ABSTRACT
Metformin, the first‐line drug to treat type 2 diabetes (T2D), inhibits mitochondrial glycerolphosphate dehydrogenase in the liver to suppress gluconeogenesis. However, the direct target and ...the underlying mechanisms by which metformin increases glucose uptake in peripheral tissues remain uncharacterized. Lipid phosphatase Src homology 2 domain‐containing inositol‐5‐phosphatase 2 (SHIP2) is upregulated in diabetic rodent models and suppresses insulin signaling by reducing Akt activation, leading to insulin resistance and diminished glucose uptake. Here, we demonstrate that metformin directly binds to and reduces the catalytic activity of the recombinant SHIP2 phosphatase domain in vitro. Metformin inhibits SHIP2 in cultured cells and in skeletal muscle and kidney of db/db mice. In SHIP2‐overexpressing myotubes, metformin ameliorates reduced glucose uptake by slowing down glucose transporter 4 endocytosis. SHIP2 overexpression reduces Akt activity and enhances podocyte apoptosis, and both are restored to normal levels by metformin. SHIP2 activity is elevated in glomeruli of patients with T2D receiving nonmetformin medication, but not in patients receiving metformin, compared with people without diabetes. Furthermore, podocyte loss in kidneys of metformin‐treated T2D patients is reduced compared with patients receiving nonmetformin medication. Our data unravel a novel molecular mechanism by which metformin enhances glucose uptake and acts renoprotectively by reducing SHIP2 activity.—Polianskyte‐Prause, Z., Tolvanen, T. A., Lindfors, S., Dumont, V., Van, M., Wang, H., Dash, S. N., Berg, M., Naams, J.‐B., Hautala, L. C., Nisen, H., Mirtti, T., Groop, P.‐H., Wähälä, K., Tienari, J., Lehtonen, S. Metformin increases glucose uptake and acts renoprotectively by reducing SHIP2 activity. FASEB J. 33, 2858–2869 (2019). www.fasebj.org
ABSTRACT
Nephrin is a core component of podocyte (glomerular epithelial cell) slit diaphragm and is required for kidney ultrafiltration. Down‐regulation or mislocalization of nephrin has been ...observed in diabetic kidney disease (DKD), characterized by albuminuria. Here, we investigate the role of protein kinase C and casein kinase 2 substrate in neurons 2 (PACSIN2), a regulator of endocytosis and recycling, in the trafficking of nephrin and development of DKD. We observe that PACSIN2 is up‐regulated and nephrin mislocalized in podocytes of obese Zucker diabetic fatty (ZDF) rats that have altered renal function. In cultured podocytes, PACSIN2 and nephrin colocalize and interact. We show that nephrin is endocytosed in PACSIN2‐positive membrane regions and that PACSIN2 overexpression increases both nephrin endocytosis and recycling. We identify rabenosyn‐5, which is involved in early endosome maturation and endosomal sorting, as a novel interaction partner of PACSIN2. Interestingly, rabenosyn‐5 expression is increased in podocytes in obese ZDF rats, and, in vitro, its overexpression enhances the association of PACSIN2 and nephrin. We also show that palmitate, which is elevated in diabetes, enhances this association. Collectively, PACSIN2 is up‐regulated and nephrin is abnormally localized in podocytes of diabetic ZDF rats. In vitro, PACSIN2 enhances nephrin turnover apparently via a mechanism involving rabenosyn‐5. The data suggest that elevated PACSIN2 expression accelerates nephrin trafficking and associates with albuminuria.—Dumont, V., Tolvanen, T. A., Kuusela, S., Wang, H., Nyman, T. A., Lindfors, S., Tienari, J., Nisen, H., Suetsugu, S., Plomann, M., Kawachi, H., Lehtonen, S. PACSIN2 accelerates nephrin trafficking and is up‐regulated in diabetic kidney disease. FASEB J. 31, 3978–3990 (2017). www.fasebj.org—Dumont, Vincent, Tolvanen, Tuomas A., Kuusela, Sara, Wang, Hong, Nyman, Tuula A., Lindfors, Sonja, Tienari, Jukka, Nisen, Harry, Suetsugu, Shiro, Plomann, Markus, Kawachi, Hiroshi, Lehtonen, Sanna PACSIN2 accelerates nephrin trafficking and is up‐regulated in diabetic kidney disease. FASEB J. 31, 3978–3990 (2017)
Protein kinase C and casein kinase substrate in neurons (PACSINs), or syndapins (synaptic dynamin‐associated proteins), are a family of proteins involved in the regulation of cell cytoskeleton, ...intracellular trafficking and signalling. Over the last twenty years, PACSINs have been mostly studied in the in vitro and ex vivo settings, and only in the last decade reports on their function in vivo have emerged. We first summarize the identification, structure and cellular functions of PACSINs, and then focus on the relevance of PACSINs in vivo. During development in various model organisms, PACSINs participate in diverse processes, such as neural crest cell development, gastrulation, laterality development and neuromuscular junction formation. In mouse, PACSIN2 regulates angiogenesis during retinal development and in human, PACSIN2 associates with monosomy and embryonic implantation. In adulthood, PACSIN1 has been extensively studied in the brain and shown to regulate neuromorphogenesis, receptor trafficking and synaptic plasticity. Several genetic studies suggest a role for PACSIN1 in the development of schizophrenia, which is also supported by the phenotype of mice depleted of PACSIN1. PACSIN2 plays an essential role in the maintenance of intestinal homeostasis and participates in kidney repair processes after injury. PACSIN3 is abundant in muscle tissue and necessary for caveolar biogenesis to create membrane reservoirs, thus controlling muscle function, and has been linked to certain genetic muscular disorders. The above examples illustrate the importance of PACSINs in diverse physiological or tissue repair processes in various organs, and associations to diseases when their functions are disturbed.
