Andrew S. Greenberg

Effect of Rosiglitazone Treatment on Nontraditional Markers of Cardiovascular Disease in Patients With Type 2 Diabetes Mellitus

Background—Markers of systemic inflammation (eg, C-reactive protein [CRP] and interleukin-6 [IL-6]) have been proposed to be “nontraditional” risk factors for cardiovascular disease in patients with type 2 diabetes mellitus. Matrix metalloproteinase-9 (MMP-9) has been implicated in the pathogenesis of atherosclerotic plaque rupture, which raises the possibility of the use of MMP-9 levels as a marker for future myocardial infarction or unstable angina. In vitro and animal studies suggest that thiazolidinediones can reduce the expression of these markers. The purpose of this analysis was to determine whether rosiglitazone alters serum concentrations of CRP, IL-6, MMP-9, and white blood cell count (WBC) and to examine the relationship of these effects with demographic and disease variables. Methods and Results—CRP, IL-6, MMP-9, and WBC were analyzed from stored frozen serum samples obtained from patients with type 2 diabetes who completed a 26-week randomized, double-blind, placebo-controlled study. After 26 weeks of rosiglitazone treatment, the percentage reductions in mean CRP, MMP-9, and WBC levels were statistically significant compared with baseline and placebo (P <0.01). The percentage reduction in mean IL-6 was small and similar in the rosiglitazone and placebo groups. The change in each inflammatory marker from baseline to week 26 was significantly correlated (P <0.05) with each of the other markers, as well as with the homeostasis model assessment estimate of insulin resistance. Conclusions—Rosiglitazone reduces serum levels of MMP-9 and the proinflammatory marker CRP in patients with type 2 diabetes, which indicates potentially beneficial effects on overall cardiovascular risk.

Adipocyte Death, Adipose Tissue Remodeling and Obesity Complications

OBJECTIVE—We sought to determine the role of adipocyte death in obesity-induced adipose tissue (AT) inflammation and obesity complications. RESEARCH DESIGN AND METHODS—Male C57BL/6 mice were fed a high-fat diet for 20 weeks to induce obesity. Every 4 weeks, insulin resistance was assessed by intraperitoneal insulin tolerance tests, and epididymal (eAT) and inguinal subcutaneous AT (iAT) and livers were harvested for histological, immunohistochemical, and gene expression analyses. RESULTS—Frequency of adipocyte death in eAT increased from <0.1% at baseline to 16% at week 12, coincident with increases in 1) depot weight; 2) AT macrophages (ATMΦs) expressing F4/80 and CD11c; 3) mRNA for tumor necrosis factor (TNF)-α, monocyte chemotactic protein (MCP)-1, and interleukin (IL)-10; and 4) insulin resistance. ATMΦs in crown-like structures surrounding dead adipocytes expressed TNF-α and IL-6 proteins. Adipocyte number began to decline at week 12. At week 16, adipocyte death reached ∼80%, coincident with maximal expression of CD11c and inflammatory genes, loss (40%) of eAT mass, widespread collagen deposition, and accelerated hepatic macrosteatosis. By week 20, adipocyte number was restored with small adipocytes, coincident with reduced adipocyte death (fourfold), CD11c and MCP-1 gene expression (twofold), and insulin resistance (35%). eAT weight did not increase at week 20 and was inversely correlated with liver weight after week 12 (r = −0. 85, P < 0.001). In iAT, adipocyte death was first detected at week 12 and remained ≤3%. CONCLUSIONS—These results implicate depot-selective adipocyte death and MΦ-mediated AT remodeling in inflammatory and metabolic complications of murine obesity.

The role of lipid droplets in metabolic disease in rodents and humans

Lipid droplets (LDs) are intracellular organelles that store neutral lipids within cells. Over the last two decades there has been a dramatic growth in our understanding of LD biology and, in parallel, our understanding of the role of LDs in health and disease. In its simplest form, the LD regulates the storage and hydrolysis of neutral lipids, including triacylglycerol and/or cholesterol esters. It is becoming increasingly evident that alterations in the regulation of LD physiology and metabolism influence the risk of developing metabolic diseases such as diabetes. In this review we provide an update on the role of LD-associated proteins and LDs in metabolic disease.

