Insulin treatment improves liver histopathology and decreases expression of inflammatory and fibrogenic genes in a hyperglycemic, dyslipidemic hamster model of NAFLD.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are highly prevalent comorbidities in patients with Type 2 diabetes. While many of these patients eventually will need treatment with insulin, little is known about the effects of insulin treatment on histopathological parameters and hepatic gene expression in diabetic patients with co-existing NAFLD and NASH. To investigate this further, we evaluated the effects of insulin treatment in NASH diet-fed hamsters with streptozotocin (STZ) -induced hyperglycemia. METHODS: Forty male Syrian hamsters were randomized into four groups (n = 10/group) receiving either a NASH-inducing (high fat, fructose and cholesterol) or control diet (CTRL) for four weeks, after which they were treated with STZ or sham-injected and from week five treated with either vehicle (CTRL, NASH, NASH-STZ) or human insulin (NASH-STZ-HI) for four weeks by continuous s.c. infusion via osmotic minipumps. RESULTS: NASH-STZ hamsters displayed pronounced hyperglycemia, dyslipidemia and more severe liver pathology compared to both CTRL and NASH groups. Insulin treatment attenuated dyslipidemia in NASH-STZ-HI hamsters and liver pathology was considerably improved compared to the NASH-STZ group, with prevention/reversal of hepatic steatosis, hepatic inflammation and stellate cell activation. In addition, expression of inflammatory and fibrotic genes was decreased compared to the NASH-STZ group. CONCLUSIONS: These results suggest that hyperglycemia is important for development of inflammation and profibrotic processes in the liver, and that insulin administration has beneficial effects on liver pathology and expression of genes related to inflammation and fibrosis in a hyperglycemic, dyslipidemic hamster model of NAFLD.

Long-acting CCK analogue NN9056 lowers food intake and body weight in obese Göttingen Minipigs.

BACKGROUND/OBJECTIVES: Cholecystokinin (CCK) is a regulator of appetite and energy intake in man. The aim of this study was to determine the effect of NN9056, a long-acting CCK-1 receptor-selective CCK analogue, on food intake and body weight (BW) in obese Göttingen Minipigs. SUBJECTS/METHODS: Tolerability of NN9056 and acute effects on food intake, pancreas histology, amylase and lipase levels were assessed in lean domestic pigs in doses up to 100 nmol/kg (n = 3-4). Subsequently, obese Göttingen Minipigs were treated subcutaneously (s.c.) once daily for 13 weeks with vehicle, NN9056 low dose (regulated from 5 to 2 nmol/kg) or NN9056 high dose (10 nmol/kg) (n = 7-8). Food intake was measured daily and BW twice weekly. At the end of the treatment period, an intravenous glucose tolerance test (IVGTT) and a 24-h exposure profile was obtained. Data are mean ± SD. RESULTS: The acute studies in domestic pigs showed significant and dose-dependent effect of NN9056 on food intake, acceptable tolerability and no histopathological signs of pancreatitis. Sub-chronic treatment in obese Göttingen Minipigs was also well tolerated and accumulated food intake was significantly lower in both treated groups compared to vehicle, with no significant difference between the dose levels of NN9056 (41.8 ± 12.6, 51.5 ± 13.8 and 86.5 ± 19.5 kg in high-dose, low-dose and vehicle groups, respectively, p = 0.012 and p < 0.0001 for low and high dose vs. vehicle, respectively). Accordingly, there was a weight loss in both treated groups vs. a weight gain in the vehicle group (-7.2 ± 4.6%, -2.3 ± 3.2% and 12.3 ± 3.9% in the high-dose, low-dose and vehicle groups, respectively, p < 0.0001 for both vs. vehicle). IVGTT data were not significantly different between groups. CONCLUSION: NN9056, a long-acting CCK-1 receptor-selective CCK analogue, significantly reduced food intake and BW in obese Göttingen Minipigs after once daily s.c. dosing for 13 weeks.

Cholecystokinin-1 receptor agonist induced pathological findings in the exocrine pancreas of non-human primates.

