Limited therapeutic efficacy of thrombopoietin on the regeneration of steatotic livers

Liver regeneration after partial hepatectomy is impaired in steatotic livers of leptin-deficient ob/ob mice. Previous studies have shown that thrombopoietin (TPO) promotes liver regeneration and improves liver cirrhosis by an increase of platelet counts and the expansion of hepatic progenitor cells. Herein we studied whether TPO exerts pro-proliferative and hepatoprotective effects and thereby improves the regenerative capacity of steatotic livers. For this purpose, we studied hepatic regeneration at day 2, 3, 7 and 10 in a model of 55% hepatectomy in obese (ob/ob) and non-obese (C57BL/6J) mice. Liver function and injury, platelet counts, weight of the regenerated liver, proliferating liver cells as well as the number of hepatic (CK19-positive) oval cells were quantified by biochemical and immunohistochemical analysis. As expected, obese mice had a markedly decreased regenerative capacity of livers compared with lean animals. Pretreatment of mice with recombinant TPO (12.5 μg/kg) had no evident effect on regeneration of fatty livers, but ameliorated acute liver damage in obese mice, as indicated by decreased liver enzyme release early after resection. TPO was unable to enhance hepatocyte proliferation, but increased proliferation of non-parenchymal cells, including CK19-positive oval cells, at later observation time points after resection. Interestingly, TPO completely inhibited the resection-induced increase of plasma triglycerides immediately after resection in non-obese mice. In conclusion, TPO slightly prevents acute liver damage after resection in obese mice, but fails to significantly enhance regeneration of fatty livers.     

STAT-3 overexpression and p21 up-regulation accompany impaired regeneration of fatty livers

Fatty liver is an important cause of morbidity in humans and is linked to impaired liver regeneration after liver injury, but the mechanisms for impaired liver regeneration remain unknown. In the normal liver, the interleukin (IL)-6/STAT-3 pathway is thought to play a central role in regeneration because this pathway is disrupted in IL-6-deficient mice that exhibit impaired liver regeneration after 70% partial hepatectomy (PH). To determine whether inhibition of STAT-3 is involved in fatty liver-related mitoinhibition, regenerative induction of STAT-3 was compared in normal mice and leptin-deficient ob/ob mice that have fatty livers and markedly impaired liver regeneration after PH. In both groups, two waves of STAT-3 activation were observed, the first in endothelia and the second in hepatocytes. Before PH, a significantly higher percentage of ob/ob endothelial and hepatocyte nuclei expressed phosphorylated (activated) STAT-3. After PH, phospho-STAT-3 accumulated in liver nuclei of lean mice and this response was markedly exaggerated in ob/ob mice. Moreover, a striking inverse correlation was noted between hepatocyte nuclear accumulation of phospho-STAT-3 and DNA synthesis (as assessed by bromodeoxyuridine labeling), as well as cyclin D1 mRNA induction and protein expression. In contrast, STAT-3 activation was positively correlated with p21 protein expression in both groups of mice. Because these results link exaggerated STAT-3 activation with impaired hepatocyte proliferation, STAT-3 inhibition cannot be a growth-arrest mechanism in ob/ob fatty livers. Rather, hyperinduction of this factor may promote mitoinhibition by up-regulating mechanisms that impede cell cycle progression.     

Expression of epidermal growth factor receptor protein in the liver of db/db mice after partial hepatectomy

Liver regeneration was impaired after partial hepatectomy (PH) in leptin receptor-deficient db/db mice with severe liver steatosis. In the present study, we analyzed the mode of epidermal growth factor receptor (EGFR) protein expression in the liver of 5- and 10-week-old db/db and age-matched control mice. In 5-week-old db/db mice, neither the expression of EGFR protein in the intact liver nor the rate of liver regeneration after PH was significantly different from that in age-matched control mice. However, in 10-week-old db/db mice, the level of EGFR protein expression was very low and liver regeneration was prominently suppressed. Histopathologically, much severer fatty change was observed in the liver of 10-week-old db/db mice than 5-week-old db/db mice. These results suggest that the down-regulation of EGFR protein expression is associated with an impairment of liver regeneration in db/db mice and that the severity of hepatic steatosis plays an indirect role in the impairment of liver regeneration by modifying EGFR expression.     