Overexpression of Perilipin A and B Blocks the Ability of Tumor Necrosis Factor α to Increase Lipolysis in 3T3-L1 Adipocytes

Perilipins, a family of phosphoproteins, are specifically located at the surface of intracellular lipid (triacylglycerol) droplets, the site of lipolysis. Stimulation of lipolysis in 3T3-L1 adipocytes by tumor necrosis factor α (TNF-α) is associated with a decrease in total cellular expression of perilipin A and B, consistent with the hypothesis that a decrease in perilipin protein expression is required for TNF-α-induced lipolysis. Adenovirus-mediated overexpression of perilipin A or B maintains perilipin protein levels on the lipid droplet and blocks TNF-α-induced lipolysis. In contrast, overexpression of perilipin A or perilipin B does not inhibit isoproterenol-stimulated lipolysis and does not alter the isoproterenol-induced migration of perilipins from the lipid droplet. These results provide the first evidence of how perilipin functions and suggest that TNF-α regulates lipolysis, in part, by decreasing perilipin protein levels at the lipid droplet surface.

Identifying the links between obesity, insulin resistance and beta-cell function: potential role of adipocyte-derived cytokines in the pathogenesis of type 2 diabetes.

A combination of insulin resistance and pancreatic β‐cell dysfunction underlies most cases of type 2 diabetes. While the interplay of these two impairments is believed to be important in the development and progression of type 2 diabetes, the mechanisms involved are unclear. A number of factors have been suggested as possibly linking insulin resistance and β‐cell dysfunction in the pathogenesis of type 2 diabetes mellitus. Pro‐inflammatory cytokines such as tumour necrosis factor‐α (TNF‐α) have deleterious effects on both glucose homeostasis and β‐cell function, and can disrupt insulin signalling pathways in both pancreatic β cells and liver and adipose tissue. The anti‐inflammatory activity of the thiazolidinedione anti‐diabetic agents is potentially beneficial, given the possible role of pro‐inflammatory cytokines in linking insulin resistance with β‐cell dysfunction.

Dynamic, M2-like Remodeling Phenotypes of CD11c+ Adipose Tissue Macrophages During High Fat Diet-Induced Obesity in Mice

OBJECTIVE To identify, localize, and determine M1/M2 polarization of epidydimal adipose tissue (eAT) macrophages (Φs) during high-fat diet (HFD)-induced obesity. RESEARCH DESIGN AND METHODS Male C57BL/6 mice were fed an HFD (60% fat kcal) or low-fat diet (LFD) (10% fat kcal) for 8 or 12 weeks. eATMΦs (F4/80+ cells) were characterized by in vivo fluorescent labeling, immunohistochemistry, fluorescence-activated cell sorting, and quantitative PCR. RESULTS Recruited interstitial macrophage galactose-type C-type lectin (MGL)1+/CD11c− and crown-like structure–associated MGL1−/CD11c+ and MGL1med/CD11c+ eATMΦs were identified after 8 weeks of HFD. MGL1med/CD11c+ cells comprised ∼65% of CD11c+ eATMΦs. CD11c+ eATMΦs expressed a mixed M1/M2 profile, with some M1 transcripts upregulated (IL-12p40 and IL-1β), others downregulated (iNOS, caspase-1, MCP-1, and CD86), and multiple M2 and matrix remodeling transcripts upregulated (arginase-1, IL-1Ra, MMP-12, ADAM8, VEGF, and Clec-7a). At HFD week 12, each eATMΦ subtype displayed an enhanced M2 phenotype as compared with HFD week 8. CD11c+ subtypes downregulated IL-1β and genes mediating antigen presentation (I-a, CD80) and upregulated the M2 hallmark Ym-1 and genes promoting oxidative metabolism (PGC-1α) and adipogenesis (MMP-2). MGL1med/CD11c+ eATMΦs upregulated additional M2 genes (IL-13, SPHK1, CD163, LYVE-1, and PPAR-α). MGL1med/CD11c+ ATMΦs expressing elevated PGC-1α, PPAR-α, and Ym-1 transcripts were selectively enriched in eAT of obese mice fed pioglitazone for 6 days, confirming the M2 features of the MGL1med/CD11c+ eATMΦ transcriptional profile and implicating PPAR activation in its elicitation. CONCLUSIONS These results 1) redefine the phenotypic potential of CD11c+ eATMΦs and 2) suggest previously unappreciated phenotypic and functional commonality between murine and human ATMΦs in the development of obesity and its complications.

