Palatability, glycemic, and insulinemic responses to various carbohydrate formulations: Alternatives for the diagnosis of insulin dysregulation in horses?

BACKGROUND: Oral glycemic challenge (GC) tests are recommended for diagnosis of insulin dysregulation (ID). Various protocols are used, but all have limitations in terms of palatability, ease of use, variable composition, geographic availability, or some combination of these. HYPOTHESIS/OBJECTIVE: To evaluate newly developed formulations with defined carbohydrate composition for use as oral GCs. ANIMALS: Thirty-four horses and ponies in various metabolic states. METHODS: Our objectives were carried out in 2 separate cross-over experiments. First, the palatability and acceptance of various GCs (2 syrups, 1 granulate) offered for free intake were compared to glucose mixed in a chaff-based diet. Subsequently, syrups were administered by syringe and compared to an oral glucose test using naso-gastric tubing (tube OGT) to investigate the glycemic and insulinemic responses. Second, these variables were compared in the best performing GC-formulations (granulate further optimized to pelleted formulation and 1 syrup) and a tube OGT. All GCs were administered with equivalent amounts of 0.5 g glycemic carbohydrates per kg body weight. RESULTS: Only the GC pellets were consumed completely by all horses (consumption time 5 ± 2 min). When administered by syringe, the GC syrup also was well accepted. The insulin concentrations at 120 min correlated significantly between tube OGT and GC pellets (r = .717; P < .001) or GC syrup (r = .913; P < .001). The new GC syrup and GC pellets discriminate between healthy and ID horses. CONCLUSIONS AND CLINICAL SIGNIFICANCE: The GC pellets (DysChEq)™ and GC syrup can be used as palatable and well-accepted oral GC tests for assessment of ID in horses.

Carbohydrate pellets to assess insulin dysregulation in horses.

BACKGROUND: A glycemic challenge test is used for the diagnosis of insulin dysregulation (ID) in horses and ponies. Different forms of the test exist where the administrative route and dose of glucose vary, which makes interpretation of results challenging. HYPOTHESIS/OBJECTIVES: To evaluate the palatability of, and blood glucose and insulin responses to, carbohydrate pellets fed as an oral glucose test (OGT), and to establish the diagnostic threshold for ID when using the pellets. ANIMALS: University and privately-owned horses and ponies (n = 157) comprised of 31 breeds and both sexes. METHODS: Multicenter cohort study. A custom-produced glycemic pellet was offered for free intake at 0.5 g/kg BW soluble carbohydrate and serum insulin and blood glucose concentrations measured before and after (60, 120, and 180 minutes) the pellets were offered. Pellet acceptance and intake time (those that finished within 10 minutes) were determined to assess palatability. RESULTS: The pellets were palatable to 132/157 animals, and ponies found the pellets more (P = .004) palatable than horses. The median intake time (4 [3-6] minutes) was positively correlated with acceptance grade (r = .51; P < .0001). Consumption of the pellets elicited peak blood glucose (6.6 [5.8-7.8] mmol/L) and serum insulin (40.5 [19-99.8] µIU/mL) responses at 120 minutes. At 120 minutes the optimal cut-off was 83 µIU/mL (95% CI: 70-99 µIU/mL) for the IMMULITE 2000XPi assay. CONCLUSIONS AND CLINICAL IMPORTANCE: The pellets were palatable and a suitable, novel carbohydrate source for the OGT.

Demographic, morphologic, hormonal and metabolic factors associated with the rate of improvement from equine hyperinsulinaemia-associated laminitis.

