Empagliflozin (an SGLT2 inhibitor), alone or in combination with linagliptin (a DPP-4 inhibitor), prevents steatohepatitis in a novel mouse model of non-alcoholic steatohepatitis and diabetes

BackgroundSodium-glucose co-transporter-2 (SGLT2) inhibitors are new oral antidiabetic drugs that reduce hyperglycemia by promoting urinary glucose excretion. Glycosuria produced by SGLT2 inhibitors is associated with weight loss, mainly due to reduced fat volume. We investigated the effects of empagliflozin (selective SGLT2 inhibitor) and linagliptin (DPP-4 inhibitor) on steatohepatitis and fibrosis in a mouse model of non-alcoholic steatohepatitis (NASH) with diabetes.MethodsA novel NASH model was generated by administration of streptozotocin to C57BL/6J mice at 2 days old, with a high-fat diet from 4 weeks. NASH mice aged 6 weeks were divided into four groups of 6 animals: vehicle, linagliptin (10 mg/kg), empagliflozin (10 mg/kg), and linagliptin + empagliflozin. The histological non-alcoholic fatty liver disease activity score was significantly lower in the empagliflozin and linagliptin + empagliflozin groups than in the vehicle or linagliptin groups. Hepatic expression of inflammatory genes (tumor necrosis factor-α, interleukin-6, and monocyte chemoattractant protein-1) was decreased in the empagliflozin and linagliptin + empagliflozin groups compared with the vehicle group. The collagen deposition with Sirius red staining was significantly reduced in the linagliptin + empagliflozin group compared with the linagliptin or the empagliflozin group. Immunohistochemistry showed that expression of α-smooth muscle actin, a marker of myofibroblasts (fibrosis), was reduced in the linagliptin + empagliflozin group compared with the vehicle group, as was expression of type 1 and 3 collagen mRNA. Linagliptin + empagliflozin decreased expression of mRNAs for genes related to fatty acid synthesis, but did not increase mRNAs for β-oxidation-related genes.ConclusionsWhile empagliflozin alone attenuates development of NASH showing anti-steatotic and anti-inflammatory effects, combined administration of empagliflozin and linagliptin can synergistically ameliorates NASH with stronger anti-fibrotic effects.

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title: Nonalcoholic fatty liver disease.
abstract: Nonalcoholic fatty liver disease (NAFLD) is present in up to one third of the general population and in the majority of patients with metabolic risk factors such as obesity and diabetes. Insulin resistance is a key pathogenic factor resulting in hepatic fat accumulation. Recent evidence demonstrates NAFLD in turn, exacerbates hepatic insulin resistance and often precedes glucose intolerance. Once hepatic steatosis is established, other factors including oxidative stress, mitochondrial dysfunction, gut-derived lipopolysaccharide and adipocytokines, may promote hepatocellular damage, inflammation and progressive liver disease. Confirmation of the diagnosis of NAFLD can usually be achieved by imaging studies, however staging the disease requires a liver biopsy. NAFLD is associated with an increased risk of all-cause death, probably because of complications of insulin resistance such as vascular disease, as well as due to cirrhosis and hepatocellular carcinoma, which occurs in a minority of patients. NAFLD is also now recognized to account for a substantial proportion of patients previously diagnosed with 'cryptogenic cirrhosis'. Diabetes, obesity and the necroinflammatory form of NAFLD known as non-alcoholic steatohepatitis, are risk factors for progressive liver disease. Current treatment relies on weight loss and exercise, although various insulin-sensitizing medications appear promising. Further research is needed to identify which patients will achieve the most benefit from therapy.

title: Prevalence of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis among a largely middle-aged population utilizing ultrasound and liver biopsy: a prospective study.
abstract: Prevalence of nonalcoholic fatty liver disease (NAFLD) has not been well established. The purpose of this study was to prospectively define the prevalence of both NAFLD and nonalcoholic steatohepatitis (NASH).



Outpatients 18 to 70 years old were recruited from Brooke Army Medical Center. All patients completed a baseline questionnaire and ultrasound. If fatty liver was identified, then laboratory data and a liver biopsy were obtained.



Four hundred patients were enrolled. Three hundred and twenty-eight patients completed the questionnaire and ultrasound. Mean age (range, 28-70 years) was 54.6 years (7.35); 62.5% Caucasian, 22% Hispanic, and 11.3% African American; 50.9% female; mean body mass index (BMI) (calculated as kg/m(2)) was 29.8 (5.64); and diabetes and hypertension prevalence 16.5% and 49.7%, respectively. Prevalence of NAFLD was 46%. NASH was confirmed in 40 patients (12.2% of total cohort, 29.9% of ultrasound positive patients). Hispanics had the highest prevalence of NAFLD (58.3%), then Caucasians (44.4%) and African Americans (35.1%). NAFLD patients were more likely to be male (58.9%), older (P = .004), hypertensive (P < .00005), and diabetic (P < .00005). They had a higher BMI (P < .0005), ate fast food more often (P = .049), and exercised less (P = 0.02) than their non-NAFLD counterparts. Hispanics had a higher prevalence of NASH compared with Caucasians (19.4% vs 9.8%; P = .03). Alanine aminotransferase, aspartate aminotransferase, BMI, insulin, Quantitative Insulin-Sensitivity Check Index, and cytokeratin-18 correlated with NASH. Among the 54 diabetic patients, NAFLD was found in 74% and NASH in 22.2%.



