with c-Fos. In solution, it binds with highest affinity to cAMP response elements but also has affinity for related sites. In cotransfection studies, LRF-1 in combination with c-Jun strongly activates a c-Jun-responsive promoter. The induction of the LRF-1 gene in regenerating liver greatly increases the potential variety of heterodimeric combinations of leucine-zipper transcription factors. While LRF-1 mRNA is rapidly induced in the absence of protein synthesis, its peak induction is later than c-fos mRNA, suggesting that LRF-1 may regulate responsive genes at a later point in the cell cycle. As such, LRF-1 may have a unique and critical role in growth regulation of regenerating liver and mitogen-stimulated cells.
MAESTRO-NAFLD-1 is a randomized, double-blind, placebo-controlled Phase 3 trial evaluating resmetirom, a thyroid hormone receptor-beta selective agonist, in NASH patients identified using noninvasive biomarkers and imaging (NCT04197479). MAESTRO-NAFLD-1 includes an open-label resmetirom arm in well-compensated NASH cirrhotic patients. Eligibility required≥3 metabolic risk factors and NASH cirrhosis (diagnosed by biopsy or accepted criteria). Primary and key secondary endpoints include safety, relative percent reduction in MRI-PDFF (Week 16), LDL-C, apoB, and triglycerides (Week 24), and fibrosis markers. All patients received 80 or 100mg resmetirom daily for 52 weeks. Cohort 1 (n=105) has completed 52 weeks of treatment. Cirrhosis stage inversely correlated with baseline MRI-PDFF. At Week 52, resmetirom reduced FibroScan CAP by 42dB/m (p<0.0001) and FibroScan VCTE (LSM) by 7.6kPa (p=0.02). In patients with baseline MRI-PDFF>5% (5%=ULN), resmetirom reduced MRI-PDFF by 37% (p<0.0057). At Week 52, resmetirom reduced MRE by 0.68kPa; 34% of patients achieved an MRE reduction≥15%. GGT and ALP were reduced with resmetirom (by 27% and 18%, respectively; p=0.04 for both). Liver volume, which was elevated at baseline, was reduced by 15.9% at Week 16 (p<0.0001), independent of baseline MRI-PDFF. Liver volume reduction correlated with reduction in MRE, MRI-PDFF, TIMP, P3NP, and SHBG. Resmetirom reduced LDL-C (20%), apoB (20%), triglycerides (21%), and Lp(a) (30%), independent of cirrhosis stage. Resmetirom was well tolerated. BP decreased by 4-5mmHg. Overall, resmetirom reduced markers of cardiovascular risk and fibrosis in NASH cirrhotic patients.
The insulin-like growth factor binding protein-1 ( IGFBP-1 ) gene is highly expressed in fetal, perinatal, and regenerating liver. Up-regulation is transcriptionally mediated in regenerating liver and occurs in the first few minutes to hours after partial hepatectomy. In transgenic mice a 970-bp region from −776 to +151 of the IGFBP-1 promoter was sufficient for tissue-specific and induced expression of the gene in fetal and hepatectomized livers. However weak and/or poorly regulated expression in some transgenic lines suggested the existence of other regulatory regions. Here, genomic clones containing large regions 5′ of the mouse IGFBP-1 gene sequence were isolated, subcloned, and sequenced. Deoxyribonuclease I (DNaseI) hypersensitivity analyses identified clusters of tissue-specific nuclease-sensitive sites in the promoter region, −100 to −300, −2,300, −3,100, and −5,000 along with other weak sites. After partial hepatectomy, enhanced sensitivity and/or novel sites were detected in the −100/−300, −5,000, and −3,100 regions, the promoter region remaining the most hypersensitive. A subset of these sites was present in fetal and perinatal livers. Novel tissue-specific sites that interacted with C/EBP and hepatic nuclear factor 3 (HNF3) transcription factors were identified in the −3,100 region. A hepatectomy-induced DNA binding complex containing the transcription factor USF1 was identified within the −100 to −300 region of the promoter. These results suggested that a complex array of tissue-specific and hepatic proliferation-induced transcription factors combine to regulate both the proximal promoter and more distal regulatory elements of the IGFBP-1 gene.
