Radmila Micanovic

FGF-21 as a novel metabolic regulator

Diabetes mellitus is a major health concern, affecting more than 5% of the population. Here we describe a potential novel therapeutic agent for this disease, FGF-21, which was discovered to be a potent regulator of glucose uptake in mouse 3T3-L1 and primary human adipocytes. FGF-21-transgenic mice were viable and resistant to diet-induced obesity. Therapeutic administration of FGF-21 reduced plasma glucose and triglycerides to near normal levels in both ob/ob and db/db mice. These effects persisted for at least 24 hours following the cessation of FGF-21 administration. Importantly, FGF-21 did not induce mitogenicity, hypoglycemia, or weight gain at any dose tested in diabetic or healthy animals or when overexpressed in transgenic mice. Thus, we conclude that FGF-21, which we have identified as a novel metabolic factor, exhibits the therapeutic characteristics necessary for an effective treatment of diabetes.

FGF-21/FGF-21 receptor interaction and activation is determined by βKlotho

Fibroblast growth factor‐21 (FGF‐21) is a metabolic regulator that can influence glucose and lipid control in diabetic rodents and primates. We demonstrate that βKlotho is an integral part of an activated FGF‐21‐βKlotho‐FGF receptor (FGFR) complex thus a critical subunit of the FGF‐21 receptor. Cells lacking βKlotho did not respond to FGF‐21; the introduction of βKlotho to these cells conferred FGF‐21‐responsiveness and recapitulated the entire scope of FGF‐21 signaling observed in naturally responsive cells. Interestingly, FGF‐21‐mediated effects are heparin independent suggesting that βKlotho plays a role in FGF‐21 activity similar to the one played by heparin in the signaling of conventional FGFs. Moreover, in addition to conferring specificity for FGF‐21, βKlotho appears to support FGF‐19 activity and mediates the receptor selectivity profile of FGF‐19. All together, these results indicate that βKlotho and FGFRs form the cognate FGF‐21 receptor complex, mediating FGF‐21 cellular specificity and physiological effects. J. Cell. Physiol. 215: 1–7, 2008.

Rational Design of a Fibroblast Growth Factor 21-Based Clinical Candidate, LY2405319

Fibroblast growth factor 21 is a novel hormonal regulator with the potential to treat a broad variety of metabolic abnormalities, such as type 2 diabetes, obesity, hepatic steatosis, and cardiovascular disease. Human recombinant wild type FGF21 (FGF21) has been shown to ameliorate metabolic disorders in rodents and non-human primates. However, development of FGF21 as a drug is challenging and requires re-engineering of its amino acid sequence to improve protein expression and formulation stability. Here we report the design and characterization of a novel FGF21 variant, LY2405319. To enable the development of a potential drug product with a once-daily dosing profile, in a preserved, multi-use formulation, an additional disulfide bond was introduced in FGF21 through Leu118Cys and Ala134Cys mutations. FGF21 was further optimized by deleting the four N-terminal amino acids, His-Pro-Ile-Pro (HPIP), which was subject to proteolytic cleavage. In addition, to eliminate an O-linked glycosylation site in yeast a Ser167Ala mutation was introduced, thus allowing large-scale, homogenous protein production in Pichia pastoris. Altogether re-engineering of FGF21 led to significant improvements in its biopharmaceutical properties. The impact of these changes was assessed in a panel of in vitro and in vivo assays, which confirmed that biological properties of LY2405319 were essentially identical to FGF21. Specifically, subcutaneous administration of LY2405319 in ob/ob and diet-induced obese (DIO) mice over 7–14 days resulted in a 25–50% lowering of plasma glucose coupled with a 10–30% reduction in body weight. Thus, LY2405319 exhibited all the biopharmaceutical and biological properties required for initiation of a clinical program designed to test the hypothesis that administration of exogenous FGF21 would result in effects on disease-related metabolic parameters in humans.

Fundamentals of FGF19 & FGF21 action in vitro and in vivo.