BACKGROUND AND AIMS: Cholecystokinin (CCK) may potentially be used to treat obesity. However, it is well-known to induce acute pancreatitis and pancreas neoplasia in rodents, but not in primates. Here we report the nonclinical safety profile of a long-acting CCK-1 receptor (CCK-1R) agonist, NN9056, in rats and monkeys to support a First-in-Man clinical trial with NN9056. METHODS: Thirteen-week toxicological studies were conducted in rats and non-human primates followed by histopathological evaluation of affected tissues. NN9056 was characterised in vitro, and CCK-1R expression was assessed by in situ hybridization in cynomolgus monkey and human pancreas tissues. RESULTS: Affinity and potency of NN9056 was comparable to native sulphated CCK-8 (CCK-8) across species on the CCK-1R while it had no effect on the CCK-2 receptor (CCK-2R). In situ hybridization demonstrated abundant expression of CCK-1Rs in the exocrine pancreas of the rat. In contrast, it was only discreetly expressed on pancreatic acinar cells in the periphery of scattered lobules in monkeys. A similar expression pattern was observed in human pancreas. 13-weeks daily dosing with NN9056 produced the expected pancreatic pathological findings in rats. In monkeys, NN9056 increased pancreas weight and induced histopathological changes despite the low expression level of CCK-1Rs. CONCLUSION: Surprisingly, chronic CCK-1R activation constitutes a risk for pancreatitis and trophic actions on the exocrine pancreas in monkeys. Since similar CCK-1R expression patterns were found in pancreas of monkeys and humans this risk is likely translatable to humans and clinical development of NN9056 was therefore halted.

Experimental non-alcoholic steatohepatitis in Göttingen Minipigs: consequences of high fat-fructose-cholesterol diet and diabetes.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in humans, and ranges from steatosis to non-alcoholic steatohepatitis (NASH), the latter with risk of progression to cirrhosis. The Göttingen Minipig has been used in studies of obesity and diabetes, but liver changes have not been described. The aim of this study was to characterize hepatic changes in Göttingen Minipigs with or without diabetes, fed a diet high in fat, fructose, and cholesterol to see if liver alterations resemble features of human NAFLD/NASH. METHODS: Fifty-four male castrated minipigs (age 6 to 7 months) were distributed into four groups and diet-fed for 13 months. Groups were: lean controls fed standard diet (SD, n = 8), a group fed high fat/fructose/cholesterol diet (FFC, n = 16), a group fed high fat/fructose/cholesterol diet but changed to standard diet after 7 months (diet normalization, FFC/SD, n = 16), and a streptozotocin-induced diabetic group fed high fat/fructose/cholesterol diet (FFCDIA, n = 14). At termination, blood samples for analyses of circulating biomarkers and liver tissue for histopathological assessment and analyses of lipids and glycogen content were collected. RESULTS: In comparison with SD and FFC/SD, FFC and FFCDIA pigs developed hepatomegaly with increased content of cholesterol, whereas no difference in triglyceride content was found. FFC and FFCDIA groups had increased values of circulating total cholesterol and triglycerides and the hepatic circulating markers alkaline phosphatase and glutamate dehydrogenase. In the histopathological evaluation, fibrosis (mainly located periportally) and inflammation along with cytoplasmic alterations (characterized by hepatocytes with pale, granulated cytoplasm) were found in FFC and FFCDIA groups compared to SD and FFC/SD. Interestingly, FFC/SD also had fibrosis, a feature not seen in SD. Only two FFC and three FFCDIA pigs had > 5% steatosis, and no hepatocellular ballooning or Mallory-Denk bodies were found in any of the pigs. CONCLUSIONS: Fibrosis, inflammation and cytoplasmic alterations were characteristic features in the livers of FCC and FFCDIA pigs. Overall, diabetes did not exacerbate the hepatic changes compared to FFC. The limited presence of the key human-relevant pathological hepatic findings of steatosis and hepatocellular ballooning and the variation in the model, limits its use in preclinical research without further optimisation.

Göttingen minipig model of diet-induced atherosclerosis: influence of mild streptozotocin-induced diabetes on lesion severity and markers of inflammation evaluated in obese, obese and diabetic, and lean control animals.

