Differential effects of bariatric surgery and caloric restriction on hepatic one-carbon and fatty acid metabolism.

Weight loss interventions, including dietary changes, pharmacotherapy, or bariatric surgery, prevent many of the adverse consequences of obesity, and may also confer intervention-specific benefits beyond those seen with decreased weight alone. We compared the molecular effects of different interventions on liver metabolism to understand the mechanisms underlying these benefits. Male rats on a high-fat, high-sucrose diet underwent sleeve gastrectomy (SG) or intermittent fasting with caloric restriction (IF-CR), achieving equivalent weight loss. The interventions were compared to ad-libitum (AL)-fed controls. Analysis of liver and blood metabolome and transcriptome revealed distinct and sometimes contrasting metabolic effects between the two interventions. SG primarily influenced one-carbon metabolic pathways, whereas IF-CR increased de novo lipogenesis and glycogen storage. These findings suggest that the unique metabolic pathways affected by SG and IF-CR contribute to their distinct clinical benefits, with bariatric surgery potentially influencing long-lasting changes through its effect on one-carbon metabolism.

Simple Continuous Glucose Monitoring in Freely Moving Mice.

Mice are a common model organism used to study metabolic diseases such as diabetes mellitus. Glucose levels are typically measured by tail-bleeding, which requires handling the mice, causes stress, and does not provide data on freely behaving mice during the dark cycle. State-of-the-art continuous glucose measurement in mice requires inserting a probe into the aortic arch of the mouse, as well as a specialized telemetry system. This challenging and expensive method has not been adopted by most labs. Here, we present a simple protocol involving the utilization of commercially available continuous glucose monitors used by millions of patients to measure glucose continuously in mice as a part of basic research. The glucose-sensing probe is inserted into the subcutaneous space in the back of the mouse through a small incision to the skin and is held in place tightly using a couple of sutures. The device is sutured to the mouse skin to ensure it remains in place. The device can measure glucose levels for up to 2 weeks and sends the data to a nearby receiver without any need to handle the mice. Scripts for the basic data analysis of glucose levels recorded are provided. This method, from surgery to computational analysis, is cost-effective and potentially very useful in metabolic research.

Mapping the metabolic reprogramming induced by sodium-glucose cotransporter 2 inhibition.

Diabetes is associated with increased risk for kidney disease, heart failure, and mortality. Sodium-glucose cotransporter 2 inhibitors (SGLT2i) prevent these adverse outcomes; however, the mechanisms involved are not clear. We generated a roadmap of the metabolic alterations that occur in different organs in diabetes and in response to SGLT2i. In vivo metabolic labeling with 13C-glucose in normoglycemic and diabetic mice treated with or without dapagliflozin, followed by metabolomics and metabolic flux analyses, showed that, in diabetes, glycolysis and glucose oxidation are impaired in the kidney, liver, and heart. Treatment with dapagliflozin failed to rescue glycolysis. SGLT2 inhibition increased glucose oxidation in all organs; in the kidney, this was associated with modulation of the redox state. Diabetes was associated with altered methionine cycle metabolism, evident by decreased betaine and methionine levels, whereas treatment with SGLT2i increased hepatic betaine along with decreased homocysteine levels. mTORC1 activity was inhibited by SGLT2i along with stimulation of AMPK in both normoglycemic and diabetic animals, possibly explaining the protective effects against kidney, liver, and heart diseases. Collectively, our findings suggest that SGLT2i induces metabolic reprogramming orchestrated by AMPK-mTORC1 signaling with common and distinct effects in various tissues, with implications for diabetes and aging.

Sleeve Gastrectomy Reduces Glycemia but Does Not Affect Cognitive Impairment in Lean 5xFAD Mice.

