Trk-fused gene plays a critical role in diet-induced adipose tissue expansion and is also involved in thyroid hormone action.

Mutations in the Trk-fused gene (TFG) cause hereditary motor and sensory neuropathy with proximal dominant involvement, which reportedly has high co-incidences with diabetes and dyslipidemia, suggesting critical roles of the TFG in metabolism as well. We found that TFG expression levels in white adipose tissues (WATs) were elevated in both genetically and diet-induced obese mice and that TFG deletion in preadipocytes from the stromal vascular fraction (SVF) markedly inhibited adipogenesis. To investigate its role in vivo, we generated tamoxifen-inducible adipocyte-specific TFG knockout (AiTFG KO) mice. While a marked down-regulation of the peroxisome proliferator-activated receptor gamma target, de novo lipogenesis (DNL), and mitochondria-related gene expressions were observed in subcutaneous WAT (scWAT) from AiTFG KO mice, these effects were blunted in SVF-derived adipocytes when the TFG was deleted after differentiation into adipocytes, implying cell nonautonomous effects. Intriguingly, expressions of thyroid hormone receptors, as well as carbohydrate responsive element-binding protein ß, which mediates the metabolic actions of thyroid hormone, were drastically down-regulated in scWAT from AiTFG KO mice. Reduced DNL and thermogenic gene expressions in AiTFG KO mice might be attributable to impaired thyroid hormone action in vivo. Finally, when adipocyte TFG was deleted in either the early or the late phase of high-fat diet feeding, the former brought about an impaired expansion of epididymal WAT, whereas the latter caused prominent adipocyte cell death. TFG deletion in adipocytes markedly exacerbated hepatic steatosis in both experimental settings. Collectively, these observations indicate that the TFG plays essential roles in maintaining normal adipocyte functions, including an enlargement of adipose tissue, thyroid hormone function, and thermogenic gene expressions, and in preserving hypertrophic adipocytes.

Prolyl isomerase Pin1 in skeletal muscles contributes to systemic energy metabolism and exercise capacity through regulating SERCA activity.

The skeletal muscle is a pivotal organ involved in the regulation of both energy metabolism and exercise capacity. There is no doubt that exercise contributes to a healthy life through the consumption of excessive energy or the release of myokines. Skeletal muscles exhibit insulin sensitivity and can rapidly uptake blood glucose. In addition, they can undergo non-shivering thermogenesis through actions of both the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) and small peptide, sarcolipin, resulting in systemic energy metabolism. Accordingly, the maintenance of skeletal muscles is important for both metabolism and exercise. Prolyl isomerase Pin1 is an enzyme that converts the cis-trans form of proline residues and controls substrate function. We have previously reported that Pin1 plays important roles in insulin release, thermogenesis, and lipolysis. However, the roles of Pin1 in skeletal muscles remains unknown. To clarify this issue, we generated skeletal muscle-specific Pin1 knockout mice. Pin1 deficiency had no effects on muscle weights, morphology and ratio of fiber types. However, they showed exacerbated obesity or insulin resistance when fed with a high-fat diet. They also showed a lower ability to exercise than wild type mice did. We also found that Pin1 interacted with SERCA and elevated its activity, resulting in the upregulation of oxygen consumption. Overall, our study reveals that Pin1 in skeletal muscles contributes to both systemic energy metabolism and exercise capacity.

Auraptene Enhances AMP-Activated Protein Kinase Phosphorylation and Thereby Inhibits the Proliferation, Migration and Expression of Androgen Receptors and Prostate-Specific Antigens in Prostate Cancer Cells.

