ADGRL1 is a glucose receptor involved in mediating energy and glucose homeostasis.

AIMS/HYPOTHESIS: The brain is a major consumer of glucose as an energy source and regulates systemic glucose as well as energy balance. Although glucose transporters such as GLUT2 and sodium-glucose cotransporter 2 (SGLT2) are known to regulate glucose homeostasis and metabolism, the identity of a receptor that binds glucose to activate glucose signalling pathways in the brain is unknown. In this study, we aimed to discover a glucose receptor in the mouse hypothalamus. METHODS: Here we used a high molecular mass glucose-biotin polymer to enrich glucose-bound mouse hypothalamic neurons through cell-based affinity chromatography. We then subjected the enriched neurons to proteomic analyses and identified adhesion G-protein coupled receptor 1 (ADGRL1) as a top candidate for a glucose receptor. We validated glucose-ADGRL1 interactions using CHO cells stably expressing human ADGRL1 and ligand-receptor binding assays. We generated and determined the phenotype of global Adgrl1-knockout mice and hypothalamus-specific Adgrl1-deficient mice. We measured the variables related to glucose and energy homeostasis in these mice. We also generated an Adgrl1Cre mouse model to investigate the role of ADGRL1 in sensing glucose using electrophysiology. RESULTS: Adgrl1 is highly expressed in the ventromedial nucleus of the hypothalamus (VMH) in mice. Lack of Adgrl1 in the VMH in mice caused fasting hyperinsulinaemia, enhanced glucose-stimulated insulin secretion and insulin resistance. In addition, the Adgrl1-deficient mice had impaired feeding responses to glucose and fasting coupled with abnormal glucose sensing and decreased physical activity before development of obesity and hyperglycaemia. In female mice, ovariectomy was necessary to reveal the contribution of ADGRL1 to energy and glucose homeostasis. CONCLUSIONS/INTERPRETATION: Altogether, our findings demonstrate that ADGRL1 binds glucose and is involved in energy as well as glucose homeostasis in a sex-dependent manner. Targeting ADGRL1 may introduce a new class of drugs for the treatment of type 2 diabetes and obesity.

Loss of function of renal Glut2 reverses hyperglycaemia and normalises body weight in mouse models of diabetes and obesity.

AIMS/HYPOTHESIS: Renal GLUT2 is increased in diabetes, thereby enhancing glucose reabsorption and worsening hyperglycaemia. Here, we determined whether loss of Glut2 (also known as Slc2a2) specifically in the kidneys would reverse hyperglycaemia and normalise body weight in mouse models of diabetes and obesity. METHODS: We used the tamoxifen-inducible CreERT2-Lox system in mice to knockout Glut2 specifically in the kidneys (Ks-Glut2 KO) to establish the contribution of renal GLUT2 to systemic glucose homeostasis in health and in insulin-dependent as well as non-insulin-dependent diabetes. We measured circulating glucose and insulin levels in response to OGTT or IVGTT under different experimental conditions in the Ks-Glut2 KO and their control mice. Moreover, we quantified urine glucose levels to explain the phenotype of the mice independently of insulin actions. We also used a transcription factor array to identify mechanisms underlying the crosstalk between renal GLUT2 and sodium-glucose cotransporter 2 (SGLT2). RESULTS: The Ks-Glut2 KO mice exhibited improved glucose tolerance and massive glucosuria. Interestingly, this improvement in blood glucose control was eliminated when we knocked out Glut2 in the liver in addition to the kidneys, suggesting that the improvement is attributable to the lack of renal GLUT2. Remarkably, induction of renal Glut2 deficiency reversed hyperglycaemia and normalised body weight in mouse models of diabetes and obesity. Longitudinal monitoring of renal glucose transporters revealed that Sglt2 (also known as Slc5a2) expression was almost abolished 3 weeks after inducing renal Glut2 deficiency. To identify a molecular basis for this crosstalk, we screened for renal transcription factors that were downregulated in the Ks-Glut2 KO mice. Hnf1α (also known as Hnf1a) was among the genes most downregulated and its recovery restored Sglt2 expression in primary renal proximal tubular cells isolated from the Ks-Glut2 KO mice. CONCLUSIONS/INTERPRETATION: Altogether, these results demonstrate a novel crosstalk between renal GLUT2 and SGLT2 in regulating systemic glucose homeostasis via glucose reabsorption. Our findings also indicate that inhibiting renal GLUT2 is a potential therapy for diabetes and obesity.

Hypothalamic MC4R regulates glucose homeostasis through adrenaline-mediated control of glucose reabsorption via renal GLUT2 in mice.