Fibroblast growth factors 19 (FGF19) and 21 (FGF21) have emerged as key regulators of energy metabolism. Several studies have been conducted to understand the mechanism of FGF19 and FGF21 action, however, the data presented has often been inconsistent and at times contradictory. Here in a single study we compare the mechanisms mediating FGF19/FGF21 actions, and how similarities/differences in actions at the cellular level between these two factors translate to common/divergent physiological outputs. Firstly, we show that in cell culture FGF19/FGF21 are very similar, however, key differences are still observed differentiating the two. In vitro we found that both FGFs activate FGFRs in the context of βKlotho (KLB) expression. Furthermore, both factors alter ERK phosphorylation and glucose uptake with comparable potency. Combination treatment of cells with both factors did not have additive effects and treatment with a competitive inhibitor, the FGF21 delta N17 mutant, also blocked FGF19s effects, suggestive of a shared receptor activation mechanism. The key differences between FGF21/FGF19 were noted at the receptor interaction level, specifically the unique ability of FGF19 to bind/signal directly via FGFR4. To determine if differential effects on energy homeostasis and hepatic mitogenicity exist we treated DIO and ob/ob mice with FGF19/FGF21. We find comparable efficacy of the two proteins to correct body weight and serum glucose in both DIO and ob/ob mice. Nevertheless, FGF21 and FGF19 had distinctly different effects on proliferation in the liver. Interestingly, in vivo blockade of FGF21 signaling in mice using ΔN17 caused profound changes in glycemia indicative of the critical role KLB and FGF21 play in the regulation of glucose homeostasis. Overall, our data demonstrate that while subtle differences exist in vitro the metabolic effects in vivo of FGF19/FGF21 are indistinguishable, supporting a shared mechanism of action for these two hormones in the regulation of energy balance.

pI-shifted insulin analogs with extended in vivo time action and favorable receptor selectivity

A long-acting (basal) insulin capable of delivering flat, sustained, reproducible glycemic control with once daily administration represents an improvement in the treatment paradigm for both type 1 and type 2 diabetes. Optimization of insulin pharmacodynamics is achievable through structural modification, but often at the expense of alterations in receptor affinity and selectivity. A series of isoelectric point (pI)-shifted insulin analogs based on the human insulin sequence or the GlyA21 acid stable variant were prepared by semi-synthetic methods. The pI shift was achieved through systematic addition of one or more arginine (Arg) or lysine (Lys) residues at the N terminus of the A chain, the N terminus of the B chain, the C terminus of the B chain, or through a combination of additions at two of the three sites. The analogs were evaluated for their affinity for the insulin and IGF-1 receptors, and aqueous solubility under physiological pH conditions. Notably, the presence of positively charged amino acid residues at the N terminus of the A chain was consistently associated with an enhanced insulin to IGF-1 receptor selectivity profile. Increased IGF-1 receptor affinity that results from Arg addition to the C terminus of the B chain was attenuated by cationic extension at the N terminus of the A chain. Analogs 10, 17, and 18 displayed in vitro receptor selectivity similar to that of native insulin and solubility at physiological pH that suggested the potential for extended time action. Accordingly, the in vivo pharmacokinetic and pharmacodynamic profiles of these analogs were established in a somatostatin-induced diabetic dog model. Analog 18 (A0:Arg, A21:Gly, B31:Arg, B32:Arg human insulin) exhibited a pharmacological profile comparable to that of analog 15 (insulin glargine) but with a 4.5-fold more favorable insulin:IGF-1 receptor selectivity. These results demonstrate that the selective combination of positive charge to the N terminus of the A chain and the C terminus of the B chain generates an insulin with sustained pharmacology and a near-native receptor selectivity profile.

Cooperativity and binding in the mechanism of cytosolic phospholipase A2.

