Chunxia Lu

A Novel Effect of Growth Hormone on Macrophage Modulates Macrophage-Dependent Adipocyte Differentiation

The GH receptor (GHR) is expressed on macrophages. However, the precise role of GH in regulation of macrophage function is unclear. We hypothesized that soluble factors including cytokines produced by macrophages in a GH-dependent manner regulate adipogenesis. We confirmed expression and functional integrity of the GHR in the J774A.1 macrophage cells. Conditioned medium (CM) from macrophages inhibited adipogenesis in a 3T3-L1 adipogenesis assay. CM from GH-treated macrophages decreased the inhibitory effect of CM from macrophages on adipogenesis. This effect on preadipocyte differentiation was active only during the first (early) phase of adipocyte differentiation. CM from stromal vascular compartment macrophages of mice with macrophage-specific deletion of the GHR exhibited more inhibitory effect on 3T3-L1 preadipocyte differentiation compared with CM from stromal vascular compartment macrophages of control mice, indicating that intact GH action in primary macrophages also increases preadipocyte differentiation. GH did not increase IGF-1 expression in macrophages. PCR array analysis identified IL-1beta as a candidate cytokine whose expression was altered by GH in macrophages. Levels of IL-1beta mRNA and protein were significantly decreased in GH-treated J774A.1 macrophages. Nuclear factor-kappaB stimulates IL-1beta gene expression, and GH induced a significant decrease in the levels of phosphorylated nuclear factor-kappaB in macrophages. IL-1beta is a known inhibitor of adipogenesis, and these results support GH-dependent down-regulation of macrophage IL-1beta expression as one mechanism for the observed increase in adipogenesis with CM from GH-treated macrophages. We conclude that GH decreases secretion of IL-1beta by the macrophage and thus in a paracrine manner increases adipocyte differentiation. These results provide a novel mechanism for GHs actions in the control of adipogenesis.

Targeted Deletion of Growth Hormone(GH) Receptor in Macrophage Reveals Novel Osteopontin-Mediated Effects of GH on Glucose Homeostasis and Insulin Sensitivity in Diet-Induced Obesity

Background: Pituitary growth hormone (GH) is a key hormone in the maintenance of glucose homeostasis. Results: GH acts on the macrophage to blunt the deleterious effects of diet-induced obesity (DIO) on insulin sensitivity. Conclusion: The effect of GH on glucose homeostasis is tissue-specific. Significance: Administration of GH could have salutary effects on DIO-associated chronic inflammation and insulin resistance in humans. We investigated GH action on macrophage (MΦ) by creating a MΦ-specific GH receptor-null mouse model (MacGHR KO). On a normal diet (10% fat), MacGHR KO and littermate controls exhibited similar growth profiles and glucose excursions on intraperitoneal glucose (ipGTT) and insulin tolerance (ITT) tests. However, when challenged with high fat diet (HFD, 45% fat) for 18 weeks, MacGHR KO mice exhibited impaired ipGTT and ITT compared with controls. In MacGHR KO, adipose-tissue (AT) MΦ abundance was increased with skewing toward M1 polarization. Expression of pro-inflammatory cytokines (IL1β, TNF-α, IL6, and osteopontin (OPN)) were increased in MacGHR KO AT stromal vascular fraction (SVF). In MacGHR KO AT, crown-like-structures were increased with decreased insulin-dependent Akt phosphorylation. The abundance of phosphorylated NF-κB and of OPN was increased in SVF and bone-marrow-derived MΦ in MacGHR KO. GH, acting via an NF-κB site in the distal OPN promoter, inhibited the OPN promoter. Thus in diet-induced obesity (DIO), lack of GH action on the MΦ exerts an unexpected deleterious effect on glucose homeostasis by accentuating AT inflammation and NF-κB-dependent activation of OPN expression. These novel results in mice support the possibility that administration of GH could have salutary effects on DIO-associated chronic inflammation and insulin resistance in humans.

New members of the insulin family : Regulators of metabolism, growth and now ... reproduction

Insulin, IGF, and relaxin are established members of the insulin protein superfamily. The application of the techniques of cellular, molecular, and computational biology has permitted the identification of new insulin-like ligands and their cognate receptors. Information regarding the biologic role is available for some of these newly identified ligand-receptor systems and indicates novel roles in diverse processes such as testicular descent, germ cell function, and cell migration.