Impaired liver regeneration after partial hepatectomy in db/db mice

Fatty liver is the most common hepatic disorder in humans and supposed to be a cause of poor prognosis after liver transplantation and hepatic resection which could be resulted from impaired liver regeneration. This study was carried out to analyze the process of liver regeneration in db/db mice which show severe steatosis because of abnormal leptin receptor. We performed 70% partial hepatectomy (PH) on db/db mice and normal +m/+m mice, and then sacrificed the animals 1, 2, 3, 5, 7 and 10 days later. The liver samples were weighed and examined histologically or immunohistochemically. As a result, the liver mass restitution was significantly inhibited in db/db mice compared with +m/+m mice. The BrdU labelling index peaked at 2 days after PH in both strains, although the value was lower in db/db mice. After that, interestingly, it decreased to the control level at 5 days in +m/+m mice while the recovery was delayed in db/db mice. Similar sequence was also observed in the PCNA labelling index. In addition, the peak time of the mitosis index was 2 days and 5 days after PH in +m/+m mice and in db/db mice, respectively. Thus, although not significant, the proliferative response of hepatocytes to PH occurred somewhat more transient and sharply in +m/+m mice while it lasted somewhat longer in db/db mice. This suggests that db/db mice may be valuable as one of the animal models for the investigation of the effects of steatosis on the liver regeneration.     

Impaired proliferation of non-parenchymal cells participates in an impairment of liver regeneration in db/db mice

In this study, we examined the possibility that impaired proliferation of non-parenchymal cells affects in an impairment of liver regeneration in db/db mice, which are congenitally deficient in receptors for leptin. Liver regeneration after a two thirds partial hepatectomy (2/3 PH) was impaired in 10-week-old female db/db mice. The proliferation of both hepatocytes and non-parenchymal cells estimated from a bromodeoxyuridine (BrdU) labeling index was suppressed, and the protein expression of vascular endothelial growth factor was blocked in db/db mice. Although the extent of fatty change and the level of epidermal growth factor receptor protein expression in the liver were improved in 5-week-old db/db mice, the regeneration of liver was impaired after 2/3 PH in both 5- and 10-week-old db/db mice. These results suggested that suppressed proliferation of non-parenchymal cells contributes to the impairment of liver regeneration in db/db mice. As leptin has also the angiogenic effect, the angiogenic inhibitor FR-118487 was administered to ICR mice to examine liver regeneration after 2/3 PH, and the rate of regeneration was affected. In conclusion, it is suggested that the suppressed proliferation of non-parenchymal cells contributes to the impairment of liver regeneration probably through a disrupted angiogenesis in db/db mice.     

Pathology of the liver in obese and diabetic ob/ob and db/db mice fed a standard or high-calorie diet

Non‐alcoholic fatty liver disease (NAFLD) is one of the commonest liver diseases in Western countries. Although leptin deficient ob/ob and db/db mice are frequently used as murine models of NAFLD, an exhaustive characterization of their hepatic lesions has not been reported to date, particularly under calorie overconsumption. Thus, liver lesions were characterized in 78 ob/ob and db/db mice fed either a standard or high‐calorie (HC) diet, for one or three months. Steatosis, necroinflammation, apoptosis and fibrosis were assessed and the NAFLD activity score (NAS) was calculated. Steatosis was milder in db/db mice compared to ob/ob mice and was more frequently microvesicular. Although necroinflammation was usually mild in both genotypes, it was aggravated in db/db mice after one month of calorie overconsumption. Apoptosis was observed in db/db mice whereas it was only detected in ob/ob mice after HC feeding. Increased apoptosis was frequently associated with microvesicular steatosis. In db/db mice fed the HC diet for three months, fibrosis was aggravated while steatosis, necroinflammation and apoptosis tended to alleviate. This was associated with increased plasma β‐hydroxybutyrate suggesting an adaptive stimulation of hepatic mitochondrial fatty acid oxidation (FAO). Nevertheless, one‐third of these db/db mice had steatohepatitis (NAS ≥ 5), whereas none of the ob/ob mice developed non‐alcoholic steatohepatitis under the same conditions. Steatosis, necroinflammation, apoptosis and fibrosis are modulated by calorie overconsumption in the context of leptin deficiency. Association between apoptosis and microvesicular steatosis in obese mice suggests common mitochondrial abnormalities. Enhanced hepatic FAO in db/db mice is associated with fibrosis aggravation.     