BACKGROUND: Although several studies have investigated factors associated with the onset and occurrence of hyperinsulinaemia-associated laminitis (HAL), few have examined the factors associated with the rate of improvement during recovery from an acute bout of the disease. This observational study sought to discover if a range of demographic, morphologic, hormonal and metabolic variables are associated with the improvement rate from HAL in 37 naturally-occurring cases identified by 16 clinics across Germany. Each case was evaluated for laminitis severity on the day of inclusion in the trial (d 0), then after 4, 9, 14, 25 and 42 d. The horses were managed according to best clinical practice including restricting exercise and prescribing a diet of hay-only, for a minimum of 9 d. Blood samples were also collected during each evaluation, except on d 9, and analysed for glucose, insulin, ACTH and leptin. RESULTS: Based on individual clinical laminitis scores plotted against time, most horses improved markedly within 2 weeks, with a 'fast group' (n = 27) having a median (interquartile range) score on a 12-point scale of 0 (0-2) by d 14. However, there was a clear disparity within the total cohort, as ~ 1 in 4 horses demonstrated much slower improvement, with a median score of 5 (4-7) by d 14, or a marked relapse thereafter ('slow group', n = 10). Horses in the slow improvement group were younger (12.5 (8.8-16.3) vs 17 (14-24) yr; P = 0.008), but were not more likely to be heavier, male, very fat, to have presented with a previous history of laminitis or elevated ACTH concentrations, or to be receiving pergolide treatment. Of the hormonal and metabolic parameters measured, glucose and insulin concentrations were within the normal range following transition to the hay-only diet, but were higher in the group that failed to improve quickly, with a small but significant difference being evident on d 4, 14 and 25 for glucose (11 to 16%; P < 0.05), and a larger difference for insulin on d 14 and 25 (51 to 55%; P < 0.05). There was no difference between the groups in ACTH or leptin concentrations throughout the study. The main limitations of this study were the small number of slow-improvement horses and an inability to control or measure certain variables, such as feed quality. CONCLUSIONS: Young age and a modest increase in blood glucose and insulin concentrations are associated with delayed laminitis improvement.

Metabolic profile distinguishes laminitis-susceptible and -resistant ponies before and after feeding a high sugar diet.

BACKGROUND: Insulin dysregulation (ID) is a key risk factor for equine endocrinopathic laminitis, but in many cases ID can only be assessed accurately using dynamic tests. The identification of other biomarkers could provide an alternative or adjunct diagnostic method, to allow early intervention before laminitis develops. The present study characterised the metabolome of ponies with varying degrees of ID using basal and postprandial plasma samples obtained during a previous study, which examined the predictive power of blood insulin levels for the development of laminitis, in ponies fed a high-sugar diet. Samples from 10 pre-laminitic (PL - subsequently developed laminitis) and 10 non-laminitic (NL - did not develop laminitis) ponies were used in a targeted metabolomic assay. Differential concentration and pathway analysis were performed using linear models and global tests. RESULTS: Significant changes in the concentration of six glycerophospholipids (adj. P ≤ 0.024) and a global enrichment of the glucose-alanine cycle (adj. P = 0.048) were found to characterise the response of PL ponies to the high-sugar diet. In contrast, the metabolites showed no significant association with the presence or absence of pituitary pars intermedia dysfunction in all ponies. CONCLUSIONS: The present results suggest that ID and laminitis risk are associated with alterations in the glycerophospholipid and glucose metabolism, which may help understand and explain some molecular processes causing or resulting from these conditions. The prognostic value of the identified biomarkers for laminitis remains to be investigated in further metabolomic trials in horses and ponies.

Component-resolved microarray analysis of IgE sensitization profiles to Culicoides recombinant allergens in horses with insect bite hypersensitivity.

BACKGROUND: Allergy to bites of blood-sucking insects, including biting midges, can affect both human and veterinary patients. Horses are often suffering from an IgE-mediated allergic dermatitis caused by bites of midges (Culicoides spp). With the aim to improve allergen immunotherapy (AIT), numerous Culicoides allergens have been produced as recombinant (r-) proteins. This study aimed to test a comprehensive panel of differently expressed Culicoides r-allergens on a cohort of IBH-affected and control horses using an allergen microarray. METHODS: IgE levels to 27 Culicoides r-allergens, including 8 previously unpublished allergens, of which 11 were expressed in more than one expression system, were determined in sera from 347 horses. ROC analyses were carried out, cut-offs selected using a specificity of 95% and seropositivity rates compared between horses affected with insect bite hypersensitivity (IBH) and control horses. The combination of r-allergens giving the best performing test was determined using logistic regression analysis. RESULTS: Seropositivity was significantly higher in IBH horses compared with controls for 25 r-allergens. Nine Culicoides r-allergens were major allergens for IBH with seven of them binding IgE in sera from > 70% of the IBH-affected horses. Combination of these top seven r-allergens could diagnose > 90% of IBH-affected horses with a specificity of > 95%. Correlation between differently expressed r-allergens was usually high (mean = 0.69, range: 0.28-0.91). CONCLUSION: This microarray will be a powerful tool for the development of component-resolved, patient-tailored AIT for IBH and could be useful for the study of allergy to biting midges in humans and other species.

Cul o 2 specific IgG3/5 antibodies predicted Culicoides hypersensitivity in a group imported Icelandic horses.