Growth Hormone (GH)-dependent expression of a natural antisense transcript induces Zinc Finger E-box-binding homeobox 2 (ZEB2) in the glomerular podocyte: a novel action of GH with implications for the pathogenesis of diabetic nephropathy

Growth hormone (GH) excess results in structural and functional changes in the kidney and is implicated as a causative factor in the development of diabetic nephropathy (DN). Glomerular podocytes are the major barrier to the filtration of serum proteins, and altered podocyte function and/or reduced podocyte number is a key event in the pathogenesis of DN. We have previously shown that podocytes are a target for GH action. To elucidate the molecular basis for the effects of GH on the podocyte, we conducted microarray and RT-quantitative PCR analyses of immortalized human podocytes and identified zinc finger E-box-binding homeobox 2 (ZEB2) to be up-regulated in a GH dose- and time-dependent manner. We established that the GH-dependent increase in ZEB2 levels is associated with increased transcription of a ZEB2 natural antisense transcript required for efficient translation of the ZEB2 transcript. GH down-regulated expression of E- and P-cadherins, targets of ZEB2, and inhibited E-cadherin promoter activity. Mutation of ZEB2 binding sites on the E-cadherin promoter abolished this effect of GH on the E-cadherin promoter. Whereas GH increased podocyte permeability to albumin in a paracellular albumin influx assay, shRNA-mediated knockdown of ZEB2 expression abrogated this effect. We conclude that GH increases expression of ZEB2 in part by increasing expression of a ZEB2 natural antisense transcript. GH-dependent increase in ZEB2 expression results in loss of P- and E-cadherins in podocytes and increased podocyte permeability to albumin. Decreased expression of P- and E-cadherins is implicated in podocyte dysfunction and epithelial-mesenchymal transition observed in DN. We speculate that the actions of GH on ZEB2 and P- and E-cadherin expression play a role in the pathogenesis of microalbuminuria of DN.

Lipopolysaccharide (LPS) directly suppresses growth hormone receptor (GHR) expression through MyD88-dependent and -independent Toll-like receptor-4/MD2 complex signaling pathways

INTRODUCTION Sepsis is associated with growth hormone (GH) insensitivity and in the intact animal the major surface component of the bacterial cell wall, lipopolysaccharide (LPS), inhibits GH receptor (GHR) gene expression. The prevailing explanation for LPS-induced effects on the GHR promoter is that this effect is indirect via generation of cytokines. Our recent studies demonstrate that saturated free fatty acids (FFAs) inhibit the activity of the murine GHR promoter. Saturated FFAs are an essential component of the lipid A moiety of LPS required for biological activity of LPS. HYPOTHESIS LPS directly modulates the activity of the dominant GHR promoter via interaction with Toll-like receptor(s) (TLR)/MD2 complex and activation of cognate signaling pathway(s). RESULTS In transient transfection experiments with RAW 264.7 cells which express endogenous TLR4 and MD2, LPS treatment inhibited GHR promoter activity. Co-transfection of dominant negative TLR4 abrogated this effect on GHR promoter activity. In HEK 293T cells, which are devoid of endogenous TLR4 or MD2, ectopic expression of TLR4 and MD2 resulted in LPS-induced inhibition of the GHR promoter activity. The inhibition of GHR promoter activity was demonstrable by 5-6h after exposure to LPS and persisted at 24h. Fatty-acid free LPS failed to elicit a similar effect on the GHR promoter and the effect of LPS was abrogated by Polymyxin B. The essential role of the cofactor MD2 on the effect of LPS on the GHR promoter was established in experiments using ectopic expression of wild type and mutant MD2. Cotransfection of CD14 in these cells failed to alter the effect of LPS on the activity of the GHR promoter. Analysis of cell culture supernatant excluded the possibility that the effect of LPS was secondary to release of cytokines from the transfected cells. The effect of LPS on the endogenous GHR promoter activity and protein expression was confirmed in F442A preadipocyte cells. In HEK 293T cells, ectopic expression of mutant MyD88 or mutant TRIF abrogated the effect of LPS on the GHR promoter, suggesting that the effect of LPS on the GHR promoter was via both MyD88-dependent and -independent pathways. CONCLUSIONS LPS acts through both MyD88-dependent and -independent TLR4 signaling pathways to directly inhibit GHR gene expression. Our results establish a novel cytokine-independent mechanism for decrease in GHR expression in bacterial sepsis.

