A report of the unusual lesions caused by Thelazia gulosa in cattle.

This is a case report of unusual lesions caused by Thelazia gulosa in cattle. After several ineffective treatments of suspected infectious keratoconjunctivitis in calves and cows, T. gulosa was found associated with retrobulbar granuloma-like masses. The masses with multiple digit-like protrusions on conjunctival surface ejected multiple worms on firm pressure on clinical examination. Samples of tissues that contained worms were surgically removed, fixed in ethanol and submitted to the parasitology and histopathology labs for morphological identification of worms and the nature of the tissue masses, respectively. The infestation was present only in young calves (<3months) and high-producing cows. Histopathology showed fibrovascular granulation tissue, containing a moderate to marked inflammatory infiltrate. Ivermectin treatment (200µg/kg, SC, once) with and without surgical excision resolved the infestation. To the best of our knowledge, this is the first report of tissue invasion by T. gulosa.

Effects of Anthocyanin and Flavanol Compounds on Lipid Metabolism and Adipose Tissue Associated Systemic Inflammation in Diet-Induced Obesity.

Background. Naturally occurring substances from the flavanol and anthocyanin family of polyphenols have been proposed to exert beneficial effects in the course of obesity. We hypothesized that their effects on attenuating obesity-induced dyslipidemia as well as the associated inflammatory sequelae especially have health-promoting potential. Methods. Male C57BL/6J mice (n = 52) received a control low-fat diet (LFD; 10 kcal% fat) for 6 weeks followed by 24 weeks of either LFD (n = 13) or high-fat diet (HFD; 45 kcal% fat; n = 13) or HFD supplemented with 0.1% w/w of the flavanol compound epicatechin (HFD+E; n = 13) or an anthocyanin-rich bilberry extract (HFD+B; n = 13). Energy substrate utilization was determined by indirect calorimetry in a subset of mice following the dietary switch and at the end of the experiment. Blood samples were collected at baseline and at 3 days and 4, 12, and 20 weeks after dietary switch and analyzed for systemic lipids and proinflammatory cytokines. Adipose tissue (AT) histopathology and inflammatory gene expression as well as hepatic lipid content were analyzed after sacrifice. Results. The switch from a LFD to a HFD lowered the respiratory exchange ratio and increased plasma cholesterol and hepatic lipid content. These changes were not attenuated by HFD+E or HFD+B. Furthermore, the polyphenol compounds could not prevent HFD-induced systemic rise of TNF-α levels. Interestingly, a significant reduction in Tnf gene expression in HFD+B mice was observed in the AT. Furthermore, HFD+B, but not HFD+E, significantly prevented the early upregulation of circulating neutrophil chemoattractant mKC. However, no differences in AT histopathology were observed between the HFD types. Conclusion. Supplementation of HFD with an anthocyanin-rich bilberry extract but not with the flavanol epicatechin may exert beneficial effects on the systemic early inflammatory response associated with diet-induced obesity. These systemic effects were transient and not observed after prolongation of HFD-feeding (24 weeks). On the tissue level, long-term treatment with bilberry attenuated TNF-α expression in adipose tissue.

Urinary ß-galactosidase stimulates Ca2+ transport by stabilizing TRPV5 at the plasma membrane.

Transcellular Ca(2+)transport in the late distal convoluted tubule and connecting tubule (DCT2/CNT) of the kidney is a finely controlled process mediated by the transient receptor potential vanilloid type 5 (TRPV5) channel. A complex-type-N-glycan bound at the extracellular residue Asn358 of TRPV5 through post-translational glycosylation has been postulated to regulate the activity of TRPV5 channels. Using in vitro Ca(2+)transport assays, immunoblot analysis, immunohistochemistry, patch clamp electrophysiology and total internal reflection fluorescence microscopy, it is demonstrated that the glycosidase ß-galactosidase (ß-gal), an enzyme that hydrolyzes galactose, stimulates TRPV5 channel activity. However, the activity of the non-glycosylated TRPV(N358Q)mutant was not altered in the presence of ß-gal, showing that the stimulation is dependent on the presence of the TRPV5N-glycan. In addition, ß-gal was found to stimulate transcellular Ca(2+)transport in isolated mouse primary DCT2/CNT cells. ß-gal expression was detected in the apical membrane of the proximal tubules, and the protein was found in mouse urine. In summary, ß-gal is present in the pro-urine from where it is thought to stimulate TRPV5 activity.

Deregulated Renal Calcium and Phosphate Transport during Experimental Kidney Failure.