Citrus hassaku extract reportedly activates AMPK. Because this extract contains an abundance of auraptene, we investigated whether pure auraptene activates AMPK and inhibits proliferation using prostate cancer cell lines. Indeed, auraptene inhibited the proliferation and migration of LNCaP cells and induced phosphorylation of AMPK or its downstream ACC in LNCaP, PC3, and HEK-293 cells, but not in DU145 cells not expressing LKB1. In addition, the mTOR-S6K pathway, located downstream from activated AMPK, was also markedly suppressed by auraptene treatment. Importantly, it was shown that auraptene reduced androgen receptor (AR) and prostate-specific antigen (PSA) expressions at both the protein and the mRNA level. This auraptene-induced downregulation of PSA was partially but significantly reversed by treatment with AMPK siRNA or the AMPK inhibitor compound C, suggesting AMPK activation to, at least partially, be causative. Finally, in DU145 cells lacking the LKB1 gene, exogenously induced LKB1 expression restored AMPK phosphorylation by auraptene, indicating the essential role of LKB1. In summary, auraptene is a potent AMPK activator that acts by elevating the AMP/ATP ratio, thereby potentially suppressing prostate cancer progression, via at least three molecular mechanisms, including suppression of the mTOR-S6K pathway, reduced lipid synthesis, and AR downregulation caused by AMPK activation.

Hepatic Pin1 Expression, Particularly in Nuclei, Is Increased in NASH Patients in Accordance with Evidence of the Role of Pin1 in Lipid Accumulation Shown in Hepatoma Cell Lines.

Our previous studies using rodent models have suggested an essential role for Pin1 in the pathogenesis of non-alcoholic steatohepatitis (NASH). In addition, interestingly, serum Pin1 elevation has been reported in NASH patients. However, no studies have as yet examined the Pin1 expression level in human NASH livers. To clarify this issue, we investigated the expression level and subcellular distribution of Pin1 in liver specimens obtained using needle-biopsy samples from patients with NASH and healthy liver donors. Immunostaining using anti-Pin1 antibody revealed the Pin1 expression level to be significantly higher, particularly in nuclei, in the livers of NASH patients than those of healthy donors. In the samples from patients with NASH, the amount of nuclear Pin1 was revealed to be negatively related to serum alanine aminotransferase (ALT), while tendencies to be associated with other serum parameters such as aspartate aminotransferase (AST) and platelet number were noted but did not reach statistical significance. Such unclear results and the lack of a significant relationship might well be attributable to our small number of NASH liver samples (n = 8). Moreover, in vitro, it was shown that addition of free fatty acids to medium induced lipid accumulation in human hepatoma HepG2 and Huh7 cells, accompanied with marked increases in nuclear Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1), in accordance with the aforementioned observations in human NASH livers. In contrast, suppression of Pin1 gene expression using siRNAs attenuated the free fatty acid-induced lipid accumulation in Huh7 cells. Taken together, these observations strongly suggest that increased expression of Pin1, particularly in hepatic nuclei, contributes to the pathogenesis of NASH with lipid accumulation.

The expression of prolyl isomerase Pin1 is expanded in the skin of patients with atopic dermatitis and facilitates IL-33 expression in HaCaT cells.

Atopic dermatitis (AD) is attributable to both a genetic predisposition and environmental factors. Among numerous cytokines involved in the pathogenesis of AD, interleukin-33 (IL-33), reportedly escaping exocytotically in response to a scratch, is abundantly expressed in the skin tissues of patients with AD and is postulated to induce inflammatory and autoimmune responses. In this study, we first demonstrated that peptidylprolyl cis/trans isomerase, NIMA-interacting 1 (Pin1), a unique enzyme which isomerizes the proline residues of target proteins, is abundantly expressed in keratinocytes, and that the areas where it is present in the skin tissues of AD patients became expanded due to hyperkeratosis. Thus, we investigated the effects of Pin1 on the regulation of IL-33 expression using the human keratinocyte cell line HaCaT. Interestingly, silencing of the Pin1 gene or treatment with Pin1 inhibitors dramatically reduced IL-33 expressions in HaCaT cells, although Pin1 overexpression did not elevate it. Subsequently, we showed that Pin1 binds to STAT1 and the nuclear factor-kappaB (NF-κB) subunit p65. Silencing the Pin1 gene with small interfering RNAs significantly reduced the phosphorylation of p65, while no marked effects of Pin1 on the STAT1 pathway were detected. Thus, it is likely that Pin1 contributes to increased expression of IL-33 via the NF-κB subunit p65 in HaCaT cells, at least modestly. Nevertheless, further study is necessary to demonstrate the pathogenic roles of Pin1 and IL-33 in AD development.

Prolyl isomerase Pin1 promotes extracellular matrix production in hepatic stellate cells through regulating formation of the Smad3-TAZ complex.