Observations of the redshift
= 7.085 quasar J1120+0641 are used to search for variations of the fine structure constant, a, over the redshift range 5:5 to 7:1. Observations at
= 7:1 probe the physics ...of the universe at only 0.8 billion years old. These are the most distant direct measurements of a to date and the first measurements using a near-IR spectrograph. A new AI analysis method is employed. Four measurements from the x-shooter spectrograph on the Very Large Telescope (VLT) constrain changes in a relative to the terrestrial value (α
). The weighted mean electromagnetic force in this location in the universe deviates from the terrestrial value by Δα/α = (α
- α
)/α
= (-2:18 ± 7:27) × 10
, consistent with no temporal change. Combining these measurements with existing data, we find a spatial variation is preferred over a no-variation model at the 3:9σ level.
The adapter protein CD2-associated protein (CD2AP) functions in various signaling and vesicle trafficking pathways, including endosomal sorting and/or trafficking and degradation pathways. Here, we ...investigated the role of CD2AP in insulin-dependent glucose transporter 4 (Glut4, also known as SLC2A4) trafficking and glucose uptake. Glucose uptake was attenuated in CD2AP(-/-) podocytes compared with wild-type podocytes in the basal state, and CD2AP(-/-) podocytes failed to increase glucose uptake in response to insulin. Live-cell imaging revealed dynamic trafficking of HA-Glut4-GFP in wild-type podocytes, whereas in CD2AP(-/-) podocytes, HA-Glut4-GFP clustered perinuclearly. In subcellular membrane fractionations, CD2AP co-fractionated with Glut4, IRAP (also known as LNPEP) and sortilin, constituents of Glut4 storage vesicles (GSVs). We further found that CD2AP forms a complex with GGA2, a clathrin adaptor, which sorts Glut4 to GSVs, suggesting a role for CD2AP in this process. We also found that CD2AP forms a complex with clathrin and connects clathrin to actin in the perinuclear region. Furthermore, clathrin recycling back to trans-Golgi membranes from the vesicular fraction containing GSVs was defective in the absence of CD2AP. This leads to reduced insulin-stimulated trafficking of GSVs and attenuated glucose uptake into CD2AP(-/-) podocytes.
Glomerular epithelial cells, podocytes, are insulin responsive and can develop insulin resistance. Here, we demonstrate that the small GTPase septin 7 forms a complex with nonmuscle myosin heavy ...chain IIA (NMHC-IIA; encoded by MYH9), a component of the nonmuscle myosin IIA (NM-IIA) hexameric complex. We observed that knockdown of NMHC-IIA decreases insulin-stimulated glucose uptake into podocytes. Both septin 7 and NM-IIA associate with SNAP23, a SNARE protein involved in GLUT4 storage vesicle (GSV) docking and fusion with the plasma membrane. We observed that insulin decreases the level of septin 7 and increases the activity of NM-IIA in the SNAP23 complex, as visualized by increased phosphorylation of myosin regulatory light chain. Also knockdown of septin 7 increases the activity of NM-IIA in the complex. The activity of NM-IIA is increased in diabetic rat glomeruli and cultured human podocytes exposed to macroalbuminuric sera from patients with type 1 diabetes. Collectively, the data suggest that the activity of NM-IIA in the SNAP23 complex plays a key role in insulin-stimulated glucose uptake into podocytes. Furthermore, we observed that septin 7 reduces the activity of NM-IIA in the SNAP23 complex and thereby hinders GSV docking and fusion with the plasma membrane.
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•Septin 7, nonmuscle myosin heavy chain IIA (NMHC-IIA) and SNAP23 form a complex.•Knockdown of septin 7 increases NM-IIA activity in the SNAP23 complex.•Insulin decreases septin 7 level and increases NM-IIA activity in the SNAP23 complex.•Septin 7 hinders GSV docking/fusion by reducing NM-IIA activity in the SNAP23 complex.
