Resistance to the stimulatory effects of insulin on glucose utilization is a key feature of type 2 diabetes, obesity, and the metabolic syndrome. Recent studies suggest that insulin resistance is primarily caused by a defect in glucose transport. GLUT4 is the main insulinresponsive glucose transporter and is expressed predominantly in muscle and adipose tissues. Whereas GLUT4 has been shown to play a critical role in maintaining systemic glucose homeostasis, the mechanisms regulating its expression are incompletely understood. We have cloned the murine homologue of KLF15, a member of the Krü ppel-like family of transcription factors. KLF15 is highly expressed in adipocytes and myocytes in vivo and is induced when 3T3-L1 preadipocytes are differentiated into adipocytes. Overexpression of KLF15 in adipose and muscle cell lines potently induces GLUT4 expression. This effect is specific to KLF15 as overexpression of two other Krü ppel-like factors, KLF2/LKLF and KLF4/GKLF, did not induce GLUT4 expression. Both basal (3.3-fold, p < 0.001) and insulin-stimulated (2.4-fold, p < 0.00001) glucose uptake are increased in KLF15-overexpressing adipocytes. In co-transfection assays, KLF15 and MEF2A, a known activator of GLUT4, synergistically activates the GLUT4 promoter. Promoter deletion and mutational analyses provide evidence that this activity requires an intact KLF15-binding site proximal to the MEF2A site. Finally, co-immunoprecipitation assays show that KLF15 specifically interacts with MEF2A. These studies indicate that KLF15 is an important regulator of GLUT4 in both adipose and muscle tissues.Glucose uptake into cells is regulated by two families of cellular transporters, the sodium-linked glucose transporters (kidney, intestine) and the facilitated glucose transporters (GLUTs). With respect to the latter, GLUT4 is the main effector of insulin-stimulated glucose transport and is located primarily in muscle and adipose tissues (1).Clinical and experimental observations suggest that insulinstimulated glucose transport via GLUT4 is critical in maintaining systemic glucose homeostasis. For example, heterozygous mice deficient in GLUT4 exhibit muscle insulin resistance and develop diabetes (2). Tissue-specific disruption of GLUT4 in adipose tissue and skeletal muscle results in the development of insulin resistance and glucose intolerance (3, 4). In human type 2 diabetic patients, impairment of insulin-stimulated glucose transport is responsible for resistance to insulin-stimulated glycogen synthesis in muscle (5-7).Studies in adipose and muscle tissues reveal that expression of the GLUT4 glucose transporter is controlled at the level of transcription (8, 9). In vitro and in vivo promoter studies support a role for members of the MADS-box family of transcription factors termed MEF2 proteins in the regulation of the GLUT4 promoter. However, these studies suggest that MEF2 binding alone is not sufficient to fully support GLUT4 expression (10 -12).The Krü ppel-like family of transcription factors are important regulators ...

BackgroundEvolutionary theories of aging propose that longevity evolves as a competition between reproduction and somatic maintenance for a finite pool of resources. Reproduction is thought to shorten lifespan by depleting resources from processes promoting somatic maintenance. Maternal yolk production, vitellogenesis, represents a significant maternal cost for reproduction and is suppressed under genetic and environmental conditions that extend lifespan. However, little is known about the pathways regulating vitellogenesis in response to prolongevity cues.ResultsIn order to identify mechanisms that suppress vitellogenesis under prolongevity conditions, we studied factors regulating vitellogenesis in C. elegans nematodes. In C. elegans, vitellogenesis is depressed in the absence of insulin-like signaling (IIS). We found that the C. elegans daf-2/IIS pathway regulates vitellogenesis through two mechanisms. vit-2 transcript levels in daf-2 mutants were indirectly regulated through a germline-dependent signal, and could be rescued by introduction of daf-2(+) sperm. However, yolk protein (YP) levels in daf-2 mutants were also regulated by germline-independent posttranscriptional mechanisms.ConclusionsC. elegans vitellogenesis is regulated transcriptionally and posttranscriptionally in response to environmental and reproductive cues. The daf-2 pathway suppressed vitellogenesis through transcriptional mechanisms reflecting reproductive phenotypes, as well as distinct posttranscriptional mechanisms. This study reveals that pleiotropic effects of IIS pathway mutations can converge on a common downstream target, vitellogenesis, as a mechanism to modulate longevity.