Reduced energy expenditure and increased inflammation are early events in the development of ovariectomy-induced obesity.

Menopause, an age-related loss of ovarian hormone production, promotes increased adiposity and insulin resistance. However, the diet-independent mechanism by which loss of ovarian function promotes increased adipose tissue mass and associated metabolic pathologies remains unclear. To address this question, we monitored food intake and weight gain of ovariectomized (OVX) mice and sham OVX (SHM) mice for 12 wk. Although food intake was similar, OVX mice gained 25% more weight than SHM mice. Moreover, the OVX mice accumulated 4.7- and 4.4-fold more perigonadal and inguinal adipose tissue by weight, respectively, with 4.4-fold (perigonadal, P < 0.001) and 5.3-fold (inguinal, P < 0.01) larger adipocytes and no change in adipocyte cell number. OVX-induced adiposity was coincident with an 18% decrease in metabolic rate during the dark phase (P = 0.001) as well as an 11% decrease during the light phase (P = 0.03). In addition, ambulatory activity levels of OVX mice were decreased only during the dark phase (40%, P = 0.008). OVX mice displayed evidence of immune infiltration and inflammation in adipose tissue, because perigonadal and inguinal adipose depots from OVX mice had increased expression of TNFalpha, iNOS, CD11c, and other hallmarks of adipose tissue inflammation. In contrast, expression of the T cell marker CD3 (3.5-fold, P = 0.03) and Th1 cytokine interferon-gamma (IFNgamma) (2.6-fold, P = 0.02) were elevated in perigonadal but not sc fat. Finally, histology revealed OVX-specific liver hepatic steatosis, coincident with increased PPARgamma gene expression and downstream lipogenic gene expression. In summary, OVX in mice decreases energy expenditure, without altering energy intake, resulting in adipocyte hypertrophy, adipose tissue inflammation, and hepatic steatosis.

Control of adipose triglyceride lipase action by serine 517 of perilipin A globally regulates protein kinase A-stimulated lipolysis in adipocytes

Phosphorylation of the lipid droplet-associated protein perilipin A (Peri A) mediates the actions of cyclic AMP-dependent protein kinase A (PKA) to stimulate triglyceride hydrolysis (lipolysis) in adipocytes. Studies addressing how Peri A PKA sites regulate adipocyte lipolysis have relied on non-adipocyte cell models, which express neither adipose triglyceride lipase (ATGL), the rate-limiting enzyme for triglyceride catabolism in mice, nor the “downstream” lipase, hormone-sensitive lipase (HSL). ATGL and HSL are robustly expressed by adipocytes that we generated from murine embryonic fibroblasts of perilipin knock-out mice. Adenoviral expression of Peri A PKA site mutants in these cells reveals that mutation of serine 517 alone is sufficient to abrogate 95% of PKA (forskolin)-stimulated fatty acid (FA) and glycerol release. Moreover, a “phosphomimetic” (aspartic acid) substitution at serine 517 enhances PKA-stimulated FA release over levels obtained with wild type Peri A. Studies with ATGL-and HSL-directed small hairpin RNAs demonstrate that 1) ATGL activity is required for all PKA-stimulated FA and glycerol release in murine embryonic fibroblast adipocytes and 2) all PKA-stimulated FA release in the absence of HSL activity requires serine 517 phosphorylation. These results provide the first demonstration that Peri A regulates ATGL-dependent lipolysis and identify serine 517 as the Peri A PKA site essential for this regulation. The contributions of other PKA sites to PKA-stimulated lipolysis are manifested only in the presence of phosphorylated or phosphomimetic serine 517. Thus, serine 517 is a novel “master regulator” of PKA-stimulated adipocyte lipolysis.