Cytosolic phospholipase A2 (cPLA2) hydrolyzes the sn-2 ester of phospholipids and is believed to be responsible for the receptor-regulated release of arachidonic acid from phospholipid pools. The enzyme was assayed using vesicles containing arachidonate-containing phospholipid substrate, such as 1-palmitoyl-2-arachidonoylphosphatidylcholine (PAPC) or 1-stearoyl-2-arachidonoylphosphatidylinositol (SAPI), dispersed within vesicles of 1,2-dimyristoylphosphatidylmethanol (DMPM). We report here that the enzyme shows an apparent cooperative effect with respect to the mole fraction of arachidonate-containing phospholipids within these covesicles. The data can be fit to a modified Hill equation yielding Hill coefficients, n, of 2-3. This effect is unusual in that it is dependent on the nature of the sn-2 ester as opposed to the phosphoglycerol head group. This cooperativity is independent of both the concentration of glycerol, which greatly increases enzyme activity and stability, and the concentration of calcium, which facilitates the fusion of the covesicles. Surprisingly, 1-palmitoyl-2-arachidonoylphosphatidylethanolamine (PAPE) does not show the same cooperative effect, although the rate at which it is hydrolyzed is much greater when PAPC is present. Moreover, PAPE has a dissociation constant from the active site (KD* = 0.7 mol %) which is comparable to that of PAPC and SAPI (KD* values of 0.3 and 0.3 mol %, respectively). These results are consistent with the presence of an allosteric site that, when occupied, induces a change in the enzyme which facilitates enzymatic hydrolysis. If so, PAPC and SAPI, but not PAPE, must be able to bind to this allosteric site. Alternatively, this effect may result from changes in the physical nature of the bilayer which result upon increasing the bilayer concentration of arachidonate-containing phospholipids. This previously unobserved effect may represent another mechanism by which cells can regulate the activity of cPLA2.

Decoy receptor 3 (DcR3) is proteolytically processed to a metabolic fragment having differential activities against Fas ligand and LIGHT.

Fas ligand (FasL) and Fas receptor are members of the tumor necrosis factor (TNF) receptor and ligand family that play an important role in regulating apoptosis in normal physiology. Decoy receptor 3 (DcR3) is a novel member of the TNF receptor superfamily, which binds to and blocks the activities of the ligands FasL and LIGHT. We have demonstrated that DcR3 was degraded rapidly to a major circulating metabolic fragment after subcutaneous administration in primates and mice. This fragment was also generated in subcutaneous tissue homogenate in vitro. Mass spectrometry and N-terminal sequencing indicated that DcR3 was proteolytically cleaved between R218 and A219 in the primary sequence to yield the fragment DcR3(1-218). While retaining its ability to bind LIGHT and inhibit LIGHT-mediated activities, DcR3(1-218) no longer bound FasL and did not inhibit FasL-mediated apoptosis in vitro. The primary sequence of DcR3 was molecularly engineered, changing the arginine residue at position 218 to glutamine to generate an analog, DcR3(R218Q), which we termed FLINT (LY498919). We demonstrated that FLINT was more stable to proteolytic degradation in vitro and in vivo and maintained its activity against both soluble FasL and soluble LIGHT in vitro. As a result, the modification in the sequence of DcR3 to produce FLINT (LY498919) should result in a pharmacologically superior molecule in the therapeutic intervention of diseases in which the pathogenesis is linked to FasL-mediated apoptotic or inflammatory events.

Tamm-Horsfall Protein Regulates Granulopoiesis and Systemic Neutrophil Homeostasis

