José M. Gallego-Escuredo

Opposite alterations in FGF21 and FGF19 levels and disturbed expression of the receptor machinery for endocrine FGFs in obese patients

Objective:Fibroblast growth factor (FGF)-21, and possibly FGF19, protect against type 2 diabetes mellitus (T2DM) and obesity in rodents. We investigated the circulating levels of FGF21 and FGF19 in obese patients with varying degrees of abnormal glucose homeostasis, and we determined gene expression for FGF receptors (FGFR1–4) and the co-receptor β-Klotho, in liver and adipose tissues.Subjects and methods:We analyzed 35 lean healthy (71% men) and 61 obese patients (49% men, median body mass index (BMI): 40.5 kg m−2, interquartile range: 34.7–46.2). Among obese patients, 36 were normoglycemic, 15 showed impaired glucose tolerance and 10 had T2DM. Biopsies from liver and visceral and subcutaneous fat from a subset of obese patients and controls were analyzed. FGF19 and FGF21 levels were measured using enzyme-linked immunosorbent assay, and tissue mRNA and protein levels by reverse transcription-polymerase chain reaction and immunoblotting.Results:FGF21 serum levels were significantly increased in obese patients compared with controls (P<0.001), whereas FGF19 levels were decreased (P<0.001). FGF21 levels were positively correlated with homeostasis model assessment of insulin resistance (P=0.0002, r=0.37) and insulin (P=0.001, r=0.32), whereas FGF19 levels were negatively correlated (P=0.007, r=−0.27; P=0.003, r=−0.28; respectively). After adjusting for BMI, the correlations of FGF21 and FGF19 levels with indicators of abnormal glucose homeostasis were not significant. In obese patients, the hepatic expression of FGF21 was increased. (P=0.04). β-Klotho transcript levels in visceral fat (P=0.002) and β-Klotho protein levels in subcutaneous (P=0.03) and visceral fat (P=0.04) were significantly reduced in obese patients, whereas hepatic levels for β-Klotho (P=0.03), FGFR1 (P=0.04) and FGFR3 (P=0.001) transcripts were significantly increased.Conclusions:Obesity is characterized by reciprocal alterations in FGF19 (decrease) and FGF21 (increase) levels. Although worsened in diabetic obese patients, obesity itself appears as the predominant determinant of the abnormalities in FGF21 and FGF19 levels. Opposite changes in β-Klotho expression in fat and liver indicate potential tissue-specific alterations in the responsiveness to endocrine FGFs in obesity.

Fibroblast growth factor-21 is expressed in neonatal and pheochromocytoma-induced adult human brown adipose tissue.

OBJECTIVE In rodents, brown (BAT) and white (WAT) adipose tissues are targets and expression sites for fibroblast growth factor-21 (FGF21). In contrast, human WAT expresses negligible levels of FGF21. We examined FGF21 expression in human BAT samples, including the induced BAT found in adult patients with pheochromocytoma, and interscapular and visceral BAT from newborns. METHODS The expression of FGF21 and uncoupling protein-1 (UCP1, a brown adipocyte marker), was determined by quantitative real-time-PCR and immunoblotting. The transcript levels of marker genes for developmentally-programmed BAT (zinc-finger-protein of the cerebellum-1, ZIC1) and inducible-BAT (cluster of differentiation-137, CD137) were also determined. RESULTS FGF21 and UCP1 are significantly expressed in visceral adipose tissue from pheochromocytoma patients, but not in visceral fat from healthy individuals. In neonates, FGF21 and UCP1 are both expressed in visceral and interscapular fat, and their expression levels show a significant positive correlation. Marker gene expression profiles suggest that inducible BAT is present in visceral fat from pheochromocytoma patients and neonates, whereas developmentally-programmed BAT is present in neonatal interscapular fat. CONCLUSIONS Human BAT, but not WAT, expresses FGF21. The expression of FGF21 is especially high in inducible, also called beige/brite, neonatal BAT, but it is also found in the interscapular, developmentally-programmed, BAT of neonates.