BACKGROUND: From a pharmacological perspective, readily-available, well-characterized animal models of cardiovascular disease, including relevant in vivo markers of atherosclerosis are important for evaluation of novel drug candidates. Furthermore, considering the impact of diabetes mellitus on atherosclerosis in human patients, inclusion of this disease aspect in the characterization of a such model, is highly relevant. The objective of this study was to evaluate the effect of mild streptozotocin-induced diabetes on ex- and in vivo end-points in a diet-induced atherosclerotic minipig model. METHODS: Castrated male Göttingen minipigs were fed standard chow (CD), atherogenic diet alone (HFD) or with superimposed mild streptozotocin-induced diabetes (HFD-D). Circulating markers of inflammation (C-reactive protein (CRP), oxidized low-density lipoprotein (oxLDL), plasminogen activator inhibitor-1, lipid and glucose metabolism were evaluated together with coronary and aortic atherosclerosis after 22 or 43 diet-weeks. Group differences were evaluated by analysis of variance for parametric data and Kruskal-Wallis test for non-parametric data. For qualitative assessments, Fisher's exact test was applied. For all analyses, p < 0.05 was considered statistically significant. RESULTS: Overall, HFD and HFD-D displayed increased CRP, oxLDL and lipid parameters compared to CD at both time points. HFD-D displayed impaired glucose metabolism as compared to HFD and CD. Advanced atherosclerotic lesions were observed in both coronary arteries and aorta of HFD and HFD-D, with more advanced plaque findings in the aorta but without differences in lesion severity or distribution between HFD and HFD-D. Statistically, triglyceride was positively (p = 0.0039), and high-density lipoprotein negatively (p = 0.0461) associated with aortic plaque area. CONCLUSIONS: In this model, advanced coronary and aortic atherosclerosis was observed, with increased levels of inflammatory markers, clinically relevant to atherosclerosis. No effect of mild streptozotocin-induced diabetes was observed on plaque area, lesion severity or inflammatory markers.

Functional and morphological renal changes in a Göttingen Minipig model of obesity-related and diabetic nephropathy.

Obesity-related glomerulopathy and diabetic nephropathy (DN) are serious complications to metabolic syndrome and diabetes. The purpose was to study effects of a fat, fructose and cholesterol-rich (FFC) diet with and without salt in order to induce hypertension on kidney function and morphology in Göttingen Minipigs with and without diabetes. Male Göttingen Minipigs were divided into 4 groups: SD (standard diet, n = 8), FFC (FFC diet, n = 16), FFC-DIA (FFC diet + diabetes, n = 14), FFC-DIA + S (FFC diet with extra salt + diabetes, n = 14). Blood and urine biomarkers, glomerular filtration rate (GFR), blood pressure (BP) and resistive index (RI) were evaluated after 6-7 months (T1) and 12-13 months (T2). Histology, electron microscopy and gene expression (excluding FFC-DIA + S) were evaluated at T2. All groups fed FFC-diet displayed obesity, increased GFR and RI, glomerulomegaly, mesangial expansion (ME) and glomerular basement membrane (GBM) thickening. Diabetes on top of FFC diet led to increased plasma glucose and urea and proteinuria and tended to exacerbate the glomerulomegaly, ME and GBM thickening. Four genes (CDKN1A, NPHS2, ACE, SLC2A1) were significantly deregulated in FFC and/or FFC-DIA compared to SD. No effects on BP were observed. Göttingen Minipigs fed FFC diet displayed some of the renal early changes seen in human obesity. Presence of diabetes on top of FFC diet exacerbated the findings and lead to changes resembling the early phases of human DN.

Semaglutide treatment attenuates vessel remodelling in ApoE-/- mice following vascular injury and blood flow perturbation.

