Disrupted hypothalamic transcriptomics and proteomics in a mouse model of type 2 diabetes exposed to recurrent hypoglycaemia.

AIMS/HYPOTHESIS: Repeated exposures to insulin-induced hypoglycaemia in people with diabetes progressively impairs the counterregulatory response (CRR) that restores normoglycaemia. This defect is characterised by reduced secretion of glucagon and other counterregulatory hormones. Evidence indicates that glucose-responsive neurons located in the hypothalamus orchestrate the CRR. Here, we aimed to identify the changes in hypothalamic gene and protein expression that underlie impaired CRR in a mouse model of defective CRR. METHODS: High-fat-diet fed and low-dose streptozocin-treated C57BL/6N mice were exposed to one (acute hypoglycaemia [AH]) or multiple (recurrent hypoglycaemia [RH]) insulin-induced hypoglycaemic episodes and plasma glucagon levels were measured. Single-nuclei RNA-seq (snRNA-seq) data were obtained from the hypothalamus and cortex of mice exposed to AH and RH. Proteomic data were obtained from hypothalamic synaptosomal fractions. RESULTS: The final insulin injection resulted in similar plasma glucose levels in the RH group and AH groups, but glucagon secretion was significantly lower in the RH group (AH: 94.5±9.2 ng/l [n=33]; RH: 59.0±4.8 ng/l [n=37]; p<0.001). Analysis of snRNA-seq data revealed similar proportions of hypothalamic cell subpopulations in the AH- and RH-exposed mice. Changes in transcriptional profiles were found in all cell types analysed. In neurons from RH-exposed mice, we observed a significant decrease in expression of Avp, Pmch and Pcsk1n, and the most overexpressed gene was Kcnq1ot1, as compared with AH-exposed mice. Gene ontology analysis of differentially expressed genes (DEGs) indicated a coordinated decrease in many oxidative phosphorylation genes and reduced expression of vacuolar H+- and Na+/K+-ATPases; these observations were in large part confirmed in the proteomic analysis of synaptosomal fractions. Compared with AH-exposed mice, oligodendrocytes from RH-exposed mice had major changes in gene expression that suggested reduced myelin formation. In astrocytes from RH-exposed mice, DEGs indicated reduced capacity for neurotransmitters scavenging in tripartite synapses as compared with astrocytes from AH-exposed mice. In addition, in neurons and astrocytes, multiple changes in gene expression suggested increased amyloid beta (Aß) production and stability. The snRNA-seq analysis of the cortex showed that the adaptation to RH involved different biological processes from those seen in the hypothalamus. CONCLUSIONS/INTERPRETATION: The present study provides a model of defective counterregulation in a mouse model of type 2 diabetes. It shows that repeated hypoglycaemic episodes induce multiple defects affecting all hypothalamic cell types and their interactions, indicative of impaired neuronal network signalling and dysegulated hypoglycaemia sensing, and displaying features of neurodegenerative diseases. It also shows that repeated hypoglycaemia leads to specific molecular adaptation in the hypothalamus when compared with the cortex. DATA AVAILABILITY: The transcriptomic dataset is available via the GEO ( http://www.ncbi.nlm.nih.gov/geo/ ), using the accession no. GSE226277. The proteomic dataset is available via the ProteomeXchange data repository ( http://www.proteomexchange.org ), using the accession no. PXD040183.

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.

Peripherally delivered hepatopreferential insulin analog insulin-406 mimics the hypoglycaemia-sparing effect of portal vein human insulin infusion in dogs.