BACKGROUND: Culicoides hypersensitivity (CH) is induced in horses by salivary allergens of Culicoides midges. In Iceland, the causal Culicoides species for CH are not present. Previous epidemiological data indicated that Icelandic horses are more susceptible to CH when they are exported from Iceland and first exposed to Culicoides at adult age. Horses born in countries where Culicoides is endemic, develop the disease less frequently. Here, we established a longitudinal allergy model to identify predictive and diagnostic serological biomarkers of CH. RESULTS: Sixteen adult Icelandic horses from Iceland were imported to the Northeastern United States (US) during the winter and were kept in the same environment with natural Culicoides exposure for the next two years. None of the horses showed clinical allergy during the first summer of Culicoides exposure. In the second summer, 9/16 horses (56%) developed CH. Allergen specific IgE and IgG isotype responses in serum samples were analysed using nine potential Culicoides allergens in a fluorescent bead-based multiplex assay. During the first summer of Culicoides exposure, while all horses were still clinically healthy, Cul o 2 specific IgG3/5 antibodies were higher in horses that developed the allergic disease in the second summer compared to those that did not become allergic (p = 0.043). The difference in Cul o 2 specific IgG3/5 antibodies between the two groups continued to be detectable through fall (p = 0.035) and winter of the first year. During the second summer, clinical signs first appeared and Cul o 3 specific IgG3/5 isotypes were elevated in allergic horses (p = 0.041). Cul o 2 specific IgG5 (p = 0.035), and Cul o 3 specific IgG3/5 (p = 0.043) were increased in late fall of year two when clinical signs started to improve again. CONCLUSIONS: Our results identified IgG5 and IgG3/5 antibodies against Cul o 2 and Cul o 3, respectively, as markers for CH during and shortly after the allergy season in the Northeastern US. In addition, Cul o 2 specific IgG3/5 antibodies may be valuable as a predictive biomarker of CH in horses that have been exposed to Culicoides but did not yet develop clinical signs.

A "modified Obel" method for the severity scoring of (endocrinopathic) equine laminitis.

BACKGROUND: Laminitis is a common equine disease characterized by foot pain, and is commonly diagnosed using a five-grade Obel system developed in 1948 using sepsis-related cases. However, endocrinopathic laminitis is now the most common form of the disease and clinical signs may be mild, or spread across two Obel grades. This paper describes a modified method which assigns scores to discreet clinical signs, providing a wider scale suitable for use in a research setting. METHODS: The "modified Obel" method was developed using an iterative process. First, a prototype method was developed during the detailed observation of 37 ponies undergoing a laminitis induction experiment. The final method was refined and validated using video footage taken during the induction study and from a clinical trial of naturally occurring endocrinopathic laminitis cases. The Obel method was deconstructed and key laminitis signs were evaluated to develop a three-stage, five criteria method that employs a severity scale of 0-12. Veterinarians (n = 28) were recruited to watch and assess 15 video recordings of cases of varying severity, using the Obel and "modified Obel" methods. The inter-observer agreement (reproducibility) was determined using Kendall's coefficient of concordance (Kendall W) and Krippendorf's alpha reliability coefficient. A total of 14 veterinarians repeated the exercise 2-4 weeks after their original assessment, to determine intra-observer agreement (repeatability), assessed using a weighted kappa statistic (kw). Agreement between methods was calculated by converting all "modified Obel" scores to Obel grades and calculating the mean and distribution of the differences. RESULTS: The "modified Obel" and Obel methods showed excellent and similar inter-observer agreement based on the Kendall W value (0.87, P < 0.001 vs. 0.85, P < 0.001) and Krippendorf's alpha (95% CI) value (0.83 [0.53-0.90] vs. 0.77 [0.55-0.85]). Based on the kw value, the "modified Obel" method also had substantial repeatability, although slightly less than the Obel method, (0.80 vs. 0.91). Excellent agreement between the methods was found, with the mean difference (95% CI), comparing the Obel grade, with the "modified Obel" score converted to an Obel grade, being -0.12 (-0.19 to -0.06) grades. The Obel and converted "modified Obel" grades were identical 62% of the time (259/420) and a difference of one grade (higher or lower) occurred in 35% of cases (148/420). CONCLUSION: Both methods show excellent agreement, reproducibility and repeatability when used to diagnose endocrinopathic laminitis. The "modified Obel" method is a three-step examination process for severity-scoring of endocrinopathic laminitis, initially proposed for use within a research setting. When using the modified method a diagnosis of laminitis also requires clinical acumen. The allocation of scores for specific clinical signs should be particularly useful in research trials monitoring laminitis recovery.