Impaired mineral homeostasis and inflammation are hallmarks of chronic kidney disease (CKD), yet the underlying mechanisms of electrolyte regulation during CKD are still unclear. Here, we applied two different murine models, partial nephrectomy and adenine-enriched dietary intervention, to induce kidney failure and to investigate the subsequent impact on systemic and local renal factors involved in Ca(2+) and Pi regulation. Our results demonstrated that both experimental models induce features of CKD, as reflected by uremia, and elevated renal neutrophil gelatinase-associated lipocalin (NGAL) expression. In our model kidney failure was associated with polyuria, hypercalcemia and elevated urinary Ca(2+) excretion. In accordance, CKD augmented systemic PTH and affected the FGF23-αklotho-vitamin-D axis by elevating circulatory FGF23 levels and reducing renal αklotho expression. Interestingly, renal FGF23 expression was also induced by inflammatory stimuli directly. Renal expression of Cyp27b1, but not Cyp24a1, and blood levels of 1,25-dihydroxy vitamin D3 were significantly elevated in both models. Furthermore, kidney failure was characterized by enhanced renal expression of the transient receptor potential cation channel subfamily V member 5 (TRPV5), calbindin-D28k, and sodium-dependent Pi transporter type 2b (NaPi2b), whereas the renal expression of sodium-dependent Pi transporter type 2a (NaPi2a) and type 3 (PIT2) were reduced. Together, our data indicates two different models of experimental kidney failure comparably associate with disturbed FGF23-αklotho-vitamin-D signalling and a deregulated electrolyte homeostasis. Moreover, this study identifies local tubular, possibly inflammation- or PTH- and/or FGF23-associated, adaptive mechanisms, impacting on Ca(2+)/Pi homeostasis, hence enabling new opportunities to target electrolyte disturbances that emerge as a consequence of CKD development.

Age-related obesity and type 2 diabetes dysregulate neuronal associated genes and proteins in humans.

Despite numerous developed drugs based on glucose metabolism interventions for treatment of age-related diseases such as diabetes neuropathies (DNs), DNs are still increasing in patients with type 1 or type 2 diabetes (T1D, T2D). We aimed to identify novel candidates in adipose tissue (AT) and pancreas with T2D for targeting to develop new drugs for DNs therapy. AT-T2D displayed 15 (e.g. SYT4 up-regulated and VGF down-regulated) and pancreas-T2D showed 10 (e.g. BAG3 up-regulated, VAV3 and APOA1 down-regulated) highly differentially expressed genes with neuronal functions as compared to control tissues. ELISA was blindly performed to measure proteins of 5 most differentially expressed genes in 41 human subjects. SYT4 protein was upregulated, VAV3 and APOA1 were down-regulated, and BAG3 remained unchanged in 1- Obese and 2- Obese-T2D without insulin, VGF protein was higher in these two groups as well as in group 3- Obese-T2D receiving insulin than 4-lean subjects. Interaction networks analysis of these 5 genes showed several metabolic pathways (e.g. lipid metabolism and insulin signaling). Pancreas is a novel site for APOA1 synthesis. VGF is synthesized in AT and could be considered as good diagnostic, and even prognostic, marker for age-induced diseases obesity and T2D. This study provides new targets for rational drugs development for the therapy of age-related DNs.

High-fat diet induced obesity primes inflammation in adipose tissue prior to liver in C57BL/6j mice.

Metabolic inflammation in adipose tissue and the liver is frequently observed as a result of diet-induced obesity in human and rodent studies. Although the adipose tissue and the liver are both prone to become chronically inflamed with prolonged obesity, their individual contribution to the development of metabolic inflammation remains speculative. Thus, we aimed to elucidate the sequence of inflammatory events in adipose and hepatic tissues to determine their contribution to the development of metabolic inflammation and insulin resistance (IR) in diet-induced obesity. To confirm our hypothesis that adipose tissue (AT) inflammation is initiated prior to hepatic inflammation, C57BL/6J male mice were fed a low-fat diet (LFD; 10% kcal fat) or high-fat diet (HFD; 45% kcal fat) for either 24, 40 or 52 weeks. Lipid accumulation and inflammation was measured in AT and liver. Glucose tolerance was assessed and plasma levels of glucose, insulin, leptin and adiponectin were measured at various time points throughout the study. With HFD, C57BL/6j mice developed a progressive obese phenotype, accompanied by IR at 24 and 40 weeks of HFD, but IR was attenuated after 52 weeks of HFD. AT inflammation was present after 24 weeks of HFD, as indicated by the increased presence of crown-like structures and up-regulation of pro-inflammatory genes Tnf, Il1ß, Mcp1 and F4/80. As hepatic inflammation was not detected until 40 weeks of HFD, we show that AT inflammation is established prior to the development of hepatic inflammation. Thus, AT inflammation is likely to have a greater contribution to the development of IR compared to hepatic inflammation.