Obesity and hyperglycemia are risk factors for cognitive decline and for the development of Alzheimer's Disease (AD). Bariatric surgery is an effective treatment for obesity that was shown to improve cognitive decline in obese patients. Bariatric surgery was shown to exert weight loss independent effects on metabolic diseases such as type 2 diabetes. We tested whether sleeve gastrectomy (SG), a common bariatric surgery, can affect the cognitive impairment in lean, normoglycemic female 5xFAD mice, a genetic model for AD. 5xFAD mice and wild-type (WT) littermates underwent SG or sham surgery at the age of 5 months and were tested for metabolic, behavioral, and molecular phenotypes 90 days later. SG led to a reduction in blood glucose levels and total plasma cholesterol levels in 5xFAD mice without inducing weight loss. However, the surgery did not affect the outcomes of long-term spatial memory tests in these mice. Analysis of ß-Amyloid plaques corroborated the behavioral studies in showing no effect of surgery on the molecular phenotype of 5xFAD mice. In conclusion, SG leads to an improved metabolic profile in lean female 5xFAD mice without inducing weight loss but does not affect the brain pathology or behavioral phenotype. Our results suggest that the positive effects of bariatric surgery on cognitive decline in obese patients are likely attributed to weight loss and improvement in obesity sequelae, and not to weight loss independent effects of surgery.

Maternal antagonism of Glp1 reverses the adverse outcomes of sleeve gastrectomy on mouse offspring.

Mothers that underwent bariatric surgery are at higher risk for delivering a small-for-gestational age (SGA) infant. This phenomenon is attributed to malabsorption and rapid weight loss following surgery. We compared pregnancy outcomes in lean mice that underwent sham surgery or sleeve gastrectomy (SG). SG led to a reduction in glucose levels and an increase in postprandial levels of glucagon-like peptide 1 (Glp1) without affecting mice weight during pregnancy. Pups of SG-operated mice (SG pups) were born SGA. The placenta and pancreas of the pups were not affected by SG, although a high-fat diet caused hepatic steatosis and glucose intolerance in male SG pups. Treatment with a Glp1 receptor antagonist during pregnancy normalized the birth weight of SG pups and diminished the adverse response to a high-fat diet without affecting glucose levels of pregnant mice. The antagonist did not affect the birth weight of pups of sham-operated mice. Our findings link elevated Glp1 signaling, rather than weight loss, to the increased prevalence of SGA births following bariatric surgery with metabolic consequences for the offspring. The long-term effects of bariatric surgery on the metabolic health of offspring of patients require further investigation.

Generation and characterization of a mouse model for one anastomosis gastric bypass surgery.

One anastomosis gastric bypass (OAGB) surgery became a common bariatric procedure in recent years. In this surgery, the distal stomach, duodenum, and proximal jejunum are bypassed, leading to weight loss, improvement in metabolic parameters, and a change in hormonal secretion. We sought to generate and characterize a mouse model for OAGB. Mice fed for 26 wk on a high-fat diet were assigned to OAGB, sham surgery, or caloric restriction and were followed for 50 more days on a high-fat diet. Physiological and histological parameters of the mice were compared during and at the end of the experiment. OAGB-operated mice lost weight and displayed low levels of plasma lipids, high insulin sensitivity, and rapid glucose metabolism compared with sham-operated mice. OAGB-operated mice had higher energy expenditure, higher levels of glucagon-like peptide (GLP-1), and lower albumin than weight-matched calorie-restricted mice. There was no difference in the histology of the endocrine pancreas. The livers of OAGB mice had little hepatic steatosis yet presented with a large number of phagocytic cells. The OAGB mouse model recapitulates many of the phenotypes described in patients that underwent OAGB and enables molecular and physiological studies on the outcome of this surgery.NEW & NOTEWORTHY A mouse model for one anastomosis gastric bypass (OAGB) surgery displays similar outcomes to clinical reports and enables to study the weight loss-dependent and -independent effects of this bariatric surgery.

Sleeve Gastrectomy Suppresses Hepatic Glucose Production and Increases Hepatic Insulin Clearance Independent of Weight Loss.

