Makoto Funaki

Bone marrow-derived human mesenchymal stem cells become quiescent on soft substrates but remain responsive to chemical or mechanical stimuli.

The microenvironment of bone marrow-derived human mesenchymal stem cells (hMSCs) strictly regulates their self-renewal and differentiation. Culturing these cells ex vivo leads to a rapid expansion followed by senescence, which is characterized by a lack of proliferation and differentiation. In this study, 250-Pa polyacrylamide gels, which mimics the elasticity of bone marrow and fat tissues, were coated with a mixture of collagen type 1 and fibronectin. When hMSCs were seeded sparsely on these gels, they halted progression through the cell cycle despite the presence of serum, but when presented with a stiff substrate, these non-proliferative cells reentered the cell cycle. Non-proliferative hMSCs on 250-Pa gels also exhibited the capability to differentiate into adipocytes when cultured in adipogenic induction medium or into osteoblasts if transferred to a stiff substrate and incubated with osteoblast induction medium. These results demonstrate that hMSCs on 250-Pa gels are quiescent but competent to resume proliferation or initiate terminal differentiation with appropriate cues. These observations suggest that mechanical signals from the elasticity of the extracellular matrix may be one of the factors that enable the bone marrow niche to maintain MSCs as a reservoir for a long period.

Altered Expression Levels and Impaired Steps in the Pathway to Phosphatidylinositol 3-Kinase Activation via Insulin Receptor Substrates 1 and 2 in Zucker Fatty Rats

To elucidate the mechanism of obesity-related insulin resistance, we investigated the impaired steps in the processes of phosphatidylinositol (PI) 3-kinase activation through binding with insulin receptor substrates 1and 2 (IRS-1 and IRS-2) in liver and muscle of Zucker fatty rats. The expressions of IRS-1 and IRS-2 were shown to be downregulated in both liver and muscle in fatty rats (hepatic IRS-1, 83%; hepatic IRS-2, 45%; muscle IRS-1, 60%; muscle IRS-2, 78%), resulting in decreased tyrosine phosphorylation in response to insulin stimulation. Despite the decrease in the tyrosine phosphorylation levels of hepatic IRS-1 and IRS-2 being mild to moderate, associated PI 3-kinase activities were dramatically decreased in fatty rats (IRS-1, 14%; IRS-2, 10%), which may suggest alteration in the sites of phosphorylated tyrosine residues of hepatic IRS-1 and IRS-2. In addition, we demonstrated that the expressions of p85α and p55α regulatory subunits of PI 3-kinase were reduced (p85α, 67%; p55α, 54%), and that the p50α regulatory subunit was markedly upregulated (176%) in the livers of fatty rats without apparent alterations in expressions of the catalytic subunits p110α and p110β. These alterations may reflect the obesity-related insulin resistance commonly observed in human NIDDM.

Overexpression of SH2-Containing Inositol Phosphatase 2 Results in Negative Regulation of Insulin-Induced Metabolic Actions in 3T3-L1 Adipocytes via Its 5′-Phosphatase Catalytic Activity

ABSTRACT Phosphatidylinositol (PI) 3-kinase plays an important role in various metabolic actions of insulin including glucose uptake and glycogen synthesis. Although PI 3-kinase primarily functions as a lipid kinase which preferentially phosphorylates the D-3 position of phospholipids, the effect of hydrolysis of the key PI 3-kinase product PI 3,4,5-triphosphate [PI(3,4,5)P3] on these biological responses is unknown. We recently cloned rat SH2-containing inositol phosphatase 2 (SHIP2) cDNA which possesses the 5′-phosphatase activity to hydrolyze PI(3,4,5)P3 to PI 3,4-bisphosphate [PI(3,4)P2] and which is mainly expressed in the target tissues of insulin. To study the role of SHIP2 in insulin signaling, wild-type SHIP2 (WT-SHIP2) and 5′-phosphatase-defective SHIP2 (ΔIP-SHIP2) were overexpressed in 3T3-L1 adipocytes by means of adenovirus-mediated gene transfer. Early events of insulin signaling including insulin-induced tyrosine phosphorylation of the insulin receptor β subunit and IRS-1, IRS-1 association with the p85 subunit, and PI 3-kinase activity were not affected by expression of either WT-SHIP2 or ΔIP-SHIP2. Because WT-SHIP2 possesses the 5′-phosphatase catalytic region, its overexpression marked by decreased insulin-induced PI(3,4,5)P3 production, as expected. In contrast, the amount of PI(3,4,5)P3 was increased by the expression of ΔIP-SHIP2, indicating that ΔIP-SHIP2 functions in a dominant-negative manner in 3T3-L1 adipocytes. Both PI(3,4,5)P3 and PI(3,4)P2 were known to possibly activate downstream targets Akt and protein kinase Cλ in vitro. Importantly, expression of WT-SHIP2 inhibited insulin-induced activation of Akt and protein kinase Cλ, whereas these activations were increased by expression of ΔIP-SHIP2 in vivo. Consistent with the regulation of downstream molecules of PI 3-kinase, insulin-induced 2-deoxyglucose uptake and Glut4 translocation were decreased by expression of WT-SHIP2 and increased by expression of ΔIP-SHIP2. In addition, insulin-induced phosphorylation of GSK-3β and activation of PP1 followed by activation of glycogen synthase and glycogen synthesis were decreased by expression of WT-SHIP2 and increased by the expression of ΔIP-SHIP2. These results indicate that SHIP2 negatively regulates metabolic signaling of insulin via the 5′-phosphatase activity and that PI(3,4,5)P3 rather than PI(3,4)P2 is important for in vivo regulation of insulin-induced activation of downstream molecules of PI 3-kinase leading to glucose uptake and glycogen synthesis.