Severely impaired insulin signaling in chronic wounds of diabetic ob/ob mice: a potential role of tumor necrosis factor-alpha

Wound-healing disorders are major complications of diabetes mellitus. Here, we investigated insulin-mediated signaling in nonwounded skin and in cutaneous tissue regeneration of healthy C57BL/6 and diabetes-impaired leptin-deficient obese/obese (ob/ob) mice. The insulin receptor (InsR) was abundantly expressed in wound margins and granulation tissue during acute healing in healthy mice. Remarkably, active signaling from the InsR, as assessed by phosphorylation of downstream targets such as protein tyrosine phosphatase-1B, glycogen synthase (GS), and GS kinase, was nearly absent in nonwounded and acutely healing skin from ob/ob mice. Systemic leptin administration to ob/ob mice reverted the diabetic phenotype and improved tissue regeneration as well as the impaired expression of InsR, insulin receptor substrate-1 and insulin receptor substrate-2, and downstream signaling (phosphorylation of GS kinase and GS) in late wounds and nonwounded skin of ob/ob mice. Importantly, tumor necrosis factor (TNF)-alpha was a mediator of insulin resistance in keratinocytes in vitro and in ob/ob wound tissue in vivo. Systemic administration of a monoclonal anti-TNF-alpha antibody (V1q) in wounded ob/ob mice attenuated wound inflammation, improved re-epithelialization, and restored InsR expression and signaling in wound tissue of ob/ob mice. These data suggest that InsR signaling in diabetes-impaired wounds is sensitive to inflammatory conditions and that anti-inflammatory approaches, such as anti-TNF-alpha strategies, improve diabetic wound healing.     

Autonomic dysregulation in ob/ob mice is improved by inhibition of angiotensin-converting enzyme

The leptin-deficient ob/ob mice are insulin resistant and obese. However, the control of blood pressure in this model is not well defined. The goal of this study was to evaluate the role of leptin and of the renin-angiotensin system in the cardiovascular abnormalities observed in obesity using a model lacking leptin. To this purpose, we measured blood pressure in ob/ob and control animals by radiotelemetry combined with fast Fourier transformation before and after both leptin and enalapril treatment. Autonomic function was assessed pharmacologically. Blood pressure during daytime was slightly higher in the ob/ob compared to control mice, while no difference in heart rate was observed. Blood pressure response to trimetaphane and heart rate response to metoprolol were greater in ob/ob mice than in control littermates indicating an activated sympathetic nervous system. Heart rate response to atropine was attenuated. Baroreflex sensitivity and heart rate variability were blunted in ob/ob mice, while low frequency of systolic blood pressure variability was found increased. Chronic leptin replacement reduced blood pressure and reversed the impaired autonomic function observed in ob/ob mice. Inhibition of angiotensin-converting enzyme by enalapril treatment had similar effects, prior to the loss of weight. These findings suggest that the renin-angiotensin system is involved in the autonomic dysfunction caused by the lack of leptin in ob/ob mice and support a role of this interplay in the pathogenesis of obesity, hypertension, and metabolic syndrome.     