AIMS/HYPOTHESIS: Melanocortin 4 receptor (MC4R) mutation is the most common cause of known monogenic obesity in humans. Unexpectedly, humans and rodents with MC4R deficiency do not develop hyperglycaemia despite chronic obesity and insulin resistance. To explain the underlying mechanisms for this phenotype, we determined the role of MC4R in glucose homeostasis in the presence and absence of obesity in mice. METHODS: We used global and hypothalamus-specific MC4R-deficient mice to investigate the brain regions that contribute to glucose homeostasis via MC4R. We performed oral, intraperitoneal and intravenous glucose tolerance tests in MC4R-deficient mice that were either obese or weight-matched to their littermate controls to define the role of MC4R in glucose regulation independently of changes in body weight. To identify the integrative pathways through which MC4R regulates glucose homeostasis, we measured renal and adrenal sympathetic nerve activity. We also evaluated glucose homeostasis in adrenaline (epinephrine)-deficient mice to investigate the role of adrenaline in mediating the effects of MC4R in glucose homeostasis. We employed a graded [13C6]glucose infusion procedure to quantify renal glucose reabsorption in MC4R-deficient mice. Finally, we measured the levels of renal glucose transporters in hypothalamus-specific MC4R-deficient mice and adrenaline-deficient mice using western blotting to ascertain the molecular mechanisms underlying MC4R control of glucose homeostasis. RESULTS: We found that obese and weight-matched MC4R-deficient mice exhibited improved glucose tolerance due to elevated glucosuria, not enhanced beta cell function. Moreover, MC4R deficiency selectively in the paraventricular nucleus of the hypothalamus (PVH) is responsible for reducing the renal threshold for glucose as measured by graded [13C6]glucose infusion technique. The MC4R deficiency suppressed renal sympathetic nerve activity by 50% in addition to decreasing circulating adrenaline and renal GLUT2 levels in mice, which contributed to the elevated glucosuria. We further report that adrenaline-deficient mice recapitulated the increased excretion of glucose in urine observed in the MC4R-deficient mice. Restoration of circulating adrenaline in both the MC4R- and adrenaline-deficient mice reversed their phenotype of improved glucose tolerance and elevated glucosuria, demonstrating the role of adrenaline in mediating the effects of MC4R on glucose reabsorption. CONCLUSIONS/INTERPRETATION: These findings define a previously unrecognised function of hypothalamic MC4R in glucose reabsorption mediated by adrenaline and renal GLUT2. Taken together, our findings indicate that elevated glucosuria due to low sympathetic tone explains why MC4R deficiency does not cause hyperglycaemia despite inducing obesity and insulin resistance. Graphical abstract.

High-fat diet-induced glucose dysregulation is independent of changes in islet ACE2 in mice.

While restoration of ACE2 activity in the pancreas leads to improvement of glycemia in experimental models of Type 2 diabetes, global deficiency in ACE2 disrupts ß-cell function and impairs glucose tolerance in mice, demonstrating the physiological role of ACE2 in glucose homeostasis. Although the contribution of pancreatic ACE2 to glucose regulation has been demonstrated in genetic models of diabetes and in models with overexpression of the renin-angiotensin system (RAS), it is unclear whether islet ACE2 is involved in glycemic control in common models of human Type 2 diabetes. To determine whether diet-induced diabetes deregulates glucose homeostasis via reduction of ACE2 in the pancreatic islets, wild-type (WT) and ACE2 knockout (KO) male mice were fed a high-fat diet (HFD) for 16 wk. ACE2 KO mice were more susceptible than WT mice to HFD-mediated glycemic dysregulation. Islet ACE2 activity and expression of various genes, including ANG II type 1a receptor (mAT1aR) were then assessed. Surprisingly, we observed no change in islet ACE2 activity and expression despite local RAS overactivity, indicated by an upregulation of mAT1aR expression. Despite a predominant expression in islet α-cells, further investigation highlighted a minor role for ACE2 on glucagon expression. Further, pancreatic ACE2 gene therapy improved glycemia in HFD-fed WT mice, leading to enhanced glucose-stimulated insulin secretion, reduced pancreatic ANG II levels, fibrosis, and ADAM17 activity. Altogether, our study demonstrates that HFD feeding increases RAS activity and mediates glycemic dysregulation likely through loss of ACE2 present outside the islets but independently of changes in islet ACE2.

Brain angiotensin-converting enzyme type 2 shedding contributes to the development of neurogenic hypertension.

RATIONALE: Overactivity of the brain renin-angiotensin system is a major contributor to neurogenic hypertension. Although overexpression of angiotensin-converting enzyme type 2 (ACE2) has been shown to be beneficial in reducing hypertension by transforming angiotensin II into angiotensin-(1-7), several groups have reported decreased brain ACE2 expression and activity during the development of hypertension. OBJECTIVE: We hypothesized that ADAM17-mediated ACE2 shedding results in decreased membrane-bound ACE2 in the brain, thus promoting the development of neurogenic hypertension. METHODS AND RESULTS: To test this hypothesis, we used the deoxycorticosterone acetate-salt model of neurogenic hypertension in nontransgenic and syn-hACE2 mice overexpressing ACE2 in neurons. Deoxycorticosterone acetate-salt treatment in nontransgenic mice led to significant increases in blood pressure, hypothalamic angiotensin II levels, inflammation, impaired baroreflex sensitivity, and autonomic dysfunction, as well as decreased hypothalamic ACE2 activity and expression, although these changes were blunted or prevented in syn-hACE2 mice. In addition, reduction of ACE2 expression and activity in the brain paralleled an increase in ACE2 activity in the cerebrospinal fluid of nontransgenic mice after deoxycorticosterone acetate-salt treatment and were accompanied by enhanced ADAM17 expression and activity in the hypothalamus. Chronic knockdown of ADAM17 in the brain blunted the development of hypertension and restored ACE2 activity and baroreflex function. CONCLUSIONS: Our data provide the first evidence that ADAM17-mediated shedding impairs brain ACE2 compensatory activity, thus contributing to the development of neurogenic hypertension.