p85α Gene Generates Three Isoforms of Regulatory Subunit for Phosphatidylinositol 3-Kinase (PI 3-Kinase), p50α, p55α, and p85α, with Different PI 3-Kinase Activity Elevating Responses to Insulin

Phosphatidylinositol 3-kinase (PI 3-kinase) is stimulated by association with a variety of tyrosine kinase receptors and intracellular tyrosine-phosphorylated substrates. We isolated a cDNA that encodes a 50-kDa regulatory subunit of PI 3-kinase with an expression cloning method using 32P-labeled insulin receptor substrate-1 (IRS-1). This 50-kDa protein contains two SH2 domains and an inter-SH2 domain of p85α, but the SH3 and bcr homology domains of p85α were replaced by a unique 6-amino acid sequence. Thus, this protein appears to be generated by alternative splicing of the p85α gene product. We suggest that this protein be called p50α. Northern blotting using a specific DNA probe corresponding to p50α revealed 6.0- and 2.8-kb bands in hepatic, brain, and renal tissues. The expression of p50α protein and its associated PI 3-kinase were detected in lysates prepared from the liver, brain, and muscle using a specific antibody against p50α. Taken together, these observations indicate that the p85α gene actually generates three protein products of 85, 55, and 50 kDa. The distributions of the three proteins (p85α, p55α, and p50α), in various rat tissues and also in various brain compartments, were found to be different. Interestingly, p50α forms a heterodimer with p110 that can as well as cannot be labeled with wortmannin, whereas p85α and p55α associate only with p110 that can be wortmannin-labeled. Furthermore, p50α exhibits a markedly higher capacity for activation of associated PI 3-kinase via insulin stimulation and has a higher affinity for tyrosine-phosphorylated IRS-1 than the other isoforms. Considering the high level of p50α expression in the liver and its marked responsiveness to insulin, p50α appears to play an important role in the activation of hepatic PI 3-kinase. Each of the three α isoforms has a different function and may have specific roles in various tissues.

14-3-3 Protein Binds to Insulin Receptor Substrate-1, One of the Binding Sites of Which Is in the Phosphotyrosine Binding Domain

Insulin binding to its receptor induces the phosphorylation of cytosolic substrates, insulin receptor substrate (IRS)-1 and IRS-2, which associate with several Src homology-2 domain-containing proteins. To identify unique IRS-1-binding proteins, we screened a human heart cDNA library with32P-labeled recombinant IRS-1 and obtained two isoforms (ε and ζ) of the 14-3-3 protein family. 14-3-3 protein has been shown to associate with IRS-1 in L6 myotubes, HepG2 hepatoma cells, Chinese hamster ovary cells, and bovine brain tissue. IRS-2, a protein structurally similar to IRS-1, was also shown to form a complex with 14-3-3 protein using a baculovirus expression system. The amount of 14-3-3 protein associated with IRS-1 was not affected by insulin stimulation but was increased significantly by treatment with okadaic acid, a potent serine/threonine phosphatase inhibitor. Peptide inhibition experiments using phosphoserine-containing peptides of IRS-1 revealed that IRS-1 contains three putative binding sites for 14-3-3 protein (Ser-270, Ser-374, and Ser-641). Among these three, the motif around Ser-270 is located in the phosphotyrosine binding domain of IRS-1, which is responsible for the interaction with the insulin receptor. Indeed, a truncated mutant of IRS-1 consisting of only the phosphotyrosine binding domain retained the capacity to bind to 14-3-3 protein in vivo. Finally, the effect of 14-3-3 protein binding on the insulin-induced phosphorylation of IRS-1 was investigated. Phosphoamino acid analysis revealed that IRS-1 coimmunoprecipitated with anti-14-3-3 antibody to be weakly phosphorylated after insulin stimulation, on tyrosine as well as serine residues, compared with IRS-1 immunoprecipitated with anti-IRS-1 antibody. Thus, the association with 14-3-3 protein may play a role in the regulation of insulin sensitivity by interrupting the association between the insulin receptor and IRS-1.