Additive effects of leptin and topiramate in reducing fat deposition in lean and obese ob/ob mice

The objective of the present study was to investigate the effects of the antiepileptic drug topiramate (TPM) on components of energy balance in lean and obese (ob/ob) mice in the presence or absence of leptin. Lean and ob/ob mice infused with either leptin or phosphate-buffered saline were treated with TPM for 7 days. TPM was mixed into the diet and administered at a dose of 60 mg/kg/day, whereas leptin was infused at the rate of 100 microg/kg/day using osmotic minipumps, which were subcutaneously implanted in the interscapular region. Food intake and body weight were monitored throughout the study. Body composition was measured prior to and following treatment with TPM and leptin, using dual-energy X-ray absorptiometry (DEXA). Glucose (glucose oxidase method) and insulin (radioimmunoassay) were also determined. TPM and leptin significantly reduced body weight gain, food intake and body fat gain in obese mice. The effects of TPM and leptin on fat gain were also statistically significant in lean animals. There was no interaction of TPM and leptin on the energy balance variables, the effects of the two substances being additive instead. Leptin abrogated hyperinsulinemia in obese mutants whereas TPM did not alter insulin levels in either lean or obese mice. The combination of leptin and TPM led to the normalization of glucose levels in obese mice. Our study demonstrates an effect of TPM in leptin-deficient animals, which suggests that TPM does not require the presence of leptin to exert its effect. They also show that the effects of leptin and TPM can be additive. The treatment with leptin in ob/ob mice neither accentuated nor blunted the effect of TPM on energy balance.     

Evaluation of liver fatty acid oxidation in the leptin-deficient obese mouse

We hypothesized that liver fatty acid oxidation (FAO) is compromised in the leptin-deficient obese (Lep(ob)/Lep(ob)) mouse model, and that this would be further challenged when these mice were fed a high-fat diet. Obese mice had a 3.8-fold increased body fat content and a 9-fold increased liver fat content as compared to control mice when both groups were fed a low-fat diet. The expression of liver FAO enzymes, carnitine palmitoyltransferase-1a, long-chain acyl-CoA dehydrogenase, medium-chain acyl-CoA dehydrogenase, and short-chain acyl-CoA dehydrogenase, was not affected in obese mice as compared to controls on either a low-fat or a high-fat diet. The expression of very-long-chain acyl-CoA dehydrogenase was elevated in obese mice on the control diet, as compared to control mice. For all measures evaluated, increasing the level of fat in the diet had a smaller effect than leptin deficiency. In summary, despite obese mice having an excess of fat available for mitochondrial beta-oxidation in liver, overall energy balance appeared to dictate that the net liver FAO remained at control levels.     

Mouse models in non-alcoholic fatty liver disease and steatohepatitis research

Non-alcoholic fatty liver disease (NAFLD) represents a histological spectrum of liver disease associated with obesity, diabetes and insulin resistance that extends from isolated steatosis to steatohepatitis and cirrhosis. As well as being a potential cause of progressive liver disease in its own right, steatosis has been shown to be an important cofactor in the pathogenesis of many other liver diseases. Animal models of NAFLD may be divided into two broad categories: those caused by genetic mutation and those with an acquired phenotype produced by dietary or pharmacological manipulation. The literature contains numerous different mouse models that exhibit histological evidence of hepatic steatosis or, more variably, steatohepatitis; however, few replicate the entire human phenotype. The genetic leptin-deficient (ob/ob) or leptin-resistant (db/db) mouse and the dietary methionine/choline-deficient model are used in the majority of published research. More recently, targeted gene disruption and the use of supra-nutritional diets to induce NAFLD have gained greater prominence as researchers have attempted to bridge the phenotype gap between the available models and the human disease. Using the physiological processes that underlie the pathogenesis and progression of NAFLD as a framework, we review the literature describing currently available mouse models of NAFLD, highlight the strengths and weaknesses of established models and describe the key findings that have furthered the understanding of disease pathogenesis.     

