Leptin enhances hypothalamic lactate dehydrogenase A (LDHA)–dependent glucose sensing to lower glucose production in high-fat–fed rats

The responsiveness of glucose sensing per se to regulate whole-body glucose homeostasis is dependent on the ability of a rise in glucose to lower hepatic glucose production and increase peripheral glucose uptake in vivo. In both rodents and humans, glucose sensing is lost in diabetes and obesity, but the site(s) of impairment remains elusive. Here, we first report that short-term high-fat feeding disrupts hypothalamic glucose sensing to lower glucose production in rats. Second, leptin administration into the hypothalamus of high-fat–fed rats restored hypothalamic glucose sensing to lower glucose production during a pancreatic (basal insulin)-euglycemic clamp and increased whole-body glucose tolerance during an intravenous glucose tolerance test. Finally, both chemical inhibition of hypothalamic lactate dehydrogenase (LDH) (achieved via hypothalamic LDH inhibitor oxamate infusion) and molecular knockdown of LDHA (achieved via hypothalamic lentiviral LDHA shRNA injection) negated the ability of hypothalamic leptin infusion to enhance glucose sensing to lower glucose production in high fat–fed rats. In summary, our findings illustrate that leptin enhances LDHA-dependent glucose sensing in the hypothalamus to lower glucose production in high-fat–fed rodents in vivo.