Hepatic stellate cells (HSCs) produce extracellular matrixes (ECMs), such as collagen and fibronectin, in response to stimulation with transforming growth factor ß (TGFß). The massive ECM accumulation in the liver due to HSCs causes fibrosis which eventually leads to hepatic cirrhosis and hepatoma development. However, details of the mechanisms underlying continuous HSC activation are as yet poorly understood. We thus attempted to elucidate the role of Pin1, one of the prolyl isomerases, in the underlying mechanism(s), using the human HSC line LX-2. Treatment with Pin1 siRNAs markedly alleviated the TGFß-induced expressions of ECM components such as collagen 1a1/2, smooth muscle actin and fibronectin at both the mRNA and the protein level. Pin1 inhibitors also decreased the expressions of fibrotic markers. In addition, it was revealed that Pin1 associates with Smad2/3/4, and that four Ser/Thr-Pro motifs in the linker domain of Smad3 are essential for binding with Pin1. Pin1 significantly regulated Smad-binding element transcriptional activity without affecting Smad3 phosphorylations or translocation. Importantly, both Yes-associated protein (YAP) and WW domain-containing transcription regulator (TAZ) also participate in ECM induction, and upregulate Smad3 activity rather than TEA domain transcriptional factor transcriptional activity. Although Smad3 interacts with both TAZ and YAP, Pin1 facilitates the Smad3 association with TAZ, but not that with YAP. In conclusion, Pin1 plays pivotal roles in ECM component productions in HSCs through regulation of the interaction between TAZ and Smad3, and Pin1 inhibitors may have the potential to ameliorate fibrotic diseases.

Par14 interacts with the androgen receptor, augmenting both its transcriptional activity and prostate cancer proliferation.

BACKGROUND: Prostate cancer (PCa) is a major cause of cancer morbidity and mortality for men globally, and androgen signaling clearly drives its onset and progression. Androgen receptor (AR) regulation is complex and remains elusive, despite several studies tackling these issues. Therefore, elucidating the mechanism(s) underlying AR regulation is a potentially promising approach to suppressing PCa. METHODS: We report that Par14, one isoform of the prolyl isomerases homologous to Pin1, is a critical regulator of AR transcriptional activity and is essential for PCa cell growth. RESULTS: Par14 was shown to be overexpressed in PCa, based on analyses of deposited data. Importantly, overexpression of Par14 significantly enhanced androgen-sensitive LNCap cell growth. In contrast, silencing of Par14 dramatically decreased cell growth in LNCap cells by causing cell cycle arrest. Mechanistically, silencing of the Par14 gene dramatically induced cyclin-dependent kinase inhibitor p21 at both the mRNA and the protein level through modulating the localization of p53. In addition, suppression of Par14 in LNCap cells was shown to downregulate the expressions of androgen response genes, at both the mRNA and the protein level, induced by dihydrotestosterone. Par14 was shown to directly associate with AR in nuclei via its DNA-binding domain and augment AR transcriptional activity. CONCLUSION: Thus, Par14 plays a critical role in PCa progression, and its enhancing effects on AR signaling are likely to be involved in the underlying molecular mechanisms. These findings suggest Par14 to be a promising therapeutic target for PCa.

Roles of peptidyl prolyl isomerase Pin1 in viral propagation.

Peptidyl-prolyl isomerase (PPIase) is a unique enzyme that promotes cis-trans isomerization of a proline residue of a target protein. Peptidyl-prolyl cis-trans isomerase NIMA (never in mitosis A)-interacting 1 (Pin1) is a PPIase that binds to the pSer/pThr-Pro motif of target proteins and isomerizes their prolines. Pin1 has been reported to be involved in cancer development, obesity, aging, and Alzheimer's disease and has been shown to promote the growth of several viruses including SARS-CoV-2. Pin1 enhances the efficiency of viral infection by promoting uncoating and integration of the human immunodeficiency virus. It has also been shown that Pin1 interacts with hepatitis B virus proteins and participates in viral replication. Furthermore, Pin1 promotes not only viral proliferation but also the progression of virus-induced tumorigenesis. In this review, we focus on the effects of Pin1 on the proliferation of various viruses and discuss the underlying molecular mechanisms.