Loss of podocytes is an early feature of diabetic nephropathy (DN) and predicts its progression. We found that treatment of podocytes with sera from normoalbuminuric type 1 diabetes patients with ...high lipopolysaccharide (LPS) activity, known to predict progression of DN, downregulated CDK2 (cyclin-dependent kinase 2). LPS-treatment of mice also reduced CDK2 expression. LPS-induced downregulation of CDK2 was prevented in vitro and in vivo by inhibiting the Toll-like receptor (TLR) pathway using immunomodulatory agent GIT27. We also observed that CDK2 is downregulated in the glomeruli of obese Zucker rats before the onset of proteinuria. Knockdown of CDK2, or inhibiting its activity with roscovitine in podocytes increased apoptosis. CDK2 knockdown also reduced expression of PDK1, an activator of the cell survival kinase Akt, and reduced Akt phosphorylation. This suggests that CDK2 regulates the activity of the cell survival pathway via PDK1. Furthermore, PDK1 knockdown reduced the expression of CDK2 suggesting a regulatory loop between CDK2 and PDK1. Collectively, our data show that CDK2 protects podocytes from apoptosis and that reduced expression of CDK2 associates with the development of DN. Preventing downregulation of CDK2 by blocking the TLR pathway with GIT27 may provide a means to prevent podocyte apoptosis and progression of DN.
Changes in the dynamic architecture of podocytes, the glomerular epithelial cells, lead to kidney dysfunction. Previous studies on protein kinase C and casein kinase 2 substrates in neurons 2 ...(PACSIN2), a known regulator of endocytosis and cytoskeletal organization, reveal a connection between PACSIN2 and kidney pathogenesis. Here, we show that the phosphorylation of PACSIN2 at serine 313 (S313) is increased in the glomeruli of rats with diabetic kidney disease. We found that phosphorylation at S313 is associated with kidney dysfunction and increased free fatty acids rather than with high glucose and diabetes alone. Phosphorylation of PACSIN2 emerged as a dynamic process that fine-tunes cell morphology and cytoskeletal arrangement, in cooperation with the regulator of the actin cytoskeleton, Neural Wiskott-Aldrich syndrome protein (N-WASP). PACSIN2 phosphorylation decreased N-WASP degradation while N-WASP inhibition triggered PACSIN2 phosphorylation at S313. Functionally, pS313-PACSIN2 regulated actin cytoskeleton rearrangement depending on the type of cell injury and the signaling pathways involved. Collectively, this study indicates that N-WASP induces phosphorylation of PACSIN2 at S313, which serves as a mechanism whereby cells regulate active actin-related processes. The dynamic phosphorylation of S313 is needed to regulate cytoskeletal reorganization.
Hot white dwarf stars are the ideal probe for a relationship between the fine-structure constant and strong gravitational fields, providing us with an opportunity for a direct observational test. We ...study a sample of hot white dwarf stars, combining far-UV spectroscopic observations, atomic physics, atmospheric modelling, and fundamental physics in the search for variation in the fine structure constant. This variation manifests as shifts in the observed wavelengths of absorption lines, such as quadruply ionized iron (FeV) and quadruply ionized nickel (NiV), when compared to laboratory wavelengths. Berengut et al. (Phys. Rev. Lett. 2013, 111, 010801) demonstrated the validity of such an analysis using high-resolution Space Telescope Imaging Spectrograph (STIS) spectra of G191-B2B. We have made three important improvements by: (a) using three new independent sets of laboratory wavelengths; (b) analysing a sample of objects; and (c) improving the methodology by incorporating robust techniques from previous studies towards quasars (the Many Multiplet method). A successful detection would be the first direct measurement of a gravitational field effect on a bare constant of nature. Here we describe our approach and present preliminary results from nine objects using both FeV and NiV.
The transgenic E1-DN mice express a kinase-negative epidermal growth factor receptor in their pancreatic islets and are diabetic from two weeks of age due to impaired postnatal growth of β-cell mass. ...Here, we characterize the development of hyperglycaemia-induced renal injury in the E1-DN mice. Homozygous mice showed increased albumin excretion rate (AER) at the age of 10 weeks; the albuminuria increased over time and correlated with blood glucose. Morphometric analysis of PAS-stained histological sections and electron microscopy images revealed mesangial expansion in homozygous E1-DN mice, and glomerular sclerosis was observed in the most hyperglycaemic mice. The albuminuric homozygous mice developed also other structural changes in the glomeruli, including thickening of the glomerular basement membrane and widening of podocyte foot processes that are typical for diabetic nephropathy. Increased apoptosis of podocytes was identified as one mechanism contributing to glomerular injury. In addition, nephrin expression was reduced in the podocytes of albuminuric homozygous E1-DN mice. Tubular changes included altered epithelial cell morphology and increased proliferation. In conclusion, hyperglycaemic E1-DN mice develop albuminuria and glomerular and tubular injury typical of human diabetic nephropathy and can serve as a new model to study the mechanisms leading to the development of diabetic nephropathy.
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