AIMS: We previously quantified the hypoglycaemia-sparing effect of portal vs peripheral human insulin delivery. The current investigation aimed to determine whether a bioequivalent peripheral vein infusion of a hepatopreferential insulin analog, insulin-406, could similarly protect against hypoglycaemia. MATERIALS AND METHODS: Dogs received human insulin infusions into either the hepatic portal vein (PoHI, n = 7) or a peripheral vein (PeHI, n = 7) for 180 minutes at four-fold the basal secretion rate (6.6 pmol/kg/min) in a previous study. Insulin-406 (Pe406, n = 7) was peripherally infused at 6.0 pmol/kg/min, a rate determined to decrease plasma glucose by the same amount as with PoHI infusion during the first 60 minutes. Glucagon was fixed at basal concentrations, mimicking the diminished α-cell response seen in type 1 diabetes. RESULTS: Glucose dropped quickly with PeHI infusion, reaching 41 ± 3 mg/dL at 60 minutes, but more slowly with PoHI and Pe406 infusion (67 ± 2 and 72 ± 4 mg/dL, respectively; P < 0.01 vs PeHI for both). The hypoglycaemic nadir (c. 40 mg/dL) occurred at 60 minutes with PeHI infusion vs 120 minutes with PoHI and Pe406 infusion. ΔAUCepinephrine during the 180-minute insulin infusion period was two-fold higher with PeHI infusion compared with PoHI and Pe406 infusion. Glucose production (mg/kg/min) was least suppressed with PeHI infusion (Δ = 0.79 ± 0.33) and equally suppressed with PoHI and Pe406 infusion (Δ = 1.16 ± 0.21 and 1.18 ± 0.17, respectively; P = NS). Peak glucose utilization (mg/kg/min) was highest with PeHI infusion (4.94 ± 0.17) and less with PoHI and Pe406 infusion (3.58 ± 0.58 and 3.26 ± 0.08, respectively; P < 0.05 vs Pe for both). CONCLUSIONS: Peripheral infusion of hepatopreferential insulin can achieve a metabolic profile that closely mimics portal insulin delivery, which reduces the risk of hypoglycaemia compared with peripheral insulin infusion.

Diabetic Phenotype in the Small Intestine of Zucker Diabetic Fatty Rats.

BACKGROUND/AIMS: In contrast to streptozotocin (STZ)-induced rodent models of diabetes, there are no thorough characterizations of the intestinal phenotype and the underlying changes in the global gene-expression of genetic models of diabetes, such as the Zucker diabetic fatty (ZDF) rat. The aim of the present study was to characterize the intestine in the ZDF rat. METHODS: The intestine of ZDF rats and lean controls was examined macroscopically and histologically, and ribonucleic acid sequencing (RNAseq) was performed in samples of jejunal mucosa. RESULTS: We observed an increased mass and length of the small and large intestines in ZDF rats. RNAseq showed an increased expression of Pdk2 and Pdk4, which are involved in the regulation of glucose and fatty acid metabolism, and increased expression of genes involved in gluconeogenesis and peroxisomal beta-oxidation in jejunal mucosa. CONCLUSION: Intestinal enlargement in ZDF rats is likely driven by increased food intake, since (i) it also occurs in obese and normoglycemic Zucker fatty rats, and (ii) insulin treatment of STZ-induced diabetic rats reduced the food intake and mass of the small intestine. Results from RNAseq indicate that small intestinal epithelial cells in ZDF rats have developed insulin resistance, and support that a normal physiological effect of insulin in the enterocytes is the regulation of glucose metabolism.

Development of small-molecule inhibitors targeting adipose triglyceride lipase.

Adipose triglyceride lipase (ATGL) is rate limiting in the mobilization of fatty acids from cellular triglyceride stores. This central role in lipolysis marks ATGL as an interesting pharmacological target as deregulated fatty acid metabolism is closely linked to dyslipidemic and metabolic disorders. Here we report on the development and characterization of a small-molecule inhibitor of ATGL. Atglistatin is selective for ATGL and reduces fatty acid mobilization in vitro and in vivo.

Structure-activity studies in the development of a hydrazone based inhibitor of adipose-triglyceride lipase (ATGL).

Adipose triglyceride lipase (ATGL) catalyzes the degradation of cellular triacylglycerol stores and strongly determines the concentration of circulating fatty acids (FAs). High serum FA levels are causally linked to the development of insulin resistance and impaired glucose tolerance, which eventually progresses to overt type 2 diabetes. ATGL-specific inhibitors could be used to lower circulating FAs, which can counteract the development of insulin resistance. In this article, we report about structure-activity relationship (SAR) studies of small molecule inhibitors of ATGL based on a hydrazone chemotype. The SAR indicated that the binding pocket of ATGL requests rather linear compounds without bulky substituents. The best inhibitor showed an IC50=10µM in an assay with COS7-cell lysate overexpressing murine ATGL.