The hallmark feature of type 2 diabetes is traditionally viewed as insulin resistance resulting in elevated hepatic glucose production and impaired glucose uptake in peripheral tissues. However, a change in glucose responsiveness as defined as the ability of glucose sensing per se in regulating glucose production and uptake could also potentially contribute to glucose dysregulation in diabetes.
For instance, studies conducted in both rodents and humans report that, independent of changes in glucoregulatory hormones during the pancreatic clamps, a doubling of circulating glucose levels inhibits glucose production and stimulates peripheral glucose uptake in healthy conditions (1)(2)(3). However, the same increment in the plasma glucose levels fails to lower glucose production in diabetic rodents (1) and humans (2,3), thereby illustrating glucose unresponsiveness as a feature in diabetic conditions. Glucose unresponsiveness in obese individuals has been determined from a mathematical minimal model analysis following a frequently sampled intravenous (i.v.) or oral glucose tolerance test (GTT). 4 Indeed, a reduction in glucose responsiveness was associated with impaired glucose tolerance in obese individuals (4). As seen in humans, obese and diabetic rodents such as the leptin-deficient ob/ob mice also display a loss of glucose sensing (5). Thus, the ability of glucose sensing per se to regulate its own metabolism is highly relevant in obesity and diabetes.
To date, the site(s) of glucose sensing impairment that leads to glucose unresponsiveness in obesity and diabetes remains elusive. In this regard, a hypothalamic glucose sensing mechanism has been documented to maintain glucose homeostasis in the context of hypoglycemia-induced counter regulation (6) as well as short-term hyperglycemia (7) in chow-fed rodents. However, whether changes in hypothalamic glucose sensing mechanisms dysregulate glucose metabolism in obesity and diabetes remains unclear. Interestingly, direct restoration of leptin signaling in the hypothalamus of the leptin receptordeficient Koletsky (fa k /fa k ) rats in parallel improves glucose tolerance (8), whereas central administration of FGF19 improves glucose effectiveness and glucose tolerance in ob/ob mice as estimated from the minimal model analysis (9). Based on these collaborative findings of glucose sensing and hormonal action in the hypothalamus, we hypothesize that hypothalamic leptin action enhances the hypothalamic glucose sensing machinery to regulate glucose metabolism in obesity and diabetes in vivo.
We next administered glucose directly into the mediobasal hypothalamus of healthy chow-fed rats for 6 h and found that, consistent with previous studies (7), the glucose infusion rate (Fig. 1F) was increased in parallel to a reduction of glucose production (Fig. 1G), but no changes were detected in glucose uptake (Fig. 1H) during the pancreatic (basal insulin)-euglycemic clamps (Table 1). Importantly, repeating hypothalamic glucose administration into 3-day high-fat-fed rats completely failed to lower plasma glucose levels because of the inability of glucose production to be inhibited (Fig. 1, F-H) independently of changes in plasma insulin and glucose levels (Table 1). Thus, we discovered, for the first time to our knowledge, that high-fat feeding disrupts glucose sensing within the hypothalamus, which lowers glucose production in vivo. It remains unknown whether the diet composition and/or the hyperphagic response of HFD is responsible for the impairment of hypothalamic glucose sensing, although it has been documented that the hyperphagic response, but not the diet composition, of HFD is responsible for the impairment of hypothalamic fatty acid sensing mechanisms (15).
Next, we assessed whether leptin can restore hypothalamic glucose sensing by infusing leptin directly into the mediobasal hypothalamus of glucose-unresponsive 3-day high-fat-fed rats Leptin and glucose sensing in the hypothalamus (Fig. 1A). During the pancreatic (basal insulin)-euglycemic clamps, we found that upon coinfusion with leptin hypothalamic glucose infusion retained its ability to lower glucose production, which led to an increase in glucose infusion rate to maintain euglycemia (Figs. 2, A and B), whereas glucose uptake remained comparable (Fig. 2C). Although such pancreatic clamp studies illustrate that leptin is able to restore hypothalamic glucose sensing in lowering glucose production independently of changes in plasma insulin and glucose levels, one would wonder whether leptin can still impact glucose metabolism while the glucoregulatory hormones are allowed to change at will within the circulation. Thus, we next evaluated whether direct leptin administration into the mediobasal hypothalamus of 3-day high-fat-fed rats alters whole-body glucose tolerance during an i.v. GTT (Fig. 2D). Despite the fact that glucoregulatory hormones are allowed to change at will, hypothalamic administration of leptin at a dosage equal to that used in the pancreatic clamp studies is sufficient to increase glucose tolerance during i.v. GTT (Fig. 2E). Collectively, these two sets of data show that hypothalamic leptin administration enhances glucoregulatory control in 3-day HFD-induced hypothalamic glucose-unresponsive rodents. Of note, hypothalamic leptin action remains intact in 3-day HFD rats, consistent with the fact that central leptin delivery activates hypothalamic STAT3 in 3-day HFD rats (16).
To begin evaluating the downstream molecular pathway of glucose sensing that is necessary for leptin to improve glucose sensing, we tested whether lactate dehydrogenase (LDH) in the hypothalamus is necessary for leptin-glucose action (Fig. 3A). We hypothesize that LDH is the primary downstream target as chemical inhibition of LDH-dependent lactate metabolism in the hypothalamus negates glucose infusion to lower glucose production in healthy rodents (7), whereas shuttling of glucosederived lactate from astrocytes into neurons serves as an important step to provide neuronal fuel ( Fig. 3A) (17)(18)(19). We reason that if leptin restores glucose sensing in the hypothalamus to control whole-body glucose metabolism then LDH-dependent lactate metabolism could be a crucial step. These LDH-targeted experiments would also be necessary to ensure that the currently described glucoregulatory effect of hypothalamic leptin infusion with glucose is specifically targeting glucose sensing mechanisms. This is because a previous study has documented that central leptin infusion into HFD rats in pancreatic (basal insulin)-euglycemic clamp conditions comparable to those in the current study still lowers glucose production in the absence of central glucose coinfusion (20), suggesting that hypothalamic leptin action lowers glucose production in hypothalamic glucose sensing-dependent and -independent pathways.
To address whether leptin enhances hypothalamic LDH-dependent glucose sensing mechanisms, chemical and molecular approaches targeted to the mediobasal hypothalamus were used. We first infused oxamate, a competitive inhibitor of both LDHA (expressed in astrocytes (21)(22)(23)(24)) and LDHB (expressed in neurons (22)(23)(24)), into the mediobasal hypothalamus (Fig.  1A) at a dose that would negate hypothalamic glucose infusion to lower glucose production in rodents (7). Here, we found that infusion of oxamate together with leptin prevented the ability of leptin to enhance hypothalamic glucose sensing in increasing the glucose infusion rate (Fig. 3B) and lowering glucose production (Fig. 3C) without any detectable differences in glucose uptake (Fig. 3D) and plasma insulin and glucose levels ( Table 2). Importantly, coinfusion of oxamate with only leptin into the hypothalamus did not negate leptin's ability to alter glucose Leptin and glucose sensing in the hypothalamus kinetics (Fig. 3, B-D). Two important implications are derived from this control experiment. (i) Consistent with previous literature, hypothalamic leptin administration into 3-day HFD rats during the pancreatic basal insulin clamp lowers glucose production (20) in the absence of glucose coinfusion into the mediobasal hypothalamus (MBH), indicating that leptin action in the brain lowers glucose production in a hypothalamic glucose sensing-independent fashion. (ii) The presence of oxamate did not negate such leptin-glucose production-lowering effect in the absence of glucose coinfusion, indicating that oxamate administration is selectively blocking the glucose sensing-dependent pathway and that leptin in the brain can also, in parallel, lower glucose production in a hypothalamic glucose sensing-dependent fashion. Thus, LDH-dependent lactate metabolism in the hypothalamus is a necessary step for leptin to enhance glucose sensing.
Next, we selectively evaluated the role of LDHA-dependent lactate metabolism by injecting a lentivirus expressing the shRNA of LDHA into the mediobasal hypothalamus of rats. We first confirmed that LDHA expression was reduced by ϳ40% in mediobasal hypothalamic tissues harvested from HFD rats injected with LDHA shRNA compared with the mismatch sequence (MM) control, whereas LDHB expression was unaltered (Fig. 4A). Second, we found that hypothalamic glucose versus saline infusion into MM-injected chow-fed rats was equally as effective as compared with non-injected rats (Fig. 1,  F-H) to increase the glucose infusion rate (Fig. 4B) and lower glucose production (Fig. 4C), independently of changes in plasma insulin and glucose levels (Table 3). However, hypothalamic glucose infusion failed to alter glucose metabolism in LDHA shRNA-injected rats (Fig. 4, B and C), illustrating for the first time that the hypothalamic LDHA-dependent pathway is necessary for glucose sensing to lower glucose production in chow-fed rats. Although hypothalamic glucose infusion failed to alter glucose metabolism in HFD-fed MM-or LDHA shRNA-injected rats (Fig. 4, B and C) as well, hypothalamic glucose-infused and LDHA shRNA-injected HFD rats also failed to respond to hypothalamic leptin administration to alter glucose metabolism (Fig. 4, B and C). In direct contrast, hypothalamic leptin infusion was effective in restoring glucose sensing in HFD MM-injected rats (Fig. 4, B and C). Glucose uptake and plasma insulin and glucose levels were comparable among groups ( Fig. 4D and Table 3). Thus, molecular knockdown of LDHA in the hypothalamus negates the effect of leptin to enhance the glucose sensing mechanism.