Prevalence of NAFLD and NASH is higher than estimated previously. Hispanics and patients with diabetes are at greatest risk for both NAFLD and NASH.

title: Non‐alcoholic fatty liver disease progresses to hepatocellular carcinoma in the absence of apparent cirrhosis
abstract: Non‐alcoholic fatty liver disease (NAFLD) is the most common liver disease in developed countries, and accumulating evidence suggests it as the hepatic manifestation of the metabolic syndrome (MS). Although the published prevalence of hepatocellular carcinoma (HCC) is low in NAFLD/NASH patients, most of these data have been derived from areas endemic for viral hepatitis. We recruited 162 adults with HCC between February 2007 and March 2008, investigated the underlying etiologies and determined the prevalence of the MS and related features within each group. Patients with NAFLD/NASH‐associated HCC exhibited a higher prevalence of metabolic features (Type 2 diabetes mellitus, hypertension, dyslipidemia, coronary artery disease) compared to non‐NAFLD/NASH‐HCC. Intriguingly, a significant number (41.7%; p < 0.005) of individuals with NAFLD/NASH‐HCC had no evidence of cirrhosis. Patients with alcohol‐induced liver disease also displayed many features (14/19, 73.7%) of the MS, although, in contrast to NAFLD/NASH‐HCC, alcohol‐associated HCC was highly associated with cirrhosis (95.0%; p = 0.064). NAFLD/NASH as the hepatic entity of the MS may itself pose a risk factor for HCC, even in the absence of cirrhosis. The MS may also promote development of HCC among those with alcoholic liver disease. Increased awareness of liver manifestations in the MS may instigate early interventions against developing HCC.

title: NASH and insulin resistance: Insulin hypersecretion and specific association with the insulin resistance syndrome
abstract: Nonalcoholic steatohepatitis (NASH) is often linked with disorders that are clearly associated with insulin resistance (IR): obesity, type 2 diabetes mellitus, and hypertriglyceridemia. We tested the hypotheses that (1) IR is an essential requirement for the development of NASH and (2) a high association between IR and liver disease is relatively specific for NASH. We measured body mass index (BMI), waist/hip ratio, and fasting serum lipid, insulin, C‐peptide, and glucose levels in 66 patients with NASH (21 with advanced fibrosis and 45 with mild fibrosis). IR was determined by the homeostasis model assessment (HOMA). We also determined the strength of the association of NASH with insulin resistance syndrome (IRS) as defined by World Health Organization criteria. To assess whether the finding of IR was relatively specific to NASH rather than simply to obesity or liver disease, we compared the results of a subset of 36 patients with less‐severe NASH with 36 age‐ and sex‐matched patients with chronic hepatitis C virus (HCV) of comparable fibrotic severity. IR was confirmed in 65 patients (98%) with NASH, and 55 (87%) fulfilled minimum criteria for IRS. IR was found in lean as well as in overweight and obese patients. The IR values and the prevalence of IRS (75% vs. 8.3%) were significantly higher in those with NASH than in comparable cases of HCV. Hyperinsulinemia was attributable to increased insulin secretion rather than decreased hepatic extraction. In conclusion, most patients with NASH have IRS, and there is a near‐universal association between NASH and IR irrespective of obesity. IR is present in mild as well as advanced cases of NASH but is unusual in chronic HCV of similar fibrotic severity.

title: Linagliptin alleviates hepatic steatosis and inflammation in a mouse model of non-alcoholic steatohepatitis
abstract: Non-alcoholic steatohepatitis (NASH) is a primary cause of cirrhosis and hepatocellular carcinoma. Dipeptidyl peptidase (DPP)-4 inhibitors are established therapies for type 2 diabetes and although DPP-4 inhibitors can reduce hepatic steatosis, their impact on local inflammation and fibrosis in NASH remains unknown. Using two different experimental treatment regimens (4- and 2-week treatments) in streptozotocin-treated neonatal mice on a high-fat diet, we show that the DPP-4 inhibitor linagliptin (10 and 30 mg/kg) significantly attenuated the NAS score from 4.9 ± 0.6 to 3.7 ± 0.4 and 3.6 ± 0.3, respectively, in the 4-week study. In the 2-week study, linagliptin 10 mg/kg significantly reduced NAS score from 4.1 ± 0.4 to 2.4 ± 0.4. Telmisartan was used as a positive control in both studies and lowered NAS score to 1.9 ± 0.7 and 1.4 ± 0.3, respectively. Due to streptozotocin treatment, elevated glucose levels were unchanged by either drug treatment. Further, linagliptin 10 mg/kg significantly reduced mRNA levels of SOCS-3 (from 1.68 ± 0.2 to 0.83 ± 0.08), IFN-γ (from 4.0 ± 0.5 to 2.3 ± 0.3), and TNF-α (from 5.7 ± 0.5 to 2.13 ± 0.3). The latter observation was confirmed by immunohistochemistry of TNF-α in liver specimens. In addition, using microautoradiography, we showed that the distribution of radiolabeled linagliptin was heterogeneous with the highest density associated with interlobular bile ducts and portal tracts (acini). In conclusion, these studies confirm that linagliptin has high exposure in hepatic tissue and has both anti-inflammatory and anti-steatotic activity in NASH.

title: Sitagliptin attenuates methionine/choline-deficient diet-induced steatohepatitis.
abstract: Accumulating evidence suggests that inhibitors of dipeptidyl peptidase-4 (DPP-4), such as sitagliptin, may play an important role in the prevention of non-alcoholic steatohepatitis (NASH). This study was conducted to elucidate whether sitagliptin could prevent steatohepatitis by inhibiting pathways involved in hepatic steatosis, inflammation, and fibrosis.



C57BL/6 mice were fed a methionine/choline-deficient (MCD) diet with or without supplement with sitagliptin for 5 weeks. Liver and adipose tissue from mice were examined histologically and immunohistochemically to estimate the effect of sitagliptin on the development of NASH.



Supplementation with sitagliptin resulted in significant improvement of MCD diet-induced fat accumulation in the liver. In addition, sitagliptin treatment lowered fatty acid uptake, expression of VLDL receptor and hepatic triglyceride content. Sitagliptin also effectively attenuated MCD diet-induced hepatic inflammation, endoplasmic reticulum (ER) stress, and liver injury, as evidenced by reduced proinflammatory cytokine levels, ER stress markers, and TUNEL staining. Expression of CYP2E1 and 4NHE were strongly increased by the MCD diet, but this effect was successfully prevented by sitagliptin treatment. Furthermore, sitagliptin significantly decreased levels of MCD diet-induced fibrosis-associated proteins such as fibronectin and α-SMA in the liver. Inflammatory and atrophic changes of adipose tissue by MCD diet were restored by sitagliptin treatment.



