Brian S. Finlin

Regulation of voltage-gated calcium channel activity by the Rem and Rad GTPases

Rem, Rem2, Rad, and Gem/Kir (RGK) represent a distinct GTPase family with largely unknown physiological functions. We report here that both Rem and Rad bind directly to Ca2+ channel β-subunits (CaVβ) in vivo. No calcium currents are recorded from human embryonic kidney 293 cells coexpressing the L type Ca2+ channel subunits CaV1.2, CaVβ2a, and Rem or Rad, but CaV1.2 and CaVβ2a transfected cells elicit Ca2+ channel currents in the absence of these small G proteins. Importantly, CaV3 (T type) Ca2+ channels, which do not require accessory subunits for ionic current expression, are not inhibited by expression of Rem. Rem is expressed in primary skeletal myoblasts and, when overexpressed in C2C12 myoblasts, wild-type Rem inhibits L type Ca2+ channel activity. Deletion analysis demonstrates a critical role for the Rem C terminus in both regulation of functional Ca2+ channel expression and β-subunit association. These results suggest that all members of the RGK GTPase family, via direct interaction with auxiliary β-subunits, serve as regulators of L type Ca2+ channel activity. Thus, the RGK GTPase family may provide a mechanism for achieving cross talk between Ras-related GTPases and electrical signaling pathways.

Omega-3 Fatty Acids Reduce Adipose Tissue Macrophages in Human Subjects With Insulin Resistance

Fish oils (FOs) have anti-inflammatory effects and lower serum triglycerides. This study examined adipose and muscle inflammatory markers after treatment of humans with FOs and measured the effects of ω-3 fatty acids on adipocytes and macrophages in vitro. Insulin-resistant, nondiabetic subjects were treated with Omega-3-Acid Ethyl Esters (4 g/day) or placebo for 12 weeks. Plasma macrophage chemoattractant protein 1 (MCP-1) levels were reduced by FO, but the levels of other cytokines were unchanged. The adipose (but not muscle) of FO-treated subjects demonstrated a decrease in macrophages, a decrease in MCP-1, and an increase in capillaries, and subjects with the most macrophages demonstrated the greatest response to treatment. Adipose and muscle ω-3 fatty acid content increased after treatment; however, there was no change in insulin sensitivity or adiponectin. In vitro, M1-polarized macrophages expressed high levels of MCP-1. The addition of ω-3 fatty acids reduced MCP-1 expression with no effect on TNF-α. In addition, ω-3 fatty acids suppressed the upregulation of adipocyte MCP-1 that occurred when adipocytes were cocultured with macrophages. Thus, FO reduced adipose macrophages, increased capillaries, and reduced MCP-1 expression in insulin-resistant humans and in macrophages and adipocytes in vitro; however, there was no measureable effect on insulin sensitivity.

Regulation of L-type Ca2+ Channel Activity and Insulin Secretion by the Rem2 GTPase

Voltage-dependent calcium (Ca2+) channels are involved in many specialized cellular functions and are controlled by a diversity of intracellular signals. Recently, members of the RGK family of small GTPases (Rem, Rem2, Rad, Gem/Kir) have been identified as novel contributors to the regulation of L-type calcium channel activity. In this study, microarray analysis of the mouse insulinoma MIN6 cell line revealed that the transcription of Rem2 gene is strongly induced by exposure to high glucose, which was confirmed by real-time reverse transcriptase-PCR and RNase protection analysis. Because elevation of intracellular Ca2+ in pancreatic β-cells is essential for insulin secretion, we tested the hypothesis that Rem2 attenuates Ca2+ currents to regulate insulin secretion. Co-expression of Rem2 with CaV 1.2 or CaV1.3 L-type Ca + channels in a heterologous expression system completely inhibits de novo Ca2+ current expression. In addition, ectopic overexpression of Rem2 both inhibited L-type Ca2+ channel activity and prevented glucose-stimulated insulin secretion in pancreatic β-cell lines. Co-immunoprecipitation studies demonstrate that Rem2 associates with a variety of CaVβ subunits. Importantly, surface biotinylation studies demonstrate that the membrane distribution of Ca2+ channels was not reduced at a time when channel activity was potently inhibited by Rem2 expression, indicating that Rem2 modulates channel function without interfering with membrane trafficking. Taken together, these data suggest that inhibition of L-type Ca2+ channels by Rem2 signaling may represent a new and potentially important mechanism for regulating Ca2+-triggered exocytosis in hormone-secreting cells, including insulin secretion in pancreatic β-cells.