Tamm-Horsfall protein (THP) is a glycoprotein uniquely expressed in the kidney. We recently showed an important role for THP in mediating tubular cross-talk in the outer medulla and in suppressing neutrophil infiltration after kidney injury. However, it remains unclear whether THP has a broader role in neutrophil homeostasis. In this study, we show that THP deficiency in mice increases the number of neutrophils, not only in the kidney but also in the circulation and in the liver, through enhanced granulopoiesis in the bone marrow. Using multiplex ELISA, we identified IL-17 as a key granulopoietic cytokine specifically upregulated in the kidneys but not in the liver of THP(-/-) mice. Indeed, neutralization of IL-17 in THP(-/-) mice completely reversed the systemic neutrophilia. Furthermore, IL-23 was also elevated in THP(-/-) kidneys. We performed real-time PCR on laser microdissected tubular segments and FACS-sorted renal immune cells and identified the S3 proximal segments, but not renal macrophages, as a major source of increased IL-23 synthesis. In conclusion, we show that THP deficiency stimulates proximal epithelial activation of the IL-23/IL-17 axis and systemic neutrophilia. Our findings provide evidence that the kidney epithelium in the outer medulla can regulate granulopoiesis. When this novel function is added to its known role in erythropoiesis, the kidney emerges as an important regulator of the hematopoietic system.

Quantitative Three-Dimensional Tissue Cytometry to Study Kidney Tissue and Resident Immune Cells

Analysis of the immune system in the kidney relies predominantly on flow cytometry. Although powerful, the process of tissue homogenization necessary for flow cytometry analysis introduces bias and results in the loss of morphologic landmarks needed to determine the spatial distribution of immune cells. An ideal approach would support three-dimensional (3D) tissue cytometry: an automated quantitation of immune cells and associated spatial parameters in 3D image volumes collected from intact kidney tissue. However, widespread application of this approach is limited by the lack of accessible software tools for digital analysis of large 3D microscopy data. Here, we describe Volumetric Tissue Exploration and Analysis (VTEA) image analysis software designed for efficient exploration and quantitative analysis of large, complex 3D microscopy datasets. In analyses of images collected from fixed kidney tissue, VTEA replicated the results of flow cytometry while providing detailed analysis of the spatial distribution of immune cells in different regions of the kidney and in relation to specific renal structures. Unbiased exploration with VTEA enabled us to discover a population of tubular epithelial cells that expresses CD11C, a marker typically expressed on dendritic cells. Finally, we show the use of VTEA for large-scale quantitation of immune cells in entire human kidney biopsies. In summary, we show that VTEA is a simple and effective tool that supports unique digital interrogation and analysis of kidney tissue from animal models or biobanked human kidney biopsies. We have made VTEA freely available to interested investigators via electronic download.

The Role of Tumor Necrosis Factor Alpha in Regulating the Expression of Tamm-Horsfall Protein (Uromodulin) in Thick Ascending Limbs during Kidney Injury

Background: Tamm-Horsfall Protein (THP) is a glycoprotein expressed exclusively by cells of the thick ascending loop (TAL) of Henle. THP has a protective role in acute kidney injury (AKI), and its expression is downregulated in the early stages of injury. Tumor necrosis factor alpha (TNFE) is a cytokine endogenously expressed by the TAL and is also induced by AKI. Therefore, we hypothesized that TNFE is a key regulator of THP expression. Methods: We used a mouse model of AKI (ischemia-reperfusion injury, IRI) and a cell culture system of a TAL cell line (MKTAL). Results: We show that TNFE is upregulated by TAL cells early after AKI in vivo. The expression of THP and its transcription factor Hepatocyte nuclear factor 1F (HNF1F) were concomitantly decreased at the peak of injury. Furthermore, recombinant TNFE inhibits significantly, and in a dose-dependent manner, the expression of THP, but not HNF1F in MKTAL cells. Interestingly, neither TNFE neutralization nor genetic deletion of TNFE increased THP or HNF levels after injury in vivo. Conclusion: Our data suggest that TNFE can inhibit the expression of THP in TAL cells via an HNF1F-independent mechanism, but the downregulation of THP expression in the early AKI does not depend on TNFE. We propose that TNFE regulates THP expression in a homeostatic setting, but the impact of TNFE on THP during kidney injury is superseded by other factors that could inhibit HNF1F-mediated expression of THP. i 2014 S. Karger AG, Basel