Perilipin Promotes Hormone-sensitive Lipase-mediated Adipocyte Lipolysis via Phosphorylation-dependent and -independent Mechanisms

Hormone-sensitive lipase (HSL) is the predominant lipase effector of catecholamine-stimulated lipolysis in adipocytes. HSL-dependent lipolysis in response to catecholamines is mediated by protein kinase A (PKA)-dependent phosphorylation of perilipin A (Peri A), an essential lipid droplet (LD)-associated protein. It is believed that perilipin phosphorylation is essential for the translocation of HSL from the cytosol to the LD, a key event in stimulated lipolysis. Using adipocytes retrovirally engineered from murine embryonic fibroblasts of perilipin null mice (Peri–/– MEF), we demonstrate by cell fractionation and confocal microscopy that up to 50% of cellular HSL is LD-associated in the basal state and that PKA-stimulated HSL translocation is fully supported by adenoviral expression of a mutant perilipin lacking all six PKA sites (Peri AΔ1–6). PKA-stimulated HSL translocation was confirmed in differentiated brown adipocytes from perilipin null mice expressing an adipose-specific Peri AΔ1–6 transgene. Thus, PKA-induced HSL translocation was independent of perilipin phosphorylation. However, Peri AΔ1–6 failed to enhance PKA-stimulated lipolysis in either MEF adipocytes or differentiated brown adipocytes. Thus, the lipolytic action(s) of HSL at the LD surface requires PKA-dependent perilipin phosphorylation. In Peri–/– MEF adipocytes, PKA activation significantly enhanced the amount of HSL that could be cross-linked to and co-immunoprecipitated with ectopic Peri A. Notably, this enhanced cross-linking was blunted in Peri–/– MEF adipocytes expressing Peri AΔ1–6. This suggests that PKA-dependent perilipin phosphorylation facilitates (either direct or indirect) perilipin interaction with LD-associated HSL. These results redefine and expand our understanding of how perilipin regulates HSL-mediated lipolysis in adipocytes.

Dietary Blueberry Attenuates Whole-Body Insulin Resistance in High Fat-Fed Mice by Reducing Adipocyte Death and Its Inflammatory Sequelae

Adipose tissue (AT) inflammation promotes insulin resistance (IR) and other obesity complications. AT inflammation and IR are associated with oxidative stress, adipocyte death, and the scavenging of dead adipocytes by proinflammatory CD11c+ AT macrophages (ATMPhi). We tested the hypothesis that supplementation of an obesitogenic (high-fat) diet with whole blueberry (BB) powder protects against AT inflammation and IR. Male C57Bl/6j mice were maintained for 8 wk on 1 of 3 diets: low-fat (10% of energy) diet (LFD), high-fat (60% of energy) diet (HFD) or the HFD containing 4% (wt:wt) whole BB powder (1:1 Vaccinium ashei and V. corymbosum) (HFD+B). BB supplementation (2.7% of total energy) did not affect HFD-associated alterations in energy intake, metabolic rate, body weight, or adiposity. We observed an emerging pattern of gene expression in AT of HFD mice indicating a shift toward global upregulation of inflammatory genes (tumor necrosis factor-alpha, interleukin-6, monocyte chemoattractant protein 1, inducible nitric oxide synthase), increased M1-polarized ATMPhi (CD11c+), and increased oxidative stress (reduced glutathione peroxidase 3). This shift was attenuated or nonexistent in HFD+B-fed mice. Furthermore, mice fed the HFD+B were protected from IR and hyperglycemia coincident with reductions in adipocyte death. Salutary effects of BB on adipocyte physiology and ATMPhi gene expression may reflect the ability of BB anthocyanins to alter mitogen-activated protein kinase and nuclear factor-kappaB stress signaling pathways, which regulate cell fate and inflammatory genes. These results suggest that cytoprotective and antiinflammatory actions of dietary BB can provide metabolic benefits to combat obesity-associated pathology.