Mitogens evoke many alterations in gene expression in eukaryotic cells. Genes which are activated rapidly and transiently, that are evolutionarily conserved and whose induction is shared by diverse cell types when exposed to different growth stimuli are likely to be of critical importance in transducing mitogenic signals and regulating cellular proliferation. c-myc and c-fos are the only known genes fulfilling these criteria. We report on the molecular cloning of a novel early growth response (egr) gene which also satisfies these conditions. In response to serum, its 3.7 kb mRNA is induced dramatically in mouse fibroblasts reaching a peak level at about 30 minutes that is ten times higher than the maximal value attained by c-fos mRNA. This transcript is induced by the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate and is "superinduced" by serum and cycloheximide together. Importantly, the gene is highly induced by different mitogens in a wide array of cell types: insulin stimulated rat hepatoma cells, adenosine diphosphate treated monkey kidney epithelial cells, and phytohemagglutinin stimulated human peripheral blood lymphocytes. Given the many properties that this gene shares with c-myc and c-fos, it may play a key role in the control of cell growth and perhaps in oncogenesis.
Fragments were isolated from subclones containing the insulin receptor cDNA described. Probe 1 as obtained from an SP64 subclone containing the 1011bp EcoRI fragment from the 5{prime} region of the U11rich cDNA. Probe 1 was a 677bp XhoI/EcoRI fragment from the {alpha} subunit region of the IR cDNA corresponding to nucleotides 334 to 1011, the putative ligand binding domain. Probe 2 was obtained from an SP64 subclone containing the 4190bp EcoRI fragment from the 3{prime} end of the U11rich cDNA. Probe 2 was a 1599 bp PstI fragment from the {beta} subunit region of the insulin receptor cDNA corresponding to nucleotides 2746 to 4345, encoding the tyrosine kinase domain. Segregation in at least one family was observed for the PstI, KpnI, and RsaI ({beta}) polymorphisms.
Insulin and insulin-like growth factor I (IGF-I) are structurally related polypeptides that stimulate DNA synthesis and cellular proliferation, probably through a common pathway. Human arterial smooth muscle cells in culture demonstrated the presence of high-affinity receptors for both these hormones. Insulin and IGF-I both exhibited cross-reactivity to each other's receptors but with an affinity that is 100-fold less than for the homologous receptor. To examine more closely the receptor responsible for producing the growth effects, we used the polyclonal antibody against the insulin receptor, B2, and a monoclonal antibody to the IGF-I receptor, αIR3 . We studied the growth effects of insulin and IGF-I as measured by stimulation of c-myc, DNA synthesis, and cellular proliferation in the presence and absence of these antibodies. F(ab') fragments of the anti-insulin-receptor antibody at a concentration of 10 μg/ml were capable of displacing >90% of the bound insulin, thus establishing an effective insulin-receptor blockade. Under such blockade, insulin and IGF-I were both capable of doubling the amount of DNA synthesis and cell number in cultured human arterial smooth muscle cells. However, in the presence of a 1:2500 dilution of the monoclonal antibody αlR3 , which caused a 90% displacement of IGF-I bound to its receptor, both the insulin and IGF-I effects on stimulating DNA synthesis or cellular proliferation were inhibited by >90%. These findings demonstrate that the IGF-I receptor is the common pathway for the growth effects of both insulin and IGF-I. Induction of the protooncogene c-myc is recognized as an early response to many mitogenic stimuli. In human smooth muscle cells, insulin (1 × 10−7M) and IGF-I (1 × 10−9M) both cause a 5- to 10-fold increase in c-myc mRNA levels. Induction of c-myc could not be assessed under conditions of selective IGF-I-receptor blockade with the antibody αIR3, because, surprisingly, the antibody itself stimulated c-myc mRNA levels. This induction of c-myc with αIR3, which does not increase DNA synthesis or cellular proliferation, suggests that c-myc induction does not entirely correlate with the growth effects of these hormones.