Involvement of neuronal and muscular Trk-fused gene (TFG) defects in the development of neurodegenerative diseases.

Trk-fused gene (TFG) mutations have been identified in patients with several neurodegenerative diseases. In this study, we attempted to clarify the effects of TFG deletions in motor neurons and in muscle fibers, using tissue-specific TFG knockout (vMNTFG KO and MUSTFG KO) mice. vMNTFG KO, generated by crossing TFG floxed with VAChT-Cre, showed deterioration of motor function and muscle atrophy especially in slow-twitch soleus muscle, in line with the predominant Cre expression in slow-twitch fatigue-resistant (S) and fast-twitch fatigue-resistant (FR) motor neurons. Consistently, denervation of the neuromuscular junction (NMJ) was apparent in the soleus, but not in the extensor digitorum longus, muscle. Muscle TFG expressions were significantly downregulated in vMNTFG KO, presumably due to decreased muscle IGF-1 concentrations. However, interestingly, MUSTFG KO mice showed no apparent impairment of muscle movements, though a denervation marker, AChRγ, was elevated and Agrin-induced AChR clustering in C2C12 myotubes was inhibited. Our results clarify that loss of motor neuron TFG is sufficient for the occurrence of NMJ degeneration and muscle atrophy, though lack of muscle TFG may exert an additional effect. Reduced muscle TFG, also observed in aged mice, might be involved in age-related NMJ degeneration, and this issue merits further study.

Prolyl isomerase Pin1 plays an essential role in SARS-CoV-2 proliferation, indicating its possibility as a novel therapeutic target.

Novel coronavirus disease 2019 (COVID-19) has emerged as a global pandemic with far-reaching societal impact. Here we demonstrate that Pin1 is a key cellular molecule necessary for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) propagation. In this study, siRNA-mediated silencing of Pin1 expression markedly suppressed the proliferation of SARS-CoV-2 in VeroE6/TMPRSS2 cells. In addition, several recently generated Pin1 inhibitors showed strong inhibitory effects on SARS-CoV-2 proliferation, measured by both viral mRNA and protein synthesis, and alleviated the cytopathic effect (CPE) on VeroE6/TMPRSS2 cells. One compound, termed H-77, was found to block SARS-CoV-2 proliferation at an EC50 below 5 µM regardless of whether it was added to the culture medium prior to or after SARS-CoV-2 infection. The inhibition of viral N protein mRNA synthesis by H-77 implies that the molecular mechanism underlying SARS-CoV-2 inhibition is likely to be associated with viral gene transcription or earlier steps. Another Pin1 inhibitor, all-trans retinoic acid (ATRA)-a commercially available drug used to treat acute promyelocytic leukemia (APL) and which both activates the retinoic acid receptor and inhibits the activity of Pin1-similarly reduced the proliferation of SARS-CoV-2. Taken together, the results indicate that Pin1 inhibitors could serve as potential therapeutic agents for COVID-19.

Impact of Plasma Xanthine Oxidoreductase Activity on the Mechanisms of Distal Symmetric Polyneuropathy Development in Patients with Type 2 Diabetes.

To unravel associations between plasma xanthine oxidoreductase (XOR) and diabetic vascular complications, especially distal symmetric polyneuropathy (DSP), we investigated plasma XOR activities using a novel assay. Patients with type 2 diabetes mellitus (T2DM) with available nerve conduction study (NCS) data were analyzed. None were currently taking XOR inhibitors. XOR activity of fasting blood samples was assayed using a stable isotope-labeled substrate and LC-TQMS. JMP Clinical version 5.0. was used for analysis. We analyzed 54 patients. Mean age was 64.7 years, mean body mass index was 26.0 kg/m2, and mean glycated hemoglobin was 9.4%. The logarithmically transformed plasma XOR activity (ln-XOR) correlated positively with hypoxanthine, xanthine, visceral fatty area, and liver dysfunction but negatively with HDL cholesterol. ln-XOR correlated negatively with diabetes duration and maximum intima-media thickness. Stepwise multiple regression analysis revealed ln-XOR to be among selected explanatory factors for various NCS parameters. Receiver operating characteristic curves showed the discriminatory power of ln-XOR. Principal component analysis revealed a negative relationship of ln-XOR with F-waves as well as positive relationships of ln-XOR with hepatic steatosis and obesity-related disorders. Taken together, our results show plasma XOR activity to be among potential disease status predictors in T2DM patients. Plasma XOR activity measurements might reliably detect pre-symptomatic DSP.