Sitagliptin attenuated MCD diet-induced hepatic steatosis, inflammation, and fibrosis in mice through amelioration of mechanisms responsible for the development of NASH, including CD36 expression, NF-κB activation, ER stress, CYP2E1 expression, and lipid peroxidation. Treatment with sitagliptin may represent an effective approach for the prevention and treatment of NASH.

title: Diet-Induced Adipose Tissue Inflammation and Liver Steatosis Are Prevented by DPP-4 Inhibition in Diabetic Mice
abstract: Diet composition alters the metabolic states of adipocytes and hepatocytes in diabetes. The effects of dipeptidyl peptidase-4 (DPP-4) inhibition on adipose tissue inflammation and fatty liver have been obscure. We investigated the extrapancreatic effects of DPP-4 inhibition on visceral fat and the liver.  We investigated diet-induced metabolic changes in β-cell–specific glucokinase haploinsufficient (Gck+/−) diabetic mice. We challenged animals with a diet containing a combination of sucrose and oleic acid (SO) or sucrose and linoleic acid (SL). Next, we assessed the effects of a DPP-4 inhibitor, des-fluoro-sitagliptin, on adipose tissue inflammation and hepatic steatosis.  The epididymal fat weight and serum leptin level were significantly higher in Gck+/− mice fed SL than in mice fed SO, although no significant differences in body weight or adipocyte size were noted. Compared with SO, SL increased the numbers of CD11c+ M1 macrophages and CD8+ T-cells in visceral adipose tissue and the expression of E-selectin, P-selectin, and plasminogen activator inhibitor-1 (PAI-1). DPP-4 inhibition significantly prevented adipose tissue infiltration by CD8+ T-cells and M1 macrophages and decreased the expression of PAI-1. The production of cytokines by activated T-cells was not affected by DPP-4 inhibition. Furthermore, DPP-4 inhibition prevented fatty liver in both wild-type and Gck+/− mice. DPP-4 inhibition also decreased the expressions of sterol regulatory element–binding protein-1c, stearoyl-CoA desaturase-1, and fatty acid synthase, and increased the expression of peroxisome proliferator–activated receptor-α in the liver.  Our findings indicated that DPP-4 inhibition has extrapancreatic protective effects against diet-induced adipose tissue inflammation and hepatic steatosis.

title: SGLT inhibitors in management of diabetes.
abstract: The two main sodium-glucose cotransporters (SGLTs), SGLT1 and SGLT2, provide new therapeutic targets to reduce hyperglycaemia in patients with diabetes. SGLT1 enables the small intestine to absorb glucose and contributes to the reabsorption of glucose filtered by the kidney. SGLT2 is responsible for reabsorption of most of the glucose filtered by the kidney. Inhibitors with varying specificities for these transporters (eg, dapagliflozin, canagliflozin, and empagliflozin) can slow the rate of intestinal glucose absorption and increase the renal elimination of glucose into the urine. Results of randomised clinical trials have shown the blood glucose-lowering efficacy of SGLT inhibitors in type 2 diabetes when administered as monotherapy or in addition to other glucose-lowering therapies including insulin. Increased renal glucose elimination also assists weight loss and could help to reduce blood pressure. Effective SGLT2 inhibition needs adequate glomerular filtration and might increase risk of urinary tract and genital infection, and excessive inhibition of SGLT1 can cause gastro-intestinal symptoms. However, the insulin-independent mechanism of action of SGLT inhibitors seems to offer durable glucose-lowering efficacy with low risk of clinically significant hypoglycaemia at any stage in the natural history of type 2 diabetes. SGLT inhibition might also be considered in conjunction with insulin therapy in type 1 diabetes.

title: Empagliflozin, a novel selective sodium glucose cotransporter‐2 (SGLT‐2) inhibitor: characterisation and comparison with other SGLT‐2 inhibitors
abstract: Empagliflozin is a selective sodium glucose cotransporter‐2 (SGLT‐2) inhibitor in clinical development for the treatment of type 2 diabetes mellitus. This study assessed pharmacological properties of empagliflozin in vitro and pharmacokinetic properties in vivo and compared its potency and selectivity with other SGLT‐2 inhibitors.

title: Efficacy and safety of empagliflozin for type 2 diabetes: a systematic review and meta‐analysis
abstract: To assess the efficacy and safety of the novel sodium‐glucose cotransporter 2 (SGLT2) inhibitor empagliflozin compared with placebo or other antidiabetic agents in patients with type 2 diabetes.

title: Effects of dapagliflozin on body weight, total fat mass, and regional adipose tissue distribution in patients with type 2 diabetes mellitus with inadequate glycemic control on metformin.
abstract: Dapagliflozin, a selective sodium-glucose cotransporter 2 (SGLT2) inhibitor, reduces hyperglycemia in patients with type 2 diabetes mellitus (T2DM) by increasing urinary glucose excretion, and weight loss is a consistent associated finding.



Our objectives were to confirm weight loss with dapagliflozin and establish through body composition measurements whether weight loss is accounted for by changes in fat or fluid components.



This was a 24-wk, international, multicenter, randomized, parallel-group, double-blind, placebo-controlled study with ongoing 78-wk site- and patient-blinded extension period at 40 sites in five countries.



Included were 182 patients with T2DM (mean values: women 63.3 and men 58.6 yr of age; hemoglobin A1c 7.17%, body mass index 31.9 kg/m2, and body weight 91.5 kg) inadequately controlled on metformin.



Dapagliflozin 10 mg/d or placebo was added to open-label metformin for 24 wk.



Primary endpoint was total body weight (TBW) change from baseline at wk 24. Key secondary endpoints were waist circumference and dual-energy x-ray absorptiometry total-body fat mass (FM) changes from baseline at wk 24, and patient proportion achieving body weight reduction of at least 5% at wk 24. In a subset of patients, magnetic resonance assessment of visceral adipose tissue (VAT) and sc adipose tissue (SAT) volume and hepatic lipid content were also evaluated.