Cholesterol-induced hepatic inflammation does not underlie the predisposition to insulin resistance in dyslipidemic female LDL receptor knockout mice.

Chronic inflammation is considered a causal risk factor predisposing to insulin resistance. However, evidence is accumulating that inflammation confined to the liver may not be causal to metabolic dysfunction. To investigate this, we assessed if hepatic inflammation explains the predisposition towards insulin resistance in low-density lipoprotein receptor knock-out (Ldlr (-/-)) mice. For this, wild type (WT) and Ldlr (-/-) mice were fed a chow diet, a high fat (HF) diet, or a high fat, high cholesterol (HFC) diet for 2 weeks. Plasma lipid levels were elevated in chow-fed Ldlr (-/-) mice compared to WT mice. Although short-term HF or HFC feeding did not result in body weight gain and adipose tissue inflammation, dyslipidemia was worsened in Ldlr (-/-) mice compared to WT mice. In addition, dyslipidemic HF-fed Ldlr (-/-) mice had a higher hepatic glucose production rate than HF-fed WT mice, while peripheral insulin resistance was unaffected. This suggests that HF-fed Ldlr (-/-) mice suffered from hepatic insulin resistance. While HFC-fed Ldlr (-/-) mice displayed the anticipated increased hepatic inflammation, this did neither exacerbate systemic nor hepatic insulin resistance. Therefore, our results show that hepatic insulin resistance is unrelated to cholesterol-induced hepatic inflammation in Ldlr (-/-) mice, indicating that hepatic inflammation may not contribute to metabolic dysfunction per se.

Lipid droplets hypertrophy: a crucial determining factor in insulin regulation by adipocytes.

Lipid droplets (LDs) hypertrophy in adipocytes is the main cause of energy metabolic system dysfunction, obesity and its afflictions such as T2D. However, the role of adipocytes in linking energy metabolic disorders with insulin regulation is unknown in humans. Human adipocytes constitutively synthesize and secrete insulin, which is biologically functional. Insulin concentrations and release are fat mass- and LDs-dependent respectively. Fat reduction mediated by bariatric surgery repairs obesity-associated T2D. The expression of genes, like PCSK1 (proinsulin conversion enzyme), GCG (Glucagon), GPLD1, CD38 and NNAT, involved in insulin regulation/release were differentially expressed in pancreas and adipose tissue (AT). INS (insulin) and GCG expression reduced in human AT-T2D as compared to AT-control, but remained unchanged in pancreas in either state. Insulin levels (mRNA/protein) were higher in AT derived from prediabetes BB rats with destructed pancreatic ß-cells and controls than pancreas derived from the same rats respectively. Insulin expression in 10 human primary cell types including adipocytes and macrophages is an evidence for extrapancreatic insulin-producing cells. The data suggest a crosstalk between AT and pancreas to fine-tune energy metabolic system or may minimize the metabolic damage during diabetes. This study opens new avenues towards T2D therapy with a great impact on public health.

Histone variants and lipid metabolism.

Within nucleosomes, canonical histones package the genome, but they can be opportunely replaced with histone variants. The incorporation of histone variants into the nucleosome is a chief cellular strategy to regulate transcription and cellular metabolism. In pathological terms, cellular steatosis is an abnormal accumulation of lipids, which reflects impairment in the turnover of triacylglycerols, affecting any organ but mainly the liver. The present review aims to summarize the experimental evidence for histone variant functions in lipid metabolism.

Increased hepatic CD36 expression with age is associated with enhanced susceptibility to nonalcoholic fatty liver disease.

CD36 has been associated with obesity and diabetes in human liver diseases, however, its role in age-associated nonalcoholic fatty liver disease (NAFLD) is unknown. Therefore, liver biopsies were collected from individuals with histologically normal livers (n=30), and from patients diagnosed with simple steatosis (NAS; n=26). Patients were divided into two groups according to age and liver biopsy samples were immunostained for CD36. NAFLD parameters were examined in young (12-week) and middle-aged (52-week) C57BL/6J mice, some fed with chow-diet and some fed with low-fat (LFD; 10% kcal fat) or high-fat diet (HFD; 60% kcal fat) for 12-weeks. CD36 expression was positively associated with age in individuals with normal livers but not in NAS patients. However, CD36 was predominantly located at the plasma membrane of hepatocytes in aged NAS patients as compared to young. In chow-fed mice, aging, despite an increase in hepatic CD36 expression, was not associated with the development of NAFLD. However, middle-aged mice did exhibit the development of HFD-induced NAFLD, mediated by an increase of CD36 on the membrane. Enhanced CD36-mediated hepatic fat uptake may contribute to an accelerated progression of NAFLD in mice and humans. Therapies to prevent the increase in CD36 expression and/or CD36 from anchoring at the membrane may prevent the development of NAFLD.