Differential gene expression indicates that ‘buffalo hump’ is a distinct adipose tissue disturbance in HIV-1-associated lipodystrophy

Objective:To elucidate the molecular basis of the progressive enlargement of dorso-cervical adipose tissue, the so-called ‘buffalo hump’, that appears in a sub-set of patients with HIV-1/HAART-associated lipodystrophy. Design:Analysis of the expression of marker genes of mitochondrial function, adipogenesis, inflammation and cell proliferation in ten ‘buffalo hump’ samples and ten subcutaneous fat samples from HIV-1-infected/HAART-treated patients, and in ten healthy controls. Methods:Quantitative real-time polymerase chain reaction analysis of mitochondrial DNA and gene transcripts, and immunoblot for specific proteins. Results:‘Buffalo hump’ patients had lower levels of mitochondrial DNA and mitochondrial DNA-encoded transcripts with respect to healthy controls. The uncoupling protein (UCP)-1 gene was expressed only in ‘buffalo hump’ fat. There were no significant changes in the expression of UCP2, UCP3 or of marker genes of adipogenesis in ‘buffalo hump’ patients relative to healthy controls. ‘Buffalo hump’ fat did not show the high expression of tumor necrosis factor-α and β2-microglobulin identified in lipoatrophic subcutaneous fat from patients. The expression of the macrophage marker CD68 was also lower in ‘buffalo hump’ than in subcutaneous fat from patients. In contrast, ‘buffalo hump’ showed a higher expression of the cell proliferation marker PCNA. Conclusions:‘Buffalo hump’ adipose tissue shows specific disturbances in gene expression with respect to subcutaneous fat from HIV-1-infected/HAART-treated patients. Mitochondrial alterations cannot explain the differential behavior of ‘buffalo hump’ with respect to adipose depots prone to lipoatrophy. The absence of a local inflammatory status in ‘buffalo hump’ may explain in part the differential behavior of this adipose tissue.

Serum FGF21 levels are elevated in association with lipodystrophy, insulin resistance and biomarkers of liver injury in HIV-1-infected patients.

Objective:HIV-1-infected patients with lipodystrophy show insulin resistance, dyslipidemia and other signs of metabolic syndrome. Fibroblast growth factor-21 (FGF21) is a novel metabolic regulator that has been suggested to exert beneficial effects on metabolic homeostasis and insulin sensitivity. Our goal was to determine the relationship between FGF21 levels and metabolic alterations in these patients. Research design and methods:Serum FGF21 levels were analyzed in 179 individuals belonging to four groups: HIV-1-infected, antiretroviral-treated patients that have developed lipodystrophy (n = 59); HIV-1-infected, antiretroviral-treated patients without lipodystrophy (n = 45); untreated (naive) HIV-1-infected patients (n = 41); and healthy control individuals (n = 34). Serum FGF21 levels were correlated with parameters indicative of altered fat distribution, metabolic and cardiovascular risk, and in relation to HIV-1 infection and antiretroviral treatment regimens. Results:Serum FGF21 levels were increased in all HIV-1-infected patients, but the increases were most marked in those with lipodystrophy. FGF21 levels showed a strong positive correlation with indicators of lipodystrophy (trunk/apendicular fat ratio, waist-to-hip ratio), insulin resistance (fasting glucose, HOMA-R), dyslipidemia (low-density lipoprotein cholesterol), and liver injury (γ-glutamyltransferase). Conclusions:FGF21 levels are increased in HIV-1-infected patients, especially in those with lipodystrophy, and this increase is closely associated with insulin resistance, metabolic syndrome and makers of liver damage. Further research will be required to determine whether the increase in FGF21 levels is caused by a compensatory response or resistance to FGF21, and to establish the potential of FGF21 as a biomarker of altered metabolism in HIV-1-infected, antiretroviral-treated patients.

Effects of nevirapine and efavirenz on human adipocyte differentiation, gene expression, and release of adipokines and cytokines

The non-nucleoside reverse transcriptase inhibitors (NNRTIs) nevirapine and efavirenz are drugs of choice for initial antiretroviral treatment for HIV-1 infection. Although NNRTIs have not traditionally been associated with the appearance of adipose alterations, recent data suggest that efavirenz may contribute to adipose tissue alterations in antiretroviral-treated patients, consistent with its ability to impair differentiation of adipocytes in cell cultures. No such effects have been reported for nevirapine, the other most commonly used NNRTI. In this study, we determined the effects of nevirapine on differentiation, gene expression and release of regulatory proteins (adipokines and cytokines) in differentiating human adipocytes, and compared them with those of efavirenz. Efavirenz caused a dose-dependent repression of adipocyte differentiation that was associated with down-regulation of the master adipogenesis regulator genes SREBP-1, PPARγ and C/EBPα, and their target genes encoding lipoprotein lipase, leptin and adiponectin, which are key proteins in adipocyte function. In contrast, nevirapine does not affect adipogenesis and causes a modest but significant coordinate increase in the expression of SREBP-1, PPARγ and C/EBPα and their target genes only at a concentration of 20 μM. Whereas efavirenz caused a significant increase in the release of pro-inflammatory cytokines (interleukin [IL]-8, IL-6, monocyte chemoattractant protein-1), plasminogen activator inhibitor type-1 and hepatocyte growth factor (HGF), nevirapine either had no effect on these factors or decreased their release (IL-6 and HGF). Nevirapine significantly increased adiponectin release, whereas efavirenz strongly repressed it. Moreover, nevirapine inhibited preadipocyte endogenous reverse transcriptase activity, whereas efavirenz did not alter it. It is concluded that, in contrast with the profound anti-adipogenic and pro-inflammatory response elicited by efavirenz, nevirapine does not impair adipogenesis.