The transport of vesicles and the retention of organelles at specific locations are fundamental processes in cells. Actin filaments and myosin motors have been shown to be required for both of these tasks. Most of the organelles in cells associate with actin filaments and some of the myosin motors required for movement on actin filaments have been identified. Myosin V has been shown to transport endoplasmic reticulum (ER) vesicles in neurons, pigment granules in melanocytes, and the vacuole in yeast. Myosin I has been shown to be involved in the transport of Golgi‐derived vesicles in epithelial cells. Myosin VI has been shown to be associated with Golgi‐derived vesicles, and cytoplasmic vesicles in living Drosophila embryos. Myosin II may be a vesicle motor but its role in vesicle transport has not been resolved. Secretory vesicles, endosomes and mitochondria appear to be transported on actin filaments but the myosin motors on these organelles have not been identified. Mitochondria in yeast may be transported by the dynamic assembly of an actin “tail.” The model that has unified all of these findings is the concept that long‐range movement of vesicles occurs on microtubules and short‐range movement on actin filaments. The details of how the microtubule‐dependent and the actin‐dependent motors are coordinated are important questions in the field. There is now strong evidence that two molecular motors, kinesin and myosin V, interact with each other and perhaps function as a complex on vesicles. An understanding of the interrelationship of microtubules and actin filaments and the motors that move cargo on them will ultimately establish how vesicles and organelles are transported to their specific locations in cells. Microsc. Res. Tech. 47:93–106, 1999. © 1999 Wiley‐Liss, Inc.

Activation of vascular smooth muscle cells (VSMCs)by proinflammatory cytokines is a key feature of atherosclerotic lesion formation. Transforming growth factor (TGF)-␤1 is a pleiotropic growth factor that can modulate the inflammatory response in diverse cell types including VSMCs. However, the mechanisms by which TGF-␤1 is able to mediate these effects remains incompletely understood. We demonstrate here that the ability of TGF-␤1 to inhibit markers of VSMC activation, inducible nitric-oxide synthase (iNOS) and interleukin (IL)-6, is mediated through its downstream effector Smad3. In reporter gene transfection studies, we found that among a panel of Smads, Smad3 could inhibit iNOS induction in an analogous manner as exogenous TGF-␤1. Adenoviral overexpression of Smad3 potently repressed inducible expression of endogenous iNOS and IL-6. Conversely, TGF-␤1 inhibition of cytokine-mediated induction of iNOS and IL-6 expression was completely blocked in Smad3-deficient VSMCs. Previous studies demonstrate that CCAAT/enhancer-binding protein (C/EBP) and NF-B sites are critical for cytokine induction of both the iNOS and IL-6 promoters. We demonstrate that the inhibitory effect of Smad3 occurs via a novel antagonistic effect of Smad3 on C/EBP DNA-protein binding and activity. Smad3 mediates this effect in part by inhibiting C/EBP-␤ and C/EBP-␦ through distinct mechanisms. Furthermore, we find that Smad3 prevents the cooperative induction of the iNOS promoter by C/EBP and NF-B. These data demonstrate that Smad3 plays an essential role in mediating TGF-␤1 anti-inflammatory response in VSMCs.

The thromhin receptor is a G protein-coupled receptor, hut the G proteins functionally coupled to this receptor in human platelets are not yet definitively identified. Thrombin stimulation of platelets leads to phospholipase C-mediated increases in intracellular calcium, and previous studies have suggested that the thrombin receptor is coupled to members of the G, family. We now demonstrate direct GTPase activation by tbrombin receptor activation peptide (TRAP) in human platelet membranes, and specific inhibition of TRAP-activated GTPase by antibodies to G,. These data demonstrate functional coupling of the thrombin receptor to a member of the G, family.