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.

The anorectic response to growth hormone in obese rats is associated with an increased rate of lipid oxidation and decreased hypothalamic galanin.

The aim of this study was to demonstrate differential effects of growth hormone (GH) on food intake in lean and obese rats and to investigate whether an anticipated anorectic response in obese rats might be associated with increased lipid oxidation and altered hypothalamic neuropeptide levels. GH (4 mg/kg/day) was administered during 5-21 days to non-obese and obese rats. Whereas GH stimulated food intake in the non-obese rats, the obese animals responded with a significantly (p<0.05) suppressed food intake for 4-5 days. On day 4, the obese rats injected with GH and those injected with vehicle consumed 9.2 ± 0.66 g and 12.7 ± 1.05 g, respectively. The suppression of food intake was associated with significantly (p<0.05) increased lipid oxidation. A similar, but statistically not verified, trend was seen in pair-fed rats not exposed to GH. However, while these animals appeared to economize their energy expenditure, the GH-exposed animals did not, thus creating a significant (p<0.05) difference between these two groups. The increased lipid oxidation and energy expenditure observed in the rats exposed to GH were associated with significantly (p<0.05) decreased levels of hypothalamic galanin (111 ± 33.2 pmol/g vs. those of the pair-fed controls: 228.5 ± 49.4 pmol/g). This difference was, however, not sustained. Thus, on day 21 both hypothalamic galanin and the food intake in the GH group were back to normal. Hypothalamic NPY remained unchanged by GH at all times. In conclusion, the present study suggests that increased lipid oxidation and decreased hypothalamic galanin are components in the mechanism by which GH inhibits food intake in an obese phenotype.

Attenuation of hyperinsulinemia by NN414, a SUR1/Kir6.2 selective K-adenosine triphosphate channel opener, improves glucose tolerance and lipid profile in obese Zucker rats.

Chronic attenuation of hyperinsulinemia by diazoxide (DZ), a K-adenosine triphosphate (ATP) channel opener and an inhibitor of glucose-mediated insulin secretion, improved glucose tolerance and lipid profile and decreased the rate of weight gain in obese Zucker rats. To determine whether suppression of hyperinsulinemia alters daily food consumption, rate of weight gain, glucose tolerance, and lipid profile, we compared the effects of NN414, a potent and SUR1/Kir6.2 selective K(atp)() channel opener, with DZ in obese and lean Zucker rats. DZ (150 mg/kg/d), low-dose (LDNN414: 10 mg/kg/d), high-dose (HDNN414: 30 mg/kg/d), and vehicle (C) were administered to 7-week-old obese and lean female Zucker rats for a period of 6 weeks. Each animal underwent an intraperitoneal glucose tolerance test (IPGTT) at the end of study period. While NN414 treatment did not affect food intake and rate of weight gain in any of the strains, DZ treatment reduced food intake (P <.001) and rate of weight gain (P <.001) in obese rats. The fasting plasma insulin levels and area under the curve (AUC) insulin response to IPGTT were significantly attenuated in LDNN414 (P <.05), HDNN414 (P <.01), and DZ (P <.01) obese and lean rats compared with their controls. This was accompanied by a significant reduction in AUC glucose only in LDNN414 (P <.05), HDNN414 (P <.01), and DZ (P <.01) obese rats compared with controls. While hemoglobin A(1c) (HbA(1c)) was not affected in LDNN414 obese rats, it was higher in HDNN414 obese animals (P <.001), LD-, HDNN414 (P <.001), and DZ (P <.005) lean rats compared with their respective controls. DZ obese rats showed lower HbA(1c) levels than C obese rats (P <.02). The plasma free fatty acid (FFA) levels were only decreased in HDNN414 (P <.05) and DZ (P <.002) obese rats, whereas plasma triglyceride (TG) levels were decreased in LDNN414 (P <.05), HDNN414 (P <.001), and DZ (P <.001) obese rats compared with controls. Finally, plasma leptin level was only decreased in DZ obese rats compared with controls (P <.001). The new SUR1/Kir6.2 selective K(atp)() channel opener, NN414, reduced hyperinsulinemia in a dose-dependent manner without a significant effect on food consumption and rate of weight gain. NN414-induced beta-cell rest in obese rats was associated with a significant improvement in glucose responsiveness, suggesting an increase in insulin sensitivity after its withdrawal. There was an overall deterioration in glycemic control at the high dose as measured by HbA(1c). There was a dose-dependent improvement in lipid profiles of obese Zucker rats. These results suggest that pharmacologic attenuation of hyperinsulinemic state by low-dose NN414 may be therapeutically beneficial in insulin-resistant states without any deterioration in overall glycemic control.

Synthesis and pharmacological evaluation of novel cis-3,4-diaryl-hydroxychromanes as high affinity partial agonists for the estrogen receptor.

The syntheses and in vitro pharmacological evaluation of a number of cis-3,4-diaryl-hydroxy-chromanes are reported, along with the results of a thorough in vivo profiling of the tissue-selective estrogen partial-agonist NNC 45-0781 [3, (-)-(3S,4R)-7-hydroxy-3-phenyl-4-(4-(2-pyrrolidinoethoxy)phenyl)chromane]. These studies showed that NNC 45-0781 is a very promising candidate for the prevention of post-menopausal osteoporosis, and the treatment of other health issues related to the loss of endogenous estrogen production.