Rem Is a New Member of the Rad- and Gem/Kir Ras-related GTP-binding Protein Family Repressed by Lipopolysaccharide Stimulation

We report the cDNA cloning and characterization of a novel GTP-binding protein, termed Rem (for Rad and Gem-related), that was identified as a product of polymerase chain reaction amplification using oligonucleotide primers derived from conserved regions of the Rad, Gem, and Kir Ras subfamily. Alignment of the full-length open reading frame of mouse Rem revealed the encoded protein to be 47% identical to the Rad, Gem, and Kir proteins. The distinct structural features of the Rad, Gem, and Kir subfamily are maintained including a series of nonconservative amino acid substitutions at positions important for GTPase activity and a unique sequence motif thought to direct membrane association. Recombinant Rem binds GTP in a specific and saturable manner. Ribonuclease protection analysis found Rem to be expressed at comparatively high levels in cardiac muscle and at moderate levels in lung, skeletal muscle, and kidney. The administration of lipopolysaccharide to mice, a potent activator of the inflammatory and immune systems, results in the general repression of Rem mRNA levels in a dose- and time-dependent manner. Thus, Rem is the first Ras-related gene whose mRNA levels have been shown to be regulated by repression.

Rem2, a new member of the Rem/Rad/Gem/Kir family of Ras-related GTPases

Here we report the molecular cloning and biochemical characterization of Rem2 (for Rem, Rad and Gem-related 2), a novel GTP-binding protein identified on the basis of its homology with the Rem, Rad, Gem and Kir (RGK) family of Ras-related small GTP-binding proteins. Rem2 mRNA was detected in rat brain and kidney, making it the first member of the RGK family to be expressed at relatively high levels in neuronal tissues. Recombinant Rem2 binds GTP saturably and exhibits a low intrinsic rate of GTP hydrolysis. Surprisingly, the guanine nucleotide dissociation constants for both Rem2 and Rem are significantly different than the majority of the Ras-related GTPases, displaying higher dissociation rates for GTP than GDP. Localization studies with green fluorescent protein (GFP)-tagged recombinant protein fusions indicate that Rem2 has a punctate, plasma membrane localization. Deletion of the C-terminal seven amino acid residues that are conserved in all RGK family members did not affect the cellular distribution of the GFP fusion protein, whereas a larger deletion, including much of the polybasic region of the Rem2 C-terminus, resulted in its redistribution to the cytosol. Thus Rem2 is a GTPase of the RGK family with distinctive biochemical properties and possessing a novel cellular localization signal, consistent with its having a unique role in cell physiology.

Analysis of the Complex between Ca2+ Channel β-Subunit and the Rem GTPase

Voltage-gated calcium channels are multiprotein complexes that regulate calcium influx and are important contributors to cardiac excitability and contractility. The auxiliary β-subunit (CaVβ) binds a conserved domain (the α-interaction domain (AID)) of the pore-forming CaVα1 subunit to modulate channel gating properties and promote cell surface trafficking. Recently, members of the RGK family of small GTPases (Rem, Rem2, Rad, Gem/Kir) have been identified as novel contributors to the regulation of L-type calcium channel activity. Here, we describe the Rem-association domain within CaVβ2a. The Rem interaction module is located in a ∼130-residue region within the highly conserved guanylate kinase domain that also directs AID binding. Importantly, CaVβ mutants were identified that lost the ability to bind AID but retained their association with Rem, indicating that the AID and Rem association sites of CaVβ2a are structurally distinct. In vitro binding studies indicate that the affinity of Rem for CaVβ2a interaction is lower than that of AID for CaVβ2a. Furthermore, in vitro binding studies indicate that Rem association does not inhibit the interaction of CaVβ2a with AID. Instead, CaVβ can simultaneously associate with both Rem and CaVα1-AID. Previous studies had suggested that RGK proteins may regulate Ca2+ channel activity by blocking the association of CaVβ subunits with CaVα1 to inhibit plasma membrane trafficking. However, surface biotinylation studies in HIT-T15 cells indicate that Rem can acutely modulate channel function without decreasing the density of L-type channels at the plasma membrane. Together these data suggest that Rem-dependent Ca2+ channel modulation involves formation of a Rem·CaVβ·AID regulatory complex without the need to disrupt CaVα1·CaVβ association or alter CaVα1 expression at the plasma membrane.

Plasma membrane targeting is essential for rem-mediated Ca2+channel inhibition

The small GTPase Rem is a potent negative regulator of high voltage-activated Ca2+ channels and a known interacting partner for Ca2+ channel accessory β subunits. The mechanism for Rem-mediated channel inhibition remains controversial, although it has been proposed that CaVβ association is required. Previous work has shown that a C-terminal truncation of Rem (Rem-(1–265)) displays reduced in vivo binding to membrane-localized β2a and lacks channel regulatory function. In this paper, we describe a role for the Rem C terminus in plasma membrane localization through association with phosphatidylinositol lipids. Moreover, Rem-(1–265) can associate with β2a in vitro and β1b in vivo, suggesting that the C terminus does not directly participate in CaVβ association. Despite demonstrated β1b binding, Rem-(1–265) was not capable of regulating a CaV1.2-β1b channel complex, indicating that β subunit binding is not sufficient for channel regulation. However, fusion of the CAAX domain from K-Ras4B or H-Ras to the Rem-(1–265) C terminus restored membrane localization and Ca2+ channel regulation, suggesting that β binding and membrane localization are independent events required for channel inhibition.

Rem GTPase interacts with the proximal CaV1.2 C-terminus and modulates calcium-dependent channel inactivation.