Bariatric operations induce weight loss, which is associated with an improvement in hepatic steatosis and a reduction in hepatic glucose production. It is not clear whether these outcomes are entirely due to weight loss, or whether the new anatomy imposed by the surgery contributes to the improvement in the metabolic function of the liver. We performed vertical sleeve gastrectomy (VSG) on obese mice provided with a high-fat high-sucrose diet and compared them to diet and weight-matched sham-operated mice (WMS). At 40 days after surgery, VSG-operated mice displayed less hepatic steatosis compared with WMS. By measuring the fasting glucose and insulin levels in the blood vessels feeding and draining the liver, we showed directly that hepatic glucose production was suppressed after VSG. Insulin levels were elevated in the portal vein, and hepatic insulin clearance was elevated in VSG-operated mice. The hepatic expression of genes associated with insulin clearance was upregulated. We repeated the experiment in lean mice and observed that portal insulin and glucagon are elevated, but only insulin clearance is increased in VSG-operated mice. In conclusion, direct measurement of glucose and insulin in the blood entering and leaving the liver shows that VSG affects glucose and insulin metabolism through mechanisms independent of weight loss and diet.

Bariatric Surgery Affects Plasma Levels of Alanine Aminotransferase Independent of Weight Loss: A Registry-Based Study.

Patients that undergo bariatric surgery experience weight loss and a reduction in the plasma levels of the hepatic enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST). We used the Israeli national bariatric registry, which includes demographic, clinical, and biochemical data on 19,403 patients, of which 1335 patients had two-year follow-up data on ALT, AST, A1C, and BMI, to test the dependence of the reduction in the levels of ALT and AST on weight loss. The data were analyzed using regression models, retrospective matching, and time course analyses. Changes in liver enzymes did not correlate with change in BMI, and linear regression models did not demonstrate that the change in ALT and AST values were dependent on pre-operative levels of BMI or the extent of weight loss. ALT and AST levels were reduced two years after surgery compared with a cohort of retrospectively matched patients for ethnicity, sex, age, BMI, and A1C. Finally, patients who regained weight displayed a reduction in levels of liver enzymes. Our results suggest that bariatric surgery affects AST and ALT levels via weight loss dependent and independent mechanisms. Mechanistic studies that will identify the nature of this effect and the clinical relevance of ALT and AST levels to the post-bariatric liver function are warranted.

Sleeve Gastrectomy Is Associated with a Greater Reduction in Plasma Liver Enzymes Than Bypass Surgeries-A Registry-Based Two-Year Follow-Up Analysis.

Bariatric surgeries may lead to an improvement in metabolic fatty liver disease, and a reduction in the levels of the hepatic enzyme Alanine Aminotransferase (ALT). We compared the effects of Sleeve Gastrectomy (SG), Roux en Y Gastric Bypass (RYGB) and One Anastomosis Gastric Bypass (OAGB) on the levels of ALT by analysis of two-year follow-up data from 4980 patients in the Israeli Bariatric Registry that included laboratory tests and demographic information. Pre-operative characteristics of patients, and particularly levels of liver enzymes, were similar across surgery types. Regression modeling and retrospective matching showed that SG was superior to RYGB and OAGB in reducing ALT levels, and in reducing the fraction of patients with abnormally high ALT levels. Two-year post-surgery, an increase in ALT levels from normal to abnormal levels was observed in 5% of SG patients, and in 18% and 23% of RYGB and OAGB patients. In conclusion, SG leads to a greater reduction in ALT levels compared with bypass surgeries and a lower incidence of post-surgical elevation of ALT levels. Further studies are required to identify the cause for the rise in liver enzymes, and to determine whether ALT levels correlate with liver pathology especially following bariatric surgery.

Biphasic dynamics of beta cell mass in a mouse model of congenital hyperinsulinism: implications for type 2 diabetes.