Discussion
The current set of findings collectively indicates that leptin enhances hypothalamic LDHA-dependent glucose sensing to lower glucose production in HFD rats in vivo and highlights several important implications. First, the defects of glucose unresponsiveness in regulating hepatic glucose production and glucose tolerance as previously reported in rodents (1, 5) and humans (2-4) with diabetes and obesity could lie within the hypothalamus, although future studies are warranted to assess hypothalamic glucose sensing mechanisms in chronic highfat-fed, obese, and/or diabetic rodent models. Second, the loss of glucose sensing in leptin-deficient ob/ob mice (5) could be restored by direct hypothalamic leptin administration as restoration of hypothalamic leptin action is effective in improving glucose tolerance in leptin receptor-deficient Koletsky (fa k / fa k ) rats (8) as well. Importantly, such postulated leptin-dependent rescue of glucose sensing could be due to an enhancement of hypothalamic glucose sensing. However, this postulation must be accepted with caution because central leptin delivery may not restore hypothalamic glucose sensing in rats fed a HFD for more than 5 weeks as central leptin action fails to lower food intake in 5-week HFD-fed rats (25), and central leptin resistance may additionally exist at the level of leptin blood-brain barrier transport (26). Third, given that LDHA is selectively expressed in astrocytes (21-24) and that leptin enhances

Leptin and glucose sensing in the hypothalamus
LDHA-dependent glucose sensing in the hypothalamus as currently described, leptin signaling and glucose-lactate metabolism could intersect within astrocytes to improve whole-body metabolic control. This postulation is consistent with findings indicating that leptin signaling in astrocytes regulates feeding (27) and that insulin signaling in astrocytes enhances central glucose sensing (28). In summary, here we report, for the first time to our knowledge, that leptin enhances LDHA-dependent hypothalamic glucose sensing to regulate glucose production in high-fat-fed in vivo conditions.