At wk 24, placebo-corrected changes with dapagliflozin were as follows: TBW, -2.08 kg [95% confidence interval (CI)=-2.84 to -1.31; P<0.0001]; waist circumference, -1.52 cm (95% CI=-2.74 to -0.31; P=0.0143); FM, -1.48 kg (95% CI=-2.22 to -0.74; P=0.0001); proportion of patients achieving weight reduction of at least 5%, +26.2% (95% CI=15.5 to 36.7; P<0.0001); VAT, -258.4 cm3 (95% CI=-448.1 to -68.6; nominal P=0.0084); SAT, -184.9 cm3 (95% CI=-359.7 to -10.1; nominal P=0.0385). In the dapagliflozin vs. placebo groups, respectively, serious adverse events were reported in 6.6 vs. 1.1%; events suggestive of vulvovaginitis, balanitis, and related genital infection in 3.3 vs. 0%; and lower urinary tract infections in 6.6 vs. 2.2%.



Dapagliflozin reduces TBW, predominantly by reducing FM, VAT and SAT in T2DM inadequately controlled with metformin.

title: Combination of the sodium-glucose cotransporter-2 inhibitor empagliflozin with orlistat or sibutramine further improves the body-weight reduction and glucose homeostasis of obese rats fed a cafeteria diet
abstract: The present study assessed the potential of the sodium glucose-linked transporter (SGLT)-2 inhibitor empagliflozin to decrease body weight when administered alone or in combination with the clinically effective weight-loss agents orlistat and sibutramine in obese rats fed a cafeteria diet. Female Wistar rats were exposed to a cafeteria diet to induce obesity. Empagliflozin was dosed once daily (10, 30, and 60 mg/kg) for 28 days. Combination studies were subsequently performed using a submaximal empagliflozin dose (10 mg/kg) with either sibutramine or orlistat. Body weight, food, and water intake were recorded daily. The effect of drug treatment on glucose tolerance, relevant plasma parameters, and carcass composition was determined. Empagliflozin dose-dependently reduced body weight, plasma leptin, and body fat though increased urinary glucose excretion. The combination of empagliflozin and orlistat significantly reduced body weight compared to animals treated with either drug alone, and significantly improved glucose tolerance, plasma insulin, and leptin compared to vehicle-treated controls. The effect of sibutramine to improve glycemic control in an oral glucose-tolerance test was also significantly increased, with empagliflozin and combination treatment leading to a reduction in carcass fat greater than that observed with either drug alone. These data demonstrate that empagliflozin reduces body weight in cafeteria-fed obese rats. In combination studies, empagliflozin further improved the body-weight or body-fat loss of animals in comparison to orlistat or sibutramine alone. Such studies may indicate improved strategies for the treatment of obese patients with prediabetes or type 2 diabetes.

title: Effects of SGLT2 selective inhibitor ipragliflozin on hyperglycemia, hyperlipidemia, hepatic steatosis, oxidative stress, inflammation, and obesity in type 2 diabetic mice.
abstract: The sodium-glucose cotransporter 2 (SGLT2) is responsible for most glucose reabsorption in the kidney and has been proposed as a novel therapeutic target for the treatment of type 2 diabetes. In the present study, the therapeutic effects of SGLT2 selective inhibitor ipragliflozin were examined in high-fat diet and streptozotocin-nicotinamide-induced type 2 diabetic mice which exhibit impaired insulin secretion, insulin resistance, hyperlipidemia, hepatic steatosis, and obesity. Single administration of ipragliflozin dose-dependently increased urinary glucose excretion, reduced blood glucose and plasma insulin levels, and improved glucose intolerance. Four-week repeated administration of ipragliflozin improved not only glucose tolerance, hyperglycemia, and hyperinsulinemia but also impaired insulin secretion, hyperlipidemia, hepatic steatosis, and obesity with a concomitant increase in urinary glucose excretion. In addition, ipragliflozin reduced plasma and liver levels of oxidative stress biomarkers (thiobarbituric acid reactive substances and protein carbonyl) and inflammatory markers (interleukin 6, tumor necrosis factor α, monocyte chemotactic protein-1, and c-reactive protein), and improved liver injury as assessed by plasma levels of aminotransferases. These results demonstrate that SGLT2 selective inhibitor ipragliflozin improves not only hyperglycemia but also diabetes/obesity-associated metabolic abnormalities in type 2 diabetic mice and suggest that ipragliflozin may be useful in treating type 2 diabetes with metabolic syndrome.

title: Ipragliflozin Improves Hepatic Steatosis in Obese Mice and Liver Dysfunction in Type 2 Diabetic Patients Irrespective of Body Weight Reduction
abstract: Type 2 diabetes mellitus (T2DM) is associated with a high incidence of non-alcoholic fatty liver disease (NAFLD) related to obesity and insulin resistance. Currently, medical interventions for NAFLD have focused on diet control and exercise to reduce body weight, and there is a requirement for effective pharmacological therapies. Sodium-glucose cotransporter 2 (SGLT2) inhibitors are oral antidiabetic drugs that promote the urinary excretion of glucose by blocking its reabsorption in renal proximal tubules. SGLT2 inhibitors lower blood glucose independent of insulin action and are expected to reduce body weight because of urinary calorie loss. Here we show that an SGLT2 inhibitor ipragliflozin improves hepatic steatosis in high-fat diet-induced and leptin-deficient (ob/ob) obese mice irrespective of body weight reduction. In the obese mice, ipragliflozin-induced hyperphagia occurred to increase energy intake, attenuating body weight reduction with increased epididymal fat mass. There is an inverse correlation between weights of liver and epididymal fat in ipragliflozin-treated obese mice, suggesting that ipragliflozin treatment promotes normotopic fat accumulation in the epididymal fat and prevents ectopic fat accumulation in the liver. Despite increased adiposity, ipragliflozin ameliorates obesity-associated inflammation and insulin resistance in epididymal fat. Clinically, ipragliflozin improves liver dysfunction in patients with T2DM irrespective of body weight reduction. These findings provide new insight into the effects of SGLT2 inhibitors on energy homeostasis and fat accumulation and indicate their potential therapeutic efficacy in T2DM-associated hepatic steatosis.