Incidence and risk factors for recurrence of endocrinopathic laminitis in horses.

BACKGROUND: Endocrinopathic laminitis is common in horses and ponies, but the recurrence rate of the disease is poorly defined. OBJECTIVES: To determine the incidence of, and risk factors for, the recurrence of endocrinopathic laminitis. ANIMALS: Privately owned horses and ponies with acute laminitis (n = 317, of which 276 cases with endocrinopathic laminitis were followed up to study completion). METHODS: This prospective cohort study collected data on veterinary-diagnosed cases of acute laminitis for 2 years. Each case was classified on acceptance to the study as endocrinopathic or non-endocrinopathic using data collected in a questionnaire completed by the animal's veterinarian. Follow-up data were collected at regular intervals to determine whether the laminitis recurred in the 2-year period after diagnosis. RESULTS: The recurrence rate for endocrinopathic laminitis was 34.1%. The risk of recurrence during the 2-year study period increased with basal, fasted serum insulin concentration (P ≤ .05), with the probability of recurrence increasing markedly as the insulin concentration increased beyond the normal range (0-20 µIU/mL) to over the threshold for normal (up to approximately 45 µIU/mL). Being previously diagnosed with laminitis (before the study; P = .05) was also a risk factor for recurrent laminitis. Cases with a higher Obel grade of laminitis were likely (P = .05) to recur sooner. CONCLUSIONS AND CLINICAL IMPORTANCE: Knowing that hyperinsulinemia and being previously diagnosed with laminitis are significant risk factors for recurrence will enable clinicians to proactively address these factors, thereby potentially reducing the risk of recurrence of laminitis.

Phenotypic, hormonal, and clinical characteristics of equine endocrinopathic laminitis.

BACKGROUND: Equine endocrinopathic laminitis is common and can be associated with an underlying endocrinopathy, such as equine metabolic syndrome (EMS), pituitary pars intermedia dysfunction (PPID), pasture consumption, or any combination of these factors. OBJECTIVES: The aim of the study was to improve the risk assessment capabilities of clinicians, and to inform management strategies, for acute endocrinopathic laminitis by prospectively examining the phenotypic, hormonal, and clinical characteristics of the disease in a large cohort. ANIMALS: Privately owned horses and ponies (n = 301) of any age, sex, or breed diagnosed with laminitis by a veterinarian. A history of laminitis was acceptable. METHODS: This was a prospective cohort study. Veterinarians provided information on each case via an online questionnaire after informed consent from the animal's owner, and all data were de-identified before analysis. Serum insulin and plasma adrenocorticotrophic hormone concentrations were measured in each case. RESULTS: Most cases were recruited in spring (109/301; 36.2%). Concurrent EMS and PPID resulted in higher basal insulin concentrations (49 [21.5-141]; P < .02) than if an animal had a single underlying cause for their laminitis. The insulin concentration was negatively correlated (r2 = -0.38; P < .001) with the animal's height, being higher in ponies (33[10-14]; P < .001) than horses (9.5 [3-25.7]) and was positively correlated (r2 = 0.12; P = .05) with their grade (severity) of laminitis. CONCLUSIONS AND CLINICAL IMPORTANCE: Horses and ponies with concurrent endocrinopathies have more marked hyperinsulinemia. Higher basal insulin concentrations were associated with more severe lameness.

Neutral endopeptidase inhibitors blunt kidney fibrosis by reducing myofibroblast formation.

