Divergent Sequence Tunes Ligand Sensitivity in Phospholipid-regulated Hormone Receptors

The members of the NR5A subfamily of nuclear receptors (NRs) are important regulators of pluripotency, lipid and glucose homeostasis, and steroidogenesis. Liver receptor homologue 1 (LRH-1; NR5A2) and steroidogenic factor 1 (SF-1; NR5A1) have therapeutic potential for the treatment of metabolic and neoplastic disease; however, a poor understanding of their ligand regulation has hampered the pursuit of these proteins as pharmaceutical targets. In this study, we dissect how sequence variation among LRH-1 orthologs affects phospholipid (PL) binding and regulation. Both human LRH-1 (hLRH-1) and mouse LRH-1 (mLRH-1) respond to newly discovered medium chain PL agonists to modulate lipid and glucose homeostasis. These PLs activate hLRH-1 by altering receptor dynamics in a newly identified alternate activation function region. Mouse and Drosophila orthologs contain divergent sequences in this region potentially altering PL-driven activation. Structural evidence suggests that these sequence differences in mLRH-1 and Drosophila FTZ-f1 (dmFTZ-f1) confer at least partial ligand independence, making them poor models for hLRH-1 studies; however, the mechanisms of ligand independence remain untested. We show using structural and biochemical methods that the recent evolutionary divergence of the mLRH-1 stabilizes the active conformation in the absence of ligand, yet does not abrogate PL-dependent activation. We also show by mass spectrometry and biochemical assays that FTZ-f1 is incapable of PL binding. This work provides a structural mechanism for the differential tuning of PL sensitivity in NR5A orthologs and supports the use of mice as viable therapeutic models for LRH-1-dependent diseases.     

Derivation of Human Induced Pluripotent Stem Cells from Patients with Maturity Onset Diabetes of the Young

Maturity onset diabetes of the young (MODY) is an autosomal dominant disease. Despite extensive research, the mechanism by which a mutant MODY gene results in monogenic diabetes is not yet clear due to the inaccessibility of patient samples. Induced pluripotency and directed differentiation toward the pancreatic lineage are now viable and attractive methods to uncover the molecular mechanisms underlying MODY. Here we report, for the first time, the derivation of human induced pluripotent stem cells (hiPSCs) from patients with five types of MODY: MODY1 (HNF4A), MODY2 (GCK), MODY3 (HNF1A), MODY5 (HNF1B), and MODY8 (CEL) with a polycistronic lentiviral vector expressing a Cre-excisable human “stem cell cassette” containing the four reprogramming factors OCT4, KLF4, SOX2, and CMYC. These MODY-hiPSCs morphologically resemble human pluripotent stem cells (hPSCs), express pluripotency markers OCT4, SOX2, NANOG, SSEA-4, and TRA-1–60, give rise to derivatives of the three germ layers in a teratoma assay, and are karyotypically normal. Overall, our MODY-hiPSCs serve as invaluable tools to dissect the role of MODY genes in the development of pancreas and islet cells and to evaluate their significance in regulating beta cell function. This knowledge will aid future attempts aimed at deriving functional mature beta cells from hPSCs.     

FOG2 Protein Down-regulation by Transforming Growth Factor-β1-induced MicroRNA-200b/c Leads to Akt Kinase Activation and Glomerular Mesangial Hypertrophy Related to Diabetic Nephropathy

Glomerular hypertrophy is a hallmark of diabetic nephropathy. Akt kinase activated by transforming growth factor-β1 (TGF-β) plays an important role in glomerular mesangial hypertrophy. However, the mechanisms of Akt activation by TGF-β are not fully understood. Recently, miR-200 and its target FOG2 were reported to regulate the activity of phosphatidylinositol 3-kinase (the upstream activator of Akt) in insulin signaling. Here, we show that TGF-β activates Akt in glomerular mesangial cells by inducing miR-200b and miR-200c, both of which target FOG2, an inhibitor of phosphatidylinositol 3-kinase activation. FOG2 expression was reduced in the glomeruli of diabetic mice as well as TGF-β-treated mouse mesangial cells (MMC). FOG2 knockdown by siRNAs in MMC activated Akt and increased the protein content/cell ratio suggesting hypertrophy. A significant increase of miR-200b/c levels was detected in diabetic mouse glomeruli and TGF-β-treated MMC. Transfection of MMC with miR-200b/c mimics significantly decreased the expression of FOG2. Conversely, miR-200b/c inhibitors attenuated TGF-β-induced decrease in FOG2 expression. Furthermore, miR-200b/c mimics increased the protein content/cell ratio, whereas miR-200b/c inhibitors abrogated the TGF-β-induced increase in protein content/cell. In addition, down-regulation of FOG2 by miR-200b/c could activate not only Akt but also ERK, which was also through PI3K activation. These data suggest a new mechanism for TGF-β-induced Akt activation through FOG2 down-regulation by miR-200b/c, which can lead to glomerular mesangial hypertrophy in the progression of diabetic nephropathy.     