The transcription factor HNF1α induces expression of angiotensin-converting enzyme 2 (ACE2) in pancreatic islets from evolutionarily conserved promoter motifs.

Pancreatic angiotensin-converting enzyme 2 (ACE2) has previously been shown to be critical for maintaining glycemia and ß-cell function. Efforts to maintain or increase ACE2 expression in pancreatic ß-cells might therefore have therapeutic potential for treating diabetes. In our study, we investigated the transcriptional role of hepatocyte nuclear factor 1α (HNF1α) and hepatocyte nuclear factor 1ß (HNF1ß) in induction of ACE2 expression in insulin-secreting cells. A deficient allele of HNF1α or HNF1ß causes maturity-onset diabetes of the young (MODY) types 3 and 5, respectively, in humans. We found that ACE2 is primarily transcribed from the proximal part of the ACE2 promoter in the pancreas. In the proximal part of the human ACE2 promoter, we further identified three functional HNF1 binding sites, as they have binding affinity for HNF1α and HNF1ß and are required for induction of promoter activity by HNF1ß in insulinoma cells. These three sites are well-conserved among mammalian species. Both HNF1α and HNF1ß induce expression of ACE2 mRNA and lead to elevated levels of ACE2 protein and ACE2 enzymatic activity in insulinoma cells. Furthermore, HNF1α dose-dependently increases ACE2 expression in primary pancreatic islet cells. We conclude that HNF1α can induce the expression of ACE2 in pancreatic islet cells via evolutionarily conserved HNF1 binding sites in the ACE2 promoter. Potential therapeutics aimed at counteracting functional HNF1α depletion in diabetes and MODY3 will thus have ACE2 induction in pancreatic islets as a likely beneficial effect.

A protocol to isolate, identify, and verify glucose- or carbohydrate-binding receptors.

Sensing, transport, and utilization of glucose is pivotal to the maintenance of energy homeostasis in animals. Although transporters involved in mobilizing glucose across different cellular compartments are fairly well known, the receptors that bind glucose to mediate its effects independently of glucose metabolism remain largely unrecognized. Establishing precise and reproducible methods to identify glucose receptors in the brain or other peripheral organs will pave the way for comprehending the role of glucose signaling pathways in maintaining, regulating, and reprogramming cellular metabolic needs. Identification of such potential glucose receptors will also likely lead to development of effective therapeutics for treatment of diabetes and related metabolic disorders. Commercially available biotin or radiolabeled glucose conjugates have low molecular weight; therefore, they do not provide enough sensitivity and density to isolate glucose receptors. Here, we describe a protocol to isolate, identify, and verify glucose-binding receptor/s using high molecular weight glucose (or other carbohydrate) conjugates. We have produced 30 kDa glucose- (or other carbohydrate-) biotin-polyacrylamide (PAA) conjugates with mole fractions of 80:5:15% respectively. These conjugates are used with biotin-streptavidin biochemistry, In-cell ELISA, and surface plasmon resonance (SPR) methods to isolate, identify, and verify glucose- or carbohydrate-binding receptors. We first demonstrate how streptavidin-coated magnetic beads are employed to immobilize glucose-biotin-PAA conjugates. Then, these beads are used to enrich and isolate glucose-binding proteins from tissue homogenates or from single-cell suspensions. The enriched or isolated proteins are subjected to mass spectrometry/proteomics to reveal the identity of top candidate proteins as potential glucose receptors. We then describe how the In-cell ELISA method is used to verify the interaction of glucose with its potential receptor through stable expression of the receptor in-vitro. We further demonstrate how a highly sensitive SPR method can be used to measure the binding kinetics of glucose with its receptor. In summary, we describe a protocol to isolate, identify, and verify glucose- or carbohydrate-binding receptors using magnetic beads, In-cell ELISA, and SPR. This protocol will form the future basis of studying glucose or carbohydrate receptor signaling pathways in health and in disease.

A kidney-hypothalamus axis promotes compensatory glucose production in response to glycosuria.