The expanding insulin family: structural, genomic, and functional considerations

Kasik JW, Lu C, Menon RK. The expanding insulin family: structural, genomic, and functional considerations. Pediatric Diabetes 2000: 1: 169–177.

Growth hormone (GH) differentially regulates NF-kB activity in preadipocytes and macrophages: implications for GH’s role in adipose tissue homeostasis in obesity

Adipose tissue remodeling in obesity involves macrophage infiltration and chronic inflammation. NF-kB-mediated chronic inflammation of the adipose tissue is directly implicated in obesity-associated insulin resistance. We have investigated the effect of growth hormone (GH) on NF-kB activity in preadipocytes (3T3-F442A) and macrophages (J774A.1). Our studies indicate that whereas GH increases NF-kB activity in preadipocytes, it decreases NF-kB activity in macrophages. This differential response of NF-kB activity to GH correlates with the GH-dependent expression of a cadre of NF-kB-activated cytokines in these two cell types. Activation of NF-kB by GH in preadipocytes heightens inflammatory response by stimulating production of multiple cytokines including TNF-α, IL-6, and MCP-1, the mediators of both local and systemic insulin resistance and chemokines that recruit macrophages. Our studies also suggest differential regulation of miR132 and SIRT1 expression as a mechanism underlying the observed variance in GH-dependent NF-kB activity and altered cytokine profile in preadipocytes and macrophages. These findings further our understanding of the complex actions of GH on adipocytes and insulin sensitivity.

Abrogation of GH action in Kupffer cells results in increased hepatic CD36 expression and exaggerated nonalcoholic fatty liver disease

OBJECTIVE To investigate the effects of GH signaling on Kupffer cells and the resulting changes in lipid homeostasis and their underlying mechanism(s) in the livers of diet-induced obese (DIO) mice. DESIGN Male macrophage specific-growth hormone receptor knockout mice (MacGHR KO) and their litter mate controls were fed a high fat diet containing 60% calories from fat for 26 weeks. Lipid content and lipid profiles in the liver and circulation were analyzed. Expression levels of CD36 in the liver were quantified by RT-PCR and Western Blot. RESULTS Increased hepatic lipid content and abundance of long-chain unsaturated fatty acids were observed in the liver of MacGHR KO mice. These findings were associated with increased steady state levels of CD36 mRNA and protein in MacGHR KO mice when compared with their litter mate controls. CONCLUSION GH action in Kupffer cells is required for maintaining hepatic lipid homeostasis, in part via regulation of hepatic CD36 expression.

Esterase 1 is a Novel Transcriptional Repressor of Growth Hormone Receptor Gene Expression: A Unique Noncatalytic Role for a Carboxyesterase Protein

The pleiotropic actions of GH result from its engagement with the GH receptor (GHR). GHR expression is regulated by free fatty acids (FFA). A cDNA phage expression library was screened to identify a phage clone expressing esterase 1 (ES1) binding to the FFA-response element (FARE), L2-D1, in the murine GHR promoter. Ectopically expressed ES1 inhibited GHR promoter activity via effects at two FARE, L2-D1 and L2-A2. Chromatin immunoprecipitation experiments demonstrated specific association of ES1 with the FARE. Catalytically inactive ES1 retained inhibitory activity on the GHR promoter and excluded the possibility that the effect on the GHR promoter was an indirect effect secondary to ES1s actions on the intracellular metabolism of FFA. Ectopically expressed ES1 inhibited the endogenous GHR mRNA and protein expression in 3T3-F442A preadipocytes. Subcellular fractionation and confocal microscopy established that ES1 localizes both to the cytoplasm and the nucleus. Experiments demonstrated chromosome region maintenance 1-dependent nuclear export and the presence of a functional nuclear export signal in ES1. The domain of ES1 responsible for the effect on the GHR promoter was localized to the C-terminal portion of the protein. The in vivo significance of ES1s effect on GHR expression was suggested by decreased liver GHR mRNA expression in mice on a high-fat diet correlating with increased steady-state abundance of liver ES1 mRNA. Our results identify and characterize ES1 as a novel transcriptional regulator of GHR gene expression, thereby establishing a unique nonenzymatic role for a carboxyesterase and expanding the potential biological roles of this protein superfamily.