Kidney fibrosis is the common pathophysiological mechanism in end-stage renal disease characterized by excessive accumulation of myofibroblast-derived extracellular matrix. Natriuretic peptides have been demonstrated to have cyclic guanosine monophosphate (cGMP)-dependent anti-fibrotic properties likely due to interference with pro-fibrotic tissue growth factor ß (TGF-ß) signaling. However, in vivo, natriuretic peptides are rapidly degraded by neutral endopeptidases (NEP). In a unilateral ureteral obstruction (UUO) mouse model for kidney fibrosis we assessed the anti-fibrotic effects of SOL1, an orally active compound that inhibits NEP and endothelin-converting enzyme (ECE). Mice (n=10 per group) subjected to UUO were treated for 1 week with either solvent, NEP-/ECE-inhibitor SOL1 (two doses), reference NEP-inhibitor candoxatril or the angiotensin II receptor type 1 (AT1)-antagonist losartan. While NEP-inhibitors had no significant effect on blood pressure, they did increase urinary cGMP levels as well as endothelin-1 (ET-1) levels. Immunohistochemical staining revealed a marked decrease in renal collagen (∼55% reduction, P<0.05) and α-smooth muscle actin (α-SMA; ∼40% reduction, P<0.05). Moreover, the number of α-SMA positive cells in the kidneys of SOL1-treated groups inversely correlated with cGMP levels consistent with a NEP-dependent anti-fibrotic effect. To dissect the molecular mechanisms associated with the anti-fibrotic effects of NEP inhibition, we performed a 'deep serial analysis of gene expression (Deep SAGE)' transcriptome and targeted metabolomics analysis of total kidneys of all treatment groups. Pathway analyses linked increased cGMP and ET-1 levels with decreased nuclear receptor signaling (peroxisome proliferator-activated receptor [PPAR] and liver X receptor/retinoid X receptor [LXR/RXR] signaling) and actin cytoskeleton organization. Taken together, although our transcriptome and metabolome data indicate metabolic dysregulation, our data support the therapeutic potential of NEP inhibition in the treatment of kidney fibrosis via cGMP elevation and reduced myofibroblast formation.

The sodium-glucose co-transporter 2 inhibitor velagliflozin reduces hyperinsulinemia and prevents laminitis in insulin-dysregulated ponies.

There are no registered veterinary drugs for treating insulin dysregulation and preventing insulin-associated laminitis in horses. Velagliflozin is a sodium-glucose co-transport 2 inhibitor that reduces renal glucose reabsorption, promotes glucosuria, and consequently, decreases blood glucose and insulin concentrations. This study aimed to determine if velagliflozin reduced hyperinsulinemia and prevented laminitis in insulin-dysregulated ponies fed a challenge diet high in non-structural carbohydrates (NSC). An oral glucose test (1 g dextrose/kg BW) was used to screen 75 ponies for insulin dysregulation, of which 49 ponies with the highest insulin concentrations were selected. These animals were assigned randomly to either a treated group (n = 12) that received velagliflozin (0.3 mg/kg BW, p.o., s.i.d.) throughout the study, or a control group (n = 37). All ponies were fed a maintenance diet of alfalfa hay for 3 weeks, before transferring to a challenge diet (12 g NSC/kg BW/d) for up to 18 d. Blood glucose and serum insulin concentrations were measured over 4 h after feeding, on d 2 of the diet. The maximum glucose concentration was 22% lower (P = 0.014) in treated animals, with a geometric mean (95% CI) of 9.4 (8.0-11.0) mM, versus 12.1 (10.7-13.7) mM in the controls. This was reflected by lower (45%) maximum insulin concentrations in the treated group (P = 0.017), of 149 (97-228) µIU/mL, versus 272 (207-356) µIU/mL for controls. The diet induced Obel grade 1 or 2 laminitis in 14 of the 37 controls (38%), whereas no velagliflozin-treated pony developed laminitis (P = 0.011). Velagliflozin was well-tolerated, with no hypoglycemia or any clinical signs of adverse effects. The main limitation of this study was the sample size. Velagliflozin shows promise as a safe and effective compound for treating insulin dysregulation and preventing laminitis by reducing the hyperinsulinemic response to dietary NSC.

Effects of Inhibiting Dipeptidyl Peptidase-4 (DPP4) in Cows with Subclinical Ketosis.