The Rem, Rem2, Rad, and Gem/Kir (RGK) GTPases, comprise a subfamily of small Ras-related GTP-binding proteins, and have been shown to potently inhibit high voltage-activated Ca2+ channel current following overexpression. Although the molecular mechanisms underlying RGK-mediated Ca2+ channel regulation remains controversial, recent studies suggest that RGK proteins inhibit Ca2+ channel currents at the plasma membrane in part by interactions with accessory channel β subunits. In this paper, we extend our understanding of the molecular determinants required for RGK-mediated channel regulation by demonstrating a direct interaction between Rem and the proximal C-terminus of CaV1.2 (PCT), including the CB/IQ domain known to contribute to Ca2+/calmodulin (CaM)-mediated channel regulation. The Rem2 and Rad GTPases display similar patterns of PCT binding, suggesting that the CaV1.2 C-terminus represents a common binding partner for all RGK proteins. In vitro Rem:PCT binding is disrupted by Ca2+/CaM, and this effect is not due to Ca2+/CaM binding to the Rem C-terminus. In addition, co-overexpression of CaM partially relieves Rem-mediated L-type Ca2+ channel inhibition and slows the kinetics of Ca2+-dependent channel inactivation. Taken together, these results suggest that the association of Rem with the PCT represents a crucial molecular determinant in RGK-mediated Ca2+ channel regulation and that the physiological function of the RGK GTPases must be re-evaluated. Rather than serving as endogenous inhibitors of Ca2+ channel activity, these studies indicate that RGK proteins may play a more nuanced role, regulating Ca2+ currents via modulation of Ca2+/CaM-mediated channel inactivation kinetics.

Pioglitazone Treatment Reduces Adipose Tissue Inflammation Through Reduction of Mast Cell and Macrophage Number and by Improving Vascularity

Context and Objective Adipose tissue in insulin resistant subjects contains inflammatory cells and extracellular matrix components. This study examined adipose pathology of insulin resistant subjects who were treated with pioglitazone or fish oil. Design, Setting and Participants Adipose biopsies were examined from nine insulin resistant subjects before/after treatment with pioglitazone 45 mg/day for 12 weeks and also from 19 subjects who were treated with fish oil (1,860 mg EPA, 1,500 mg DHA daily). These studies were performed in a clinical research center setting. Results Pioglitazone treatment increased the cross-sectional area of adipocytes by 18% (p = 0.01), and also increased capillary density without affecting larger vessels. Pioglitazone treatment decreased total adipose macrophage number by 26%, with a 56% decrease in M1 macrophages and an increase in M2 macrophages. Mast cells were more abundant in obese versus lean subjects, and were decreased from 24 to 13 cells/mm2 (p = 0.02) in patients treated with pioglitazone, but not in subjects treated with FO. Although there were no changes in total collagen protein, pioglitazone increased the amount of elastin protein in adipose by 6-fold. Conclusion The PPARγ agonist pioglitazone increased adipocyte size yet improved other features of adipose, increasing capillary number and reducing mast cells and inflammatory macrophages. The increase in elastin may better permit adipocyte expansion without triggering cell necrosis and an inflammatory reaction.

Increasing adipocyte lipoprotein lipase improves glucose metabolism in high fat diet-induced obesity.

Background: Lipoprotein lipase regulates fat uptake into adipose tissue. Results: A mouse model with increased adipose tissue lipoprotein lipase has improved glucose metabolism when challenged with a high fat diet. Conclusion: Increasing adipose tissue lipoprotein lipase improves adipose tissue function. Significance: Adipose tissue lipoprotein lipase protects against obesity-induced glucose and insulin intolerance. Lipid accumulation in liver and skeletal muscle contributes to co-morbidities associated with diabetes and obesity. We made a transgenic mouse in which the adiponectin (Adipoq) promoter drives expression of lipoprotein lipase (LPL) in adipocytes to potentially increase adipose tissue lipid storage. These mice (Adipoq-LPL) have improved glucose and insulin tolerance as well as increased energy expenditure when challenged with a high fat diet (HFD). To identify the mechanism(s) involved, we determined whether the Adipoq-LPL mice diverted dietary lipid to adipose tissue to reduce peripheral lipotoxicity, but we found no evidence for this. Instead, characterization of the adipose tissue of the male mice after HFD challenge revealed that the mRNA levels of peroxisome proliferator-activated receptor-γ (PPARγ) and a number of PPARγ-regulated genes were higher in the epididymal fat pads of Adipoq-LPL mice than control mice. This included adiponectin, whose mRNA levels were increased, leading to increased adiponectin serum levels in the Adipoq-LPL mice. In many respects, the adipose phenotype of these animals resembles thiazolidinedione treatment except for one important difference, the Adipoq-LPL mice did not gain more fat mass on HFD than control mice and did not have increased expression of genes in adipose such as glycerol kinase, which are induced by high affinity PPAR agonists. Rather, there was selective induction of PPARγ-regulated genes such as adiponectin in the adipose of the Adipoq-LPL mice, suggesting that increasing adipose tissue LPL improves glucose metabolism in diet-induced obesity by improving the adipose tissue phenotype. Adipoq-LPL mice also have increased energy expenditure.