The kidneys facilitate energy conservation through reabsorption of nutrients including glucose. Almost all of the filtered blood glucose is reabsorbed by the kidneys. Loss of glucose in urine (glycosuria) is offset by an increase in endogenous glucose production to maintain normal energy supply in the body. How the body senses this glucose loss and consequently enhances glucose production is unclear. Using renal Glut2 knockout mice, we demonstrate that elevated glycosuria activates the hypothalamic-pituitary-adrenal axis, which in turn drives endogenous glucose production. This phenotype was attenuated by selective afferent renal denervation, indicating the involvement of the afferent nerves in promoting the compensatory increase in glucose production. In addition, through plasma proteomics analyses we observed that acute phase proteins - which are usually involved in body's defense mechanisms against a threat - were the top candidates which were either upregulated or downregulated in renal Glut2 KO mice. Overall, afferent renal nerves contribute to promoting endogenous glucose production in response to elevated glycosuria and loss of glucose in urine is sensed as a biological threat in mice. These findings may be useful in improving efficiency of drugs like SGLT2 inhibitors that are intended to treat hyperglycemia by enhancing glycosuria, but are met with a compensatory increase in endogenous glucose production.

Pancreatic angiotensin-converting enzyme 2 improves glycemia in angiotensin II-infused mice.

An overactive renin-angiotensin system (RAS) is known to contribute to type 2 diabetes mellitus (T2DM). Although ACE2 overexpression has been shown to be protective against the overactive RAS, a role for pancreatic ACE2, particularly in the islets of Langerhans, in regulating glycemia in response to elevated angiotensin II (Ang II) levels remains to be elucidated. This study examined the role of endogenous pancreatic ACE2 and the impact of elevated Ang II levels on the enzyme's ability to alleviate hyperglycemia in an Ang II infusion mouse model. Male C57bl/6J mice were infused with Ang II or saline for a period of 14 days. On the 7th day of infusion, either an adenovirus encoding human ACE2 (Ad-hACE2) or a control adenovirus (Ad-eGFP) was injected into the mouse pancreas. After an additional 7-8 days, glycemia and plasma insulin levels as well as RAS components expression and oxidative stress were assessed. Ang II-infused mice exhibited hyperglycemia, hyperinsulinemia, and impaired glucose-stimulated insulin secretion from pancreatic islets compared with control mice. This phenotype was associated with decreased ACE2 expression and activity, increased Ang II type 1 receptor (AT1R) expression, and increased oxidative stress in the mouse pancreas. Ad-hACE2 treatment restored pancreatic ACE2 expression and compensatory activity against Ang II-mediated impaired glycemia, thus improving ß-cell function. Our data suggest that decreased pancreatic ACE2 is a link between overactive RAS and impaired glycemia in T2DM. Moreover, maintenance of a normal endogenous ACE2 compensatory activity in the pancreas appears critical to avoid ß-cell dysfunction, supporting a therapeutic potential for ACE2 in controlling diabetes resulting from an overactive RAS.

Angiotensin converting enzyme 2: a new important player in the regulation of glycemia.

In spite of the novel antidiabetic drugs available on the market, type 2 diabetes mellitus (T2DM) affects nearly 25 million people in the USA and causes about 5% of all deaths globally each year. Given the rate and proportion by which T2DM is affecting human beings, it is indispensable to identify new therapeutic targets that can control the disease. Recent preclinical and clinical studies suggest that attenuating the activity of the renin-angiotensin system (RAS) could improve glycemia in diabetic patients. Angiotensin-converting enzyme 2 (ACE2) counteracts RAS overactivity by degrading angiotensin-II (Ang-II), a vasoconstrictor, to Ang-(1-7) which is a vasodilator. A decrease in ACE2 and an increase in A disintegrin and metalloproteinase (ADAM17)-mediated shedding activity have been observed with the progression of T2DM, suggesting the importance of this mechanism in the disease. Indeed, restoration of ACE2 improves glycemia in db/db and Ang-II-infused mice. The beneficial effects of ACE2 can be attributed to reduced oxidative stress and ADAM17 expression in the islets of Langerhans in addition to the improvement of blood flow to the ß-cells. The advantage of ACE2 over other RAS blockers is that ACE2 not only counteracts the negative effects of Ang-II but also increases Ang-(1-7)/Mas receptor (MasR) [a receptor through which Ang-(1-7) produces its actions] signaling in the cells. Increased Ang-(1-7)/MasR signaling has been reported to improve insulin sensitivity and glycemia in diabetic animals. Altogether, ACE2/Ang-(1-7)/MasR axis of the RAS appears to be protective in T2DM and strategies to restore ACE2 levels in the disease seem to be a promising therapy for Ang-II-mediated T2DM.

Osmotic Minipump Implantation for Increasing Glucose Concentration in Mouse Cerebrospinal Fluid.