The inhibition of dipeptidyl peptidase-4 (DPP4) via specific inhibitors is known to result in improved glucose tolerance and insulin sensitivity and decreased accumulation of hepatic fat in type II diabetic human patients. The metabolic situation of dairy cows can easily be compared to the status of human diabetes and non-alcoholic fatty liver. For both, insulin sensitivity is reduced, while hepatic fat accumulation increases, characterized by high levels of non-esterified fatty acids (NEFA) and ketone bodies.Therefore, in the present study, a DPP4 inhibitor was employed (BI 14332) for the first time in cows. In a first investigation BI 14332 treatment (intravenous injection at dosages of up to 3 mg/kg body weight) was well tolerated in healthy lactating pluriparous cows (n = 6) with a significant inhibition of DPP4 in plasma and liver. Further testing included primi- and pluriparous lactating cows suffering from subclinical ketosis (ß-hydroxybutyrate concentrations in serum > 1.2 mM; n = 12). The intension was to offer effects of DPP4 inhibition during comprehensive lipomobilisation and hepatosteatosis. The cows of subclinical ketosis were evenly allocated to either the treatment group (daily injections, 0.3 mg BI 14332/kg body weight, 7 days) or the control group. Under condition of subclinical ketosis, the impact of DPP4 inhibition via BI 14332 was less, as in particular ß-hydroxybutyrate and the hepatic lipid content remained unaffected, but NEFA and triglyceride concentrations were decreased after treatment. Owing to lower NEFA, the revised quantitative insulin sensitivity check index (surrogate marker for insulin sensitivity) increased. Therefore, a positive influence on energy metabolism might be quite possible. Minor impacts on immune-modulating variables were limited to the lymphocyte CD4+/CD8+ ratio for which a trend to decreased values in treated versus control animals was noted. In sum, the DPP4 inhibition in cows did not affect glycaemic control like it is shown in humans, but was able to impact hyperlipemia, as NEFA and TG decreased.

Effects of elevated parameters of subclinical ketosis on the immune system of dairy cows: in vivo and in vitro results.

Using an established model in which subclinical ketosis is induced, the response of differential blood counts and levels of various haematological variables, including the inflammatory marker haptoglobin (Hp), were tested over the last six weeks of parturition until the 56th day post-partum in cows with lower or higher body condition scores (LBC and HBC, respectively; n = 9/group). Animals in the HBC group evidenced subclinical ketosis whereas LBC animals were metabolically healthy. For in vitro examination with ß-hydroxybutyrate (BHB) as a further stimulus, peripheral blood mononuclear cell (PBMC) counts of cows with and without subclinical ketosis (n = 5/group) were observed. Counts of leucocytes, granulocytes and lymphocytes (LY) peaked at day 1 post-partum in HBC cows, with a more marked increase in heifers. In subclinical ketosis LY count increased again, with significantly higher values in the HBC group. The red blood cell (RBC) profile was affected by parity (counts were higher in heifers). Hp showed a positive linear correlation with BHB and non-esterified fatty acids (NEFA; R(2) = 0.41). PBMC from cows that were not pre-stressed with subclinical ketosis were more sensitive to increasing levels of BHB in vitro, as evidenced by both their higher proliferative capability and increased release of nitric oxide (NO). In summary, cows with subclinical ketosis showed a heightened immune response compared with metabolically healthy individuals, based on increased LY counts, increasing stimulative properties of PBMC and a relationship between Hp and typically increased values of BHB and NEFA. Concentrations of BHB in vivo during subclinical ketosis did not alter the proliferative capability of bovine PBMC in vitro, which was first significantly decreased at a dosage of 5 mM BHB.

Effects of prepartal body condition score and peripartal energy supply of dairy cows on postpartal lipolysis, energy balance and ketogenesis: an animal model to investigate subclinical ketosis.

Subclinical ketosis is a metabolic disorder which often goes undiagnosed and leads to constricted performance and an impairment of general condition. In the current study subclinical ketosis was characterised by a ß-hydroxybutyrate (BHB) concentration of >1·2 mmol/l in blood serum. To generate this metabolic situation, an animal model was created. The model, based on group-specific interaction of dietary energy supply and body condition, is appropriate for testing the medical effectiveness of treating this kind of ketosis and its concomitants. During the trial, 18 dairy cows (primiparous and pluriparous) were assigned, according to their body condition score (BCS) 6 weeks before expected parturition, to a normal [6.78 MJ net energy for lactation (NEL)/kg dry matter; 20% concentrate] or to a high-energy feeding group (7·71 MJ NEL/kg dry matter; 60% concentrate). Therefore cows with the highest BCS were allocated to the high-energy group to enhance the contrast with the control group. Statistical analysis was done using the MIXED procedure of SAS. Effects were declared significant when P-values were ⩽0.05. Owing to the higher energy concentration and dry matter intake, the energy intake and balance was significantly higher in the high-energy feeding group, with strong effects on lipid metabolism and health in blood and liver post partum. Within the first 2 weeks after calving, 8 out of 9 cows (89%) of the high-energy group had BHB values indicative of subclinical ketosis. These cows also had significantly higher values of non-esterified fatty acids (NEFA), aspartate transaminase (AST) and glutamate dehydrogenase (GLDH) post partum, as well as a raised total lipid content of the liver. RQUICKI, a calculated parameter which is based on serum concentrations of glucose, insulin and NEFA to assess the insulin sensitivity, was not affected by treatment. Therefore, RQUICKI does not seem to be the right parameter for diagnosing decreased insulin sensitivity in cows affected by subclinical ketosis. The milk fat and the fat:protein ratio of the high-energy group was also higher, even though there was no decrease in milk yield for cows with subclinical BHB values.