title: Treatment with the SGLT2 inhibitor luseogliflozin improves nonalcoholic steatohepatitis in a rodent model with diabetes mellitus
abstract: BackgroundInsulin resistance with elevated glucose is a risk factor for non-alcoholic steatohepatitis (NASH). We investigated the effects of the sodium glucose cotransporter 2 (SGLT2) inhibitor luseogliflozin on NASH development using a rodent model.MethodsMice were treated with both nicotinamide and streptozotocin (NA/STZ) to reduce insulin secretory capacity, and then fed a high fat diet containing trans fatty acids (HFDT) for 8 weeks. The NA/STZ HFDT-fed mice were divided into two groups, either treated with luseogliflozin or untreated, during this period. The glucose elevations in the NA/STZ-treated and HFDT-fed mice were significantly improved by luseogliflozin administration. While HFDT feeding induced NASH development as shown by liver weight gain with lipid accumulation and increased serum alanine aminotransferase, these changes were all attenuated in the group treated with luseogliflozin. In addition, fibrotic change and increases in collagen deposition with upregulations of collagen1 and smooth muscle actin and inflammatory cytokine expressions observed in the HFDT-fed mouse livers were also normalized by luseogliflozin administration.ConclusionsTaken together, these results obtained in mice demonstrate the favorable effects of administering SGLT2 inhibitors, for the treatment of NASH associated with diabetes mellitus. We anticipate that these agents would be applicable to humans.

title: A murine model for non-alcoholic steatohepatitis showing evidence of association between diabetes and hepatocellular carcinoma
abstract: Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related deaths. In addition to hepatitis viral infections, several cohort studies have shown that diabetes mellitus is a risk factor of HCC, making the incidence alarming high. However, it has not been demonstrated directly how diabetes develops to HCC, because of its difficulty to follow changes of liver histology in diabetic populations. Here, we report that non-alcoholic steatohepatitis (NASH) is pivotal to link diabetes with HCC by establishing a novel, reproducible NASH–HCC model in mice. Neonatal male mice exposed to low-dose streptozotocin (STZ) developed liver steatosis with diabetes 1 week after feeding high-fat diet (HFD). Continuous HFD decreased hepatic fat deposit whilst increased lobular inflammation with foam cell-like macrophages, showing NASH pathology. In parallel with decreased phagocytosis of macrophages, fibroblasts accumulated to form “chicken-wired” fibrosis. All mice developed multiple HCC later. Female mice treated with STZ–HFD and male mice treated with STZ alone showed diabetes but never developed HCC by the absence of NASH-based fibrosis. Thus, the present study provides the evidence in novel mouse model that NASH-based fibrosis is an essential histological process for diabetic populations to accelerate the development of HCC.

title: The SGLT2 inhibitor empagliflozin improves insulin sensitivity in db/db mice both as monotherapy and in combination with linagliptin.
abstract: Combining different drug classes to improve glycemic control is one treatment strategy for type 2 diabetes. The effects on insulin sensitivity of long-term treatment with the sodium glucose co-transporter 2 (SGLT2) inhibitor empagliflozin alone or co-administered with the dipeptidyl peptidase-4 inhibitor linagliptin (both approved antidiabetes drugs) were investigated in mice using euglycemic-hyperinsulinemic clamps.



db/db mice (n=15/group) were treated for 8weeks with 10mg/kg/day empagliflozin monotherapy, 10mg/kg/day empagliflozin plus 3mg/kg/day linagliptin combination therapy, or 3mg/kg/day linagliptin monotherapy. At the end of the study, euglycemic-hyperinsulinemic clamp studies were performed 4days after the last dose of treatment.



HbA1c and 2-hour fasting glucose concentrations were improved with empagliflozin monotherapy and combination therapy compared with vehicle and linagliptin monotherapy. During the clamp, glucose disposal rates increased and hepatic glucose production decreased with empagliflozin monotherapy and combination therapy compared with vehicle and linagliptin monotherapy. Glucose uptake in liver and kidney was higher with empagliflozin monotherapy and combination therapy compared with vehicle; glucose uptake into both muscle and adipose tissue was only affected by linagliptin treatment. Empagliflozin and combination therapy altered the expression of genes involved in the inflammatory response, fatty acid synthesis and oxidation.



These findings suggest that the insulin-sensitizing effects of SGLT2 inhibition contribute to improvements in glycemic control in insulin-resistant states.

title: Serum Dipeptidyl Peptidase-4 Activity in Insulin Resistant Patients with Non-Alcoholic Fatty Liver Disease: A Novel Liver Disease Biomarker
abstract: In a cross-sectional study we studied the fasting serum DPP-4 enzymatic activity (sDPP-4) and the insulin resistance index (HOMA2-IR) in gliptin naïve patients with type 2 diabetes and in non-alcoholic fatty liver disease (NAFLD) and in healthy controls (CNTRL).  was measured by kinetic assay in 39 NAFLD (F/M:19/20, mean age: 47.42 yrs) and 82 type 2 diabetes (F/M:48/34, 62.8 yrs) patients and 26 (F/M:14/12, 35.3 yrs) controls. Definition of T2D group as patients with type 2 diabetes but without clinically obvious liver disease created non-overlapping study groups. Diagnosis of NAFLD was based on ultrasonography and the exclusion of other etiololgy. Patients in T2D and NAFLD groups were similarly obese. 75 g CH OGTT in 39 NAFLD patients: 24-NGT, 4-IGT or IFG (“prediabetes”), 11-type 2 diabetes. HOMA2-IR: CNTRL: 1.44; T2D-group: 2.62 (p = 0.046 vs CNTRL, parametric tests); NAFLD(NGTonly): 3.23 (p = 0.0013 vs CNTRL); NAFLD(IFG/IGT/type 2 diabetes): 3.82 (p<0.001 vs CNTRL, p = 0.049 vs 2TD group). sDPP-4 activity was higher in NAFLD both with NGT (mean:33.08U/L) and abnormal glucose metabolism (30.38U/L) than in CNTRL (25.89U/L, p<0.001 and p = 0.013) or in T2D groups (23.97U/L, p<0.001 and p = 0.004). Correlations in NAFLD among sDPP-4 and ALT: r = 0.4637,p = 0.0038 and γGT: r = 0.4991,p = 0.0017 and HOMA2-IR: r = 0.5295,p = 0.0026 and among HOMA2-IR and ALT: r = 0.4340,p = 0.0147 and γGT: r = 0.4128,p = 0.0210.  The fasting serum DPP-4 activity was not increased in T2D provided that patients with liver disease were intentionally excluded. The high serum DPP-4 activities in NAFLD were correlated with liver tests but not with the fasting plasma glucose or HbA1C supporting that the excess is of hepatic origin and it might contribute to the speedup of metabolic deterioration. The correlation among γGT, ALT and serum DPP-4 activity and also between serum DPP-4 activity and HOMA2-IR in NAFLD strongly suggests that serum DPP-4 activity should be considered as a novel liver disease biomarker.