Diabetes increases the risk of cognitive decline and impairs brain function. Whether or not this relationship between high glucose and cognitive deficits is causal remains elusive. Moreover, whether these deficits are mediated by an increase in glucose levels in cerebrospinal fluid (CSF) and/or blood is also unclear. There are very few studies investigating the direct effects of high CSF glucose levels on central nervous system (CNS) function, especially on learning and memory, since current diabetes models are not sufficiently developed to address such research questions. This article describes a method to chronically increase CSF glucose levels for 4 weeks by continuously infusing glucose into the lateral ventricle using osmotic minipumps in mice. The protocol was validated by measuring glucose levels in CSF. This protocol increased CSF glucose levels to ~328 mg/dL after infusion of a 50% glucose solution at a 0.25 µL/h flow rate, compared to a CSF glucose concentration of ~56 mg/dL in mice that received artificial cerebrospinal fluid (aCSF). Furthermore, this protocol did not affect blood glucose levels. Therefore, this method can be used to determine the direct effects of high CSF glucose on brain function or a specific neural pathway independently of changes in blood glucose levels. Overall, the approach described here will facilitate the development of animal models for testing the role of high CSF glucose in mediating features of Alzheimer's disease and/or other neurodegenerative disorders associated with diabetes.

Normal ß-Cell Glut2 Expression Is not Required for Regulating Glucose-Stimulated Insulin Secretion and Systemic Glucose Homeostasis in Mice.

OBJECTIVE: Glucose transporter 2 (GLUT2) is expressed in the pancreatic ß-cell, intestine, liver, and kidney in mice. Although GLUT2 is considered as a major regulator of insulin secretion, in vivo contribution of ß-cell Glut2 to glucose-stimulated insulin secretion and systemic glucose homeostasis is undefined. Therefore, the main objective of this study is to determine the role of ß-cell Glut2 in regulating insulin secretion and blood glucose levels in mice. METHODS: We produced mice in which we can knock down Glut2 at a desired time specifically in ß-cells (ß-Glut2 KD) by crossing Glut2LoxP/LoxP mice with Ins1CreERT2 mouse strain and using the Cre-Lox recombination technique. We measured fasting blood glucose levels, glucose tolerance, and glucose-stimulated insulin secretion in the ß-Glut2 KD mice. We used qRT-PCR and immunofluorescence to validate the deficiency of ß-cell Glut2 in ß-Glut2 KD mice. RESULTS: We report that both male and female ß-Glut2 KD mice have normal glucose-stimulated insulin secretion. Moreover, the ß-Glut2 KD mice exhibit normal fasting blood glucose levels and glucose tolerance. The ß-Glut2 KD mice have upregulated GLUT1 in islets. CONCLUSIONS: Our findings demonstrate that normal ß-cell Glut2 expression is not essential for regulating glucose-stimulated insulin secretion and systemic glucose homeostasis in mice. Therefore, the currently assumed role of ß-cell GLUT2 in regulating insulin secretion and blood glucose levels needs to be recalibrated. This will allow an opportunity to determine the contribution of other ß-cell glucose transporters or factors whose normal expression may be necessary for mediating glucose stimulated insulin secretion.

The acute effects of a DASH diet and whole food, plant-based diet on insulin requirements and related cardiometabolic markers in individuals with insulin-treated type 2 diabetes.

AIMS: There is limited research regarding insulin dosing changes following adoption of plant-based diets. We conducted a nonrandomized crossover trial utilizing two plant-based diets (Dietary Approaches to Stop Hypertension, or DASH, and Whole Food, Plant-Based, or WFPB) to assess acute changes in insulin requirements and associated markers among individuals with insulin-treated type 2 diabetes. METHODS: Participants (n = 15) enrolled in a 4-week trial with sequential, one-week phases: Baseline, DASH 1, WFPB, and DASH 2. Each diet was ad libitum and meals were provided. RESULTS: Compared to baseline, daily insulin usage was 24%, 39%, and 30% lower after DASH 1, WFPB, and DASH 2 weeks respectively (all p < 0.01). Insulin resistance (HOMA-IR) was 49% lower (p < 0.01) and the insulin sensitivity index was 38% higher (p < 0.01) at the end of the WFPB week before regressing toward baseline during DASH 2. Total, LDL, and HDL cholesterol, leptin, urinary glucose, and hsCRP decreased to a nadir at the end of the WFPB week before increasing during DASH 2. CONCLUSIONS: Adopting a DASH or WFPB diet can result in significant, rapid changes in insulin requirements, insulin sensitivity, and related markers among individuals with insulin-treated type 2 diabetes, with larger dietary changes producing larger benefits.

Species-specific inhibitor sensitivity of angiotensin-converting enzyme 2 (ACE2) and its implication for ACE2 activity assays.