Background and aims: Randomized clinical studies have shown a reduction in cardiovascular outcomes with glucagon-like peptide 1 receptor agonist (GLP-1RA) treatment with the hypothesized mechanisms being an underlying effect on atherosclerosis. Here, we aimed to assess the pharmacological effects of semaglutide in an atheroprone murine model that recapitulates central mechanisms related to vascular smooth muscle cell (VSMC) phenotypic switching and endothelial dysfunction known to operate within the atherosclerotic plaque. Methods: In study A, we employed an electrical current to the carotid artery in ApoE-/- mice to induce severe VSMC injury and death, after which the arteries were allowed to heal for 4 weeks. In study B, a constrictive cuff was added for 6 h at the site of the healed segment to induce a disturbance in blood flow. Results: Compared to vehicle, semaglutide treatment reduced the intimal and medial area by ∼66% (p = 0.007) and ∼11% (p = 0.0002), respectively. Following cuff placement, expression of the pro-inflammatory marker osteopontin and macrophage marker Mac-2 was reduced (p < 0.05) in the semaglutide-treated group compared to vehicle. GLP-1R were not expressed in murine carotid artery and human coronary vessels with and without atherosclerotic plaques, and semaglutide treatment did not affect proliferation of cultured primary human VSMCs. Conclusions: Semaglutide treatment reduced vessel remodelling following electrical injury and blood flow perturbation in an atheroprone mouse model. This effect appears to be driven by anti-inflammatory and -proliferative mechanisms independent of GLP-1 receptor-mediated signalling in the resident vascular cells. This mechanism of action may be important for cardiovascular protection.

Oral delivery of systemic monoclonal antibodies, peptides and small molecules using gastric auto-injectors.

Oral administration provides a simple and non-invasive approach for drug delivery. However, due to poor absorption and swift enzymatic degradation in the gastrointestinal tract, a wide range of molecules must be parenterally injected to attain required doses and pharmacokinetics. Here we present an orally dosed liquid auto-injector capable of delivering up to 4-mg doses of a bioavailable drug with the rapid pharmacokinetics of an injection, reaching an absolute bioavailability of up to 80% and a maximum plasma drug concentration within 30 min after dosing. This approach improves dosing efficiencies and pharmacokinetics an order of magnitude over our previously designed injector capsules and up to two orders of magnitude over clinically available and preclinical chemical permeation enhancement technologies. We administered the capsules to swine for delivery of clinically relevant doses of four commonly injected medications, including adalimumab, a GLP-1 analog, recombinant human insulin and epinephrine. These multi-day dosing experiments and oral administration in awake animal models support the translational potential of the system.

A neutralizing antibody against DKK1 does not reduce plaque formation in classical murine models of atherosclerosis: Is the therapeutic potential lost in translation?

BACKGROUND AND AIMS: Clinical interventions targeting nonlipid risk factors are needed given the high residual risk of atherothrombotic events despite effective control of dyslipidemia. Dickkopf-1 (DKK1) plays a lipid-independent role in vascular pathophysiology but its involvement in atherosclerosis development and its therapeutic attractiveness remain to be established. METHODS: Patient data, in vitro studies and pharmacological intervention in murine models of atherosclerosis were utilized. RESULTS: In patients' material (n = 127 late stage plaque specimens and n = 10 control vessels), DKK1 mRNA was found to be higher in atherosclerotic plaques versus control arteries. DKK1 protein was detected in the luminal intimal area and in the necrotic core of plaques. DKK1 was released from isolated primary human platelets (~12 - 21-fold) and endothelial cells (~1.4-2.5-fold) upon stimulation with different pathophysiological stimuli. In ApoE-/- and Ldlr-/- mice, plasma DKK1 concentrations were similar to those observed in humans, whereas DKK1 expression in different atheroprone arterial segments was very low/absent. Chronic treatment with a neutralizing DKK1 antibody effectively reduced plasma concentrations, however, plaque lesion area was not reduced in ApoE-/- and Ldlr-/- mice fed a western diet for 14 and 16 weeks. Anti-DKK1 treatment increased bone volume and bone mineral content. CONCLUSIONS: Functional inhibition of DKK1 with an antibody does not alter atherosclerosis progression in classical murine models. This may reflect the absence of DKK1 expression in plaques and more advanced animal disease models could be needed to evaluate the role and therapeutic attractiveness of DKK1 in late stage complications such as plaque destabilization, calcification, rupture and thrombosis.