title: Serum level of soluble CD26/dipeptidyl peptidase-4 (DPP-4) predicts the response to sitagliptin, a DPP-4 inhibitor, in patients with type 2 diabetes controlled inadequately by metformin and/or sulfonylurea.
abstract: Dipeptidyl peptidase 4 (DPP-4) inhibitors is a new class of antihyperglycemic agents that is now available for the treatment of type 2 diabetes. We investigated the relationship between the baseline serum level of soluble CD 26/DPP-4 and the response to treatment with sitagliptin, a DPP-4 inhibitor, over 24 weeks in patients who had type 2 diabetes inadequately controlled by metformin and/or sulfonylurea therapy. We studied 52 consecutive patients with type 2 diabetes who had poor glycemic control despite treatment with metformin and/or sulfonylurea. All patients were given 50 mg/day of sitagliptin and were followed at monthly intervals for 24 weeks. Treatment with sitagliptin decreased significantly hemoglobin A1c (HbA1c) from 7.91 ± 1.08% at baseline to 6.96 ± 1.18% at 8 weeks, 7.04 ± 0.77% at 16 weeks, and 7.08 ± 0.80% at 24 weeks. The baseline serum level of sCD26 was correlated positively with HbA1c at both 16 weeks and 24 weeks. Furthermore, the serum sCD26 level at baseline was also correlated positively with the changes from baseline of HbA1c at 16 and 24 weeks (r = 0.318, P = 0.0296 and r = 0.516, P = 0.0003, respectively). In a multivariate logistic regression model that explained 56.1% (R(2) = 0.561) of the variation of the changes from baseline of HbA1c at 24 weeks, the baseline HbA1c (β = -0.638, P < 0.001) and serum sCD26 (β = 0.357, P = 0.041) were independent determinants of the change of HbA1c at 24 weeks. In conclusions, a higher serum level of sCD26 is associated with a worse response to sitagliptin in patients with type 2 diabetes controlled inadequately by metformin and/or sulfonylurea therapy.

title: Nonalcoholic fatty liver disease and lipids
abstract: This article reviews the mechanisms leading to the development of nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) and the effects of hypoglycaemic and lipid-lowering therapies on NAFLD/NASH.  The interaction of lipogenesis, fatty acid oxidation, inflammation, endoplasmic reticulum stress and hepatic insulin resistance contribute to the pathogenesis of NAFLD/NASH. Few large scale clinical trials exist with biopsy or magnetic resonance endpoints as opposed to ultrasonographic and transaminase endpoints. Trial evidence that exists supports the utility of weight loss, metformin, thiazolidinediones, fibrates, niacin, ezetimibe and statins in improving the steatosis component of NAFLD/NASH though with less or minimal effects on the fibrotic component of NASH.  Hypoglycaemic and lipid-lowering therapies may have a role in the treatment of NAFLD/NASH but large scale endpoint trials remain to be performed.

title: Impact of current treatments on liver disease, glucose metabolism and cardiovascular risk in non-alcoholic fatty liver disease (NAFLD): a systematic review and meta-analysis of randomised trials
abstract: liver disease (NAFLD) encompasses a spectrum ranging from simple steatosis to non-alcoholic steatohepatitis (NASH): NAFLD causes an increased risk of cardiovascular disease, diabetes and liver-related complications (the latter confined to NASH). The effect of proposed treatments on liver disease, glucose metabolism and cardiovascular risk in NAFLD is unknown. We reviewed the evidence for the management of liver disease and cardio-metabolic risk in NAFLD.MethodsPublications through November 2011 were systematically reviewed by two authors. Outcomes evaluated though standard methods were: histological/radiological/biochemical features of NAFLD, variables of glucose metabolism and cardiovascular risk factors. Seventy-eight randomised trials were included (38 in NASH, 40 in NAFLD): 41% assessed post-treatment histology, 71% assessed glucose metabolism and 88% assessed cardiovascular risk factors. Lifestyle intervention, thiazolidinediones, metformin and antioxidants were most extensively evaluated.ResultsLifestyle-induced weight loss was safe and improved cardio-metabolic risk profile; a weight loss ≥7% improved histological disease activity, but was achieved by <50% patients. Statins and polyunsaturated fatty acids improved steatosis, but their effects on liver histology are unknown. Thiazolidinediones improved histological disease activity, glucose, lipid and inflammatory variables and delayed fibrosis progression. Pioglitazone also improved blood pressure. Weight gain (up to 4.8%) was common. Antioxidants yielded mixed histological results: vitamin E improved histological disease activity when administered for 2 years, but increased insulin resistance and plasma triacylglycerols. loss is safe, and improves liver histology and cardio-metabolic profile. For patients not responding to lifestyle intervention, pioglitazone improves histological disease activity, slows fibrosis progression and extensively ameliorates cardio-metabolic endpoints. Further randomised controlled trials (RCTs) of adequate size and duration will assess long-term safety and efficacy of proposed treatments on clinical outcomes.