MKK6/3 and p38 MAPK Pathway Activation Is Not Necessary for Insulin-induced Glucose Uptake but Regulates Glucose Transporter Expression

p38 mitogen-activated protein kinase (MAPK), which is situated downstream of MAPK kinase (MKK) 6 and MKK3, is activated by mitogenic or stress-inducing stimuli, as well as by insulin. To clarify the role of the MKK6/3-p38 MAPK pathway in the regulation of glucose transport, dominant negative p38 MAPK and MKK6 mutants and constitutively active MKK6 and MKK3 mutants were overexpressed in 3T3-L1 adipocytes and L6 myotubes using an adenovirus-mediated transfection procedure. Constitutively active MKK6/3 mutants up-regulated GLUT1 expression and down-regulated GLUT4 expression, thereby significantly increasing basal glucose transport but diminishing transport induced by insulin. Similar effects were elicited by chronic (24 h) exposure to tumor necrosis factor α, interleukin-1β, or 200 mm sorbitol, all activate the MKK6/3-p38 MAPK pathway. SB203580, a specific p38 MAPK inhibitor, attenuated these effects, further confirming that both MMK6 and MMK3 act via p38 MAPK, whereas they had no effect on the increase in glucose transport induced by a constitutively active MAPK kinase 1 (MEK1) mutant or by myristoylated Akt. In addition, suppression of p38 MAPK activation by overexpression of a dominant negative p38 MAPK or MKK6 mutant did not diminish insulin-induced glucose uptake by 3T3-L1 adipocytes. It is thus apparent that activation of p38 MAPK is not essential for insulin-induced increases in glucose uptake. Rather, p38 MAPK activation leads to a marked down-regulation of insulin-induced glucose uptake via GLUT4, which may underlie cellular stress-induced insulin resistance caused by tumor necrosis factor α and other factors.

Cell Permeant Polyphosphoinositide-binding Peptides That Block Cell Motility and Actin Assembly

Polyphosphoinositides (PPIs) affect the localization and activities of many cellular constituents, including actin-modulating proteins. Several classes of polypeptide sequences, including pleckstrin homology domains, FYVE domains, and short linear sequences containing predominantly hydrophobic and cationic residues account for phosphoinositide binding by most such proteins. We report that a ten-residue peptide derived from the phosphatidylinositol 4,5-bisphosphate (PIP2) binding region in segment 2 of gelsolin, when coupled to rhodamine B has potent PIP2 binding activity in vitro; crosses the cell membrane of fibroblasts, platelets, melanoma cells, and neutrophils by a process not involving endocytosis; and blocks cell motility. This peptide derivative transiently disassembles actin filament structures in GFP-actin-expressing NIH3T3 fibroblasts and prevents thrombin- or chemotactic peptide-stimulated actin assembly in platelets and neutrophils, respectively, but does not block the initial [Ca2+] increase caused by these agonists. The blockage of actin assembly and motility is transient, and cells recover motility within an hour after their immobilization by 5–20 μmpeptide. This class of reagents confirms the critical relation between inositol lipids and cytoskeletal structure and may be useful to probe the location and function of polyphosphoinositides in vivo.