Contraction-induced lipolysis is not impaired by inhibition of hormone-sensitive lipase in skeletal muscle.

In skeletal muscle hormone-sensitive lipase (HSL) has long been accepted to be the principal enzyme responsible for lipolysis of intramyocellular triacylglycerol (IMTG) during contractions. However, this notion is based on in vitro lipase activity data, which may not reflect the in vivo lipolytic activity. We investigated lipolysis of IMTG in soleus muscles electrically stimulated to contract ex vivo during acute pharmacological inhibition of HSL in rat muscles and in muscles from HSL knockout (HSL-KO) mice. Measurements of IMTG are complicated by the presence of adipocytes located between the muscle fibres. To circumvent the problem with this contamination we analysed intramyocellular lipid droplet content histochemically. At maximal inhibition of HSL in rat muscles, contraction-induced breakdown of IMTG was identical to that seen in control muscles (P < 0.001). In response to contractions IMTG staining decreased significantly in both HSL-KO and WT muscles (P < 0.05). In vitro TG hydrolase activity data revealed that adipose triglyceride lipase (ATGL) and HSL collectively account for ∼98% of the TG hydrolase activity in mouse skeletal muscle, other TG lipases accordingly being of negligible importance for lipolysis of IMTG. The present study is the first to demonstrate that contraction-induced lipolysis of IMTG occurs in the absence of HSL activity in rat and mouse skeletal muscle. Furthermore, the results suggest that ATGL is activated and plays a major role in lipolysis of IMTG during muscle contractions.

Standard procedures for blood withdrawal in conscious male rats induce stress and profoundly affect glucose tolerance and secretion of glucoregulatory hormones.

OBJECTIVE: A fundamental difference between physiological and pharmacological studies in rats and humans is that withdrawal of blood from conscious rats necessitates restraint which inevitably inflicts a higher level of stress. We investigated the impact of handling on acute glucose regulation and secretion of glucoregulatory hormones in rats. METHODS: Fasted male Sprague Dawley rats (375-400 g, n = 11) were given an oral glucose tolerance test (OGTT) by gavage (2 g/kg). Blood was sampled frequently until 90 min after challenge by handheld sampling (HS) or by automated sampling (AS). In the HS experiment, blood was withdrawn by restraint and sublingual vein puncture; two weeks later, samples were obtained by AS through an implanted catheter in a carotid artery, allowing sampling without disturbing the animals. RESULTS: On the day of HS, post challenge glucose AUCs were ∼17% higher (P < 0.0001), despite gastric emptying (AUC) being reduced by ∼30% (P < 0.0001). Plasma insulin AUC was 3.5-fold lower (P < 0.001), and glucose-dependent insulinotropic peptide (GIP) AUC was reduced by ∼36% but glucagon-like peptide-1 concentrations were not affected. Glucagon concentrations were higher both before and after challenge (fold difference in AUCs = 3.3). Adrenocorticotropin (ACTH) and corticosterone AUCs were 2.4-fold and 3.6-fold higher (P < 0.001), respectively. DISCUSSION AND CONCLUSION: Our study highlights that sampling of blood from conscious rats by sublingual vein puncture inflicts stress which reduces glucose absorption and glucose tolerance and blunts secretion of insulin and GIP. As blood sampling in humans are less stressful, standard procedures of conducting OGTT's in rats by HS presumably introduce an interspecies difference that may have negative consequences for translatability of test results.

The critical role of neutral cholesterol ester hydrolase 1 in cholesterol removal from human macrophages.