Differential Effects of Efavirenz and Lopinavir/Ritonavir on Human Adipocyte Differentiation, Gene Expression and Release of Adipokines and Pro-Inflammatory Cytokines.

In the present study, a comparative assessment of the effects of efavirenz (EFV) and lopinavir/ritonavir (LPV/r; 4:1) on human adipocytes in culture has been performed. Human pre-adipocytes were treated with EFV or LPV/r during or after adipogenic differentiation. Acquisition of adipocyte morphology, expression of gene markers of mitochondrial toxicity, adipogenesis and inflammation, and release of adipokines and cytokines to the medium were measured. Results indicated that EFV and LPV/r impaired adipocyte differentiation in association with a reduction in transcript levels for adipogenic differentiation genes (adiponectin, lipoprotein lipase, leptin) and master regulators of adipogenesis (PPAR, C/EBP). The effects were greater with EFV than LPV/r. Both LPV/r and EFV induced increases in monocytechemoattactant protein-1 (MCP-1) mRNA levels, but the effect was greater with EFV. Similarly, the release of proinflammatory cytokines and other inflammation-related molecules (interleukins 6 and 8, MCP-1, PAI-1) was enhanced to a much higher degree by EFV than by LPV/r. Adiponectin and leptin release by adipocytes was reduced by both drugs, although to a higher extent by EFV. Neither drug affected mitochondrial DNA levels, transcripts encoding mitochondrial proteins or lactate release by adipocytes. In previously differentiated adipocytes, EFV caused a significant reduction in PPARγ and adiponectin expression, whereas LPV/r did not. We conclude that both EFV and LPV/r impair human adipogenesis, reduce adipokine release and increase the expression and release of inflammation-related cytokines, but the overall effects are greater with EFV. These findings may have implications for the pathogenesis of HIV-1-associated lipodystrophy and the development of HIV-1 therapies.

Differentially altered molecular signature of visceral adipose tissue in HIV-1-associated lipodystrophy.

Objective:Lipodystrophy in HIV-1–infected antiretroviral-treated patients is often associated with opposite alterations in adipose tissue depots as follows: lipoatrophy of subcutaneous adipose tissue (SAT) versus lipohypertrophy of visceral adipose tissue (VAT). We determined the specific molecular alterations in VAT relative to SAT in patients. Design:We analyzed the expression of marker genes of mitochondrial function, adipogenesis, and inflammation in a unique collection of 8 biopsies of omental VAT from HIV-1–infected antiretroviral-treated patients with lipodystrophy. For comparison, we analyzed SAT from 10 patients, and SAT and VAT from 10 noninfected individuals. Methods:Quantitative real-time polymerase chain reaction of mitochondrial DNA and gene transcripts; immunoblot and multiplex for quantification of specific proteins. Results:Similar mitochondrial DNA depletion and abnormal increases in mitochondrial protein levels were found in VAT and SAT from patients. Transcript levels of adipogenesis and metabolism marker genes were unaltered in VAT but were decreased in SAT. Tumor necrosis factor &agr; and CD68 were similarly induced in both adipose depots from patients, but other markers of inflammation-related pathways showed distinct alterations as follows: interleukin 18 and interleukin 1 receptor antagonist were induced only in SAT, whereas interleukin 6, interleukin 8, and monocyte chemoattractant protein 1 expression was reduced in VAT but not in SAT. Conclusions:Mitochondrial alterations are similar in VAT and SAT from patients, whereas adipogenic gene expression is decreased in SAT but unaltered in VAT, highlighting the relevance of adipogenic processes in the differential alterations of fat depots. Specific disturbances in inflammatory status in VAT relative to SAT are present. Milder induction of proinflammatory signaling in VAT could be involved in preventing fat wasting in this depot.