Angiotensin-converting enzyme 2 (ACE2) is a component of the renin-angiotensin system, and its expression and activity have been shown to be reduced in cardiovascular diseases. Enzymatic activity of ACE2 is commonly measured by hydrolysis of quenched fluorescent substrates in the absence or presence of an ACE2-specific inhibitor, such as the commercially available inhibitor DX600. Whereas recombinant human ACE2 is readily detected in mouse tissues using 1 µM DX600 at pH 7.5, the endogenous ACE2 activity in mouse tissues is barely detectable. We compared human, mouse, and rat ACE2 overexpressed in cell lines for their sensitivity to inhibition by DX600. ACE2 from all three species could be inhibited by DX600, but the half maximal inhibitory concentration (IC(50)) for human ACE2 was much lower (78-fold) than for rodent ACE2. Following optimization of pH, substrate concentration, and antagonist concentration, rat and mouse ACE2 expressed in a cell line could be accurately quantified with 10 µM DX600 (>95% inhibition) but not with 1 µM DX600 (<75% inhibition). Validation that the optimized method robustly quantifies ACE2 in mouse tissues (kidney, brain, heart, and plasma) was performed using wild-type and ACE2 knockout mice. This study provides a reliable method for measuring human, as well as endogenous ACE2 activity in rodents. Our data underscore the importance of validating the effect of DX600 on ACE2 from each particular species at the experimental conditions employed.

Hypothalamic POMC deficiency increases circulating adiponectin despite obesity.

OBJECTIVE: The steep rise in the prevalence of obesity and its related metabolic syndrome have become a major worldwide health concerns. Melanocortin peptides from hypothalamic arcuate nucleus (Arc) POMC neurons induce satiety to limit food intake. Consequently, Arc Pomc-deficient mice (ArcPomc-/-) exhibit hyperphagia and obesity. Previous studies demonstrated that the circulating levels of adiponectin, a protein abundantly produced and secreted by fat cells, negatively correlate with obesity in both rodents and humans. However, we found that ArcPomc-/- mice have increased circulating adiponectin levels despite obesity. Therefore, we investigated the physiological function and underlying mechanisms of hypothalamic POMC in regulating systemic adiponectin levels. METHODS: Circulating adiponectin was measured in obese ArcPomc-/- mice at ages 4-52 weeks. To determine whether increased adiponectin was a direct result of ArcPomc deficiency or a secondary effect of obesity, we examined plasma adiponectin levels in calorie-restricted mice with or without a history of obesity and in ArcPomc-/- mice before and after genetic restoration of Pomc expression in the hypothalamus. To delineate the mechanisms causing increased adiponectin in ArcPomc-/- mice, we determined sympathetic outflow to adipose tissue by assessing epinephrine, norepinephrine, and tyrosine hydroxylase protein levels and measured the circulating adiponectin in the mice after acute norepinephrine or propranolol treatments. In addition, adiponectin mRNA and protein levels were measured in discrete adipose tissue depots to ascertain which fat depots contributed the most to the high level of adiponectin in the ArcPomc-/- mice. Finally, we generated compound Adiopoq-/-:ArcPomc-/- mice and compared their growth, body composition, and glucose homeostasis to the individual knockout mouse strains and their wild-type controls. RESULTS: Obese ArcPomc-/- female mice had unexpectedly increased plasma adiponectin compared to wild-type siblings at all ages greater than 8 weeks. Despite chronic calorie restriction to achieve normal body weights, higher adiponectin levels persisted in the ArcPomc-/- female mice. Genetic restoration of Pomc expression in the Arc or acute treatment of the ArcPomc-/- female mice with melanotan II reduced adiponectin levels to control littermate values. The ArcPomc-/- mice had defective thermogenesis and decreased epinephrine, norepinephrine, and tyrosine hydroxylase protein levels in their fat pads, indicating reduced sympathetic outflow to adipose tissue. Injections of norepinephrine into the ArcPomc-/- female mice reduced circulating adiponectin levels, whereas injections of propranolol significantly increased adiponectin levels. Despite the beneficial effects of adiponectin on metabolism, the deletion of adiponectin alleles in the ArcPomc-/- mice did not exacerbate their metabolic abnormalities. CONCLUSION: In summary, to the best of our knowledge, this study provides the first evidence that despite obesity, the ArcPomc-/- mouse model has high circulating adiponectin levels, which demonstrated that increased fat mass is not necessarily correlated with hypoadiponectinemia. Our investigation also found a previously unknown physiological pathway connecting POMC neurons via the sympathetic nervous system to circulating adiponectin, thereby shedding light on the biological regulation of adiponectin.

Hypothalamic POMC or MC4R deficiency impairs counterregulatory responses to hypoglycemia in mice.