A Novel 55-kDa Regulatory Subunit for Phosphatidylinositol 3-Kinase Structurally Similar to p55PIK Is Generated by Alternative Splicing of the p85 Gene

Phosphatidylinositol 3-kinase, which is composed of a 110-kDa catalytic subunit and a regulatory subunit, plays important roles in various cellular signaling mechanisms. We screened a rat brain cDNA expression library with P-labeled human IRS-1 protein and cloned cDNAs that were very likely to be generated by alternative splicing of p85α gene products. These cDNAs were demonstrated to encode a 55-kDa protein (p55α) containing two SH2 domains and an inter-SH2 domain of p85α but neither a bcr domain nor a SH3 homology domain. Interestingly, p55α contains a unique 34-amino acid sequence at its NH terminus, which is not included in the p85α amino acid sequence. This 34-amino acid portion was revealed to be comparable with p55PIK (p55) in length, with a high homology between the two, suggesting that these NH-terminal domains of p55α and p55 may have a specific role that p85 does not. The expression of p55α mRNA is most abundant in the brain, but expression is ubiquitous in most rat tissues. Furthermore, it should be noted that the expression of p85α mRNA in muscle is almost undetectably low by Northern blotting with a cDNA probe coding for the p85α SH3 domain, while the expression of p55α can be readily detected. These results suggest that p55α may play an unique regulatory role for phosphatidylinositol 3-kinase in brain and muscle.

Enhanced insulin-stimulated activation of phosphatidylinositol 3-kinase in the liver of high-fat-fed rats.

Insulin receptor substrate (IRS)-1 and IRS-2, which mediate phosphatidylinositol (PI) 3-kinase activation, play essential roles in insulin-induced translocation of GLUT4 and in glycogen synthesis. In this study, we investigated the process of PI 3-kinase activation via binding with IRS-1 and -2 in liver, muscle, and fat of high-fat-fed rats, a model of insulin-resistant diabetes. In the liver of high-fat-fed rats, insulin increased the PI 3-kinase regulatory subunit p85alpha and the PI 3-kinase activities associated with IRS-1 3.6- and 2.4-fold, and with IRS-2, 4.7- and 3.0-fold, respectively, compared with those in control rats. The tyrosine phosphorylation levels of IRS-1 and IRS-2 were not significantly altered, however. In contrast with the liver, tyrosine phosphorylation levels and associated PI 3-kinase proteins and activities were decreased in the muscle and adipose tissue of high-fat-fed rats. Thus, high-fat feeding appears to cause insulin resistance in the liver by a mechanism different from the impaired PI 3-kinase activation observed in muscle and adipose tissue. Taking into consideration that hepatic PI 3-kinase activation is severely impaired in obese diabetic models such as Zucker fatty rats, it is possible that the mechanism by which a high-fat diet causes insulin resistance is quite different from that associated with obesity and overeating due to abnormality in the leptin system. This is the first report to show increased PI 3-kinase activation by insulin in an insulin-resistant diabetic animal model. These findings may be important for understanding the mechanism of insulin resistance in human NIDDM, since a high-fat diet is considered to be one of the major factors exacerbating insulin insensitivity in humans.

Imidapril, an angiotensin-converting enzyme inhibitor, improves insulin sensitivity by enhancing signal transduction via insulin receptor substrate proteins and improving vascular resistance in the Zucker fatty rat☆

Angiotensin-converting enzyme (ACE) inhibitors are antihypertensive agents, that inhibit the conversion of angiotensin I to angiotensin II, resulting in smooth-muscle relaxation and a reduction of vascular resistance. Recently, it has been suggested that ACE inhibitors improve insulin resistance in diabetic patients. To investigate the effect of an ACE inhibitor on insulin sensitivity, insulin signaling, and circulation, imidapril was administered orally or intraduodenally to Zucker fatty rats. Oral administration of imidapril improved insulin sensitivity based on the results of an oral glucose tolerance test (OGTT) and a decrease in urinary glucose secretion. Phosphatidylinositol 3-kinase (PI 3-kinase) activity associated with hepatic insulin receptor substrate-1 (IRS-1) in the insulin-stimulated condition was significantly enhanced 110% without a significant alteration in tyrosine phosphorylation of IRS-1 in the imidapril-treated group. In muscle, IRS-1 tyrosine phosphorylation and PI 3-kinase activity associated with IRS-1 in the insulin-stimulated condition were enhanced 70% and 20%, respectively, in the imidapril-treated group. In contrast, an alteration of the IRS-2 pathway was observed only in liver; a significant insulin-induced increase in the IRS-2-associated PI 3-kinase over the basal level was observed in the imidapril-treated group but not in the control. In addition, treatment with imidapril was shown to significantly reduce blood pressure and increase blood flow in the liver and muscle. These results suggest that the ACE inhibitor imidapril may improve insulin sensitivity not only by acting directly on the insulin signaling pathway but also by increasing blood flow in tissues via normalization of vascular resistance, a major cause of hypertension.