SIRT1-metabolite binding histone macroH2A1.1 protects hepatocytes against lipid accumulation.

Non-alcoholic-fatty-liver-disease (NAFLD) encompasses conditions associated to fat deposition in the liver, which are generally deteriorated during the aging process. MacroH2A1, a variant of histone H2A, is a key transcriptional regulator involved in tumorigenic processes and cell senescence, and featuring two alternatively splicing isoforms, macroH2A1.1 and macroH2A1.2. MacroH2A1.1 binds with high affinity O-acetyl ADP ribose, a small metabolite produced by the reaction catalysed by NAD+-dependent deacetylase SIRT1, whereas macroH2A1.2 is unable to do so. The functional significance of this binding is unknown. We previously reported that the hepatic levels of macroH2A1.1 and macroH2A1.2 are differentially expressed in mice models of NAFLD. Here we show that over-expression of macroH2A1.1, but not of macroH2A1.2, is able to protect hepatocytes against lipid accumulation. MacroH2A1.1 over-expressing cells display ameliorated glucose metabolism, reduced expression of lipidogenic genes and fatty acids content. SIRT1/macroH2A1.1-dependent epigenetic regulation of lipid metabolism may be relevant to NAFLD development.

Cholesterol-induced hepatic inflammation does not contribute to the development of insulin resistance in male LDL receptor knockout mice.

OBJECTIVE: It is generally assumed that hepatic inflammation in obesity is linked to the pathogenesis of insulin resistance. Several recent studies have shed doubt on this view, which questions the causality of this association. This study focuses on Kupffer cell-mediated hepatic inflammation as a possible driver of insulin resistance in the absence and presence of obesity. METHODS: We used male mice deficient for the low-density lipoprotein receptor (Ldlr(-/-)) and susceptible to cholesterol-induced hepatic inflammation. Whole body and hepatic insulin resistance was measured in mice fed 4 diets for 2 and 15 weeks, i.e., chow, high-fat (HF), HF-cholesterol (HFC; 0.2% cholesterol) and HF without cholesterol (HFnC). Biochemical parameters in plasma and liver were measured and inflammation was determined using immunohistochemistry and RT-PCR. RESULTS: At 2 weeks, we did not find significant metabolic effects in either diet group, except for the mice fed a HFC diet which showed pronounced hepatic inflammation (p < 0.05) but normal insulin sensitivity. At 15 weeks, a significant increase in insulin levels, HOMA-IR, and hepatic insulin resistance was observed in mice fed a HFC, HFnC, and HF diet compared to chow-fed mice (p < 0.05). Regardless of the level of hepatic inflammation (HFC > HF, HFnC; p < 0.05) insulin resistance in mice fed HFC was no worse compared to mice on a HFnC and HF diet. CONCLUSION: These data show that cholesterol-induced hepatic inflammation does not contribute to the development of insulin resistance in male Ldlr(-/-) mice. This study suggests that Kupffer cell-driven hepatic inflammation is a consequence, not a cause, of metabolic dysfunction in obesity.

Liver diseases and aging: friends or foes?

The liver is the only internal human organ capable of natural regeneration of lost tissue, as little as 25% of a liver can regenerate into a whole liver. The process of aging predisposes to hepatic functional and structural impairment and metabolic risk. Therefore, understanding how aging could affect the molecular pathology of liver diseases is particularly important, and few studies to date have tackled this complex process. The most common liver disease, affecting one-third of the overall population, is nonalcoholic fatty liver disease (NAFLD), characterized by an intrahepatic accumulation of lipids. NAFLD can evolve into nonalcoholic steatohepatitis (NASH) in the presence of oxidative stress and inflammation. NASH is a serious risk factor for disabling and deadly liver diseases such as cirrhosis and hepatocellular carcinoma (HCC). Old age seems to favor NAFLD, NASH, and ultimately HCC, in agreement with the inflamm-aging theory, according to which aging accrues inflammation. However, the incidence of HCC drops significantly in the very elderly (individuals aged more than 70) and the relationship between the progression of NAFLD/NASH/HCC and very old age is obscure. In this review, we discuss the literature and we argue that there might be an age window in which the liver becomes resistant to the development of injury; this needs to be studied to understand fully the interaction between age and liver diseases from a therapeutic perspective.