Genetic and Functional Mitochondrial Assessment of HIV-Infected Patients Developing HAART-Related Hyperlactatemia

Background:Mitochondrial damage of HIV and antiretrovirals, especially nucleoside-analogue interference on mitochondrial DNA (mtDNA) replication, is reported to underlay highly active antiretroviral therapy (HAART)-related hyperlactatemia, but scarce approaches have been performed to correlate clinical manifestations and mitochondrial abnormalities. Methods:We obtained lymphocytes and monocytes of 26 HIV-infected and treated patients who developed hyperlactatemia and after recovery, 28 nonhyperlactatemic HIV subjects on HAART, 31 naive individuals, and 20 uninfected controls. Mitochondrial replication and transcription analysis were performed by quantitative real-time PCR, mitochondrial translation quantification by western blot and mitochondrial enzymatic activities by spectrophotometry. Results:Mitochondrial parameters decreased during hyperlactatemia and improved at recovery. Mitochondrial replication and transcription species were reduced (P = 0.16 and P = 0.71), but the most significant decay was observed on mitochondrial protein content (P < 0.05) and mitochondrial complexes III and IV activities (P < 0.01 and P < 0.001). During hyperlactatemia lactate level correlated complexes III and IV function (P < 0.05). After recovery mitochondrial parameters achieved values of nonhyperlactatemic HIV individuals, which were lower than ranges of naive subjects and uninfected controls. Conclusions:HIV and HAART-related hyperlactatemia is associated with a general mitochondrial impairment which reverts after recovery. Mitochondrial biochemistry show a better correlation with lactate levels than mitochondrial genetics suggesting that mitochondrial function could be a better marker of hyperlactatemia development than mtDNA content.

Lipotoxicity on the basis of metabolic syndrome and lipodystrophy in HIV-1-infected patients under antiretroviral treatment.

The development of efficacious antiretroviral drugs that minimize adverse effects is a current challenge in HIV-1 therapy. Metabolic alterations reminiscent of the metabolic syndrome and overt lipodystrophy appear often in HIV-1-infected patients undergoing antiretroviral treatment. The etiopathogenesis of these alterations is complex, but lipotoxicity has recently emerged as a key concept for explaining the metabolic syndrome in HIV-1-infected patients, similarly to what has been observed in diseases such as obesity and genetic lipodystrophies. Antiretroviral drugs from distinct drug families may directly elicit such lipotoxic phenomena, via increased lipolysis, enhanced adipocyte apoptosis and impaired adipogenesis, which collectively lead to a reduced capacity of subcutaneous adipose tissue to enlarge to meet fat storage requirements. Thus, fatty acids that cannot be properly stored as triglycerides in subcutaneous adipose tissue are expected to accumulate in visceral fat as well as in organs and tissues, such as the pancreas, muscle and liver, leading to the pattern of metabolic alterations associated with abnormal ectopic fat accumulation, mainly insulin resistance. Inflammatory responses, evoked by the combined effects of antiretroviral drugs and the underlying HIV-1 infection, also contribute to lipotoxicity, reflecting the action of pro-inflammatory cytokines that enhance lipolytic activity in adipose tissue and impair adipogenesis. Minimizing the lipotoxic action of antiretroviral drugs is ultimately essential in reducing metabolic alterations in treated patients. Moreover, pharmacological strategies that reduce lipotoxicity and promote adipose tissue expandability can be expected to ameliorate the overall metabolic abnormalities in HIV-1-infected, antiretroviral-treated patients.

Fibroblast growth factor 15/19 (FGF15/19) protects from diet-induced hepatic steatosis: development of an FGF19-based chimeric molecule to promote fatty liver regeneration

Objective Fibroblast growth factor 15/19 (FGF15/19), an enterokine that regulates synthesis of hepatic bile acids (BA), has been proposed to influence fat metabolism. Without FGF15/19, mouse liver regeneration after partial hepatectomy (PH) is severely impaired. We studied the role of FGF15/19 in response to a high fat diet (HFD) and its regulation by saturated fatty acids. We developed a fusion molecule encompassing FGF19 and apolipoprotein A-I, termed Fibapo, and evaluated its pharmacological properties in fatty liver regeneration. Design Fgf15−/− mice were fed a HFD. Liver fat and the expression of fat metabolism and endoplasmic reticulum (ER) stress-related genes were measured. Influence of palmitic acid (PA) on FGF15/19 expression was determined in mice and in human liver cell lines. In vivo half-life and biological activity of Fibapo and FGF19 were compared. Hepatoprotective and proregenerative activities of Fibapo were evaluated in obese db/db mice undergoing PH. Results Hepatosteatosis and ER stress were exacerbated in HFD-fed Fgf15−/− mice. Hepatic expression of Pparγ2 was elevated in Fgf15−/− mice, being reversed by FGF19 treatment. PA induced FGF15/19 expression in mouse ileum and human liver cells, and FGF19 protected from PA-mediated ER stress and cytotoxicity. Fibapo reduced liver BA and lipid accumulation, inhibited ER stress and showed enhanced half-life. Fibapo provided increased db/db mice survival and improved regeneration upon PH. Conclusions FGF15/19 is essential for hepatic metabolic adaptation to dietary fat being a physiological regulator of Pparγ2 expression. Perioperative administration of Fibapo improves fatty liver regeneration.