RATIONALE: Hydrolysis of intracellular cholesterol ester (CE) is the key step in the reverse cholesterol transport in macrophage foam cells. We have recently shown that neutral cholesterol ester hydrolase (Nceh)1 and hormone-sensitive lipase (Lipe) are key regulators of this process in mouse macrophages. However, it remains unknown which enzyme is critical in human macrophages and atherosclerosis. OBJECTIVE: We aimed to identify the enzyme responsible for the CE hydrolysis in human macrophages and to determine its expression in human atherosclerosis. METHODS AND RESULTS: We compared the expression of NCEH1, LIPE, and cholesterol ester hydrolase (CES1) in human monocyte-derived macrophages (HMMs) and examined the effects of inhibition or overexpression of each enzyme in the cholesterol trafficking. The pattern of expression of NCEH1 was similar to that of neutral CE hydrolase activity during the differentiation of HMMs. Overexpression of human NCEH1 increased the hydrolysis of CE, thereby stimulating cholesterol mobilization from THP-1 macrophages. Knockdown of NCEH1 specifically reduced the neutral CE hydrolase activity. Pharmacological inhibition of NCEH1 also increased the cellular CE in HMMs. In contrast, LIPE was barely detectable in HMMs, and its inhibition did not decrease neutral CE hydrolase activity. Neither overexpression nor knockdown of CES1 affected the neutral CE hydrolase activity. NCEH1 was expressed in CD68-positive macrophage foam cells of human atherosclerotic lesions. CONCLUSIONS: NCEH1 is expressed in human atheromatous lesions, where it plays a critical role in the hydrolysis of CE in human macrophage foam cells, thereby contributing to the initial part of reverse cholesterol transport in human atherosclerosis.

An integrated platform approach enables discovery of potent, selective and ligand-competitive cyclic peptides targeting the GIP receptor.

In any drug discovery effort, the identification of hits for further optimisation is of crucial importance. For peptide therapeutics, display technologies such as mRNA display have emerged as powerful methodologies to identify these desired de novo hit ligands against targets of interest. The diverse peptide libraries are genetically encoded in these technologies, allowing for next-generation sequencing to be used to efficiently identify the binding ligands. Despite the vast datasets that can be generated, current downstream methodologies, however, are limited by low throughput validation processes, including hit prioritisation, peptide synthesis, biochemical and biophysical assays. In this work we report a highly efficient strategy that combines bioinformatic analysis with state-of-the-art high throughput peptide synthesis to identify nanomolar cyclic peptide (CP) ligands of the human glucose-dependent insulinotropic peptide receptor (hGIP-R). Furthermore, our workflow is able to discriminate between functional and remote binding non-functional ligands. Efficient structure-activity relationship analysis (SAR) combined with advanced in silico structural studies allow deduction of a thorough and holistic binding model which informs further chemical optimisation, including efficient half-life extension. We report the identification and design of the first de novo, GIP-competitive, incretin receptor family-selective CPs, which exhibit an in vivo half-life up to 10.7 h in rats. The workflow should be generally applicable to any selection target, improving and accelerating hit identification, validation, characterisation, and prioritisation for therapeutic development.

Temporal Development of Dyslipidemia and Nonalcoholic Fatty Liver Disease (NAFLD) in Syrian Hamsters Fed a High-Fat, High-Fructose, High-Cholesterol Diet.

The use of translationally relevant animal models is essential, also within the field of nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). Compared to frequently used mouse and rat models, the hamster may provide a higher degree of physiological similarity to humans in terms of lipid profile and lipoprotein metabolism. However, the effects in hamsters after long-term exposure to a NASH diet are not known. Male Syrian hamsters were fed either a high-fat, high-fructose, high-cholesterol diet (NASH diet) or control diets for up to 12 months. Plasma parameters were assessed at two weeks, one, four, eight and 12 months and liver histopathology and biochemistry was characterized after four, eight and 12 months on the experimental diets. After two weeks, hamsters on NASH diet had developed marked dyslipidemia, which persisted for the remainder of the study. Hepatic steatosis was present in NASH-fed hamsters after four months, and hepatic stellate cell activation and fibrosis was observed within four to eight months, respectively, in agreement with progression towards NASH. In summary, we demonstrate that hamsters rapidly develop dyslipidemia when fed a high-fat, high-fructose, high-cholesterol diet. Moreover, within four to eight months, the NASH-diet induced hepatic changes with resemblance to human NAFLD.