Leptin regulates ACE activity in mice

Leptin is a hormone related to metabolism. It also influences blood pressure, but the mechanisms triggered in this process are not yet elucidated. Angiotensin-I converting enzyme (ACE) regulates cardiovascular functions and recently has been associated with metabolism control and obesity. Here, we used ob/ob mice, a model lacking leptin, to answer the question whether ACE and leptin could interact to influence blood pressure, thereby linking the renin-angiotensin system and obesity. These mice are obese and diabetic but have normal 24 h mean arterial pressure. Our results show that plasma and lung ACE activities as well as ACE mRNA expression were significantly decreased in ob/ob mice. In agreement with these findings, the hypotensive effect produced by enalapril administration was attenuated in the obese mice. Plasma renin, angiotensinogen, angiotensin I, bradykinin, and angiotensin 1–7 were increased, whereas plasma angiotensin II concentration was unchanged in obese mice. Chronic infusion of leptin increased renin activity and angiotensin II concentration in both groups and increased ACE activity in ob/ob mice. Acute leptin infusion restored ACE activity in leptin-deficient mice. Moreover, the effect of an ACE inhibitor on blood pressure was not changed in ob/+ mice during leptin treatment but increased four times in obese mice. In summary, our findings show that the renin–angiotensin system is altered in ob/ob mice, with markedly reduced ACE activity, which suggests a possible connection between the renin–angiotensin system and leptin. These results point to an important interplay between the angiotensinergic and the leptinergic systems, which may play a role in the pathogenesis of obesity, hypertension, and metabolic syndrome.     

Leptin deficiency, not obesity, protects mice from Con A-induced hepatitis

Leptin-deficient ob/ob mice are protected from Con A-induced hepatitis. However, it is unclear whether leptin deficiency or obesity itself is responsible for this protection. To address this question, wild-type (WT) obese mice with high serum leptin levels were generated by injection of gold thioglucose (WT GTG). Both Con A-injected WT and WT GTG mice developed hepatitis, whereas no hepatic damage was observed in ob/ob mice. Moreover, TNF-alpha and IFN-gamma levels as well as expression of the activation marker CD69 were elevated in liver mononuclear cells of WT and WT GTG mice, but not in ob/ob mice following administration of Con A. The liver of WT and WT GTG mice had the same percentage of NK T cells, a lymphocyte population involved in Con A-induced hepatitis. This population decreased equally in both WT and WT GTG mice after Con A injection. In contrast, the liver of ob/ob mice contained 50% less NK T cells compared to WT and WT GTG mice. Furthermore, no decrease in NK T cells was observed in Con A-injected ob/ob mice. We conclude that leptin-deficiency, not obesity, is responsible for protection from Con A-induced hepatitis.     

Pulmonary Mycobacterium tuberculosis infection in leptin-deficient ob/ob mice

The development of active tuberculosis after infection with Mycobacterium tuberculosis is almost invariably caused by a persistent or transient state of relative immunodeficiency. Leptin, the product of the obese (ob) gene, is a pleiotropic protein produced mainly by adipocytes and is down-regulated during malnutrition and starvation, conditions closely connected with active tuberculosis. To investigate the role of leptin in tuberculosis, we intranasally infected wild-type (Wt) and leptin-deficient ob/ob mice with live virulent M. tuberculosis. Ob/ob mice displayed higher mycobacterial loads in the lungs after 5 and 10 weeks of infection, although the difference with Wt mice remained 1 log of M. tuberculosis colony forming unit. Nevertheless, ob/ob mice were less able to form well-shaped granuloma and lung lymphocyte numbers were reduced compared with Wt mice early during infection. In addition, ob/ob mice had a reduced capacity to produce the protective cytokine IFNgamma at the site of the infection early during infection and upon antigen-specific recall stimulation, and showed reduced delayed-type hypersensitivity reaction to intra-dermal tuberculin purified protein derivative. Leptin replacement restored the reduced IFNgamma response observed in ob/ob mice. Mortality did not differ between ob/ob and Wt mice. These data suggest that leptin plays a role in the early immune response to pulmonary tuberculosis.     