Assessment of impaired vascular reactivity in a rat model of diabetic nephropathy: effect of nitric oxide synthesis inhibition on intrarenal diffusion and oxygenation measured by magnetic resonance imaging.

Diabetes is associated with impaired vascular reactivity and the development of diabetic nephropathy. In a rat model of streptozotocin-induced diabetic nephropathy, the effects of systemic nitric oxide (NO) synthesis inhibition on intrarenal diffusion and oxygenation were determined by noninvasive magnetic resonance diffusion tensor imaging and blood O2 level-dependent (BOLD) imaging, respectively. Eight weeks after the induction of diabetes, 21 rats [n = 7 rats each in the untreated control group, diabetes mellitus (DM) group, and DM with uninephrectomy (DM UNX) group] were examined by MRI. Diffusion tensor imaging and BOLD sequences were acquired before and after NO synthesis inhibition with N-nitro-L-arginine methyl ester (L-NAME). In the same rats, mean arterial pressure and vascular conductance were determined with and without the influence of L-NAME. In control animals, NO synthesis inhibition was associated with a significant increase of mean arterial pressure of 33.8 ± 4.3 mmHg (P < 0.001) and a decrease of vascular conductance of -17.8 ± 2.0 µl·min(-1)·100 mmHg(-1) (P < 0.001). These changes were attenuated in both DM and DM UNX groups with no significant difference between before and after L-NAME measurements in DM UNX animals. Similarly, L-NAME challenge induced a significant reduction of renal transverse relaxation time (T2*) at MRI in control animals, indicating reduced renal oxygenation after L-NAME injection compared with baseline. DM UNX animals did not show a significant T2* reduction after NO synthesis inhibition in the renal cortex and attenuated T2* reduction in the outer medulla. MRI parameters of tissue diffusion were not affected by L-NAME in all groups. In conclusion, BOLD imaging proved valuable to noninvasively measure renal vascular reactivity upon NO synthesis inhibition in control animals and to detect impaired vascular reactivity in animals with diabetic nephropathy.

Magnetic resonance diffusion tensor imaging for evaluation of histopathological changes in a rat model of diabetic nephropathy.

OBJECTIVES: The aim of this study was to investigate whether magnetic resonance (MR) diffusion tensor imaging (DTI) allows assessment of renal pathologies in a rat model of diabetic nephropathy. MATERIALS AND METHODS: Twenty-one male Sprague-Dawley rats were divided into 3 groups: (1) untreated controls, (2) diabetes (DM), (3) diabetes with uninephrectomy (DM UNX) to accelerate renal impairment. Eight weeks after diabetes induction with streptozotocin, MR imaging was performed in a 1.5-T scanner using an 8-channel wrist coil. Morphological proton density images and echoplanar DTI were obtained (b = 0 and 300 s/mm, 6 diffusion directions). Renal apparent diffusion coefficient (ADC) and fractional anisotropy (FA) values were calculated for each of the different anatomical layers of the kidney. Imaging results, laboratory parameters of diabetic state and kidney function, and renal histopathological changes (glomerulosclerosis, tubular dilatation, and renal fibrosis) were compared between groups. Correlations between FA and histopathological changes were evaluated. RESULTS: All diabetic animals developed hyperglycemia and hypoinsulinemia. Uremia, albuminuria, and histopathological changes were most pronounced in DM UNX animals. Fractional anisotropy was significantly reduced in DM UNX animals in the cortex (CO) (0.167; confidence interval [CI], 0.151-0.184; P < 0.001), outer stripe of the outer medulla (OS) (0.254; CI, 0.225-0.283; P = 0.038), and inner medulla (IM) (0.459; CI, 0.395-0.523; P = 0.008) compared with control animals (CO, 0.251; CI, 0.224-0.277; OS, 0.309; CI, 0.267-0.350; IM, 0.559; CI, 0.515-0.603). In DM-without-UNX animals, only cortical FA was significantly lower than in controls (P < 0.001). Between groups, ADC values were not different, except for cortical ADC, which was higher in DM UNX animals than in controls. Significant negative correlations were observed between the FA of different anatomical layers and the extent of glomerulosclerosis (CO, P = 0.003, r = -0.65; and OS, P = 0.022, r = -0.52), tubulointerstitial fibrosis (IM, P = 0.028, r = -0.50), and tubular dilatation (CO, P = 0.015, r = -0.55; and IM, P = 0.006, r = -0.61), respectively. CONCLUSIONS: Magnetic resonance DTI by reduction of FA identified renal pathologies of diabetic nephropathy such as glomerulosclerosis, interstitial fibrosis, and tubular damage. Representing different stages of disease, DM and DM UNX animals could be differentiated. Thus, MR DTI may be valuable for noninvasive detection and monitoring of renal pathology in patients with diabetes.