An ingestible self-orienting system for oral delivery of macromolecules.

Biomacromolecules have transformed our capacity to effectively treat diseases; however, their rapid degradation and poor absorption in the gastrointestinal (GI) tract generally limit their administration to parenteral routes. An oral biologic delivery system must aid in both localization and permeation to achieve systemic drug uptake. Inspired by the leopard tortoise's ability to passively reorient, we developed an ingestible self-orienting millimeter-scale applicator (SOMA) that autonomously positions itself to engage with GI tissue. It then deploys milliposts fabricated from active pharmaceutical ingredients directly through the gastric mucosa while avoiding perforation. We conducted in vivo studies in rats and swine that support the applicator's safety and, using insulin as a model drug, demonstrated that the SOMA delivers active pharmaceutical ingredient plasma levels comparable to those achieved with subcutaneous millipost administration.

Long-term Western diet fed apolipoprotein E-deficient rats exhibit only modest early atherosclerotic characteristics.

In the apolipoprotein E-deficient mouse, the gut microbiota has an impact on the development of atherosclerosis, but whether such correlations are also present in rats requires investigation. Therefore, we studied female SD-Apoe tm1sage (Apoe-/-) rats fed either a Western diet or a low-fat control diet with or without gluten, which is known to promote gut microbiota changes, until 20 weeks of age. We hypothesized that the manifestation of atherosclerosis would be more severe in Apoe-/- rats fed the Western high-fat diet, as compared with rats fed the low-fat diet, and that atherosclerosis would be accelerated by gluten. Both Western diet-feeding and gluten resulted in significant changes in gut microbiota, but the microbiota impact of gluten was transient. Compared with Apoe-/- rats fed a low-fat diet, Western diet-fed Apoe-/- rats were heavier and became glucose intolerant with increased levels of oxidative stress. They developed early fatty streak lesions in their aortic sinus, while there was no evidence of atherosclerosis in the thoracic aorta. No conclusions could be made on the impact of gluten on atherosclerosis. Although Western diet-fed Apoe-/- rats exhibited a more human-like LDL dominated blood lipid profile, signs of obesity, type 2 diabetes and cardiovascular disease were modest.

Modulating the Gut Microbiota Improves Glucose Tolerance, Lipoprotein Profile and Atherosclerotic Plaque Development in ApoE-Deficient Mice.

The importance of the gut microbiota (GM) in disease development has recently received increased attention, and numerous approaches have been made to better understand this important interplay. For example, metabolites derived from the GM have been shown to promote atherosclerosis, the underlying cause of cardiovascular disease (CVD), and to increase CVD risk factors. Popular interest in the role of the intestine in a variety of disease states has now resulted in a significant proportion of individuals without coeliac disease switching to gluten-free diets. The effect of gluten-free diets on atherosclerosis and cardiovascular risk factors is largely unknown. We therefore investigated the effect of a gluten-free high-fat cholesterol-rich diet, as compared to the same diet in which the gluten peptide gliadin had been added back, on atherosclerosis and several cardiovascular risk factors in apolipoprotein E-deficient (Apoe-/-) mice. The gluten-free diet transiently altered GM composition in these mice, as compared to the gliadin-supplemented diet, but did not alter body weights, glucose tolerance, insulin levels, plasma lipids, or atherosclerosis. In parallel, other Apoe-/- mice fed the same diets were treated with ampicillin, a broad-spectrum antibiotic known to affect GM composition. Ampicillin-treatment had a marked and sustained effect on GM composition, as expected. Furthermore, although ampicillin-treated mice were slightly heavier than controls, ampicillin-treatment transiently improved glucose tolerance both in the absence or presence of gliadin, reduced plasma LDL and VLDL cholesterol levels, and reduced aortic atherosclerotic lesion area. These results demonstrate that a gluten-free diet does not seem to have beneficial effects on atherosclerosis or several CVD risk factors in this mouse model, but that sustained alteration of GM composition with a broad-spectrum antibiotic has beneficial effects on CVD risk factors and atherosclerosis. These findings support the concept that altering the microbiota might provide novel treatment strategies for CVD.