title: Weight Loss Induced by Chronic Dapagliflozin Treatment Is Attenuated by Compensatory Hyperphagia in Diet‐Induced Obese (DIO) Rats
abstract: Dapagliflozin is a potent and selective sodium glucose cotransporter‐2 (SGLT2) inhibitor which promotes urinary glucose excretion and induces weight loss. Since metabolic compensation can offset a negative energy balance, we explored the potential for a compensatory physiological response to the weight loss induced by dapagliflozin. Dapagliflozin was administered (0.5–5 mpk; p.o.) to diet‐induced obese (DIO) rats with or without ad libitum access to food for 38 days. Along with inducing urinary glucose excretion, chronic administration of dapagliflozin dose‐dependently increased food and water intake relative to vehicle‐treated controls. Despite this, it reduced body weight by 4% (relative to controls) at the highest dose. The degree of weight loss was increased by an additional 9% if hyperphagia was prevented by restricting food intake to that of vehicle controls. Neither oxygen consumption (vO2) or the respiratory exchange ratio (RER) were altered by dapagliflozin treatment alone. Animals treated with dapagliflozin and pair‐fed to vehicle controls (5 mpk PF‐V) showed a reduction in RER and an elevation in nonfasting β‐hydroxybutyrate (BHBA) relative to ad libitum‐fed 5 mpk counterparts. Fasting BHBA was elevated in the 1 mpk, 5 mpk, and 5 mpk PF‐V groups. Serum glucose was reduced in the fasted, but not the unfasted state. Insulin was reduced in the non‐fasted state. These data suggest that in rodents, the persistent urinary glucose excretion induced by dapagliflozin was accompanied by compensatory hyperphagia, which attenuated the weight loss induced by SGLT2 inhibition. Therefore, it is possible that dapagliflozin‐induced weight loss could be enhanced with dietary intervention.

title: Fatty acid synthase and liver triglyceride metabolism: housekeeper or messenger?
abstract: Fatty acid synthase (FAS) catalyzes the de novo synthesis of fatty acids. In the liver, FAS has long been categorized as a housekeeping protein, producing fat for storage of energy when nutrients are present in excess. Most previous studies of FAS regulation have focused on the control of gene expression. However, recent findings suggest that hepatic FAS may also be involved in signaling processes that include activation of peroxisome proliferator-activated receptor α (PPARα). Moreover, reports of rapid alterations in FAS activity as well as findings of post-translational modifications of the FAS protein support the notion that dynamic events in addition to transcription impact FAS regulation. These results indicate that FAS enzyme activity can impact liver physiology through signaling as well as energy storage and that its regulation may be complex. This article is part of a Special Issue entitled Triglyceride Metabolism and Disease.

title: The value of α‐SMA in the evaluation of hepatic fibrosis severity in hepatitis B infection and cirrhosis development: a histopathological and immunohistochemical study
abstract: To investigate the value of α‐smooth muscle actin (α‐SMA), an indicator of stellate cell activation, in predicting fibrosis in chronic hepatitis B (CHB) patients.

title: Nonalcoholic fatty liver disease: From steatosis to cirrhosis
abstract: Nonalcoholic steatohepatitis (NASH), the lynchpin between steatosis and cirrhosis in the spectrum of nonalcoholic fatty liver disorders (NAFLD), was barely recognized in 1981. NAFLD is now present in 17% to 33% of Americans, has a worldwide distribution, and parallels the frequency of central adiposity, obesity, insulin resistance, metabolic syndrome and type 2 diabetes. NASH could be present in one third of NAFLD cases. Age, activity of steatohepatitis, and established fibrosis predispose to cirrhosis, which has a 7‐ to 10‐year liver‐related mortality of 12% to 25%. Many cases of cryptogenic cirrhosis are likely endstage NASH. While endstage NAFLD currently accounts for 4% to 10% of liver transplants, this may soon rise. Pathogenic concepts for NAFLD/NASH must account for the strong links with overnutrition and underactivity, insulin resistance, and genetic factors. Lipotoxicity, oxidative stress, cytokines, and other proinflammatory mediators may each play a role in transition of steatosis to NASH. The present “gold standard” management of NASH is modest weight reduction, particularly correction of central obesity achieved by combining dietary measures with increased physical activity. Whether achieved by “lifestyle adjustment” or anti‐obesity surgery, this improves insulin resistance and reverses steatosis, hepatocellular injury, inflammation, and fibrosis. The same potential for “unwinding” fibrotic NASH is indicated by studies of the peroxisome proliferation activator receptor (PPAR)‐γ agonist “glitazones,” but these agents may improve liver disease at the expense of worsening obesity. Future challenges are to approach NAFLD as a preventive public health initiative and to motivate affected persons to adopt a healthier lifestyle.

title: Non‐alcoholic steatohepatitis: Pathogenesis and novel therapeutic approaches
abstract: Non-alcoholic fatty liver disease (NAFLD) refers to a disease spectrum, ranging from mere hepatic steatosis to hepatic necroinflammation (NASH, non-alcoholic steatohepatitis). NASH often leads to fibrosis, which can progress to cirrhosis with a high risk of liver failure and hepatocellular carcinoma. The course of NAFLD is highly variable, and only a minority of patients (2-3%) progress to end-stage liver disease. However, due to a dramatic increase of the risk factors for NAFLD, that is obesity and insulin resistance/type 2 diabetes, that affect 15-30% and 7-15% of subjects, in most industrialized countries, respectively, NAFLD has become the most frequent liver disease and is even considered a pace setter of the metabolic syndrome. Sedentary lifestyle, modern Western nutrition, and genetic predispositions have been identified as major causes of NAFLD. These lead to liver injury via insulin resistance and an excess of free fatty acids in hepatocytes, resulting in oxidant stress and lipotoxicity that promote the activation of intracellular stress kinases and apoptosis or necroapoptosis (NASH). The damaged hepatocytes directly trigger inflammation and fibrogenesis, but can also lead to the emergence of fibrogenic progenitor cells. Moreover, NASH is linked to inflammation in peripheral adipose tissues that involves mainly macrophages and humoral factors, such as adipokines and cytokines. The most efficient treatment is by weight loss and exercise, but (adjunctive) pharmacological strategies are urgently needed. Here, we highlight the aspects of NAFLD epidemiology and pathophysiology that are beginning to lead to novel pharmacological approaches to address this growing health-care challenge.