Molecular Engineering of Insulin Icodec, the First Acylated Insulin Analog for Once-Weekly Administration in Humans.

Here, we describe the molecular engineering of insulin icodec to achieve a plasma half-life of 196 h in humans, suitable for once-weekly subcutaneously administration. Insulin icodec is based on re-engineering of the ultra-long oral basal insulin OI338 with a plasma half-life of 70 h in humans. This systematic re-engineering was accomplished by (1) further increasing the albumin binding by changing the fatty diacid from a 1,18-octadecanedioic acid (C18) to a 1,20-icosanedioic acid (C20) and (2) further reducing the insulin receptor affinity by the B16Tyr → His substitution. Insulin icodec was selected by screening for long intravenous plasma half-life in dogs while ensuring glucose-lowering potency following subcutaneous administration in rats. The ensuing structure-activity relationship resulted in insulin icodec. In phase-2 clinical trial, once-weekly insulin icodec provided safe and efficacious glycemic control comparable to once-daily insulin glargine in type 2 diabetes patients. The structure-activity relationship study leading to insulin icodec is presented here.

Insulin Treatment Attenuates Small Nerve Fiber Damage in Rat Model of Type 2 Diabetes.

INTRODUCTION: Current clinical guidelines for management of diabetic peripheral neuropathy (DPN) emphasize good glycemic control. However, this has limited effect on prevention of DPN in type 2 diabetic (T2D) patients. This study investigates the effect of insulin treatment on development of DPN in a rat model of T2D to assess the underlying causes leading to DPN. METHODS: Twelve-week-old male Sprague-Dawley rats were allocated to a normal chow diet or a 45% kcal high-fat diet. After eight weeks, the high-fat fed animals received a mild dose of streptozotocin to induce hyperglycemia. Four weeks after diabetes induction, the diabetic animals were allocated into three treatment groups receiving either no insulin or insulin-releasing implants in a high or low dose. During the 12-week treatment period, blood glucose and body weight were monitored weekly, whereas Hargreaves' test was performed four, eight, and 12 weeks after treatment initiation. At study termination, several blood parameters, body composition, and neuropathy endpoints were assessed. RESULTS: Insulin treatment lowered blood glucose in a dose-dependent manner. In addition, both doses of insulin lowered lipids and increased body fat percentage. High-dose insulin treatment attenuated small nerve fiber damage assessed by Hargreaves' test and intraepidermal nerve fiber density compared to untreated diabetes and low-dose insulin; however, neuropathy was not completely prevented by tight glycemic control. Linear regression analysis revealed that glycemic status, circulating lipids, and sciatic nerve sorbitol level were all negatively associated with the small nerve fiber damage observed. CONCLUSION: In summary, our data suggest that high-dose insulin treatment attenuates small nerve fiber damage. Furthermore, data also indicate that both poor glycemic control and dyslipidemia are associated with disease progression. Consequently, this rat model of T2D seems to fit well with progression of DPN in humans and could be a relevant preclinical model to use in relation to research investigating treatment opportunities for DPN.

Variation in diagnostic NAFLD/NASH read-outs in paired liver samples from rodent models.

INTRODUCTION: In animal models of non-alcoholic fatty liver disease (NAFLD), assessment of disease severity and treatment effects of drugs rely on histopathological scoring of liver biopsies. However, little is known about the sampling variation in liver samples from animal models of NAFLD, even though several histopathological hallmarks of the disease are known to be affected by sampling variation in patients. The aim of this study was to assess the sampling variation in multiple paired liver biopsies from three commonly used diet-induced rodent models of NAFLD. METHODS: Eight male C57BL/6 mice, 8 male Sprague Dawley rats and 16 female Hartley guinea pigs were fed a NAFLD-inducing high-fat diet for 16 weeks (mice and rats), 20 or 24 weeks (guinea pigs). After the initial diet period, liver sections were sampled and subsequently assessed by histopathological scoring and biochemical analyses. RESULTS: Fibrosis was heterogeneously distributed throughout the liver in mice, manifesting as both intra- and interlobular statistically significant differences. Hepatic triglyceride content showed interlobular differences in mice, and both intra- and interlobular differences in guinea pigs (24-week time point) all of which were statistically significant. Also, hepatic cholesterol content was subject to significant intra-lobular sampling variation in mice, and hepatic glycogen content differed significantly between lobes in mice and guinea pigs. DISCUSSION: Dependent on animal model, both histopathological and biochemical end-points differed between sampling sites in the liver. Based on these findings, we recommend that sample site location is highly standardized and properly reported in order to minimize potential sampling variation and to optimize reproducibility and meaningful comparisons of preclinical studies of NAFLD.