Development of intestinal inflammation in double IL-10- and leptin-deficient mice

Leptin-deficient (ob/ob) mice are resistant in different models of autoimmunity and inflammation, suggesting that leptin regulates immunity and inflammation. To investigate whether leptin deficiency modulates the spontaneous intestinal inflammation observed in interleukin (IL)-10-deficient mice, double IL-10- and leptin-deficient [IL-10 knockout (KO) ob/ob] mice were generated and compared with single IL-10 KO mice for colitis severity. Body weight in IL-10 KO ob/ob mice was significantly reduced compared with that of ob/ob mice. However, when compared with wild-type or IL-10 KO mice, IL-10 KO ob/ob mice were still markedly obese. IL-10 KO and IL-10 KO ob/ob mice developed colitis with a comparable time-course and severity in terms of macroscopic and histologic scores. Likewise, production of interferon-gamma, IL-6, and IL-13 from colon cultures and splenocytes did not differ among these two groups. Conversely, rates of apoptosis were higher in lamina propria lymphocytes obtained from the colon of IL-10 KO ob/ob compared with IL-10 KO mice. In conclusion, although leptin deficiency has been associated with resistance in models of autoimmunity and inflammation induced by exogenous stimuli, leptin appears not to play a significant role in the spontaneous colitis of IL-10 KO mice, although it modulates survival of intestinal lymphocytes.     

Leptin-deficient mice are protected from accelerated nephrotoxic nephritis

Leptin is an adipose tissue-derived hormone that signals nutritional status to the hypothalamus. Recent evidence indicates that leptin modifies proinflammatory immune responses and may provide a key link between nutritional deficiency and immune dysfunction. To study the influence of leptin deficiency on immune-mediated renal disease, susceptibility to accelerated nephrotoxic nephritis was examined in leptin-deficient C57BL/6-ob/ob mice and C57BL/6 wild-type controls. The model was induced with sheep anti-mouse glomerular basement membrane antibody injected to mice preimmunized against sheep IgG, and mice were sacrificed 8 days after induction of disease. The leptin-deficient ob/ob mice were strongly protected from glomerular crescent formation, macrophage infiltration, glomerular thrombosis, and albuminuria in this model. Our findings suggest that leptin is required for the induction and maintenance of immune-mediated glomerulonephritis, and that blockade of the leptin axis might provide an attractive therapeutic possibility in human autoimmune disease.     

Participation of liver progenitor cells in liver regeneration: lack of evidence in the AAF/PH rat model

When hepatocyte proliferation is impaired, liver progenitor cells (LPC) are activated to participate in liver regeneration. We used the 2-acetaminofluorene/partial hepatectomy (AAF/PH) model to evaluate the contribution of LPC to liver cell replacement and function restoration. Fischer rats subjected to AAF/PH (or PH alone) were investigated 7, 10 and 14 days post-hepatectomy. Liver mass recovery (LMR) was estimated, and the liver mass to body weight ratio calculated. We used serum albumin and bilirubin levels, and liver albumin mRNA levels to assess the liver function. LPC expansion was analyzed by cytokeratin 19 (CK19), glutathione S-transferase protein (GSTp) immunohistochemistry and by CK19, CD133, transforming growth factor-β1 and hepatocyte growth factor mRNA expression in livers. Cell proliferation was evaluated by Ki67 and BrdU immunostaining. Compared with PH alone where LMR was ～100% 14 days post-PH, LMR was defective in AAF/PH rats (64.1±15.5%, P=0.0004). LPC expansion was scarce in PH livers (0.5±0.4% of CK19(+) area), but significant in AAF/PH livers (8.5±7.2% of CK19(+)), and inversely correlated to LMR (r(2)=0.63, P<0.0001). A quarter of AAF/PH animals presented liver failure (low serum albumin and high serum bilirubin) 14 days post-PH. Compared with animals with preserved function, this was associated with a lower LMR (50±6.8 vs 74.6±9.4%, P=0.0005), a decreased liver to body weight ratio (2±0.3 vs 3.5±0.6%, P=0.001), and a larger LPC expansion such as proliferating Ki67(+) LPC covered 17.4±4.2% of the liver parenchyma vs 3.1±1.5%, (P<0.0001). Amongst those, rare LPC with an intermediate hepatocyte-like phenotype were seen. Also, less than 2% of hepatocytes were engaged into the cell cycle (Ki67(+)), while more numerous (～25% of hepatocytes) in the livers with preserved function. These observations suggest that, in this model, the efficient recovery of the liver function was ensured rather by the proliferation of mature hepatocytes than by the LPC expansion and differentiation into hepatocytes.