Pathological Role of Pin1 in the Development of DSS-Induced Colitis.

Inflammatory bowel diseases (IBDs) are serious disorders of which the etiologies are not, as yet, fully understood. In this study, Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) protein was shown to be dramatically upregulated in the colons of dextran sodium sulfate (DSS)-induced ulcerative colitis model mice. Interestingly, Pin1 knockout (KO) mice exhibited significant attenuation of DSS-induced colitis compared to wild-type (WT) mice, based on various parameters, including body weight, colon length, microscopic observation of the intestinal mucosa, inflammatory cytokine expression, and cleaved caspase-3. In addition, a role of Pin1 in inflammation was suggested because the percentage of M1-type macrophages in the colon was decreased in the Pin1 KO mice while that of M2-type macrophages was increased. Moreover, Pin1 KO mice showed downregulation of both Il17 and Il23a expression in the colon, both of which have been implicated in the development of colitis. Finally, oral administration of Pin1 inhibitor partially but significantly prevented DSS-induced colitis in mice, raising the possibility of Pin1 inhibitors serving as therapeutic agents for IBD.

Carnosic Acid and Carnosol Activate AMPK, Suppress Expressions of Gluconeogenic and Lipogenic Genes, and Inhibit Proliferation of HepG2 Cells.

Carnosic acid (CA), carnosol (CL) and rosmarinic acid (RA), components of the herb rosemary, reportedly exert favorable metabolic actions. This study showed that both CA and CL, but not RA, induce significant phosphorylation of AMP-dependent kinase (AMPK) and its downstream acetyl-CoA carboxylase 1 (ACC1) in HepG2 hepatoma cells. Glucose-6-phosphatase (G6PC) and phosphoenolpyruvate carboxykinase 1 (PCK1), rate-limiting enzymes of hepatic gluconeogenesis, are upregulated by forskolin stimulation, and this upregulation was suppressed when incubated with CA or CL. Similarly, a forskolin-induced increase in CRE transcriptional activity involved in G6PC and PCK1 regulations was also stymied when incubated with CA or CL. In addition, mRNA levels of ACC1, fatty acid synthase (FAS) and sterol regulatory element-binding protein 1c (SREBP-1c) were significantly reduced when incubated with CA or CL. Finally, it was shown that CA and CL suppressed cell proliferation and reduced cell viability, possibly as a result of AMPK activation. These findings raise the possibility that CA and CL exert a protective effect against diabetes and fatty liver disease, as well as subsequent cases of hepatoma.

Prolyl isomerase Pin1 interacts with adipose triglyceride lipase and negatively controls both its expression and lipolysis.

BACKGROUND: Lipolysis is essential for the supply of nutrients during fasting, the control of body weight, and remodeling of white adipose tissues and thermogenesis. In the obese state, lipolysis activity and the expression of adipose triglyceride lipase (ATGL), a rate-limiting enzyme, is suppressed. However, the mechanism underlying the regulation of ATGL remains largely unknown. We previously reported that a high-fat diet obviously increases protein levels of the prolyl isomerase, Pin1, in epididymal white adipose tissue (epiWAT) of mice and that Pin1 KO mice are resistant to developing obesity. RESULTS: The present study found that deletion of the Pin1 gene in epiWAT upregulated lipolysis and increased ATGL protein expression by ~2-fold. In addition, it was demonstrated that Pin1 directly associated with ATGL and enhanced its degradation through the ubiquitin proteasome system. Indeed, Pin1 overexpression decreased ATGL expression levels, whereas Pin1 knockdown by siRNA treatment upregulated ATGL protein levels without altering mRNA levels. Moreover, under a high fat diet (HFD)-fed condition, adipocyte-specific Pin1 KO (adipoPin1 KO) mice had 2-fold increase lipolytic activity and upregulated ß-oxidation-related gene expressions. These mice also gained less body weight, and had better glucose metabolism according to the results of glucose and insulin tolerance tests. CONCLUSION: Taken together, these results showed that Pin1 directly interacted with and degraded ATGL via a ubiquitin-proteasome system, consequently causing the downregulation of lipolysis. Therefore, Pin1 could be considered a target for the treatment of dyslipidemia and related disorders.