Heligmosomoides polygyrus infection reduces severity of type 1 diabetes induced by multiple low-dose streptozotocin in mice via STAT6- and IL-10-independent mechanisms

Some parasitic helminths are known to protect their hosts from allergic and autoimmune disorders. Here, we tested the effects of a gastrointestinal nematode, Heligmosomoides polygyrus (Hp), on streptozotocin (STZ)-induced type 1 diabetes (T1D) in mice. Hp infection significantly suppressed hyperglycemia induced by multiple low-dose administration of STZ, but did not affect hyperglycemia induced by single high-dose administration of STZ. In the multiple low dose model, Hp infection prevented a decrease in pancreatic islet size. The augmentation of TNF-α and IL-1β expression in the pancreas was abrogated by Hp infection. The genetic absence of IL-10 or STAT6 did not abrogate the anti-hyperglycemic effect of Hp. Hp has a suppressive effect on immune mechanism-mediated experimental T1D via Th2 polarization-independent mechanisms.     

Genetic disruption of protein phosphatase 5 in mice prevents high-fat diet feeding-induced weight gain

The role of serine/threonine protein phosphatase 5 (PP5) in the development of obesity and insulin resistance associated with high-fat diet-feeding (HFD) was examined using PP5-deficient mice (Ppp5c^−/−). Despite similar caloric intake, Ppp5c^−/− mice on HFD gained markedly less weight and did not accumulate visceral fat compared to wild-type littermates (Ppp5c^+/+). On a control diet, Ppp5c^−/− mice had markedly improved glucose control compared to Ppp5c^+/+ mice, an effect diminished by HFD. However, even after 10 weeks of HFD glucose control in Ppp5c^−/− mice was similar to that observed in Ppp5c^+/+ mice on the control diet. Thus, PP5 deficiency confers protection against HFD-induced weight gain in mice.     

Oxidative damage associated with obesity is prevented by overexpression of CuZn- or Mn-superoxide dismutase

The development of insulin resistance is the primary step in the etiology of type 2 diabetes mellitus. There are several risk factors associated with insulin resistance, yet the basic biological mechanisms that promote its development are still unclear. There is growing literature that suggests mitochondrial dysfunction and/or oxidative stress play prominent roles in defects in glucose metabolism. Here, we tested whether increased expression of CuZn-superoxide dismutase (Sod1) or Mn-superoxide dismutase (Sod2) prevented obesity-induced changes in oxidative stress and metabolism. Both Sod1 and Sod2 overexpressing mice were protected from high fat diet-induced glucose intolerance. Lipid oxidation (F₂-isoprostanes) was significantly increased in muscle and adipose with high fat feeding. Mice with increased expression of either Sod1 or Sod2 showed a significant reduction in this oxidative damage. Surprisingly, mitochondria from the muscle of high fat diet-fed mice showed no significant alteration in function. Together, our data suggest that targeting reduced oxidative damage in general may be a more applicable therapeutic target to prevent insulin resistance than is improving mitochondrial function.     

Placenta mesenchymal stem cell accelerates wound healing by enhancing angiogenesis in diabetic Goto-Kakizaki (GK) rats

Multipotent mesenchymal stem cells have recently emerged as an attractive cell type for the treatment of diabetes-associated wounds. The purpose of this study was to examine the potential biological function of human placenta-derived mesenchymal stem cells (PMSCs) in wound healing in diabetic Goto-Kakizaki (GK) rats. PMSCs were isolated from human placenta tissue and characterized by flow cytometry. A full-thickness circular excisional wound was created on the dorsum of each rat. Red fluorescent CM-DiI-labeled PMSCs were injected intradermally around the wound in the treatment group. After complete wound healing, full-thickness skin samples were taken from the wound sites for histological evaluation of the volume and density of vessels. Our data showed that the extent of wound closure was significantly enhanced in the PMSCs group compared with the no-graft controls. Microvessel density in wound bed biopsy sites was significantly higher in the PMSCs group compared with the no-graft controls. Most surprisingly, immunohistochemical studies confirmed that transplanted PMSCs localized to the wound tissue and were incorporated into recipient vasculature with improved angiogenesis. Notably, PMSCs secreted comparable amounts of proangiogenic molecules, such as VEGF, HGF, bFGF, TGF-β and IGF-1 at bioactive levels. This study demonstrated that PMSCs improved the wound healing rate in diabetic rats. It is speculated that this effect can be attributed to the PMSCs engraftment resulting in vascular regeneration via direct de novo differentiation and paracrine mechanisms. Thus, placenta-derived mesenchymal stem cells are implicated as a potential angiogenesis cell therapy for repair-resistant chronic wounds in diabetic patients.     

Flaxseed (Linum usitatissimum L.) extract as well as (+)-secoisolariciresinol diglucoside and its mammalian derivatives are potent inhibitors of α-amylase activity

Type 2 diabetes mellitus (T2DM) is one of the common global diseases. Flaxseed is by far the richest source of the dietary lignans (i.e., secoisolariciresinol diglucoside) which have been shown to delay the development of T2DM in animal models. Herein, we propose the first evidences for a mechanism of action involving the inhibition of the pancreatic α-amylase (EC 3.2.1.1) by flaxseed-derived lignans that could therefore constitute a promising nutraceutical for the prevention and the treatment of T2DM.     

Design and synthesis of 2-substituted benzoxazoles as novel PTP1B inhibitors

A series of benzoxazole compounds containing oxamic acid were synthesized and screened for the PTP1B inhibition. Compound 31d showed best biochemical potency (K_i) of 6.7 μM. Structure–activity relationship were explained with the help of molecular modeling approach.