Animal preparation and surgeries
Adult male Sprague-Dawley rats (Charles River Laboratories, Saint-Constant, Quebec, Canada) initially weighing 280 -300 g were studied. Rats were housed in individual cages and subjected to a standard light-dark cycle (7 a.m. light, 7 p.m. dark) and had ad libitum access to drinking water and regular chow or HFD. The regular chow (Teklad Diet 7002, Harlan Laboratories, Madison, WI) contained 18% fat, 33% protein,

Leptin and glucose sensing in the hypothalamus
and 49% carbohydrate content (3.1 kcal/g total metabolizable energy), whereas the 10% lard oil-enriched HFD (TestDiet 571R, Purina Mills) contained 34% fat, 22% protein, and 44% carbohydrate (3.9 kcal/g total metabolizable energy). Ketamine (60 mg/kg) and xylazine (8 mg/kg) were used to anesthetize rats during surgeries. A 26-gauge stainless steel bilateral guide catheter (C235G, Plastics One Inc.) was stereotaxically placed into the MBH using the coordinates 3.1 mm posterior to bregma, 0.4 mm lateral of midline, and 9.6 mm below skull surface as described (29). After recovery for 7-8 days, vascular catheters were inserted into the internal jugular vein and carotid artery for infusion and blood sampling (29). Postsurgical body weight and food intake were checked daily, and only those rats that attained a minimum of 90% of their prevascular surgery body weight underwent subsequent in vivo studies. All procedures complied with the rules of the Institutional Animal Care and Use Committee of University Health Network.

Lentivirus injection
Immediately following brain surgery, a group of rats received 3 l of lentivirus expressing shRNA to LDHA (1.0 ϫ 10 6 infectious units; sc-270631-V, Santa Cruz Biotechnology, Inc., Dallas, TX) or a mismatch sequence as a control (1.0 ϫ 10 6 infectious units; sc-108080, Santa Cruz Biotechnology, Inc.) through each side of the MBH catheters that target the arcuate nucleus (29,30). Eight days after MBH cannulation and virus injection, vascular catheterization was performed as described above in rats that would undergo clamp experiments.

Pancreatic (basal insulin)-euglycemic clamps
Four days following vascular catheterization, rats were subjected to a 4 -6-h fast before the clamp experiments. On the day of the clamp, conscious, unrestrained rats received the following MBH infusions at 0.33 l/h (CMA 400 syringe pump, CMA Microdialysis, Inc., North Chelmsford, MA): (i) 0.9% saline, (ii) glucose (Sigma-Aldrich; 2 mM for 6 h; previously documented to lower glucose production (7)), (iii) glucose (2 mM) ϩ leptin (R&D Systems, Minneapolis, MN; 46 ng/l), (iv) lactate dehydrogenase blocker oxamate (Millipore Sigma; 50 mM; first given as bolus (0.33 l)), (v) glucose (2 mM) ϩ oxamate (50 mM), or (vi) glucose (2 mM) ϩ leptin (46 ng/l) ϩ oxamate (50 mM). Leptin was infused at 33 ng/h for 5 h, and this dose was chosen based on a previous study that reports leptin administered at 33 ng/h into the MBH for 6 h exhibits metabolic control (31). In addition, we chose to infuse leptin 1 h after glucose was started as we attempted to have the HFD rats first engage in glucose sensing mechanisms before examining whether leptin alters glucose sensing. Oxamate concentration was based on a study that reported MBH oxamate at 50 mM sufficiently negated glucose sensing in healthy rats (7). Infusions of oxamate were started at t ϭ Ϫ180 min followed by glucose ϩ oxamate at t ϭ Ϫ150 min. Leptin ϩ glucose ϩ oxamate was then started at t ϭ Ϫ90 min and maintained until the end of the clamps, t ϭ 210 min. Clamp methodology was performed as follows. At t ϭ 0 min, a primed, continuous infusion (PHD2000 syringe pump, Harvard Apparatus, Saint Laurent, Quebec, Canada) of [3-3 H]glucose (PerkinElmer Life Sciences; 40-Ci bolus ϩ 0.4 Ci min Ϫ1 infusion) was started and maintained until the end Table 3 Plasma insulin and glucose concentrations during the basal and clamp periods relating to