AIMS/HYPOTHESIS: Acute hyperglycaemia stimulates pancreatic beta cell proliferation in the mouse whereas chronic hyperglycaemia appears to be toxic. We hypothesise that this toxic effect is mediated by increased beta cell workload, unrelated to hyperglycaemia per se. METHODS: To test this hypothesis, we developed a novel mouse model of cell-autonomous increased beta cell glycolytic flux caused by a conditional heterozygous beta cell-specific mutation that activates glucokinase (GCK), mimicking key aspects of the rare human genetic disease GCK-congenital hyperinsulinism. RESULTS: In the mutant mice, we observed random and fasting hypoglycaemia (random 4.5-5.4 mmol/l and fasting 3.6 mmol/l) that persisted for 15 months. GCK activation led to increased beta cell proliferation as measured by Ki67 expression (2.7% vs 1.5%, mutant and wild-type (WT), respectively, p < 0.01) that resulted in a 62% increase in beta cell mass in young mice. However, by 8 months of age, mutant mice developed impaired glucose tolerance, which was associated with decreased absolute beta cell mass from 2.9 mg at 1.5 months to 1.8 mg at 8 months of age, with preservation of individual beta cell function. Impaired glucose tolerance was further exacerbated by a high-fat/high-sucrose diet (AUC 1796 vs 966 mmol/l × min, mutant and WT, respectively, p < 0.05). Activation of GCK was associated with an increased DNA damage response and an elevated expression of Chop, suggesting metabolic stress as a contributor to beta cell death. CONCLUSIONS/INTERPRETATION: We propose that increased workload-driven biphasic beta cell dynamics contribute to decreased beta cell function observed in long-standing congenital hyperinsulinism and type 2 diabetes.

Sleeve Gastrectomy Improves Glycemia Independent of Weight Loss by Restoring Hepatic Insulin Sensitivity.

Bariatric surgery dramatically improves glycemic control, yet the underlying molecular mechanisms remain controversial because of confounding weight loss. We performed sleeve gastrectomy (SG) on obese and diabetic leptin receptor-deficient mice (db/db). One week postsurgery, mice weighed 5% less and displayed improved glycemia compared with sham-operated controls, and islets from SG mice displayed reduced expression of diabetes markers. One month postsurgery SG mice weighed more than preoperatively but remained near-euglycemic and displayed reduced hepatic lipid droplets. Pair feeding of SG and sham db/db mice showed that surgery rather than weight loss was responsible for reduced glycemia after SG. Although insulin secretion profiles from islets of sham and SG mice were indistinguishable, clamp studies revealed that SG causes a dramatic improvement in muscle and hepatic insulin sensitivity accompanied by hepatic regulation of hepatocyte nuclear factor-α and peroxisome proliferator-activated receptor-α targets. We conclude that long-term weight loss after SG requires leptin signaling. Nevertheless, SG elicits a remarkable improvement in glycemia through insulin sensitization independent of reduced feeding and weight loss.

PAX6 regulates melanogenesis in the retinal pigmented epithelium through feed-forward regulatory interactions with MITF.

During organogenesis, PAX6 is required for establishment of various progenitor subtypes within the central nervous system, eye and pancreas. PAX6 expression is maintained in a variety of cell types within each organ, although its role in each lineage and how it acquires cell-specific activity remain elusive. Herein, we aimed to determine the roles and the hierarchical organization of the PAX6-dependent gene regulatory network during the differentiation of the retinal pigmented epithelium (RPE). Somatic mutagenesis of Pax6 in the differentiating RPE revealed that PAX6 functions in a feed-forward regulatory loop with MITF during onset of melanogenesis. PAX6 both controls the expression of an RPE isoform of Mitf and synergizes with MITF to activate expression of genes involved in pigment biogenesis. This study exemplifies how one kernel gene pivotal in organ formation accomplishes a lineage-specific role during terminal differentiation of a single lineage.

The expression of the beta cell-derived autoimmune ligand for the killer receptor nkp46 is attenuated in type 2 diabetes.