OBJECTIVE: Life-threatening hypoglycemia is a major limiting factor in the management of diabetes. While it is known that counterregulatory responses to hypoglycemia are impaired in diabetes, molecular mechanisms underlying the reduced responses remain unclear. Given the established roles of the hypothalamic proopiomelanocortin (POMC)/melanocortin 4 receptor (MC4R) circuit in regulating sympathetic nervous system (SNS) activity and the SNS in stimulating counterregulatory responses to hypoglycemia, we hypothesized that hypothalamic POMC as well as MC4R, a receptor for POMC derived melanocyte stimulating hormones, is required for normal hypoglycemia counterregulation. METHODS: To test the hypothesis, we induced hypoglycemia or glucopenia in separate cohorts of mice deficient in either POMC or MC4R in the arcuate nucleus (ARC) or the paraventricular nucleus of the hypothalamus (PVH), respectively, and measured their circulating counterregulatory hormones. In addition, we performed a hyperinsulinemic-hypoglycemic clamp study to further validate the function of MC4R in hypoglycemia counterregulation. We also measured Pomc and Mc4r mRNA levels in the ARC and PVH, respectively, in the streptozotocin-induced type 1 diabetes mouse model and non-obese diabetic (NOD) mice to delineate molecular mechanisms by which diabetes deteriorates the defense systems against hypoglycemia. Finally, we treated diabetic mice with the MC4R agonist MTII, administered stereotaxically into the PVH, to determine its potential for restoring the counterregulatory response to hypoglycemia in diabetes. RESULTS: Stimulation of epinephrine and glucagon release in response to hypoglycemia or glucopenia was diminished in both POMC- and MC4R-deficient mice, relative to their littermate controls. Similarly, the counterregulatory response was impaired in association with decreased hypothalamic Pomc and Mc4r expression in the diabetic mice, a phenotype that was not reversed by insulin treatment which normalized glycemia. In contrast, infusion of an MC4R agonist in the PVH restored the counterregulatory response in diabetic mice. CONCLUSION: In conclusion, hypothalamic Pomc as well as Mc4r, both of which are reduced in type 1 diabetic mice, are required for normal counterregulatory responses to hypoglycemia. Therefore, enhancing MC4R function may improve hypoglycemia counterregulation in diabetes.

Electrical stimulation of renal nerves for modulating urine glucose excretion in rats.

BACKGROUND: The role of the kidney in glucose homeostasis has gained global interest. Kidneys are innervated by renal nerves, and renal denervation animal models have shown improved glucose regulation. We hypothesized that stimulation of renal nerves at kilohertz frequencies, which can block propagation of action potentials, would increase urine glucose excretion. Conversely, we hypothesized that low frequency stimulation, which has been shown to increase renal nerve activity, would decrease urine glucose excretion. METHODS: We performed non-survival experiments on male rats under thiobutabarbital anesthesia. A cuff electrode was placed around the left renal artery, encircling the renal nerves. Ureters were cannulated bilaterally to obtain urine samples from each kidney independently for comparison. Renal nerves were stimulated at kilohertz frequencies (1-50 kHz) or low frequencies (2-5 Hz), with intravenous administration of a glucose bolus shortly into the 25-40-min stimulation period. Urine samples were collected at 5-10-min intervals, and colorimetric assays were used to quantify glucose excretion and concentration between stimulated and non-stimulated kidneys. A Kruskal-Wallis test was performed across all stimulation frequencies (α = 0.05), followed by a post-hoc Wilcoxon rank sum test with Bonferroni correction (α = 0.005). RESULTS: For kilohertz frequency trials, the stimulated kidney yielded a higher average total urine glucose excretion at 33 kHz (+ 24.5%; n = 9) than 1 kHz (- 5.9%; n = 6) and 50 kHz (+ 2.3%; n = 14). In low frequency stimulation trials, 5 Hz stimulation led to a lower average total urine glucose excretion (- 40.4%; n = 6) than 2 Hz (- 27.2%; n = 5). The average total urine glucose excretion between 33 kHz and 5 Hz was statistically significant (p < 0.005). Similar outcomes were observed for urine flow rate, which may suggest an associated response. No trends or statistical significance were observed for urine glucose concentrations. CONCLUSION: To our knowledge, this is the first study to investigate electrical stimulation of renal nerves to modulate urine glucose excretion. Our experimental results show that stimulation of renal nerves may modulate urine glucose excretion, however, this response may be associated with urine flow rate. Future work is needed to examine the underlying mechanisms and identify approaches for enhancing regulation of glucose excretion.

Reduced renal sympathetic nerve activity contributes to elevated glycosuria and improved glucose tolerance in hypothalamus-specific Pomc knockout mice.

OBJECTIVE: Hypothalamic arcuate nucleus-specific pro-opiomelanocortin deficient (ArcPomc-/-) mice exhibit improved glucose tolerance despite massive obesity and insulin resistance. We demonstrated previously that their improved glucose tolerance is due to elevated glycosuria. However, the underlying mechanisms that link glucose reabsorption in the kidney with ArcPomc remain unclear. Given the function of the hypothalamic melanocortin system in controlling sympathetic outflow, we hypothesized that reduced renal sympathetic nerve activity (RSNA) in ArcPomc-/- mice could explain their elevated glycosuria and consequent enhanced glucose tolerance. METHODS: We measured RSNA by multifiber recording directly from the nerves innervating the kidneys in ArcPomc-/- mice. To further validate the function of RSNA in glucose reabsorption, we denervated the kidneys of WT and diabetic db/db mice before measuring their glucose tolerance and urine glucose levels. Moreover, we performed western blot and immunohistochemistry to determine kidney GLUT2 and SGLT2 levels in either ArcPomc-/- mice or the renal-denervated mice. RESULTS: Consistent with our hypothesis, we found that basal RSNA was decreased in ArcPomc-/- mice relative to their wild type (WT) littermates. Remarkably, both WT and db/db mice exhibited elevated glycosuria and improved glucose tolerance after renal denervation. The elevated glycosuria in obese ArcPomc-/-, WT and db/db mice was due to reduced renal GLUT2 levels in the proximal tubules. Overall, we show that renal-denervated WT and diabetic mice recapitulate the phenotype of improved glucose tolerance and elevated glycosuria associated with reduced renal GLUT2 levels observed in obese ArcPomc-/- mice. CONCLUSION: Hence, we conclude that ArcPomc is essential in maintaining basal RSNA and that elevated glycosuria is a possible mechanism to explain improved glucose tolerance after renal denervation in drug resistant hypertensive patients.