Some cannabinoid receptor ligands and their distomers are direct-acting openers of SUR1 K(ATP) channels.

Here, we examined the chronic effects of two cannabinoid receptor-1 (CB1) inverse agonists, rimonabant and ibipinabant, in hyperinsulinemic Zucker rats to determine their chronic effects on insulinemia. Rimonabant and ibipinabant (10 mg·kg⁻¹·day⁻¹) elicited body weight-independent improvements in insulinemia and glycemia during 10 wk of chronic treatment. To elucidate the mechanism of insulin lowering, acute in vivo and in vitro studies were then performed. Surprisingly, chronic treatment was not required for insulin lowering. In acute in vivo and in vitro studies, the CB1 inverse agonists exhibited acute K channel opener (KCO; e.g., diazoxide and NN414)-like effects on glucose tolerance and glucose-stimulated insulin secretion (GSIS) with approximately fivefold better potency than diazoxide. Followup studies implied that these effects were inconsistent with a CB1-mediated mechanism. Thus effects of several CB1 agonists, inverse agonists, and distomers during GTTs or GSIS studies using perifused rat islets were unpredictable from their known CB1 activities. In vivo rimonabant and ibipinabant caused glucose intolerance in CB1 but not SUR1-KO mice. Electrophysiological studies indicated that, compared with diazoxide, 3 µM rimonabant and ibipinabant are partial agonists for K channel opening. Partial agonism was consistent with data from radioligand binding assays designed to detect SUR1 K(ATP) KCOs where rimonabant and ibipinabant allosterically regulated ³H-glibenclamide-specific binding in the presence of MgATP, as did diazoxide and NN414. Our findings indicate that some CB1 ligands may directly bind and allosterically regulate Kir6.2/SUR1 K(ATP) channels like other KCOs. This mechanism appears to be compatible with and may contribute to their acute and chronic effects on GSIS and insulinemia.

Cholinergic and noncholinergic innervation of the smooth muscle layers in the bovine abomasum.

The intrinsic innervation of muscle layers in the mammalian gastrointestinal tract has been mainly studied in nonruminants. The aim of this study was to identify intrinsic motor neurones in the bovine abomasum that innervate the circular and longitudinal muscles. Circular (CMN) and longitudinal muscle motor neurones (LMN) were selectively labeled by application of the retrograde tracer 1,1'-didodecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate (DiI) onto the muscle layers. The transmitter phenotype was determined by immunohistochemical detection of choline acetyltransferase (ChAT), nitric oxide synthase (NOS), and neurone-specific enolase (NSE). On average, the myenteric ganglia contained 61 +/- 19 NSE-positive cell bodies, of which 89% were ChAT-positive and 10% were NOS-positive. Only 0.7% of NSE-positive neurones (41 of 5,777) contained both ChAT and NOS. Application of DiI onto the circular and longitudinal muscles revealed on average 60 +/- 27 (n = 4) and 68 +/- 36 (n = 4), respectively, labeled cell bodies in the myenteric plexus. For the circular and longitudinal muscles the proportions of ascending to descending neurones were 76 : 24% and 54 : 46%, respectively. While most ascending CMN were ChAT-positive (96%), 51% of the descending CMN were ChAT-negative. All ascending and 95% of descending LMN were ChAT-positive. It was concluded that cholinergic excitatory innervation is predominant in both muscle layers of the abomasum. Whereas the circular muscle receives cholinergic excitatory and nitrergic inhibitory innervation, the longitudinal muscle is only innervated by cholinergic pathways. This innervation pattern is different from that in gastric muscle layers in monogastric animals.