Development of Pin1 Inhibitors and their Potential as Therapeutic Agents.

The prolyl isomerase Pin1 is a unique enzyme, which isomerizes the cis-trans conformation between pSer/pThr and proline and thereby regulates the function, stability and/or subcellular distribution of its target proteins. Such regulations by Pin1 are involved in numerous physiological functions as well as the pathogenic mechanisms underlying various diseases. Notably, Pin1 deficiency or inactivation is a potential cause of Alzheimer's disease, since Pin1 induces the degradation of Tau. In contrast, Pin1 overexpression is highly correlated with the degree of malignancy of cancers, as Pin1 controls a number of oncogenes and tumor suppressors. Accordingly, Pin1 inhibitors as anti-cancer drugs have been developed. Interestingly, recent intensive studies have demonstrated Pin1 to be responsible for the onset or development of nonalcoholic steatosis, obesity, atherosclerosis, lung fibrosis, heart failure and so on, all of which have been experimentally induced in Pin1 deficient mice. In this review, we discuss the possible applications of Pin1 inhibitors to a variety of diseases including malignant tumors and also introduce the recent advances in Pin1 inhibitor research, which have been reported.

Prolyl isomerase Pin1 in metabolic reprogramming of cancer cells.

Pin1 is one member of a group consisting of three prolyl isomerases. Pin1 interacts with the motif containing phospho-Ser/Thr-Pro of substrates and enhances cis-trans isomerization of peptide bonds, thereby controlling the functions of these substrates. Importantly, the Pin1 expression level is highly upregulated in most cancer cells and correlates with malignant properties, and thereby with poor outcomes. In addition, Pin1 was revealed to promote the functions of multiple oncogenes and to abrogate tumor suppressors. Accordingly, Pin1 is well recognized as a master regulator of malignant processes. Recent studies have shown that Pin1 also binds to a variety of metabolic regulators, such as AMP-activated protein kinase, acetyl CoA carboxylase and pyruvate kinase2, indicating Pin1 to have major impacts on lipid and glucose metabolism in cancer cells. In this review, we focus on the roles of Pin1 in metabolic reprogramming, such as "Warburg effects", of cancer cells. Our aim is to introduce these important roles of Pin1, as well as to present evidence supporting the possibility of Pin1 inhibition as a novel anti-cancer strategy.

Pin1 Plays Essential Roles in NASH Development by Modulating Multiple Target Proteins.

Pin1 is one of the three known prolyl-isomerase types and its hepatic expression level is markedly enhanced in the obese state. Pin1 plays critical roles in favoring the exacerbation of both lipid accumulation and fibrotic change accompanying inflammation. Indeed, Pin1-deficient mice are highly resistant to non-alcoholic steatohepatitis (NASH) development by either a high-fat diet or methionine-choline-deficient diet feeding. The processes of NASH development can basically be separated into lipid accumulation and subsequent fibrotic change with inflammation. In this review, we outline the molecular mechanisms by which increased Pin1 promotes both of these phases of NASH. The target proteins of Pin1 involved in lipid accumulation include insulin receptor substrate 1 (IRS-1), AMP-activated protein kinase (AMPK) and acetyl CoA carboxylase 1 (ACC1), while the p60 of the NF-kB complex and transforming growth factor ß (TGF-ß) pathway appear to be involved in the fibrotic process accelerated by Pin1. Interestingly, Pin1 deficiency does not cause abnormalities in liver size, appearance or function. Therefore, we consider the inhibition of increased Pin1 to be a promising approach to treating NASH and preventing hepatic fibrosis.

Xanthine Oxidase Inhibitor Febuxostat Exerts an Anti-Inflammatory Action and Protects against Diabetic Nephropathy Development in KK-Ay Obese Diabetic Mice.