Reprogramming the body weight set point by a reciprocal interaction of hypothalamic leptin sensitivity and Pomc gene expression reverts extreme obesity.

OBJECTIVE: A major challenge for obesity treatment is the maintenance of reduced body weight. Diet-induced obese mice are resistant to achieving normoweight once the obesogenic conditions are reversed, in part because lowered circulating leptin leads to a reduction in metabolic rate and a rebound of hyperphagia that defend the previously elevated body weight set point. Because hypothalamic POMC is a central leptin target, we investigated whether changes in circulating leptin modify Pomc expression to maintain normal energy balance in genetically predisposed obese mice. METHODS: Mice with reversible Pomc silencing in the arcuate nucleus (ArcPomc (-/-)) become morbidly obese eating low-fat chow. We measured body composition, food intake, plasma leptin, and leptin sensitivity in ArcPomc (-/-) mice weight-matched to littermate controls by calorie restriction, either from weaning or after developing obesity. Pomc was reactivated by tamoxifen-dependent Cre recombinase transgenes. Long acting PASylated leptin was administered to weight-reduced ArcPomc (-/-) mice to mimic the super-elevated leptin levels of obese mice. RESULTS: ArcPomc (-/-) mice had increased adiposity and leptin levels shortly after weaning. Despite chronic calorie restriction to achieve normoweight, ArcPomc (-/-) mice remained moderately hyperleptinemic and resistant to exogenous leptin's effects to reduce weight and food intake. However, subsequent Pomc reactivation in weight-matched ArcPomc (-/-) mice normalized plasma leptin, leptin sensitivity, adiposity, and food intake. In contrast, extreme hyperleptinemia induced by PASylated leptin blocked the full restoration of hypothalamic Pomc expression in calorie restricted ArcPomc (-/-) mice, which consequently regained 30% of their lost body weight and attained a metabolic steady state similar to that of tamoxifen treated obese ArcPomc (-/-) mice. CONCLUSIONS: Pomc reactivation in previously obese, calorie-restricted ArcPomc (-/-) mice normalized energy homeostasis, suggesting that their body weight set point was restored to control levels. In contrast, massively obese and hyperleptinemic ArcPomc (-/-) mice or those weight-matched and treated with PASylated leptin to maintain extreme hyperleptinemia prior to Pomc reactivation converged to an intermediate set point relative to lean control and obese ArcPomc (-/-) mice. We conclude that restoration of hypothalamic leptin sensitivity and Pomc expression is necessary for obese ArcPomc (-/-) mice to achieve and sustain normal metabolic homeostasis; whereas deficits in either parameter set a maladaptive allostatic balance that defends increased adiposity and body weight.

Hypothalamic POMC Deficiency Improves Glucose Tolerance Despite Insulin Resistance by Increasing Glycosuria.

Hypothalamic proopiomelanocortin (POMC) is essential for the physiological regulation of energy balance; however, its role in glucose homeostasis remains less clear. We show that hypothalamic arcuate nucleus (Arc)POMC-deficient mice, which develop severe obesity and insulin resistance, unexpectedly exhibit improved glucose tolerance and remain protected from hyperglycemia. To explain these paradoxical phenotypes, we hypothesized that an insulin-independent pathway is responsible for the enhanced glucose tolerance. Indeed, the mutant mice demonstrated increased glucose effectiveness and exaggerated glycosuria relative to wild-type littermate controls at comparable blood glucose concentrations. Central administration of the melanocortin receptor agonist melanotan II in mutant mice reversed alterations in glucose tolerance and glycosuria, whereas, conversely, administration of the antagonist Agouti-related peptide (Agrp) to wild-type mice enhanced glucose tolerance. The glycosuria of ArcPOMC-deficient mice was due to decreased levels of renal GLUT 2 (rGLUT2) but not sodium-glucose cotransporter 2 and was associated with reduced renal catecholamine content. Epinephrine treatment abolished the genotype differences in glucose tolerance and rGLUT2 levels, suggesting that reduced renal sympathetic nervous system (SNS) activity is the underlying mechanism for the observed glycosuria and improved glucose tolerance in ArcPOMC-deficient mice. Therefore, the ArcPOMC-SNS-rGLUT2 axis is potentially an insulin-independent therapeutic target to control diabetes.