Pancreatic beta-cell overexpression of the glucagon receptor gene results in enhanced beta-cell function and mass.

In addition to its primary role in regulating glucose production from the liver, glucagon has many other actions, reflected by the wide tissue distribution of the glucagon receptor (Gcgr). To investigate the role of glucagon in the regulation of insulin secretion and whole body glucose homeostasis in vivo, we generated mice overexpressing the Gcgr specifically on pancreatic beta-cells (RIP-Gcgr). In vivo and in vitro insulin secretion in response to glucagon and glucose was increased 1.7- to 3.9-fold in RIP-Gcgr mice compared with controls. Consistent with the observed increase in insulin release in response to glucagon and glucose, the glucose excursion resulting from both a glucagon challenge and intraperitoneal glucose tolerance test (IPGTT) was significantly reduced in RIP-Gcgr mice compared with controls. However, RIP-Gcgr mice display similar glucose responses to an insulin challenge. beta-Cell mass and pancreatic insulin content were also increased (20 and 50%, respectively) in RIP-Gcgr mice compared with controls. When fed a high-fat diet (HFD), both control and RIP-Gcgr mice developed similar degrees of obesity and insulin resistance. However, the severity of both fasting hyperglycemia and impaired glucose tolerance (IGT) were reduced in RIP-Gcgr mice compared with controls. Furthermore, the insulin response of RIP-Gcgr mice to an IPGTT was twice that of controls when fed the HFD. These data indicate that increased pancreatic beta-cell expression of the Gcgr increased insulin secretion, pancreatic insulin content, beta-cell mass, and, when mice were fed a HFD, partially protected against hyperglycemia and IGT.

Targeting insulin to the liver corrects defects in glucose metabolism caused by peripheral insulin delivery.

Peripheral hyperinsulinemia resulting from subcutaneous insulin injection is associated with metabolic defects which include abnormal glucose metabolism. The first aim of this study was to quantify the impairments in liver and muscle glucose metabolism that occur when insulin is delivered via a peripheral vein compared to when it is given through its endogenous secretory route (the hepatic portal vein) in overnight fasted conscious dogs. The second aim was to determine if peripheral delivery of a hepato-preferential insulin analog could restore the physiologic response to insulin that occurs under meal feeding conditions. This study is the first to show that hepatic glucose uptake correlates with insulin's direct effects on the liver under hyperinsulinemic-hyperglycemic conditions. In addition, glucose uptake was equally divided between the liver and muscle when insulin was infused into the portal vein, but when it was delivered into a peripheral vein the percentage of glucose taken up by muscle was 4-times greater than that going to the liver, with liver glucose uptake being less than half of normal. These defects could not be corrected by adjusting the dose of peripheral insulin. On the other hand, hepatic and non-hepatic glucose metabolism could be fully normalized by a hepato-preferential insulin analog.

Dietary fat stimulates development of NAFLD more potently than dietary fructose in Sprague-Dawley rats.