Hyperuricemia has been recognized as a risk factor for insulin resistance as well as one of the factors leading to diabetic kidney disease (DKD). Since DKD is the most common cause of end-stage renal disease, we investigated whether febuxostat, a xanthine oxidase (XO) inhibitor, exerts a protective effect against the development of DKD. We used KK-Ay mice, an established obese diabetic rodent model. Eight-week-old KK-Ay mice were provided drinking water with or without febuxostat (15 µg/mL) for 12 weeks and then subjected to experimentation. Urine albumin secretion and degrees of glomerular injury judged by microscopic observations were markedly higher in KK-Ay than in control lean mice. These elevations were significantly normalized by febuxostat treatment. On the other hand, body weights and high serum glucose concentrations and glycated albumin levels of KK-Ay mice were not affected by febuxostat treatment, despite glucose tolerance and insulin tolerance tests having revealed febuxostat significantly improved insulin sensitivity and glucose tolerance. Interestingly, the IL-1ß, IL-6, MCP-1, and ICAM-1 mRNA levels, which were increased in KK-Ay mouse kidneys as compared with normal controls, were suppressed by febuxostat administration. These data indicate a protective effect of XO inhibitors against the development of DKD, and the underlying mechanism likely involves inflammation suppression which is independent of hyperglycemia amelioration.

Possible involvement of normalized Pin1 expression level and AMPK activation in the molecular mechanisms underlying renal protective effects of SGLT2 inhibitors in mice.

BACKGROUND: Recently, clinical studies have shown the protective effects of sodium glucose co-transporter2 (SGLT2) inhibitors against progression of diabetic nephropathy, but the underlying molecular mechanisms remain unclear. METHODS: Diabetic mice were prepared by injecting nicotinamide and streptozotocin, followed by high-sucrose diet feeding (NA/STZ/Suc mice). The SGLT2 inhibitor canagliflozin was administered as a 0.03% (w/w) mixture in the diet for 4 weeks. Then, various parameters and effects of canagliflozin on diabetic nephropathy were investigated. RESULTS: Canagliflozin administration to NA/STZ/Suc mice normalized hyperglycemia as well as elevated renal mRNA of collagen 1a1, 1a2, CTGF, TNFα and MCP-1. Microscopic observation revealed reduced fibrotic deposition in the kidneys of canagliflozin-treated NA/STZ/Suc mice. Interestingly, the protein level of Pin1, reportedly involved in the inflammation and fibrosis affecting several tissues, was markedly increased in the NA/STZ/Suc mouse kidney, but this was normalized with canagliflozin treatment. The cells showing increased Pin1 expression in the kidney were mainly mesangial cells, along with podocytes, based on immunohistochemical analysis. Furthermore, it was revealed that canagliflozin induced AMP-activated kinase (AMPK) activation concentration-dependently in CRL1927 mesangial as well as THP-1 macrophage cell lines. AMPK activation was speculated to suppress mesangial cell proliferation and exert anti-inflammatory effects in hematopoietic cells. CONCLUSION: Therefore, we can reasonably suggest that normalized Pin1 expression and AMPK activation contribute to the molecular mechanisms underlying SGLT2 inhibitor-induced suppression of diabetic nephropathy, possibly at least in part by reducing inflammation and fibrotic change.

Prolyl Isomerase Pin1 Suppresses Thermogenic Programs in Adipocytes by Promoting Degradation of Transcriptional Co-activator PRDM16.

Non-shivering thermogenesis in adipocytes provides defense against low temperatures and obesity development, but the underlying regulatory mechanism remains to be fully clarified. Based on both markedly increased Pin1 expression in states of excess nutrition and resistance to obesity development in Pin1 null mice, we speculated that adipocyte Pin1 may play a role in thermogenic programs. Adipose-specific Pin1 knockout (adPin1 KO) mice showed enhanced transcription of thermogenic genes and tolerance to hypothermia when exposed to cold. In addition, adPin1 KO mice were resistant to high-fat diet-induced obesity and glucose intolerance. A series of experiments revealed that Pin1 binds to PRDM16 and thereby promotes its degradation through the ubiquitin-proteasome system. Consistent with these results, Pin1 deletion in differentiated adipocytes showed enhancement of thermogenic programs in response to the ß3 agonist CL316243 through the upregulation of PRDM16 proteins. These observations indicate that Pin1 is a negative regulator of non-shivering thermogenesis.