NK cells rapidly kill tumor cells, virus infected cells and even self cells. This is mediated via killer receptors, among which NKp46 (NCR1 in mice) is prominent. We have recently demonstrated that in type 1 diabetes (T1D) NK cells accumulate in the diseased pancreas and that they manifest a hyporesponsive phenotype. In addition, we found that NKp46 recognizes an unknown ligand expressed by beta cells derived from humans and mice and that blocking of NKp46 activity prevented diabetes development. Here we investigated the properties of the unknown NKp46 ligand. We show that the NKp46 ligand is mainly located in insulin granules and that it is constitutively secreted. Following glucose stimulation the NKp46 ligand translocates to the cell membrane and its secretion decreases. We further demonstrate by using several modalities that the unknown NKp46 ligand is not insulin. Finally, we studied the expression of the NKp46 ligand in type 2 diabetes (T2D) using 3 different in vivo models and 2 species; mice and gerbils. We demonstrate that the expression of the NKp46 ligand is decreased in all models of T2D studied, suggesting that NKp46 is not involved in T2D.

Cdx1 is essential for the initiation of HoxC8 expression during early embryogenesis.

The Hox genes pattern the anterior-posterior axis in developing embryos through tightly regulated, partially overlapping, temporal and spatial expression domains. Initial regulation of Hox expression is important to establish these overlapping transcription domains. The Cdx homeodomain factors have been proposed as Hox regulators, but their precise role and mechanism during this regulatory interaction remain unclear. In Xenopus embryos, HoxC8 transcripts begin to accumulate during mid/late gastrula. Cdx1 overexpression and knockdown lead to precocious or slower HoxC8 expression, respectively. The mouse HoxC8 early enhancer when introduced into Xenopus embryos recapitulates the endogenous XHoxC8 temporal expression pattern and shows the same responsiveness to Cdx1 regulation. Three pairs of conserved Cdx binding sites were identified within the HoxC8 early enhancer. We demonstrate that Cdx1 binds directly these responsive elements during embryogenesis, as part of the mechanism for the timely activation of HoxC8 expression. We define the function and mechanism of Cdx1 regulation on HoxC8 expression and suggest the possibility that the temporal changes in Cdx activity levels during gastrulation, combined with differential DNA binding affinity, might play a role in the establishment of Hox sequential activation.

Negative autoregulation of Oct3/4 through Cdx1 promotes the onset of gastrulation.

Gastrulation marks the onset of germ layer formation from undifferentiated precursor cells maintained by a network including the Pou5f1 gene, Oct3/4. Negative regulation of the undifferentiated state is a prerequisite for germ layer formation and subsequent development. A novel cross-regulatory network was characterized including the Pou5f1 and Cdx1 genes as part of the signals controlling the onset of gastrulation. Of particular interest was the observation that, preceding gastrulation, the Xenopus Oct3/4 factors, Oct60, Oct25, and Oct91, positively regulate Cdx1 expression through FGF signaling, and during gastrulation the Oct3/4 factors become repressors of Cdx1. Cdx1 negatively regulates the Pou5f1 genes during gastrulation, thus contributing to the repression of the network maintaining the undifferentiated state and promoting the onset of gastrulation. These regulatory interactions suggest that Oct3/4 initiates its own negative autoregulation through Cdx1 up-regulation to begin the repression of pluripotency in preparation for the onset of gastrulation and germ layer differentiation.

Multiple RTK pathways downregulate Groucho-mediated repression in Drosophila embryogenesis.

RTK pathways establish cell fates in a wide range of developmental processes. However, how the pathway effector MAPK coordinately regulates the expression of multiple target genes is not fully understood. We have previously shown that the EGFR RTK pathway causes phosphorylation and downregulation of Groucho, a global co-repressor that is widely used by many developmentally important repressors for silencing their various targets. Here, we use specific antibodies that reveal the dynamics of Groucho phosphorylation by MAPK, and show that Groucho is phosphorylated in response to several RTK pathways during Drosophila embryogenesis. Focusing on the regulation of terminal patterning by the Torso RTK pathway, we demonstrate that attenuation of Groucho's repressor function via phosphorylation is essential for the transcriptional output of the pathway and for terminal cell specification. Importantly, Groucho is phosphorylated by an efficient mechanism that does not alter its subcellular localisation or decrease its stability; rather, modified Groucho endures long after MAPK activation has terminated. We propose that phosphorylation of Groucho provides a widespread, long-term mechanism by which RTK signals control target gene expression.