BACKGROUND: In humans and animal models, excessive intake of dietary fat, fructose and cholesterol has been linked to the development of non-alcoholic fatty liver disease (NAFLD). However, the individual roles of the dietary components remain unclear. To investigate this further, we compared the effects of a high-fat diet, a high-fructose diet and a combination diet with added cholesterol on the development of NAFLD in rats. METHODS: Forty male Sprague-Dawley rats were randomized into four groups receiving either a control-diet (Control: 10% fat); a high-fat diet (HFD: 60% fat, 20% carbohydrate), a high-fructose diet [HFr: 10% fat, 70% carbohydrate (mainly fructose)] or a high-fat/high-fructose/high-cholesterol-diet (NASH: 40% fat, 40% carbohydrate (mainly fructose), 2% cholesterol) for 16 weeks. RESULTS: After 16 weeks, liver histology revealed extensive steatosis and inflammation in both NASH- and HFD-fed rats, while hepatic changes in HFr-rats were much more subtle. These findings were corroborated by significantly elevated hepatic triglyceride content in both NASH- (p < 0.01) and HFD-fed rats (p < 0.0001), elevated hepatic cholesterol levels in NASH-fed rats (p < 0.0001), but no changes in HFr-fed rats, compared to Control. On the contrary, only HFr-fed rats developed dyslipidemia as characterized by higher levels of plasma triglycerides compared to all other groups (p < 0.0001). Hepatic dysfunction and inflammation was confirmed in HFD-fed rats by elevated levels of hepatic MCP-1 (p < 0.0001), TNF-alpha (p < 0.001) and plasma ß-hydroxybutyrate (p < 0.0001), and in NASH-fed rats by elevated levels of hepatic MCP-1 (p < 0.01), increased hepatic macrophage infiltration (p < 0.001), and higher plasma levels of alanine aminotransferase (p < 0.0001) aspartate aminotransferase (p < 0.05), haptoglobin (p < 0.001) and TIMP-1 (p < 0.01) compared to Control. CONCLUSION: These findings show that dietary fat and cholesterol are the primary drivers of NAFLD development and progression in rats, while fructose mostly exerts its effect on the circulating lipid pool.

Synthesis and structure-activity relationship for a novel class of potent and selective carbamate-based inhibitors of hormone selective lipase with acute in vivo antilipolytic effects.

Hormone-sensitive lipase (HSL) is an intracellular enzyme that has a central role in the regulation of fatty acid metabolism. The enzyme, therefore, is a potentially interesting pharmacological target for the treatment of insulin resistance and dyslipidemic disorders. Based on a high throughput screening, a carbamate based HSL inhibitor was identified and optimized into the selective HSL inhibitors 4-hydroxymethyl-piperidine-1-carboxylic acid 4-(5-trifluoromethylpyridin-2-yloxy)-phenyl ester (13f) and 4-hydroxy-piperidine-1-carboxylic acid 4-(5-trifluoromethylpyridin-2-yloxy)-phenyl ester (13g), with IC50 values of 110 and 500 nM, respectively. Both inhibitors were active in acute antilipolytic experiments in vivo and none of the inhibitors inhibited the cytochrome P450 (CYP) isoforms 2D6, 3A4, and 1A2.

Oral administration of glucose promotes intracellular partitioning of fatty acid toward storage in white but not in red muscle.

Lipid accumulation in non-adipose tissues is strongly associated with the metabolic syndrome, possibly due to aberrant partitioning of intracellular fatty acids between storage and oxidation. In the present study, we administered the non-metabolizable fatty acid analog [9,10-(3)H]-(R)-2-bromopalmitate, and authentic (14)C-palmitate to conscious rats, in order to directly examine the initial intracellular fate of fatty acids in a range of insulin-sensitive tissues, including white and red muscles, liver, white adipose tissue, and heart. Rats were studied after administration of an oral glucose load to examine the effect of physiological elevation of glucose and insulin. The tracer results showed that glucose administration partitioned fatty acid toward storage in white muscle (storage:uptake ratios, vehicle vs glucose; 0.64 +/- 0.02 vs 0.92 +/- 0.09, P < 0.05), and in liver (0.66 +/- 0.07 vs 0.98 +/- 0.04, P < 0.05), but not in red muscle (1.18 +/- 0.07 vs 1.36 +/- 0.11, P = not significant). These results demonstrate the physiological relevance of the so-called 'reverse' Randle cycle, but surprisingly show that it may be more important in white rather than oxidative red muscle.