Leptin and glucose sensing in the hypothalamus
of the clamps (t ϭ 210 min) to measure glucose kinetics using tracer-dilution methodology. The glucose turnover was calculated using steady-state formulas where the rate of appearance of glucose is calculated using [3-3 H]glucose. The total rate of appearance of endogenous glucose production is equivalent to the rate of glucose utilization during the basal period (t ϭ 60 -90 min). The pancreatic (basal insulin)-euglycemic clamp was initiated at t ϭ 90 min with a primed, continuous infusion of insulin (1.2 milliunits kg Ϫ1 min Ϫ1 ) and somatostatin (3 g kg Ϫ1 min Ϫ1 ) and a variable infusion of 25% glucose to maintain glycemia at a similar level to the basal period and maintained until t ϭ 210 min as described (29). Plasma samples were obtained every 10 min for the determination of [3-3 H]glucose concentrations. At the end of the experiment, rats were anesthetized and injected with 3 l of bromphenol blue through each side of the MBH catheter to verify the correct placement of the catheter. The MBH wedges were then collected, frozen in liquid nitrogen, and stored at Ϫ80°C for subsequent analysis.

Intravenous glucose tolerance test
One day after vascular catheterization, separate groups of MBH rats were placed on HFD for 3 consecutive days and subsequently subjected to an overnight (16 -18-h) fast to undergo the i.v. GTT as described (32,33). MBH infusions (0.33 l/h; CMA 400 syringe pump, CMA Microdialysis, Inc., North Chelmsford, MA) of 0.9% saline or leptin (33 ng/h for 5 h) were commenced at t ϭ Ϫ240 min and maintained until the end of the experiment at t ϭ 60 min to ensure that rats received the same duration of MBH leptin treatment as clamp experiments. After t ϭ 0 min, blood samples were obtained, an intravenous bolus of glucose (20% glucose; 0.25 g/kg) was injected followed by flushing with saline. The jugular vein catheter was used to administer the injections, and the carotid artery catheter was used to sample blood to measure plasma glucose for 60 min following the bolus glucose injection.

Body composition
On the morning before the clamp experiments, the body composition (fat mass, lean mass, free water, and total body water) of the rats was assessed with an EchoMRI TM (Echo Medical Systems, Houston, TX).

Western blot analyses
MBH samples from rats that received lentivirus injections of LDHA shRNA or MM were lysed on ice with a handheld homogenizer in a lysis buffer containing 50 mM Tris-HCl (pH 7.5), 1 mM EGTA, 1 mM EDTA, 1% (w/v) Nonidet P40, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 0.27 M sucrose, 1 M dithiothreitol (DTT), and protease inhibitor mixture (Roche Applied Science). The protein concentration of homogenized tissues was determined using the Pierce 660 nm Protein Assay (Thermo Scientific). Lysates (10 -30 g) obtained as described above were subjected to electrophoresis on a 10% polyacrylamide gel and transferred to nitrocellulose membranes. The membranes were incubated with blocking solution (5% BSA in Tris-buffered saline containing 0.2% Tween 20 (TBS-T)) for 1 h at room temperature followed by primary antibody incubation with the following antibodies diluted 1:1,000 in 5% BSA in TBS-T: anti-LDHA (ab135366 rabbit, Abcam, Cambridge, MA; same antibody as reported previously (34)) overnight at 4°C or anti-LDHB (ab85318 mouse, Abcam) and anti-␤-tubulin (MAB1637 mouse, Millipore Sigma) for 2 h at 4°C. The blots were then washed four times with TBS-T and incubated with secondary HRP-conjugated antibodies in 5% skimmed milk for 1 h. After repeating the washing steps, the signal was detected with enhanced chemiluminescence (ECL) reagent. Immunoblots were detected using a MicroChemi 4.2 chemiluminescence imaging system and quantified with GelQuant image analysis software (DNR Bio-Imaging Systems, Jerusalem, Israel).

Statistics
Unpaired Student's t tests were performed in the statistical analysis of two groups. Where comparisons were made across more than two groups, analysis of variance was performed and, if significant, followed by Tukey's post hoc tests. Significance was accepted as p Ͻ 0.05. For clamp experiments, the time period of 60 -90 min was averaged for the basal condition, and the period of 180 -210 min was averaged for the clamp condition.