title: Linagliptin Improves Insulin Sensitivity and Hepatic Steatosis in Diet-Induced Obesity
abstract: Linagliptin (tradjenta™) is a selective dipeptidyl peptidase-4 (DPP-4) inhibitor. DPP-4 inhibition attenuates insulin resistance and improves peripheral glucose utilization in humans. However, the effects of chronic DPP-4 inhibition on insulin sensitivity are not known. The effects of long-term treatment (3–4 weeks) with 3 mg/kg/day or 30 mg/kg/day linagliptin on insulin sensitivity and liver fat content were determined in diet-induced obese C57BL/6 mice. Chow-fed animals served as controls. DPP-4 activity was significantly inhibited (67–89%) by linagliptin (P<0.001). Following an oral glucose tolerance test, blood glucose concentrations (measured as area under the curve) were significantly suppressed after treatment with 3 mg/kg/day (–16.5% to –20.3%; P<0.01) or 30 mg/kg/day (–14.5% to –26.4%; P<0.05) linagliptin (both P<0.01). Liver fat content was significantly reduced by linagliptin in a dose-dependent manner (both doses P<0.001). Diet-induced obese mice treated for 4 weeks with 3 mg/kg/day or 30 mg/kg/day linagliptin had significantly improved glycated hemoglobin compared with vehicle (both P<0.001). Significant dose-dependent improvements in glucose disposal rates were observed during the steady state of the euglycemic–hyperinsulinemic clamp: 27.3 mg/kg/minute and 32.2 mg/kg/minute in the 3 mg/kg/day and 30 mg/kg/day linagliptin groups, respectively; compared with 20.9 mg/kg/minute with vehicle (P<0.001). Hepatic glucose production was significantly suppressed during the clamp: 4.7 mg/kg/minute and 2.1 mg/kg/minute in the 3 mg/kg/day and 30 mg/kg/day linagliptin groups, respectively; compared with 12.5 mg/kg/minute with vehicle (P<0.001). In addition, 30 mg/kg/day linagliptin treatment resulted in a significantly reduced number of macrophages infiltrating adipose tissue (P<0.05). Linagliptin treatment also decreased liver expression of PTP1B, SOCS3, SREBP1c, SCD-1 and FAS (P<0.05). Other tissues like muscle, heart and kidney were not significantly affected by the insulin sensitizing effect of linagliptin. Long-term linagliptin treatment reduced liver fat content in animals with diet-induced hepatic steatosis and insulin resistance, and may account for improved insulin sensitivity.

title: SOCS-1 and SOCS-3 Block Insulin Signaling by Ubiquitin-mediated Degradation of IRS1 and IRS2*
abstract: Inflammation associates with peripheral insulin resistance, which dysregulates nutrient homeostasis and leads to diabetes. Inflammation induces the expression of SOCS proteins. We show that SOCS1 or SOCS3 targeted IRS1 and IRS2, two critical signaling molecules for insulin action, for ubiquitin-mediated degradation. SOCS1 or SOCS3 bound both recombinant and endogenous IRS1 and IRS2 and promoted their ubiquitination and subsequent degradation in multiple cell types. Mutations in the conserved SOCS box of SOCS1 abrogated its interaction with the elongin BC ubiquitin-ligase complex without affecting its binding to IRS1 or IRS2. The SOCS1 mutants also failed to promote the ubiquitination and degradation of either IRS1 or IRS2. Adenoviral-mediated expression of SOCS1 in mouse liver dramatically reduced hepatic IRS1 and IRS2 protein levels and caused glucose intolerance; by contrast, expression of the SOCS1 mutants had no effect. Thus, SOCS-mediated degradation of IRS proteins, presumably via the elongin BC ubiquitin-ligase, might be a general mechanism of inflammation-induced insulin resistance, providing a target for therapy.

title: Suppressor of Cytokine Signaling-3 (SOCS-3), a Potential Mediator of Interleukin-6-dependent Insulin Resistance in Hepatocytes*
abstract: Interleukin-6 (IL-6) is one of several pro-inflammatory cytokines implicated in insulin resistance during infection, cachexia, and obesity. We recently demonstrated that IL-6 inhibits insulin signaling in hepatocytes (Senn, J. J., Klover, P. J., Nowak, I. A., and Mooney, R. A. (2002) Diabetes51, 3391–3399). Members of the suppressors of cytokine signaling (SOCS) family associate with the insulin receptor (IR), and their ectopic expression inhibits IR signaling. Since several SOCS proteins are induced by IL-6, a working hypothesis is that IL-6-dependent insulin resistance is mediated, at least in part, by induction of SOCS protein(s) in insulin target cells. To examine the involvement of SOCS protein(s) in IL-6-dependent inhibition of insulin receptor signaling, HepG2 cells were treated with IL-6 (20 ng/ml) for periods from 1 min to 8 h. IL-6 induced SOCS-3 transcript at 30 min with a maximum effect at 1 h. SOCS-3 protein levels were also markedly elevated at 1 h. Transcript and protein levels returned to near basal levels by 2 h. SOCS-3 induction by IL-6 paralleled IL-6-dependent inhibition of IR signal transduction. Ectopically expressed SOCS-3 associated with the IR and suppressed insulin-dependent receptor autophosphorylation, insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation, association of IRS-1 with the p85 subunit of phosphatidylinositol 3-kinase, and activation of Akt. SOCS-3 was also a direct inhibitor of insulin receptor autophosphorylation in vitro. In mice exposed to IL-6 for 60–90 min, hepatic SOCS-3 expression was increased. This was associated with inhibition of hepatic insulin-dependent receptor autophosphorylation and IRS-1 tyrosine phosphorylation. These data suggest that induction of SOCS-3 in liver may be an important mechanism of IL-6-mediated insulin resistance.